Patent Publication Number: US-2023159961-A1

Title: Biosynthesis of chemically diversified non-natural terpene products

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is U.S. National Stage Filing under 35 U.S.C. 371 of International Patent Application No. PCT/US2020/059144, filed Nov. 5, 2020, and published WO 2021/092200 A1 on May 14, 2021, which claims the benefit of U.S. Provisional Patent Appl. Ser. No. 62/930,898, filed Nov. 5, 2019, which is incorporated by reference as if fully set forth herein. 
     Incorporation by Reference of Sequence Listing Provided as a Text File A Sequence Listing is provided herewith as a text file, “2089186.txt” created on Nov. 5, 2020 and having a size of 303,104 bytes. The contents of the text file are incorporated by reference herein in their entirety. 
    
    
     BACKGROUND 
     Plant diterpenes occupy a unique molecular space with critical pharmaceutical applications over a diverse spectrum including anti-cancer, anti-microbial and immunomodulatory properties. In addition, plant-derived terpenoids have a wide range of commercial and industrial uses. Examples of uses for terpenoids include specialty fuels, agrochemicals, fragrances, nutraceuticals and pharmaceuticals. However, currently available methods for synthesis, extraction, and purification of terpenoids from the native plant sources have limited economic sustainability. Moreover, currently available methods for do not provide the substrates and methods for biosynthesis of non-natural terpenoids. 
     The enzymes of terpene synthesis pathways are evolutionarily optimized to deliver bioactive molecules, novel molecular scaffolds and chemistry. Yet, cost-effective synthesis and access to analogs of plant diterpenoids and their derivatives is technologically limited on the levels of isolation, purification, detection and synthesis. 
     SUMMARY 
     While the terpene biosynthetic enzymes catalyze some of nature&#39;s most complex chemistries, the natural entry into terpenoid pathways is limited to a single precursor, geranylgeranyl diphosphate (GGPP). And although GGPP is a compound having all-trans double bonds, it has been recently shown that the cis-prenyl is also relevant in other plant species. See, e.g., https://doi.org/10.1111/tpj.14957. However, as described herein a variety of non-natural substrates can be used by terpene biosynthetic enzymes to produce structurally diverse unnatural diterpene analogs and unnatural terpene key intermediates for further functionalization. Products formed using the non-natural substrates and methods described herein are bioactive and compared to related natural compounds they have modulated specificity against their molecular targets. 
     For example, methods are described herein that include contacting an unnatural substrate with one or more enzymes that can synthesize a terpene to generate a primary terpene product. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG.  1 A- 1 C  illustrate a process for evaluating unnatural substrates as candidates to produce novel diterpene-inspired drug candidates.  FIG.  1 A  illustrates building and screening unnatural substrates for cyclization into unnatural decalin-core and irregular scaffolds.  FIG.  1 B  illustrates combinatorial biosynthesis of unnatural decalin-core scaffolds.  FIG.  1 C  illustrates bioprocessing of unnatural forskolin and jolkinol c compounds. The decalin-core representative, forskolin, and an irregular jolkinol C structure are shown. Enzyme families are delineated by dashed lines. 
         FIG.  2    illustrates the modular biosynthesis of diterpenes from the substrate geranylgeranyl diphosphate (GGPP). 
         FIG.  3    schematically illustrates development of unnatural terpene scaffolds where the diversity of diterpenes that can be formed from geranylgeranyl diphosphate (GGPP) using a variety of different enzymes (represented as building blocks) and unnatural substrates. Such unnatural substrates can be converted into novel diterpenes through combinatorial biochemistry. 
         FIG.  4    is a schematic diagram of the strategy and process for making a library of unnatural substrates for terpenoid synthesis. 
         FIG.  5 A- 5 D  illustrate in vitro conversion of unGGPP by a casbene synthase to a macrocyclic product with a fragmentation pattern and an increase in m/z that was predicted by the inventors.  FIG.  5 A  illustrates the retention time of the product formed by casbene synthase with geranylgeranyl diphosphate (GGPP) as substrate, as detected by gas chromatography.  FIG.  5 B  illustrates the retention time of the product formed by casbene synthase with an unnatural methyl derivative of geranylgeranyl diphosphate (unGGPP) as substrate, as detected by gas chromatography.  FIG.  5 C  illustrates the mass (m/z) of fragments of the product formed by casbene synthase with geranylgeranyl diphosphate (GGPP) as substrate, as detected by GC-MS.  FIG.  5 D  illustrates the mass (m/z) of fragments of the product formed by casbene synthase with unnatural methyl derivative of geranylgeranyl diphosphate (unGGPP) as substrate, as detected by GC-MS. 
         FIG.  6 A- 6 B  illustrate which enzymes can produce a product after enzymatic action on unnatural variants of GGPP (unGGPP).  FIG.  6 A  shows structures of unnatural variants of GGPP (unGGPP) and lists their names. Three classes of chemistries are represented by different hatching overlays for the different unGGPP substrates.  FIG.  6 B  shows which of fifteen heterologously expressed diTPS produce novel unnatural product analogs (indicated by cross-hatched circle), where the type of cross-hatching overlay corresponds to the substrate types listed in  FIG.  6 B . GC-MS analyses from in vitro assays were used to analyze which of the fifteen diTPS enzymes generate novel unnatural product analogs generated. Top nine rows were labdane-type class II diTPS assayed with  Salvia sclarea  sclareol synthase, SsSCS. Lower six rows were class I irregular diTPS that were analyzed directly (without SsSCS). 
         FIG.  7    illustrates typical cyclo-isomerization of diphosphate intermediates by class I diTPS. Ar,  Ajuga reptans;  Ll,  Leonotis leonorus;  Ms,  Mentha  spicata; Nm,  Nepeta mussini;  Om,  Origanum majorana;  Pa,  Perovskia atriplicifolia;  Pv,  Prunella vulgare;  So,  Salvia officinalis.    
         FIG.  8    illustrates the biosynthetic pathway to Jolkinol C within  Euphorbia.  GGPP was cyclized to the irregular diterpene scaffold Casbene, which was subsequently oxidized and further re-arranged by P450s and an ADH1. 
         FIG.  9 A- 9 C  illustrate the substrate promiscuity of P450s of the CYP76 family.  FIG.  9 A  shows that P450 enzymes from Salvia and Rosemary oxidize the non-native heteroatom-containing manoyl oxide as detected by GC/MS analysis of 13R-manoyl oxide and miltiradiene derived diterpenoids.  FIG.  9 B  shows diterpene structures.  FIG.  9 C  illustrates that CYP76AH15 from  Coleus  quantitatively converts the non-native miltiradiene to ferruginol. Ro,  Rosmarinus officinalis;  Sf,  Salvia fruticosa;  Cf,  Coleus forskohlii.    
         FIG.  10    illustrates detection of new methyl-diterpene product, with a structure similar to sclareol, when the  Coleus forskohlii  CfTPS2 and  Salvia sclarea  SsSCS enzymes are coupled together in an in vitro assay where the starting substrate is the unnatural methyl-GGDP (C21) substrate. 
     
    
    
     DETAILED DESCRIPTION 
     New substrates for terpene biosynthesis and methods for making new types of terpenes are described herein. Diterpenes occupy a unique molecular space with critical pharmaceutical applications over a diverse spectrum including anti-microbial, anti-cancer, immunomodulatory and psychoactive properties. Many diterpenoids are currently recognized as “drugs” (351 of over 12,500 are listed in the Dictionary of Natural Products, Taylor and Francis Group, DNP 28.1). A key challenge, however, is optimization of these compounds, and derivatization is usually not synthetically tractable. 
     While terpene synthase enzymes catalyze some of nature&#39;s most complex chemistries, the natural entry into the pathways is limited to a single precursor, geranylgeranyl diphosphate (GGPP), a precursor to almost all of natural diterpenes. Small molecule libraries for novel and promising leads for further manipulation are in demand as in vitro tools to investigate disease mechanisms, as in vivo probes, and to serve as starting points for the development of effective drugs. New compound libraries with high sp 3 -character, rather than the sp 2 -character typically observed in existing libraries, are generally missed by current technologies for library production (Karaki et al. Chem Med Chem (2019)). A unique three-dimensional space, or molecular complexity is correlated with success in the transition from discovery, to clinical testing, to approved drugs (Lovering, Medchemcomm 4: 515-519 (2013); Lovering et al. J. Med. Chem. 52, 6752-6756 (2009)). Complexity is measured by two descriptors, the fraction of tetrahedral sp 3  carbons (Fsp 3 ) where Fsp 3  equals the number of sp 3  hybridized carbons by total carbon count, and the chiral carbon count. 
     As described herein the terpene synthesis pathway is unexpectedly modular and the enzymes involved in terpene synthesis are surprisingly promiscuous. Unique, novel substrates for terpenes are described herein that are useful for making diverse types of new terpenoids. 
     Terpenes 
     Terpenes are the oldest and structurally most complex family of specialized metabolites on the planet. The class of diterpenes with their characteristic C20 scaffold is structurally diverse with over 12,500 compounds reported with a significant spectrum of pharmaceutical applications (Banerjee &amp; Hamberger,  P 450 s controlling metabolic bifurcations in plant terpene specialized metabolism.  Phytochem. Rev. (2017)). Their molecular weight, extraordinary high fraction of sp a  centers (Fsp 3  often &gt;0.8), number of stereogenic centers, and regiospecific and stereospecific heteroatom functionalization (exceeding 95% with 2+ oxygens) makes them superior candidates for the discovery and development of novel therapeutics. The structural complexity of a representative diterpenoid is illustrated by the diterpene scaffold of stevioside shown below, which has an Fsp 3  of 0.9. 
     
       
         
         
             
             
         
       
     
     The enzymes of terpene synthesis pathways are evolutionarily optimized to deliver bioactive molecules, novel molecular scaffolds, and novel chemistries, with pharmaceutical targets and modes of action identified only for a few, due to their limited availability (e.g., Picato®, Taxol®, forskolin, and salvinorin). 
     
       
         
         
             
             
         
       
     
     Cost-effective synthesis and access to analogs of plant diterpenoids and their derivatives is technologically limited by the levels of isolation, purification, detection and synthesis. Isolation and purification for screening of their pharmaceutical properties and clinical development are severely impeded by a lack of sustainable supply through their natural sources where diterpenoids accumulate in complex mixtures of closely related, but unwanted compounds. Formal chemical synthesis is economically challenging, as targets are still deconstructed one at a time, and even the most elegant biomimetic routes can be mind-bending in their complexity (Jorgensen et al. Science 341: 878-882 (2013); Appendino et al. Angew. Chemie Int. Ed. 53, 927-929 (2014)). 
     Synthetic Biology can alleviate the bottleneck of access. However, despite earlier successes by others (C15 anti-malaria drug artemisinin, Paddon et al. Nature 496: 528— 32 (2013)) and by the inventors (C20 drugs forskolin and phorbol-ester lead molecule jolkinol C, Luo et al. Proc. Natl. Acad. Sci. 113(34): E5082-9 (2016); Pateraki et al. Elife 6, (2017)), these approaches were limited to single targets and are incompatible with the need to generate diversified libraries that can be structurally manipulated by terpene synthases and other enzymes. 
     Jolkinol C represents the scaffold of the class of lathyrane-type phorbol esters with a macrocyclic, irregular structure. Compounds of this class exhibit potent antineoplastic activities against multidrug-resistant carcinoma lines. The NF-KB transcription factor provides a model system to study the posttranslational activation of a phorbol-ester-inducible transcription factor. The induction of NF-KB proceeds directly from protein kinase C upon binding of phorbol esters. The labdane-type diterpene forskolin is an important tool to raise cellular levels of cyclic AMP, a second messenger necessary for responses to hormones and cell communication. The mechanism proceeds via direct activation of all membrane bound isoforms of the adenylate cyclase. Acyl-analogs of forskolin were shown to strongly modulate the potency. The inventors have found that individual enzymes of both pathways, when probed with a small number of substrates, showed multifunctionality and promising promiscuity. In view of the utility of compounds similar to jolkinol C and forskolin the inventors have defined jolkinol C and forskolin functionalization pathways and identified diterpene scaffolds derived from GGPP, for biosynthesis using unnatural substrate scaffolds. 
     Described herein is a chemical strategy to bioprocess libraries of plant-inspired small molecules of the diterpene class. Novel synthetic substrate analogs are provided (i) to interrogate the intricate mechanism and substrate tolerance of terpene cyclization leading to unnatural decalin-core and irregular terpenes, (ii) to generate a panel of unnatural terpene key intermediates for functionalization through two pharmaceutically relevant pathways, and (iii) to characterize the function of such compounds with bioassays. 
     Despite their structural complexity, the biosynthesis routes of diterpenes are modular. This is illustrated in  FIG.  2   . For example, as shown in  FIG.  2   , pairs of enzymes or single enzymes (diterpene synthases, diTPS), cyclize the diterpene scaffold, followed by cytochromes P450 (P450s) that functionalize the scaffold in regiospecific and stereospecific fashion, thereby creating molecular handles for further modification such as acylation or further cyclization (acyl transferases, ACTs; aldehyde dehydrogenases, ADHs). The typical natural substrate all-trans (E,E,E)-geranylgeranyl diphosphate (GGPP) for diterpenes is a shared acyclic, achiral C 20 -building block. Such hierarchical organization and shared entry are not found in other pathways, including those leading to alkaloids or polyketides. 
     Terpene Substrates 
     Enzymatic bioprocessing of novel pharmaceutical candidates is increasingly important for securing access to relevant chemistries, scalability of production, and long-term reduction in cost for synthesis of scaffolds. Genetic information was used to reconstruct the pathways to the pharmacologically active cyclic AMP booster forskolin, and jolkinol C (shown in  FIG.  8   ), precursors of phorbol esters drugs with unique anti-cancer, anti-HIV and analgesic activities (Luo et al. Proc. Natl. Acad. Sci. 113(34): E5082-9 (2016); Pateraki et al. Elife 6, (2017); Pateraki et al. Plant Physiol. 164, 1222-36 (2014)). 
     A degree of substrate promiscuity was unexpectedly observed on all three hierarchical levels of the biosynthetic route, indicating that the enzymes involved in such biosynthesis have an ability to act on substrates that they do not normally encounter and that the enzymes can convert a broader range of intermediates to diverse end products. 
     Taking advantage of natural substrate promiscuity, precursor-directed biosynthesis was used to generate variants of the drugs in the family of non-ribosomal peptides, polyketides and non-natural indole alkaloids. Modification of natural products can provide analogs with improved or novel medicinal properties. To that end, the disclosure relates to substrates of the formula (I) or (II): 
     
       
         
         
             
             
         
       
     
     wherein: m is an integer from 0 to 3 (e.g., 1 or 2), with the understanding that if m is 2 or 3, each repeating subunit can be the same or different; 
     n is an integer from 0 to 1; 
     the dashed lines   represent a double bond when R 3′  and R 4′  are absent or when R 5′  and R 6′  are absent , 
     A and A′ are each independently cycloalkyl, aryl or heterocyclyl, each of which can be optionally substituted; 
     X 1  is a heteroatom, —X 3 -alkyl, -alkyl-X 3 — or alkyl, wherein X 3  is a heteroatom or alkyl or X 1  is: 
     
       
         
         
             
             
         
       
     
     R 1  and R 2  form a double bond or an epoxide; 
     each R′, R 1′ , R 2 , R 2′ , and R 3 —R 6  is, independently, H, alkyl, halo, aryl, and alkylaryl; 
     R 3′  and R 4′  are absent or R 3′  and R 4′ , together with the carbon atoms to which they are attached, form an epoxide, a cycloalkyl group, an aryl group or a heterocyclyl group; 
     R 5′  and R 6′  are absent or R 5′  and R 6′ , together with the carbon atoms to which they are attached, form an epoxide, a cycloalkyl group, an aryl group or a heterocyclyl group; 
     X 2  is a bond, alkenyl or acyl; and 
     X 4  is a absent, a heteroatom or alkyl; 
     with the proviso that the compound of the formula (I) is not a compound of the formula: 
     
       
         
         
             
             
         
       
     
     Examples of compounds of the formula (I) include compounds of the formula: 
     
       
         
         
             
             
         
       
     
     Examples of the formula (II) include compounds of the formula: 
     
       
         
         
             
             
         
       
     
     Examples of compounds of the formula (I) include compounds wherein if X 1  is a heteroatom, the heteroatom is oxygen. Other examples of compounds of the formula (I) include compounds wherein X 3  is oxygen or C 1 -C 5 -alkyl, such as —CH 2 — and C 2 -C 3 -alkyl. Still other examples of compounds of the formula (I) include compounds wherein R 3 -R 6  are each H or C 1 -C 5 -alkyl, such as methyl and C 2 -C 3 -alkyl. Still other examples of compounds of the formula (I) include compounds wherein R 3  and R 5  are each H or C 1 -C 5 -alkyl, such as methyl and C 2 -C 3 -alkyl; and R 4  and R 6  are each H. Yet other examples of compounds of the formula (I) include compounds wherein m is 1 or 2. In other examples, m is 0. Other examples of compound of the formula (I) include compounds wherein X 2  is an alkenyl group of the formula: 
     
       
         
         
             
             
         
       
     
     or an acyl group of the formula: 
     
       
         
         
             
             
         
       
     
     Examples of compounds of the formula (I) include compounds of the formulae: 
     
       
         
         
             
             
         
       
     
     The compounds of the formula (I) or (II) can be enzymatically transformed into terpenoids having compound cores of the formula: 
     
       
         
         
             
             
         
       
     
     which correspond to the cores of stevioside, Taxol®, Forskolin, Picato®, and Salvinorin, Casbene, CPP respectively; or the core shared by CPP, LPP, PgPP, and KPP, namely: 
     
       
         
         
             
             
         
       
     
     and derivatives thereof, wherein derivatives can comprise additional double bonds, alkyl groups, hydroxy groups, acyl groups, and the like, dispersed about the cores. 
     As used herein, the term “heteroatom” refers to heteroatom such as, but not limited to, NR 7 , O, and SO, wherein R 7  is H, alkyl or arylalkyl, and x is 0, 1 or 2. 
     The term “alkyl” as used herein refers to substituted or unsubstituted straight chain, branched and cyclic, saturated mono- or bi-valent groups having from 1 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to about 10 carbon atoms, 1 to 10 carbons atoms, 1 to 8 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 1 to 6 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 1 to 3 carbon atoms. Examples of straight chain mono-valent (C 1 -C 20 -alkyl groups include those with from 1 to 8 carbon atoms such as methyl (i.e., CH 3 ), ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl groups. Examples of branched mono-valent (C 1 -C 20 -alkyl groups include isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, and isopentyl. Examples of straight chain bi-valent (C 1 -C 20 )alkyl groups include those with from 1 to 6 carbon atoms such as —CH 2 —, —CH 2 CH 2 —, —CH 2 CH 2 CH 2 —, —CH 2 CH 2 CH 2 CH 2 —, and —CH 2 CH 2 CH 2 CH 2 CH 2 —. Examples of branched bi-valent alkyl groups include —CH(CH 3 )CH 2 — and —CH 2 CH(CH 3 )CH 2 —. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, and bicyclo[2.2.1]heptyl. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. In some embodiments, alkyl includes a combination of substituted and unsubstituted alkyl. As an example, alkyl, and also (C 1 )alkyl, includes methyl and substituted methyl. As a particular example, (C 1 )alkyl includes benzyl. As a further example, alkyl can include methyl and substituted (C 2 -C 8 )alkyl. Alkyl can also include substituted methyl and unsubstituted (C 2 -C 8 )alkyl. In some embodiments, alkyl can be methyl and C 2 -C 8  linear alkyl. In some embodiments, alkyl can be methyl and C 2 -C 8  branched alkyl. The term methyl is understood to be —CH 3 , which is not substituted. The term methylene is understood to be —CH 2 -, which is not substituted. For comparison, the term (C 1 )alkyl is understood to be a substituted or an unsubstituted —CH 3  or a substituted or an unsubstituted —CH 2 —. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, cycloalkyl, heterocyclyl, aryl, amino, haloalkyl, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. As further example, representative substituted alkyl groups can be substituted one or more fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamido. In some embodiments, representative substituted alkyl groups can be substituted from a set of groups including amino, hydroxy, cyano, carboxy, nitro, thio and alkoxy, but not including halogen groups. 
     The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. 
     The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl, group or the like. 
     The term “alkenyl” as used herein refers to substituted or unsubstituted straight chain, branched and cyclic, saturated mono- or bi-valent groups having at least one carbon-carbon double bond and from 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to about 10 carbon atoms, 2 to 10 carbons atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. The double bonds can be trans or cis orientation. The double bonds can be terminal or internal. The alkenyl group can be attached via the portion of the alkenyl group containing the double bond, e.g., vinyl, propen-1-yl and buten-1-yl, or the alkenyl group can be attached via a portion of the alkenyl group that does not contain the double bond, e.g., penten-4-yl. Examples of mono-valent (C 2 -C 20 )-alkenyl groups include those with from 1 to 8 carbon atoms such as vinyl, propenyl, propen-1-yl, propen-2-yl, butenyl, buten-1-yl, buten-2-yl, sec-buten-1-yl, sec-buten-3-yl, pentenyl, hexenyl, heptenyl and octenyl groups. Examples of branched mono-valent (C 2 -C 20 )-alkenyl groups include isopropenyl, iso-butenyl, sec-butenyl, t-butenyl, neopentenyl, and isopentenyl. Examples of straight chain bi-valent (C 2 -C 2 o)alkenyl groups include those with from 2 to 6 carbon atoms such as —CHCH—, —CHCHCH 2 —, —CHCHCH 2 CH 2 —, and —CHCHCH 2 CH 2 CH 2 —. Examples of branched bi-valent alkyl groups include —C(CH 3 )CH— and —CHC(CH 3 )CH 2 —. Examples of cyclic alkenyl groups include cyclopentenyl, cyclohexenyl and cyclooctenyl. It is envisaged that alkenyl can also include masked alkenyl groups, precursors of alkenyl groups or other related groups. As such, where alkenyl groups are described it, compounds are also envisaged where a carbon-carbon double bond of an alkenyl is replaced by an epoxide or aziridine ring. Substituted alkenyl also includes alkenyl groups which are substantially tautomeric with a non-alkenyl group. For example, substituted alkenyl can be 2-aminoalkenyl, 2-alkylaminoalkenyl, 2-hydroxyalkenyl, 2-hydroxyvinyl, 2-hydroxypropenyl, but substituted alkenyl is also understood to include the group of substituted alkenyl groups other than alkenyl which are tautomeric with non-alkenyl containing groups. In some embodiments, alkenyl can be understood to include a combination of substituted and unsubstituted alkenyl. For example, alkenyl can be vinyl and substituted vinyl. For example, alkenyl can be vinyl and substituted (C 3 -C 8 )alkenyl. Alkenyl can also include substituted vinyl and unsubstituted (C 3 -C 8 )alkenyl. Representative substituted alkenyl groups can be substituted one or more times with any of the groups listed herein, for example, monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio, alkoxy, and halogen groups. As further example, representative substituted alkenyl groups can be substituted one or more fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamido. In some embodiments, representative substituted alkenyl groups can be substituted from a set of groups including monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio and alkoxy, but not including halogen groups. Thus, in some embodiments, alkenyl can be substituted with a non-halogen group. In some embodiments, representative substituted alkenyl groups can be substituted with a fluoro group, substituted with a bromo group, substituted with a halogen other than bromo, or substituted with a halogen other than fluoro. For example, alkenyl can be 1-fluorovinyl, 2-fluorovinyl, 1,2-difluorovinyl, 1,2,2-trifluorovinyl, 2,2-difluorovinyl, trifluoropropen-2-yl, 3,3,3-trifluoropropenyl, 1-fluoropropenyl, 1-chlorovinyl, 2-chlorovinyl, 1,2-dichlorovinyl, 1,2,2-trichlorovinyl or 2,2-dichlorovinyl. In some embodiments, representative substituted alkenyl groups can be substituted with one, two, three or more fluoro groups or they can be substituted with one, two, three or more non-fluoro groups. 
     The term “alkynyl” as used herein, refers to substituted or unsubstituted straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 50 carbon atoms, 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to about 10 carbon atoms, 2 to 10 carbons atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples include, but are not limited to ethynyl, propynyl, propyn-1-yl, propyn-2-yl, butynyl, butyn-1-yl, butyn-2-yl, butyn-3-yl, butyn-4-yl, pentynyl, pentyn-1-yl, hexynyl, Examples include, but are not limited to —C≡CH, —C≡C(CH 3 ), —C≡C(CH 2 CH 3 ), —CH 2 C≡CH, —CH 2 C≡C(CH 3 ), and —CH 2 C≡C(CH 2 CH 3 ) among others. 
     The term “aryl” as used herein refers to substituted or unsubstituted univalent groups that are derived by removing a hydrogen atom from an arene, which is a cyclic aromatic hydrocarbon, having from 6 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 20 carbon atoms, 6 to about 10 carbon atoms or 6 to 8 carbon atoms. Examples of (C 6 -C 20 )aryl groups include phenyl, napthalenyl, azulenyl, biphenylyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, anthracenyl groups. Examples include substituted phenyl, substituted napthalenyl, substituted azulenyl, substituted biphenylyl, substituted indacenyl, substituted fluorenyl, substituted phenanthrenyl, substituted triphenylenyl, substituted pyrenyl, substituted naphthacenyl, substituted chrysenyl, and substituted anthracenyl groups. Examples also include unsubstituted phenyl, unsubstituted napthalenyl, unsubstituted azulenyl, unsubstituted biphenylyl, unsubstituted indacenyl, unsubstituted fluorenyl, unsubstituted phenanthrenyl, unsubstituted triphenylenyl, unsubstituted pyrenyl, unsubstituted naphthacenyl, unsubstituted chrysenyl, and unsubstituted anthracenyl groups. Aryl includes phenyl groups and also non-phenyl aryl groups. From these examples, it is clear that the term (C 6 -C 20 )aryl encompasses mono- and polycyclic (C 6 -C 20 )aryl groups, including fused and non-fused polycyclic (C 6 -C 20 )aryl groups. The term “heterocyclyl” as used herein refers to substituted aromatic, unsubstituted aromatic, substituted non-aromatic, and unsubstituted non-aromatic rings containing 3 or more atoms in the ring, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. In some embodiments, heterocyclyl groups include heterocyclyl groups that include 3 to 8 carbon atoms (C 3 -C 8 ), 3 to 6 carbon atoms (C 3 -C 6 ) or 6 to 8 carbon atoms (C 6 -C 8 ). A heterocyclyl group designated as a C 2 -heterocyclyl can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms and so forth. Likewise, a C 4 -heterocyclyl can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. Representative heterocyclyl groups include, but are not limited to piperidynyl, piperazinyl, morpholinyl, furanyl, pyrrolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, imidazolyl, triazyolyl, tetrazolyl, benzoxazolinyl, and benzimidazolinyl groups. For example, heterocyclyl groups include, without limitation: 
     
       
         
         
             
             
         
       
     
     wherein X 5  represents H, (C 1 -C 20 )alkyl, (C 6 -C 20 )aryl or an amine protecting group (e.g., a t-butyloxycarbonyl group) and wherein the heterocyclyl group can be substituted or unsubstituted. A nitrogen-containing heterocyclyl group is a heterocyclyl group containing a nitrogen atom as an atom in the ring. In some embodiments, the heterocyclyl is other than thiophene or substituted thiophene. In some embodiments, the heterocyclyl is other than furan or substituted furan. 
     The term “aralkyl” and “arylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl, biphenylmethyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. 
     The term “substituted” as used herein refers to a group that is substituted with one or more groups including, but not limited to, the following groups: halogen (e.g., F, Cl, Br, and I), R, OR, ROH (e.g., CH 2 OH), OC(O)N(R) 2 , CN, NO, NO 2 , ONO 2 , azido, CF 3 , OCF 3 , methylenedioxy, ethylenedioxy, (C 3 -C 20 )heteroaryl, N(R) 2 , Si(R) 3 , SR, SOR, SO 2 R, SO 2 N(R) 2 , SO 3 R, P(O)(OR) 2 , OP(O)(OR) 2 , C(O)R, C(O)C(O)R, C(O)CH 2 C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R) 2 , C(O)N(R)OH, OC(O)N(R) 2 , C(S)N(R) 2 , (CH 2 ) 0-2 N(R)C(O)R, (CH 2 ) 0-2 N(R)N(R) 2 , N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R) 2 , N(R)SO 2 R, N(R)SO 2 N(R) 2 , N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R) 2 , N(R)C(S)N(R) 2 , N(COR)COR, N(OR)R, C(═NH)N(R) 2 , C(O)N(OR)R, or C(═NOR)R wherein R can be hydrogen, (C 1 -C 20 )alkyl, (C 6 -C 20 )aryl, heterocyclyl or polyalkylene oxide groups, such as polyalkylene oxide groups of the formula —(CH 2 CH 20 ) f —R—OR, —(CH 2 CH 2 CH 20 ) g —R—OR, —(CH 2 CH 20 ) f (CH 2 CH 2 CH 20 ) g —R—OR each of which can, in turn, be substituted or unsubstituted and wherein f and g are each independently an integer from 1 to 50 (e.g., 1 to 10, 1 to 5, 1 to 3 or 2 to 5). Substituted also includes a group that is substituted with one or more groups including, but not limited to, the following groups: fluoro, chloro, bromo, iodo, amino, amido, alkyl, hydroxy, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamido. Where there are two or more adjacent substituents, the substituents can be linked to form a carbocyclic or heterocyclic ring. Such adjacent groups can have a vicinal or germinal relationship, or they can be adjacent on a ring in, e.g., an ortho-arrangement. Each instance of substituted is understood to be independent. For example, a substituted aryl can be substituted with bromo and a substituted heterocycle on the same compound can be substituted with alkyl. It is envisaged that a substituted group can be substituted with one or more non-fluoro groups. As another example, a substituted group can be substituted with one or more non-cyano groups. As another example, a substituted group can be substituted with one or more groups other than haloalkyl. As yet another example, a substituted group can be substituted with one or more groups other than tert-butyl. As yet a further example, a substituted group can be substituted with one or more groups other than trifluoromethyl. As yet even further examples, a substituted group can be substituted with one or more groups other than nitro, other than methyl, other than methoxymethyl, other than dialkylaminosulfonyl, other than bromo, other than chloro, other than amido, other than halo, other than benzodioxepinyl, other than polycyclic heterocyclyl, other than polycyclic substituted aryl, other than methoxycarbonyl, other than alkoxycarbonyl, other than thiophenyl, or other than nitrophenyl, or groups meeting a combination of such descriptions. Further, substituted is also understood to include fluoro, cyano, haloalkyl, tert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxy carbonyl, alkoxycarbonyl, thiophenyl, and nitrophenyl groups. 
     Enzymes 
     A variety of enzymes can be used to convert the substrates into useful products. Examples of enzymes that can be used include terpene synthases. For example, the enzymes employed can be those that naturally convert geranylgeranyl diphosphate (GGPP) into biosynthesis of gibberellins, carotenoids, chlorophylls, isoprenoid quinones, and geranylgeranylated proteins. However, the enzymes are also promiscuous and can accept unnatural substrates such as the unnatural GGPP analogs or derivatives described herein. Additional enzymes can also be employed that convert the products formed from the unnatural substrates (e.g., the primary products) into other products (e.g., secondary products). 
     For example, the enzymes can be from organisms such as  Tripterygium wilfordii  (Tw),  Euphorbia peplus  (Ep),  Coleus forskohlii  (Cf),  Ajuga reptans  (Ar),  Perovskia atriciplifolia  (Pa),  Nepeta mussini  (Nm),  Origanum majorana  (Om),  Hyptis suaveolens  (Hs),  Grindelia robusta  (Gr),  Leonotis leonurus  (Ll),  Marrubium vulgare  (Mv),  Vitex agnus - castus  (Vac),  Euphorbia peplus  (Ep),  Ricinus communis  (Rc),  Daphne genkwa  (Dg),  Zea mays  (Zm), and other organisms. 
     The enzymes can in some cases, for example, be type I or type II enzymes. In general, a type II enzyme can catalyze transformation of an unnatural substrate derivative of geranylgeranyl diphosphate (GGPP) to a primary terpene product, while the type I enzymes can modify such a terpene product to generate a second terpene product. 
     The enzymes can be used in single step reactions, or in multi-step reactions when mixed together or when used sequentially. Multi-step reactions can occur by enzyme coupling. Enzyme coupling refers to one enzyme catalyzing a reaction to produce a product that is a substrate for a second enzyme. For example, the type II and type I enzymes can be coupled together, where a type II enzyme can accept and enzymatically convert an unnatural substrate to a first product and where a type I enzyme accepts the first product as a substrate for enzymatic conversion to generate a second product. Such enzyme coupling is demonstrated in the Examples. In some cases, an unnatural substrate can undergo efficient conversion to a first product by one enzyme without producing side products or undesirable fragments that could undermine the efficiency of a second enzyme to produce desirable yields of a second product. 
     Examples of enzymes that can be used include those that naturally produce ent-CPP (e.g., TwTPS3, EpTPS7, ZmAN2), shown below. 
     
       
         
         
             
             
         
       
     
     Examples of enzymes that can be used include those that naturally produce (+)-CPP (e.g., CfTPS1, ArTPS1, PaTPS1, NmTPS1, OmTPS1, TwTPS9 and CfTPS16), shown below. 
     
       
         
         
             
             
         
       
     
     Examples of enzymes that can be used include those that naturally produce (13E)-labda-7,13-dien-15-yl diphosphate (i.e., (7,13)-LPP) (e.g., HsTPS1, GrTPS), shown below. 
     
       
         
         
             
             
         
       
     
     Examples of enzymes that can be used include those that naturally produce peregrinol diphosphate (PGPP) (e.g., LlTPS1, MvCPS1, VacTPS1), shown below. 
     
       
         
         
             
             
         
       
     
     Examples of enzymes that can be used include those that naturally produce (−)-kolavenyl diphosphate (KPP) (e.g., TwTPS10, TwTPS14, VacTPS5), shown below. 
     
       
         
         
             
             
         
       
     
     Examples of enzymes that can be used include those that naturally produce casbene (e.g., EpCBS, RcCBS, DgTPS1), shown below. 
     
       
         
         
             
             
         
       
     
     Approximately 30 functional diTPS of the mint family have been identified and isolated by the inventors as having both labdane-type and irregular diterpene biosynthetic activities. These enzymes represent a repository of enzymes that can be used in the methods and reaction mixtures described herein. 
     For example, an  Ajuga reptans  miltiradiene synthase (ArTPS3), a  Leonotis leonurus  sandaracopimaradiene synthase (L1TPS4), a  Mentha spicata  class I diterpene synthase (MsTPS1), an  Origanum majorana  trans-abienol synthase (OmTPS3), an  Origanum majorana  manool synthase (OmTPS4), an  Origanum majorana  palustradiene synthase (OmTPS5),  Perovskia atriplicifolia  miltiradiene synthase (PaTPS3),  Prunella vulgaris  miltiradiene synthase (PvTPS1),  Salvia officinalis  miltiradiene synthase (SoTPS1) were identified and isolated. 
     Eight of these enzymes, ArTPS3, L1TPS4, MsTPS1, OmTPS4, OmTPS5, PaTPS3, PvTPS1, and SoTPS1 can convert a labda-13-en-8-ol diphosphate ((+) LPP) [compound 10]) to 13R-(+)-manoyl oxide [8]. 
     
       
         
         
             
             
         
       
     
     The ArTPS3, L1TPS4, OmTPS4, OmTPS5, PaTPS3, PvTPS1, and SoYPS1 enzymes can also convert peregrinol diphosphate (PgPP) [5] to a combination of compounds 1, 2, and 3, as illustrated below. 
     
       
         
         
             
             
         
       
     
     However, MsTPS1 produced only compound 3 from compound 5, while the OmTPS3 enzyme produced only 1, and 2. The OmTPS4 enzyme produced compound 4 (shown below) in addition to compounds 1, 2, and 3. 
     
       
         
         
             
             
         
       
     
     The ArTPS3, PaTPS3, PvTPS1, and SoTPS1 enzymes can also convert (+)-copalyl diphosphate ((+)-CPP) [31]) to miltiradiene [32]. 
     
       
         
         
             
             
         
       
     
     However, LlTPS4 and MsTPS1 converted (+)-copalyl diphosphate ((+)-CPP) [31]) to sadaracopimaradiene [27], while OmTPS3 converted (+)-copalyl diphosphate ((+)-CPP) [31]) to trans-biformene [34]. 
     
       
         
         
             
             
         
       
     
     The  Ajuga reptans  miltiradiene synthase (ArTPS3) has the amino acid sequence shown below (SEQ ID NO:1). 
                                    1   MSLSETIKVT   PFSGQRVHSS   TESFPIQQFP   TITTKSAMAV               41   KCSSLSTATV   SFQDFVGKIR   DTINGKVDNS   PAATTIHPAD               81   IPSNLCVVDT   LQRLGVDRYF   QSEIDSVLND   TYRFWQQKGE               121   DIFTDVACRA   MAFRLLRVKG   YEVSSDELAS   YAEQEHVNLQ               161   PSDITTVIEL   YRASQTRLYE   DEGNLEKLHT   WTSNFLKQQL               201   QSETISDEKL   HKQVEYYLKN   YHGILDRAGV   RQSLDLYDIN               241   QYQNLKSTDR   FPTLSNEDLL   EFAKQDFNFC   QAQHQKELQQ               281   LQRWYADCKL   DTLTYGRDVV   RVASFLTAAI   FGEPEFSDAR               321   LAFAKHIILV   TRIDDFFDHG   GSIEESYKIL   DLVKEWEDKP               361   AEEYPSKEVE   ILFTAVYNTV   NDLAEMAYIE   QGRSIKPLLI               401   KLWVEILTSF   KKELDSWTED   TELTLEEYLA   SSWVSIGCRI               441   CSLNSLQFLG   ITLSEEMLSS   EECMELCRHV   SSVDRLLNDV               481   QTFEKERLEN   TINSVSLQLA   EAQREGRTIT   EEEAMSKIKD               521   LADYHRRQLM   QMVYKDGTIF   PRQCKDVFLR   VCRIGYYLYA               561   SGDEFTTPQQ   MMGDMKSLVY   EPLNTSSS                
A nucleic acid encoding the  Ajuga reptans  miltiradiene synthase (ArTPS3) with SEQ ID NO:1 is shown below as SEQ ID NO:2.
 
     
       
         
           
               
               
               
               
               
            
               
                 1 
                 ATGTCACTCT 
                 CGTTCACCAT 
                 CAAAGTCACC 
                 CCCTTTTCGG 
               
               
                   
               
               
                 41 
                 GCCAGAGAGT 
                 TCACAGCAGC 
                 ACAGAAAGCT 
                 TTCCAATCCA 
               
               
                   
               
               
                 81 
                 ACAATTTCCA 
                 ACGATCACCA 
                 CCAAATCCGC 
                 CATGGCTGTC 
               
               
                   
               
               
                 121 
                 AAATGCAGCA 
                 GCCTCAGTAC 
                 CGCAACAGTA 
                 AGCTTCCAGG 
               
               
                   
               
               
                 161 
                 ATTTCGTCGG 
                 AAAAATCAGA 
                 GATACGATCA 
                 ACGGGAAAGT 
               
               
                   
               
               
                 201 
                 TGACAATTCT 
                 CCAGCAGCGA 
                 CCACTATTCA 
                 TCCTGCAGAT 
               
               
                   
               
               
                 241 
                 ATACCCTCCA 
                 ATCTCTGCGT 
                 GGTGGATACC 
                 CTCCAAAGAT 
               
               
                   
               
               
                 281 
                 TGGGAGTTGA 
                 CCGTTACTTC 
                 CAATCTGAAA 
                 TCGACAGCGT 
               
               
                   
               
               
                 321 
                 TCTTAACGAC 
                 ACATACAGGT 
                 TCTGGCAGCA 
                 GAAAGGAGAA 
               
               
                   
               
               
                 361 
                 GATATCTTCA 
                 CTGATGTTGC 
                 TTGTCGTGCA 
                 ATGGCATTTC 
               
               
                   
               
               
                 401 
                 GACTTTTGCG 
                 AGTTAAAGGA 
                 TATGAAGTTT 
                 CATCAGATGA 
               
               
                   
               
               
                 521 
                 ACTGGCTTCG 
                 TATGCTGAAC 
                 AAGAGCATGT 
                 TAACCTGCAA 
               
               
                   
               
               
                 561 
                 CCAAGTGACA 
                 TAACTACGGT 
                 TATCGAGCTT 
                 TACAGAGCAT 
               
               
                   
               
               
                 601 
                 CACAGACAAG 
                 ATTATATGAA 
                 GACGAGGGCA 
                 ATCTTGAGAA 
               
               
                   
               
               
                 641 
                 GTTACATACT 
                 TGGACTAGCA 
                 ATTTTCTGAA 
                 GCAACAATTG 
               
               
                   
               
               
                 681 
                 CAGAGTGAAA 
                 CTATTTCTGA 
                 CGAGAAATTG 
                 CACAAACAGG 
               
               
                   
               
               
                 721 
                 TGGAGTATTA 
                 CTTGAAGAAC 
                 TACCACGGCA 
                 TACTAGACCG 
               
               
                   
               
               
                 761 
                 TGCTGGAGTT 
                 AGACAAAGTC 
                 TCGATTTATA 
                 TGACATAAAC 
               
               
                   
               
               
                 801 
                 CAATACCAGA 
                 ATCTAAAATC 
                 TACAGATAGA 
                 TTCCCTACTT 
               
               
                   
               
               
                 841 
                 TAAGTAACGA 
                 AGATTTACTT 
                 GAATTCGCGA 
                 AGCAAGATTT 
               
               
                   
               
               
                 881 
                 TAACTTTTGC 
                 CAAGCTCAAC 
                 ACCAGAAAGA 
                 GCTTCAGCAA 
               
               
                   
               
               
                 921 
                 CTGCAAAGGT 
                 GGTATGCGGA 
                 TTGTAAATTG 
                 GATACATTGA 
               
               
                   
               
               
                 961 
                 CTTACGGAAG 
                 AGATGTGGTA 
                 CGTGTTGCAA 
                 GTTTCCTGAC 
               
               
                   
               
               
                 1001 
                 AGCTGCAATT 
                 TTTGGTGAGC 
                 CTGAATTCTC 
                 TGATGCTCGT 
               
               
                   
               
               
                 1041 
                 CTAGCCTTCG 
                 CCAAACACAT 
                 CATCCTCGTG 
                 ACACGTATTG 
               
               
                   
               
               
                 1081 
                 ATGATTTCTT 
                 CGATCATGGT 
                 GGGTCTATAG 
                 AAGAGTCATA 
               
               
                   
               
               
                 1121 
                 CAAGATCCTG 
                 GATTTAGTAA 
                 AAGAATGGGA 
                 AGATAAGCCA 
               
               
                   
               
               
                 1161 
                 GCTGAGGAAT 
                 ATCCTTCCAA 
                 GGAAGTTGAA 
                 ATCCTCTTTA 
               
               
                   
               
               
                 1201 
                 CAGCAGTATA 
                 TAATACAGTA 
                 AATGACTTGG 
                 CAGAAATGGC 
               
               
                   
               
               
                 1241 
                 TTATATTGAG 
                 CAAGGCCGTT 
                 CCATTAAACC 
                 TCTTCTAATT 
               
               
                   
               
               
                 1281 
                 AAACTGTGGG 
                 TTGAAATACT 
                 GACAAGTTTC 
                 AAGAAAGAAC 
               
               
                   
               
               
                 1321 
                 TGGATTCATG 
                 GACAGAAGAC 
                 ACAGAACTAA 
                 CCTTGGAGGA 
               
               
                   
               
               
                 1361 
                 GTACTTGGCT 
                 TCCTCCTGGG 
                 TGTCGATCGG 
                 TTGCAGAATC 
               
               
                   
               
               
                 1401 
                 TGCAGTCTCA 
                 ATTCGCTGCA 
                 GTTCCTTGGT 
                 ATAACATTAT 
               
               
                   
               
               
                 1441 
                 CCGAAGAAAT 
                 GCTTTCAAGC 
                 GAAGAGTGCA 
                 TGGAGTTGTG 
               
               
                   
               
               
                 1481 
                 TAGGCATGTT 
                 TCTTCAGTCG 
                 ACAGGCTACT 
                 CAATGACGTG 
               
               
                   
               
               
                 1521 
                 CAAACTTTCG 
                 AGAAGGAACG 
                 CCTAGAAAAT 
                 ACGATAAACA 
               
               
                   
               
               
                 1561 
                 GTGTGAGCCT 
                 ACAGCTAGCA 
                 GAAGCTCAGA 
                 GAGAAGGAAG 
               
               
                   
               
               
                 1601 
                 AACCATTACA 
                 GAAGAGGAGG 
                 CTATGTCAAA 
                 GATTAAAGAC 
               
               
                   
               
               
                 1641 
                 CTGGCTGATT 
                 ATCACAGGAG 
                 ACAACTGATG 
                 CAGATGGTTT 
               
               
                   
               
               
                 1681 
                 ATAAGGATGG 
                 GACCATATTT 
                 CCGAGACAAT 
                 GCAAAGATGT 
               
               
                   
               
               
                 1721 
                 CTTTTTGAGG 
                 GTATGCAGGA 
                 TTGGCTACTA 
                 CTTATACGCG 
               
               
                   
               
               
                 1761 
                 AGCGGCGATG 
                 AATTCACTAC 
                 TCCACAACAA 
                 ATGATGGGGG 
               
               
                   
               
               
                 1801 
                 ATATGAAATC 
                 ATTGGTTTAT 
                 GAACCCCTAA 
                 ACACTTCATC 
               
               
                   
               
               
                 1841 
                 CTCTTGA 
                   
                   
                   
               
            
           
         
       
     
     The  Leonotis leonurus  sandaracopimaradiene synthase (L1TPS4) has the amino acid sequence shown below (SEQ ID NO:3). 
                                    1   MSVAFNLIVV   RFPGHGIQSS   RETFPAKIIT   RTKSSMRFQS               41   SLNTSTDFVG   KIREMIRGKT   DNSINPLDIP   STLCVIDTLH               81   SFGIDRYFQS   EINSVLHHTY   RLWNDRNNII   FKDVICCAIA               121   FRLLRVKGYQ   VSSDELAPFA   QQQVTGLQTS   DIATILELYR               161   ASQERLHEDD   DTLDKLHDWS   SNLLKLHLLN   ENIPDHKLHK               201   RVGYFLKNYH   GMLDRVAVRR   NIDLHNINHY   QIPEVADRFP               241   TEAFLEFSRQ   DFNICQAQHQ   KELQQLHRWY   ADCRLDTLNH               281   GTDVVHFANF   LTSAIFGEPE   FSEARLAFAK   QVILITRMDD               321   FFDHDGSREE   SHKILHLVQQ   WKEKPAEEYG   SKEVEILFTA               361   VYTTVNSLAE   KACMEQGRSV   KQLLIKLWVE   LLTSFKKELD               401   SWTEKMALTL   DEYLSFSWVS   IGCRLCILNS   LQFLGIKLSE               441   EMLWSQECLD   LCRHVSSVVR   LLNDLQTFKK   ERIENTINGV               481   DVQLAARKGE   RAITEEEAMS   KIKEMADHHR   RKLMQIVYKE               521   GTIFPRECKD   VFLRVCRIGY   YLYSGDELTS   PQQMKEDMKA               561   LVHESSS                        
A nucleic acid encoding the  Leonotis leonurus  sandaracopimaradiene synthase (L1TPS4) with SEQ ID NO:3 is shown below as SEQ ID NO:4.
 
     
       
         
           
               
               
               
               
               
            
               
                 1 
                 ATGTCGGTGG 
                 CGTTCAACCT 
                 CATAGTCGTC 
                 CGTTTTCCGG 
               
               
                   
               
               
                 41 
                 GCCATGGAAT 
                 TCAGAGCAGT 
                 AGAGAAACTT 
                 TTCCAGCCAA 
               
               
                   
               
               
                 81 
                 AATTATTACC 
                 AGAACTAAAT 
                 CAAGCATGAG 
                 ATTCCAAAGC 
               
               
                   
               
               
                 121 
                 AGCCTCAACA 
                 CTTCAACAGA 
                 TTTCGTGGGA 
                 AAAATAAGAG 
               
               
                   
               
               
                 161 
                 AGATGATCAG 
                 AGGGAAAACT 
                 GATAATTCTA 
                 TTAATCCCCT 
               
               
                   
               
               
                 201 
                 GGATATTCCC 
                 TCCACTCTAT 
                 GCGTAATCGA 
                 CACCCTACAC 
               
               
                   
               
               
                 241 
                 AGCTTCGGAA 
                 TTGATCGCTA 
                 CTTTCAATCC 
                 GAAATCAACT 
               
               
                   
               
               
                 281 
                 CTGTTCTTCA 
                 CCACACATAC 
                 AGATTATGGA 
                 ACGACAGAAA 
               
               
                   
               
               
                 321 
                 TAATATCATC 
                 TTCAAAGATG 
                 TCATTTGCTG 
                 CGCAATTGCC 
               
               
                   
               
               
                 361 
                 TTTAGACTTT 
                 TGCGAGTGAA 
                 AGGATATCAA 
                 GTCTCATCAG 
               
               
                   
               
               
                 401 
                 ATGAACTGGC 
                 GCCATTTGCC 
                 CAACAACAGG 
                 TGACTGGACT 
               
               
                   
               
               
                 441 
                 ACAAACAAGC 
                 GACATTGCCA 
                 CGATTCTAGA 
                 GCTCTACAGA 
               
               
                   
               
               
                 481 
                 GCATCACAGG 
                 AGAGATTACA 
                 CGAAGACGAC 
                 GACACTCTTG 
               
               
                   
               
               
                 521 
                 ACAAACTACA 
                 TGATTGGAGC 
                 AGCAACCTTC 
                 TGAAGCTGCA 
               
               
                   
               
               
                 561 
                 TCTGCTGAAT 
                 GAGAACATTC 
                 CTGATCATAA 
                 ACTGCACAAA 
               
               
                   
               
               
                 601 
                 CGGGTGGGGT 
                 ATTTCTTGAA 
                 GAACTACCAT 
                 GGCATGCTAG 
               
               
                   
               
               
                 641 
                 ATCGCGTTGC 
                 GGTTAGACGA 
                 AACATCGACC 
                 TTCACAACAT 
               
               
                   
               
               
                 681 
                 AAACCATTAC 
                 CAAATCCCAG 
                 AAGTTGCAGA 
                 TAGGTTCCCT 
               
               
                   
               
               
                 721 
                 ACTGAAGCTT 
                 TTCTTGAATT 
                 TTCAAGGCAA 
                 GATTTTAATA 
               
               
                   
               
               
                 761 
                 TTTGCCAAGC 
                 TCAACACCAG 
                 AAAGAACTTC 
                 AGCAACTGCA 
               
               
                   
               
               
                 801 
                 TAGGTGGTAT 
                 GCAGATTGTA 
                 GATTGGACAC 
                 ACTGAATCAC 
               
               
                   
               
               
                 841 
                 GGAACAGACG 
                 TAGTACATTT 
                 TGCTAATTTT 
                 CTAACTTCAG 
               
               
                   
               
               
                 881 
                 CAATTTTCGG 
                 AGAGCCTGAA 
                 TTCTCCGAGG 
                 CTCGTCTAGC 
               
               
                   
               
               
                 921 
                 CTTTGCTAAA 
                 CAGGTTATCC 
                 TAATAACACG 
                 TATGGATGAT 
               
               
                   
               
               
                 961 
                 TTCTTCGATC 
                 ACGATGGGTC 
                 TAGAGAAGAA 
                 TCACACAAGA 
               
               
                   
               
               
                 1001 
                 TCCTCCATCT 
                 AGTTCAACAA 
                 TGGAAAGAGA 
                 AGCCCGCCGA 
               
               
                   
               
               
                 1041 
                 AGAATATGGT 
                 TCAAAGGAAG 
                 TTGAGATCCT 
                 CTTTACAGCA 
               
               
                   
               
               
                 1081 
                 GTGTACACTA 
                 CAGTAAATAG 
                 CTTGGCAGAA 
                 AAGGCTTGTA 
               
               
                   
               
               
                 1121 
                 TGGAGCAAGG 
                 CCGTAGTGTC 
                 AAACAACTTC 
                 TAATTAAGCT 
               
               
                   
               
               
                 1161 
                 GTGGGTCGAG 
                 CTGCTAACAA 
                 GTTTCAAGAA 
                 AGAATTGGAT 
               
               
                   
               
               
                 1201 
                 TCATGGACGG 
                 AGAAGATGGC 
                 GCTAACCTTG 
                 GATGAGTACT 
               
               
                   
               
               
                 1241 
                 TGTCTTTCTC 
                 CTGGGTGTCA 
                 ATTGGCTGCA 
                 GACTCTGCAT 
               
               
                   
               
               
                 1281 
                 TCTCAATTCC 
                 CTGCAATTTC 
                 TTGGGATAAA 
                 ATTATCTGAA 
               
               
                   
               
               
                 1321 
                 GAAATGCTGT 
                 GGAGTCAAGA 
                 GTGTCTGGAT 
                 TTATGCCGGC 
               
               
                   
               
               
                 1361 
                 ATGTTTCATC 
                 AGTGGTTCGC 
                 CTGCTCAACG 
                 ATTTACAAAC 
               
               
                   
               
               
                 1401 
                 TTTCAAGAAG 
                 GAGCGCATAG 
                 AAAATACGAT 
                 AAACGGTGTG 
               
               
                   
               
               
                 1441 
                 GACGTTCAGC 
                 TAGCTGCTCG 
                 TAAAGGCGAA 
                 AGAGCCATTA 
               
               
                   
               
               
                 1481 
                 CAGAAGAGGA 
                 GGCCATGTCC 
                 AAGATTAAGG 
                 AAATGGCTGA 
               
               
                   
               
               
                 1521 
                 CCATCACAGG 
                 AGAAAACTGA 
                 TGCAAATTGT 
                 GTATAAAGAA 
               
               
                   
               
               
                 1561 
                 GGAACCATTT 
                 TTCCAAGAGA 
                 ATGCAAAGAT 
                 GTGTTTTTGA 
               
               
                   
               
               
                 1601 
                 GAGTGTGCAG 
                 GATTGGCTAC 
                 TATCTCTACT 
                 CGGGCGATGA 
               
               
                   
               
               
                 1641 
                 GTTAACTTCT 
                 CCACAACAAA 
                 TGAAGGAGGA 
                 TATGAAAGCG 
               
               
                   
               
               
                 1681 
                 TTGGTACATG 
                 AATCATCCTC 
                 TTGA 
                   
               
            
           
         
       
     
     The  Mentha spicata  class I diterpene synthase (MsTPS1) has the amino acid sequence shown below (SEQ ID NO:5). 
                                    1   MSSIRNLSLH   IDLPKAEKKL   VEKIRERIRN   GRVEMSPSAY               41   DTAWVAMVPS   RGYSGRPGFP   ECVDWIIENQ   NPDGSWGLDS               81   DQPLLVKDSL   SSTLACLLAL   RKWKTHNQLV   QRGMEFIDSR               121   GWAATDDDNQ   ISPIGFNIAF   PAMINYAKEL   NLTLPLHPPS               161   IHSLLHIRDS   EIRKRNWEYV   AEGVVDDTSN   WKQIIGTHQR               201   NNGSLFNSPA   TTAAAVIHSH   DDKCFRYLIS   TLENSNGGWV               241   PTIYPYDIYA   PLCMIDTLER   LGIHTYFEVE   LSGIFDDIYR               281   NWQEREEEIF   CNVMCRALAF   RLLRMRGYHV   SSDELAEFVD               321   KEEFFNSVSM   QESGEGTVLE   LYRASLTKIN   EEERILDKIH               361   AWTKPFLKHQ   LLNRSIRDKR   LEKQVEYDLK   NFYGALVRFQ               401   NRRTIDSYDA   KSIQISKTAY   RCSTVYNEDF   IHLSVEDFKI               441   SRAQYLKELE   EMNKWYSDCR   LDLLTKGRNA   CRESYILTAA               481   IIVDPHESMA   RISYAQSILL   ITVFDDFFDH   YGSKEEALNI               521   IDLVKEWKPA   GSYCSKEVEI   LFTALHDTIN   EIAAKADAEQ               561   GFSSKQQLIN   MWVELLESAV   REKDSLSXNK   VSTLEEYLSF               601   APITIGCKLC   VLTSVHFLGI   KLSEEIWTSE   ELSSLCRHGN               641   WCRLLNDLK   TYEREREENT   LNSVSVQTVG   GGVSEEEAVT               681   KVEEVLEFHR   RKVMQLACRR   GGSSVPRECK   ELVWKTCTIG               721   YCLYGHDGGD   ELSSPKDILK   DINAMMFEPL   K            
A nucleic acid encoding the  Mentha spicata  class I diterpene synthase (MsTPS1) with SEQ ID NO:5 is shown below as SEQ ID NO:6.
 
     
       
         
           
               
               
               
               
               
            
               
                 1 
                 ATGAGTTCCA 
                 TTCGAAATTT 
                 AAGTTTGCAT 
                 ATTGATCTGC 
               
               
                   
               
               
                 41 
                 CAAAGGCCGA 
                 GAAGAAGTTG 
                 GTTGAGAAAA 
                 TCAGAGAGAG 
               
               
                   
               
               
                 81 
                 GATAAGAAAT 
                 GGGAGGGTGG 
                 AGATGTCGCC 
                 GTCGGCTTAC 
               
               
                   
               
               
                 121 
                 GACACCGCGT 
                 GGGTGGCCAT 
                 GGTGCCGTCT 
                 CGAGGATATT 
               
               
                   
               
               
                 161 
                 CCGGCAGGCC 
                 GGGTTTCCCG 
                 GAGTGCGTGG 
                 ATTGGATAAT 
               
               
                   
               
               
                 201 
                 CGAGAACCAG 
                 AATCCGGACG 
                 GGTCGTGGGG 
                 TTTGGATTCG 
               
               
                   
               
               
                 241 
                 GATCAACCAC 
                 TTCTGGTCAA 
                 AGACTCCCTC 
                 TCGTCCACCT 
               
               
                   
               
               
                 281 
                 TGGCATGCCT 
                 ACTTGCCCTG 
                 CGTAAATGGA 
                 AAACACACAA 
               
               
                   
               
               
                 321 
                 CCAACTAGTG 
                 CAAAGGGGCA 
                 TGGAGTTCAT 
                 CGACTCCCGT 
               
               
                   
               
               
                 361 
                 GGTTGGGCTG 
                 CAACTGATGA 
                 TGACAATCAG 
                 ATTTCTCCTA 
               
               
                   
               
               
                 401 
                 TTGGATTCAA 
                 TATTGCCTTT 
                 CCTGCAATGA 
                 TTAATTACGC 
               
               
                   
               
               
                 441 
                 CAAAGAGCTT 
                 AATTTAACTC 
                 TGCCTCTACA 
                 TCCACCTTCG 
               
               
                   
               
               
                 481 
                 ATTCATTCAT 
                 TGTTACACAT 
                 TAGAGATTCA 
                 GAAATAAGAA 
               
               
                   
               
               
                 521 
                 AGCGAAACTG 
                 GGAATACGTA 
                 GCTGAAGGAG 
                 TAGTCGACGA 
               
               
                   
               
               
                 561 
                 TACAAGCAAT 
                 TGGAAGCAAA 
                 TAATCGGCAC 
                 GCATCAAAGA 
               
               
                   
               
               
                 601 
                 AATAATGGAT 
                 CCTTGTTCAA 
                 CTCACCTGCT 
                 ACCACTGCAG 
               
               
                   
               
               
                 641 
                 CTGCTGTTAT 
                 TCACTCTCAC 
                 GACGATAAAT 
                 GTTTCCGATA 
               
               
                   
               
               
                 681 
                 TTTGATCTCC 
                 ACTCTTGAGA 
                 ATTCTAACGG 
                 TGGATGGGTA 
               
               
                   
               
               
                 721 
                 CCAACTATCT 
                 ATCCATACGA 
                 TATATACGCT 
                 CCTCTCTGCA 
               
               
                   
               
               
                 761 
                 TGATCGATAC 
                 GCTAGAAAGA 
                 TTAGGAATAC 
                 ACACATATTT 
               
               
                   
               
               
                 801 
                 TGAAGTTGAA 
                 CTCAGCGGCA 
                 TTTTTGATGA 
                 CATATACAGG 
               
               
                   
               
               
                 841 
                 AATTGGCAAG 
                 AGAGAGAAGA 
                 AGAGATCTTT 
                 TGTAATGTTA 
               
               
                   
               
               
                 881 
                 TGTGTCGAGC 
                 TCTGGCATTT 
                 CGGCTTCTAC 
                 GAATGAGGGG 
               
               
                   
               
               
                 921 
                 ATATCATGTT 
                 TCATCTGATG 
                 AACTAGCAGA 
                 ATTTGTGGAC 
               
               
                   
               
               
                 961 
                 AAGGAGGAGT 
                 TTTTTAATAG 
                 CGTGAGCATG 
                 CAAGAGAGCG 
               
               
                   
               
               
                 1001 
                 GCGAAGGCAC 
                 AGTGCTTGAG 
                 CTTTACAGAG 
                 CTTCACTCAC 
               
               
                   
               
               
                 1041 
                 AAAAATCAAC 
                 GAAGAAGAAA 
                 GGATTCTCGA 
                 CAAAATTCAT 
               
               
                   
               
               
                 1081 
                 GCATGGACCA 
                 AACCATTTCT 
                 CAAGCACCAG 
                 CTTCTCAACC 
               
               
                   
               
               
                 1121 
                 GCAGCATTCG 
                 CGACAAACGA 
                 TTAGAGAAGC 
                 AGGTGGAATA 
               
               
                   
               
               
                 1161 
                 CGACTTGAAG 
                 AACTTCTACG 
                 GCGCACTAGT 
                 CCGATTCCAG 
               
               
                   
               
               
                 1201 
                 AACAGAAGAA 
                 CCATCGACTC 
                 ATACGATGCT 
                 AAATCAATCC 
               
               
                   
               
               
                 1241 
                 AAATTTCGAA 
                 AACAGCATAT 
                 AGGTGCTCTA 
                 CAGTTTACAA 
               
               
                   
               
               
                 1281 
                 TGAAGACTTC 
                 ATCCATTTAT 
                 CCGTTGAGGA 
                 CTTCAAAATC 
               
               
                   
               
               
                 1321 
                 TCCCGAGCAC 
                 AATACCTAAA 
                 AGAACTTGAA 
                 GAAATGAACA 
               
               
                   
               
               
                 1361 
                 AGTGGTACTC 
                 TGATTGTAGG 
                 TTGGACCTCT 
                 TAACTAAAGG 
               
               
                   
               
               
                 1401 
                 AAGAAATGCA 
                 TGTCGAGAAT 
                 CTTACATTTT 
                 AACAGCTGCA 
               
               
                   
               
               
                 1441 
                 ATCATTGTCG 
                 ATCCTCACGA 
                 ATCCATGGCT 
                 CGAATCTCTT 
               
               
                   
               
               
                 1481 
                 ACGCTCAATC 
                 TATTCTTCTT 
                 ATAACTGTTT 
                 TCGACGACTT 
               
               
                   
               
               
                 1521 
                 TTTCGATCAT 
                 TATGGGTCTA 
                 AAGAAGAGGC 
                 TCTCAATATT 
               
               
                   
               
               
                 1561 
                 ATTGATCTAG 
                 TCAAGGAATG 
                 GAAGCCAGCT 
                 GGCAGTTACT 
               
               
                   
               
               
                 1601 
                 GCTCCAAAGA 
                 AGTGGAGATT 
                 TTGTTTACTG 
                 CATTACACGA 
               
               
                   
               
               
                 1641 
                 CACGATAAAT 
                 GAGATTGCAG 
                 CCAAGGCTGA 
                 TGCAGAGCAA 
               
               
                   
               
               
                 1681 
                 GGCTTTTCTT 
                 CCAAACAACA 
                 GCTTATCAAC 
                 ATGTGGGTGG 
               
               
                   
               
               
                 1721 
                 AGCTACTTGA 
                 GAGCGCCGTG 
                 AGAGAAAAGG 
                 ACTCGCTGAG 
               
               
                   
               
               
                 1761 
                 TGGNAACAAA 
                 GTGTCGACTC 
                 TAGAAGAGTA 
                 CTTATCTTTC 
               
               
                   
               
               
                 1801 
                 GCACCAATCA 
                 CCATCGGCTG 
                 CAAACTTTGC 
                 GTCCTGACGT 
               
               
                   
               
               
                 1841 
                 CTGTCCATTT 
                 CCTCGGAATC 
                 AAACTGTCCG 
                 AGGAAATCTG 
               
               
                   
               
               
                 1881 
                 GACTTCCGAG 
                 GAGTTGAGCA 
                 GTCTGTGCAG 
                 GCACGGCAAT 
               
               
                   
               
               
                 1921 
                 GTTGTCTGCA 
                 GACTGCTCAA 
                 CGACCTCAAG 
                 ACTTACGAGA 
               
               
                   
               
               
                 1961 
                 GAGAGCGCGA 
                 AGAGAACACG 
                 CTCAACAGCG 
                 TGAGCGTGCA 
               
               
                   
               
               
                 2001 
                 GACAGTGGGA 
                 GGAGGCGTTT 
                 CGGAGGAAGA 
                 GGCGGTGACG 
               
               
                   
               
               
                 2041 
                 AAGGTGGAGG 
                 AGGTGTTGGA 
                 ATTTCATAGA 
                 AGAAAAGTGA 
               
               
                   
               
               
                 2081 
                 TGCAGCTCGC 
                 GTGTCGAAGA 
                 GGAGGAAGCA 
                 GTGTTCCGAG 
               
               
                   
               
               
                 2121 
                 AGAATGTAAG 
                 GAGCTGGTGT 
                 GGAAGACGTG 
                 CACGATAGGT 
               
               
                   
               
               
                 2161 
                 TACTGCTTGT 
                 ACGGTCACGA 
                 CGGAGGCGAT 
                 GAGTTATCGT 
               
               
                   
               
               
                 2201 
                 CTCCGAAGGA 
                 TATTCTAAAG 
                 GACATTAATG 
                 CAATGATGTT 
               
               
                   
               
               
                 2241 
                 TGAGCCTCTC 
                 AAGTGA 
                   
                   
               
            
           
         
       
     
     A  Nepeta mussinii  ent-kaurene synthase (NmTPS2) was identified and isolated. This NmTPS2 enzyme was identified as an ent-kaurene synthase, which converts ent-CPP [16] into ent-kaurene [19]. 
     
       
         
         
             
             
         
       
     
     The  Nepeta mussinii  ent-kaurene synthase (NmTPS2) has the amino acid sequence shown below (SEQ ID NO:7). 
                                    1   MSLPLSSCVL   FPPNDSRFPV   SRFSRASASL   EVGLQGATSA               41   KVSSQSSCFE   ETKRRITKLF   HKDELSVSTY   DTAWVAMVPS               81   PTSSEEPCFP   GCLTWLLENQ   CRDGSWARPH   HHSLLKKDVL               121   SSTLACILAL   KKWGVGEEQI   NKGLHFIELN   CASATEKCQI               161   TPVGFDIIFP   AMLDYARDFS   LNLRLEPTTF   NDLMDKRDLE               201   LKRCYQNYTP   EREAYLAYIV   EGMGRLQDWE   LVMKYQRKNG               241   SLFNCPSTTA   AAFIALRDSA   CLNYLNLSLK   KFGNAVPAVY               281   PLDIYSQLCT   VDNLERLGIN   QYFIAEIQSV   LDETYRCWIQ               321   GNEDIFLDTS   TCALAFRILR   MNGYDVTSDS   LTKILEECFS               361   SSFRGNMTDI   NTTLDLYRAS   ELMLYPDEKD   LEKHNLRLKL               401   LLKQKLSTVL   IQSFQLGRNI   NEEVKQTLEH   PFYASLDRIA               441   KRKNIEHYNF   DNTRILKTSY   CSPNFGNKDF   FFLSIEDFNW               481   CQVIHRQELA   ELERWLIENR   LDELKFARSK   SAYCYFSAAA               521   TFFAPELSDA   RMSWAKSGVL   TTVVDDFFDV   GGSMEELKNL               561   IQLVELWDVD   ASTKCSSHNV   HIIFSALRRT   IYEIGNKGFK               601   LQGRNITNHI   IDIWLDLLNS   MMKETEWARD   NFVPTIDEYM               641   SNAYTSFALG   PIVLPTLYLV   GPKLSEEMIN   HSEYHNLFKL               681   MSTCGRLLND   IRGYERELKD   GKLNALSLYI   INNGGKVSKE               721   AGISEMKSWI   EAQRRELLRL   VLESNKSVLP   KSCKELFWHM               761   CSVVHLFYCK   DDGFTSQDLI   QVVNAVIHEP   IALKDFKVHE            
A nucleic acid encoding the  Nepeta mussinii  ent-kaurene synthase (NmTPS2) with SEQ ID NO:7 is shown below as SEQ ID NO:8.
 
     
       
         
           
               
               
               
               
               
            
               
                 1 
                 ATGTCTCTTC 
                 CGCTCTCCTC 
                 TTGTGTCTTA 
                 TTTCCCCCCA 
               
               
                   
               
               
                 41 
                 ATGACTCACG 
                 TTTTCCGGTC 
                 TCCCGCTTTT 
                 CTCGCGCTTC 
               
               
                   
               
               
                 81 
                 AGCTTCTTTG 
                 GAAGTCGGGC 
                 TTCAAGGAGC 
                 TACTTCAGCA 
               
               
                   
               
               
                 121 
                 AAAGTCTCCT 
                 CACAATCATC 
                 GTGTTTTGAG 
                 GAGACAAAGA 
               
               
                   
               
               
                 161 
                 GAAGGATAAC 
                 AAAGTTGTTT 
                 CATAAGGACG 
                 AACTTTCGGT 
               
               
                   
               
               
                 201 
                 TTCGACATAT 
                 GACACAGCAT 
                 GGGTTGCTAT 
                 GGTCCCTTCT 
               
               
                   
               
               
                 241 
                 CCAACTTCTT 
                 CAGAGGAACC 
                 TTGCTTCCCA 
                 GGTTGTTTGA 
               
               
                   
               
               
                 281 
                 CTTGGTTGCT 
                 TGAAAACCAG 
                 TGTCGAGATG 
                 GTTCATGGGC 
               
               
                   
               
               
                 321 
                 TCGTCCCCAC 
                 CATCACTCTT 
                 TGTTAAAAAA 
                 AGATGTCCTT 
               
               
                   
               
               
                 361 
                 TCTTCTACCT 
                 TGGCATGCAT 
                 TCTCGCACTT 
                 AAAAAATGGG 
               
               
                   
               
               
                 401 
                 GGGTTGGTGA 
                 AGAACAAATC 
                 AACAAGGGTT 
                 TGCATTTTAT 
               
               
                   
               
               
                 441 
                 AGAGCTAAAT 
                 TGTGCTTCAG 
                 CTACCGAGAA 
                 GTGTCAAATT 
               
               
                   
               
               
                 481 
                 ACTCCCGTGG 
                 GGTTTGACAT 
                 TATATTTCCT 
                 GCCATGCTTG 
               
               
                   
               
               
                 521 
                 ATTATGCAAG 
                 AGACTTCTCT 
                 TTGAACTTGC 
                 GTTTAGAGCC 
               
               
                   
               
               
                 561 
                 AACTACGTTT 
                 AATGATTTGA 
                 TGGATAAAAG 
                 GGATTTAGAG 
               
               
                   
               
               
                 601 
                 CTCAAAAGGT 
                 GTTACCAAAA 
                 TTACACACCG 
                 GAGAGGGAAG 
               
               
                   
               
               
                 641 
                 CATACTTGGC 
                 ATATATAGTT 
                 GAAGGAATGG 
                 GAAGATTGCA 
               
               
                   
               
               
                 681 
                 AGATTGGGAA 
                 TTGGTGATGA 
                 AATATCAAAG 
                 AAAGAATGGA 
               
               
                   
               
               
                 721 
                 TCTCTTTTCA 
                 ATTGTCCATC 
                 TACAACTGCA 
                 GCAGCTTTTA 
               
               
                   
               
               
                 761 
                 TTGCCCTTCG 
                 GGATTCTGCG 
                 TGCCTCAACT 
                 ATCTGAATTT 
               
               
                   
               
               
                 801 
                 GTCTTTGAAA 
                 AAGTTCGGGA 
                 ATGCAGTTCC 
                 TGCAGTTTAT 
               
               
                   
               
               
                 841 
                 CCTCTAGATA 
                 TATATTCTCA 
                 ACTTTGCACG 
                 GTTGATAATC 
               
               
                   
               
               
                 881 
                 TTGAAAGGCT 
                 GGGGATCAAC 
                 CAATATTTTA 
                 TAGCAGAAAT 
               
               
                   
               
               
                 921 
                 TCAGAGTGTG 
                 TTGGATGAAA 
                 CGTACAGATG 
                 TTGGATACAG 
               
               
                   
               
               
                 961 
                 GGAAACGAAG 
                 ACATATTTTT 
                 GGACACCTCA 
                 ACTTGTGCTT 
               
               
                   
               
               
                 1001 
                 TAGCATTCCG 
                 AATATTGAGA 
                 ATGAATGGCT 
                 ATGATGTGAC 
               
               
                   
               
               
                 1041 
                 TTCAGATTCA 
                 CTTACAAAAA 
                 TCCTAGAAGA 
                 GTGCTTTTCA 
               
               
                   
               
               
                 1081 
                 AGTTCCTTTC 
                 GTGGAAATAT 
                 GACAGACATT 
                 AACACAACTC 
               
               
                   
               
               
                 1121 
                 TTGACTTATA 
                 TAGGGCATCA 
                 GAACTTATGT 
                 TATATCCAGA 
               
               
                   
               
               
                 1161 
                 TGAAAAGGAT 
                 CTGGAGAAAC 
                 ATAATTTAAG 
                 GCTTAAACTC 
               
               
                   
               
               
                 1201 
                 TTACTTAAGC 
                 AAAAACTATC 
                 CACTGTTTTA 
                 ATCCAATCAT 
               
               
                   
               
               
                 1241 
                 TTCAACTTGG 
                 AAGAAATATC 
                 AATGAAGAGG 
                 TGAAACAGAC 
               
               
                   
               
               
                 1281 
                 TCTCGAGCAT 
                 CCCTTTTATG 
                 CAAGTTTGGA 
                 TAGGATTGCA 
               
               
                   
               
               
                 1321 
                 AAGCGGAAAA 
                 ATATAGAGCA 
                 TTACAACTTT 
                 GATAACACAA 
               
               
                   
               
               
                 1361 
                 GAATTCTTAA 
                 AACTTCATAT 
                 TGTTCGCCAA 
                 ATTTTGGCAA 
               
               
                   
               
               
                 1401 
                 CAAGGATTTC 
                 TTTTTTCTTT 
                 CCATAGAAGA 
                 CTTCAATTGG 
               
               
                   
               
               
                 1441 
                 TGTCAAGTCA 
                 TACATCGACA 
                 AGAACTCGCA 
                 GAACTTGAAA 
               
               
                   
               
               
                 1481 
                 GATGGTTAAT 
                 TGAAAATAGA 
                 TTGGATGAGC 
                 TGAAGTTTGC 
               
               
                   
               
               
                 1521 
                 AAGGAGTAAG 
                 TCTGCATACT 
                 GTTATTTTTC 
                 TGCGGCAGCA 
               
               
                   
               
               
                 1561 
                 ACTTTTTTTG 
                 CTCCAGAATT 
                 GTCGGATGCC 
                 CGCATGTCAT 
               
               
                   
               
               
                 1601 
                 GGGCTAAAAG 
                 TGGTGTTCTA 
                 ACCACAGTGG 
                 TAGATGACTT 
               
               
                   
               
               
                 1641 
                 TTTTGATGTT 
                 GGAGGTTCTA 
                 TGGAGGAATT 
                 GAAGAACTTA 
               
               
                   
               
               
                 1681 
                 ATTCAATTGG 
                 TTGAACTATG 
                 GGATGTGGAT 
                 GCTAGCACAA 
               
               
                   
               
               
                 1721 
                 AATGCTCTTC 
                 TCATAATGTC 
                 CATATAATAT 
                 TTTCAGCACT 
               
               
                   
               
               
                 1761 
                 TAGGCGCACC 
                 ATCTATGAGA 
                 TAGGGAACAA 
                 AGGATTTAAG 
               
               
                   
               
               
                 1801 
                 CTACAAGGAC 
                 GTAACATTAC 
                 CAATCATATA 
                 ATTGACATTT 
               
               
                   
               
               
                 1841 
                 GGCTAGATTT 
                 ACTAAACTCT 
                 ATGATGAAAG 
                 AAACCGAATG 
               
               
                   
               
               
                 1881 
                 GGCCAGAGAC 
                 AACTTTGTCC 
                 CAACAATTGA 
                 TGAATACATG 
               
               
                   
               
               
                 1921 
                 AGCAATGCAT 
                 ATACATCGTT 
                 TGCTCTGGGG 
                 CCAATTGTCC 
               
               
                   
               
               
                 1961 
                 TTCCAACTCT 
                 CTATCTTGTC 
                 GGGCCCAAGC 
                 TCTCAGAAGA 
               
               
                   
               
               
                 2001 
                 GATGATTAAC 
                 CACTCCGAAT 
                 ACCATAACCT 
                 ATTCAAATTG 
               
               
                   
               
               
                 2041 
                 ATGAGTACGT 
                 GCGGACGTCT 
                 TCTAAATGAC 
                 ATCCGTGGTT 
               
               
                   
               
               
                 2081 
                 ATGAGAGAGA 
                 ACTGAAAGAT 
                 GGTAAATTGA 
                 ACGCGTTATC 
               
               
                   
               
               
                 2121 
                 ATTGTACATA 
                 ATTAATAATG 
                 GTGGTAAAGT 
                 AAGTAAAGAA 
               
               
                   
               
               
                 2161 
                 GCTGGCATCT 
                 CGGAGATGAA 
                 AAGTTGGATC 
                 GAGGCACAAC 
               
               
                   
               
               
                 2201 
                 GAAGAGAGTT 
                 ACTGAGATTA 
                 GTTTTGGAGA 
                 GCAACAAAAG 
               
               
                   
               
               
                 2241 
                 CGTCCTTCCG 
                 AAGTCGTGCA 
                 AGGAATTGTT 
                 TTGGCATATG 
               
               
                   
               
               
                 2281 
                 TGCTCAGTGG 
                 TGCATCTATT 
                 CTACTGCAAA 
                 GATGATGGAT 
               
               
                   
               
               
                 2321 
                 TCACCTCGCA 
                 GGATTTGATT 
                 CAAGTTGTAA 
                 ATGCAGTTAT 
               
               
                   
               
               
                 2361 
                 TCATGAACCT 
                 ATTGCTCTCA 
                 AGGATTTTAA 
                 GGTGCATGAA 
               
               
                   
               
               
                 2401 
                 TAA 
                   
                   
                   
               
            
           
         
       
     
     An  Origanum majorana  trans-abienol synthase (OmTPS3) was identified and isolated. When this OmTPS3 enzyme was expressed in  N. benthamiana  with  Hyptis suaveolens  labda-7,13E-dienyl diphosphate synthase (HsTPS1) a new compound, labda-7,12E,14-triene [24], was produced. The HsTPS1 enzyme produced labda-7,13(16),14-triene [22] when HsTPS1 was expressed in  N. benthamiana.    
     
       
         
         
             
             
         
       
     
     OmTPS3 also produced trans-abienol [11] from labda-13-en-8-ol diphosphate ((+)-8-LPP) [10]). 
     
       
         
         
             
             
         
       
     
     The  Origanum majorana  trans-abienol synthase (OmTPS3) has the amino acid sequence shown below (SEQ ID NO:9). 
                                MASLAETPGA   ATFSGNVVRR   RKDNFPVHGF   PTTIRSSVSV               TVKCYVSTTN   LMVKIKEKFK   GKNVNSLTVE   AADDDMPSNL               CIIDTLQRLG   IDRYFQPQVD   SVLDHAYKLW   QGKEKDTVYS               DISIHAMAFR   LLRVKGYQVS   SEELDPYIDV   ERMKKLKTVD               VPTVIELYRA   AQERMYEEEG   SLERLHVWST   NFLMHQLQAN               SIPDEKLHKL   VEYYLKNYHG   ILDRVGVRRN   LDLFDISHYP               TLRARVPNLC   TEDFLSFAKE   DFNTCQAQHQ   KEHEQLQRWF               EDCRFDTLKF   GRETAVGAAH   FLSSAILGES   ELCNVRLALA               KHMVLVVFID   DFFDHYGSRE   DSFKILHLLK   EWKEKPAGEY               GSEEVEILFT   AVYNTVNELA   EMAHVEQGRN   IKGFLIELWV               EIVSIFKIEL   DTWSNDTTLT   LDEYLSSSWV   SVGCRICILV               SMQLLGVQLT   DEMLLSDECI   NLCKHVSMVD   RLLNDVGTFE               KERKENTGNS   VSLLLAAAVK   EGRPITEEEA   IIKIKKMAEN               ERRKLMQIVY   KRESVFPRKC   KDMFLKVCRI   GCYLYASGDE               FTSPQKMKED   VKSLIYESL                    
A nucleic acid encoding the  Origanum majorana  trans-abienol synthase (OmTPS3) with SEQ ID NO:9 is shown below as SEQ ID NO:10.
 
     
       
         
           
               
               
            
               
                   
                 ATGGCGTCGC TCGCGTTCAC ACCCGGAGCC GCCACTTTCT 
               
               
                   
               
               
                   
                 CCGGCAACGT AGTTCGGAGG AGGAAAGATA ACTTTCCGGT 
               
               
                   
               
               
                   
                 CCACGGATTT CCGACGACGA TCAGGTCATC GGTCTCCGTC 
               
               
                   
               
               
                   
                 ACCGTCAAAT GCTACGTCAG TACAACGAAT TTGATGGTGA 
               
               
                   
               
               
                   
                 AAATCAAAGA GAAGTTCAAG GGTAAAAACG TCAATTCGCT 
               
               
                   
               
               
                   
                 GACAGTTGAA GCTGCTGATG ACGATATGCC CTCTAATCTG 
               
               
                   
               
               
                   
                 TGCATAATTG ACACCCTCCA ACGATTGGGA ATCGACCGTT 
               
               
                   
               
               
                   
                 ACTTCCAACC CCAAGTCGAC TCTGTTCTCG ACCACGCCTA 
               
               
                   
               
               
                   
                 CAAACTATGG CAAGGGAAAG AGAAAGATAC GGTGTATTCG 
               
               
                   
               
               
                   
                 GACATTAGTA TTCATGCGAT GGCATTTAGA CTTTTACGAG 
               
               
                   
               
               
                   
                 TCAAAGGCTA TCAAGTCTCT TCGGAGGAAC TGGATCCATA 
               
               
                   
               
               
                   
                 CATCGATGTG GAGCGAATGA AGAAACTGAA AACAGTTGAT 
               
               
                   
               
               
                   
                 GTTCCGACGG TTATCGAACT GTACAGAGCG GCACAGGAGA 
               
               
                   
               
               
                   
                 GAATGTATGA AGAAGAAGGT AGCCTTGAGA GACTCCATGT 
               
               
                   
               
               
                   
                 TTGGAGCACC AACTTCCTCA TGCACCAGCT GCAGGCTAAC 
               
               
                   
               
               
                   
                 TCAATTCCTG ATGAAAAGCT ACACAAACTG GTGGAATACT 
               
               
                   
               
               
                   
                 ACTTGAAGAA CTACCATGGC ATACTGGATA GAGTTGGAGT 
               
               
                   
               
               
                   
                 TCGACGAAAC CTCGACCTAT TCGACATAAG CCATTATCCA 
               
               
                   
               
               
                   
                 ACACTCAGAG CTAGGGTTCC GAACCTATGT ACCGAAGATT 
               
               
                   
               
               
                   
                 TTCTATCGTT CGCGAAGGAA GATTTCAATA CTTGCCAAGC 
               
               
                   
               
               
                   
                 CCAACACCAG AAAGAACATG AGCAACTACA AAGGTGGTTC 
               
               
                   
               
               
                   
                 GAAGATTGTA GGTTCGATAC GTTGAAGTTC GGAAGGGAGA 
               
               
                   
               
               
                   
                 CAGCCGTAGG CGCTGCTCAT TTTCTATCTT CAGCAATACT 
               
               
                   
               
               
                   
                 TGGTGAATCT GAACTATGTA ATGTTCGTCT TGCCCTTGCT 
               
               
                   
               
               
                   
                 AAGCATATGG TGCTTGTGGT ATTCATCGAT GACTTCTTCG 
               
               
                   
               
               
                   
                 ACCATTATGG CTCTAGAGAA GACTCCTTCA AGATCCTCCA 
               
               
                   
               
               
                   
                 CCTCTTAAAA GAATGGAAAG AGAAGCCGGC CGGAGAATAC 
               
               
                   
               
               
                   
                 GGTTCCGAGG AAGTCGAAAT CCTCTTCACA GCCGTATACA 
               
               
                   
               
               
                   
                 ATACAGTAAA CGAGTTGGCG GAGATGGCTC ATGTCGAACA 
               
               
                   
               
               
                   
                 AGGACGTAAT ATCAAAGGAT TTCTAATTGA ATTGTGGGTT 
               
               
                   
               
               
                   
                 GAAATAGTGT CAATTTTCAA GATAGAACTG GATACATGGA 
               
               
                   
               
               
                   
                 GCAATGACAC AACACTAACC TTGGATGAGT ACTTGTCCTC 
               
               
                   
               
               
                   
                 CTCATGGGTG TCGGTCGGTT GCAGAATCTG CATCCTCGTC 
               
               
                   
               
               
                   
                 TCAATGCAGC TCCTCGGTGT ACAACTAACC GACGAAATGC 
               
               
                   
               
               
                   
                 TTCTGAGCGA CGAGTGCATA AACCTGTGTA AGCATGTCTC 
               
               
                   
               
               
                   
                 GATGGTCGAT CGCCTCCTCA ACGACGTCGG AACATTCGAG 
               
               
                   
               
               
                   
                 AAGGAACGGA AGGAGAATAC AGGAAACAGT GTGAGCCTTC 
               
               
                   
               
               
                   
                 TGCTAGCAGC AGCTGTGAAA GAAGGAAGGC CTATTACCGA 
               
               
                   
               
               
                   
                 AGAGGAAGCT ATTATTAAAA TTAAAAAAAT GGCGGAAAAC 
               
               
                   
               
               
                   
                 GAGAGGAGGA AACTAATGCA GATTGTGTAT AAAAGAGAGA 
               
               
                   
               
               
                   
                 GTGTTTTCCC CAGAAAATGC AAGGATATGT TCTTGAAGGT 
               
               
                   
               
               
                   
                 GTGTAGAATT GGGTGCTATC TATACGCGAG CGGCGACGAA 
               
               
                   
               
               
                   
                 TTTACGTCTC CTCAGAAAAT GAAGGAAGAT GTGAAATCCT 
               
               
                   
               
               
                   
                 TAATTTATGA ATCCTTGTAG 
               
            
           
         
       
     
     The  Origanum majorana  manool synthase (OmTPS4) can also convert ent-copalyl diphosphate (ent-CPP) [16] to ent-manool [20]. 
     
       
         
         
             
             
         
       
     
     In addition,  Origanum majorana  manool synthase (OmTPS4) can also convert (+)-copalyl diphosphate ((+)-CPP) [31]) to manool [33]. 
     
       
         
         
             
             
         
       
     
     The  Origanum majorana  manool synthase (OmTPS4) can have the amino acid sequence shown below (SEQ ID NO:11). 
                            MSLAFSHVST FFSGQRVVGS RREIIPVNGV PTTANKPSFA                   VKCNLTTKDL MVKMKEKLKG QDGNLTVGVA DMPSSLCVID                   TLERLGVDRY FRSEIHVILH DTYRLWQQKD KDICSNVTTH                   AMAFRLLRVN GYEVSSEELA PYANLEHFSQ QKVDTAMAIE                   LYRAAQERIH EDESGLDKIL AWTTTFLEQQ LLTNSILDNK                   LHKLVEYYLN NYHGQTNRVG ARRHLDLYEM SHYQNLKPSH                   SLCNEDLLAF AKQGFRDFQI QQQKEFEQLQ RWYEDCRLDK                   LSYGRDVVKI SSFMASILMD DPELADVRLS IAKQMVLVTR                   IDDFFDHGGS REDSYKIIEL VKEWKEKAEY DSEEVKILFT                   AVYTTVNELA EACVQQGRNS TTVKEFLVQL WIEILSAFKV                   ELDTWSDGTE VSLDEYLSWS WISNGCRVSI VTTMHLLPTK                   LCSDEMLRSE ECKDLCRHVS MVGRLLNDIH SFEKEHEENT                   GNSVSILVAG EDTEEEAIGK IKEIVEYERR KLMQIVYKRG                   TILPRECKDI FLKACRATFY VYSSTDEFTS PRQVMEDMKT                   LSS            
A nucleic acid encoding  Origanum majorana  manool synthase (OmTPS4) with SEQ ID NO:11 is shown below as SEQ ID NO:12.
 
     
       
         
           
               
               
            
               
                   
                 ATGTCACTCG CCTTCAGCCA TGTTAGTACC TTTTTCTCCG 
               
               
                   
               
               
                   
                 GCCAAAGAGT CGTCGGAAGC AGGAGAGAGA TTATTCCAGT 
               
               
                   
               
               
                   
                 TAACGGAGTT CCGACGACGG CCAATAAGCC GTCGTTCGCC 
               
               
                   
               
               
                   
                 GTTAAGTGCA ACCTTACTAC AAAGGATTTG ATGGTGAAAA 
               
               
                   
               
               
                   
                 TGAAGGAGAA GTTGAAGGGG CAAGACGGTA ATTTGACTGT 
               
               
                   
               
               
                   
                 CGGAGTAGCC GATATGCCCT CTAGCCTGTG CGTGATCGAC 
               
               
                   
               
               
                   
                 ACTCTTGAAA GGTTGGGAGT TGACCGATAC TTCCGATCTG 
               
               
                   
               
               
                   
                 AAATCCACGT TATTCTACAC GACACTTACC GGTTATGGCA 
               
               
                   
               
               
                   
                 ACAAAAGGAC AAAGATATAT GTTCCAACGT TACTACTCAT 
               
               
                   
               
               
                   
                 GCAATGGCGT TTAGACTTCT GAGAGTGAAT GGATACGAGG 
               
               
                   
               
               
                   
                 TTTCATCAGA GGAACTGGCT CCATATGCTA ACCTAGAGCA 
               
               
                   
               
               
                   
                 CTTTAGCCAG CAAAAAGTTG ATACTGCAAT GGCTATAGAG 
               
               
                   
               
               
                   
                 CTCTACAGAG CAGCACAGGA GAGAATACAC GAAGACGAGA 
               
               
                   
               
               
                   
                 GCGGTCTCGA CAAAATACTT GCTTGGACCA CCACTTTTCT 
               
               
                   
               
               
                   
                 CGAGCAACAG CTGCTCACTA ACTCCATTCT TGACAATAAA 
               
               
                   
               
               
                   
                 TTGCATAAAC TGGTGGAGTA CTACTTGAAC AACTACCACG 
               
               
                   
               
               
                   
                 GCCAAACGAA TAGGGTCGGA GCTAGACGAC ACCTCGACCT 
               
               
                   
               
               
                   
                 ATATGAGATG AGCCATTACC AAAATCTAAA ACCTTCACAT 
               
               
                   
               
               
                   
                 AGTCTATGCA ATGAAGACCT TCTAGCATTT GCAAAGCAAG 
               
               
                   
               
               
                   
                 GTTTTCGAGA TTTTCAAATC CAGCAGCAGA AAGAATTCGA 
               
               
                   
               
               
                   
                 GCAACTGCAA AGGTGGTATG AAGATTGCAG GTTGGACAAG 
               
               
                   
               
               
                   
                 TTGAGTTATG GGAGAGATGT AGTAAAAATT TCTAGTTTCA 
               
               
                   
               
               
                   
                 TGGCTTCAAT ATTGATGGAT GATCCAGAAT TAGCCGATGT 
               
               
                   
               
               
                   
                 TCGTCTCTCC ATCGCCAAAC AGATGGTGCT CGTGACACGT 
               
               
                   
               
               
                   
                 ATCGATGATT TCTTCGACCA CGGTGGCTCT AGAGAAGACT 
               
               
                   
               
               
                   
                 CCTACAAGAT CATTGAACTA GTAAAAGAAT GGAAGGAGAA 
               
               
                   
               
               
                   
                 GGCaGAATAC GATTCCGAGG AAGTAAAAAT CCTTTTTACA 
               
               
                   
               
               
                   
                 GCAGTATACA CCACAGTAAA TGAGCTAGCA GAGGCTTGTG 
               
               
                   
               
               
                   
                 TTCAACAAGG AAGGAATAGT ACTACTGTCA AAGAATTCCT 
               
               
                   
               
               
                   
                 AGTTCAGTTG TGGATTGAAA TACTATCAGC TTTCAAGGTC 
               
               
                   
               
               
                   
                 GAGCTAGATA CGTGGAGCGA TGGCACGGAA GTAAGCCTGG 
               
               
                   
               
               
                   
                 ACGAGTACTT GTCGTGGTCG TGGATTTCGA ATGGCTGCAG 
               
               
                   
               
               
                   
                 AGTGTCTATA GTAACGACGA TGCATTTGCT CCCTACGAAA 
               
               
                   
               
               
                   
                 TTATGCAGTG ATGAAATGCT TAGGAGTGAA GAGTGCAAGG 
               
               
                   
               
               
                   
                 ATTTGTGTAG GCATGTTTCT ATGGTTGGCC GCTTGCTCAA 
               
               
                   
               
               
                   
                 CGACATCCAC TCTTTTGAGA AGGAGCATGA GGAGAATACG 
               
               
                   
               
               
                   
                 GGAAACAGTG TGAGCATTCT AGTAGCAGGT GAGGATACCG 
               
               
                   
               
               
                   
                 AAGAGGAAGC TATTGGAAAG ATCAAAGAGA TAGTTGAGTA 
               
               
                   
               
               
                   
                 TGAGAGGAGA AAATTGATGC AAATTGTGTA CAAGAGAGGA 
               
               
                   
               
               
                   
                 ACCATTCTCC CAAGAGAATG CAAAGACATA TTCTTGAAGG 
               
               
                   
               
               
                   
                 CGTGTAGGGC TACATTTTAC GTGTACTCGA GCACGGATGA 
               
               
                   
               
               
                   
                 GTTTACGTCT CCTCGACAAG TGATGGAAGA TATGAAAACC 
               
               
                   
               
               
                   
                 CTAAGCTCCT AG 
               
            
           
         
       
     
       Origanum majorana  palustradiene synthase (OmTPS5) can also convert (+)-copalyl diphosphate ((+)-CPP) [M]) to palustradiene [29]. 
     
       
         
         
             
             
         
       
     
     The  Origanum majorana  palustradiene synthase (OmTPS5) can have the amino acid sequence shown below (SEQ ID NO:13). 
                            MVSACLKLKN NPFLDHRFRK SSNGFSVNFP ATMLTTVKCS                   RDNSEDLIAK IKERMNEKFV TVPAREYSVI EHRNPKPAWC                   GGLQSKTVIE EEVCSRLFLV EHLQDLGVDR FFQSEIQHIL                   HHTFRLWQQK DEQVFKDVTC RAMAFRLLRL EGYHVSSGEL                   GEYVDEEKFF RTVRLEWRST DTILELYKAS QVRLPEDDND                   NSNILKNLHE WTFIFLKEQL RRKTILDKGL ERKVEFYLKN                   YHGILDAVKH RRSLDHTRFW KTTAYNPAVY DEDLFRLSAQ                   DFMARQAQSQ KELEMLLKWY DECRLDKMEY GRNVIHVSHF                   LNANNFPDPR LSETRLSFAK TMTLVTRLDD FFDHHGSRED                   SVLIIELIRQ WNEPSTITTI FPSEEVEILY SALHSTVTDI                   AEKAYPIQGR CIKSLIIHLW VEILSSFMSE MDSCTAETQP                   DFHEYLGFAW ISIGCRICIL IAIHFLGEKV SQQMVMGAEC                   TELCRHVSTI ARLLNDLQTF KKEREERKVN SVIIQLKGDK                   ISEEVAVSNI ERMVEYHRKE LLKMVVRREG SLVPKRCKDV                   FWKSCNIAYY LYAFTDEFTS PQQMKEDMKL LFRDPINCVP                   SIPS            
A nucleic acid encoding the  Origanum majorana  palustradiene synthase (OmTPS5) with SEQ ID NO:13 is shown below as SEQ ID NO:14.
 
     
       
         
           
               
               
            
               
                   
                 ATGGTATCTG CATGTCTAAA ACTCAAAAAT AATCCTTTCT 
               
               
                   
               
               
                   
                 TGGACCATCG ATTCAGGAAA AGCAGCAATG GATTTTCAGT 
               
               
                   
               
               
                   
                 TAATTTTCCG GCGACCATGC TCACCACTGT CAAGTGCAGC 
               
               
                   
               
               
                   
                 CGCGATAATT CAGAAGACTT GATAGCAAAG ATAAAAGAAA 
               
               
                   
               
               
                   
                 GGATGAATGA AAAATTTGTT ACGGTGCCGG CGAGGGAATA 
               
               
                   
               
               
                   
                 TTCCGTCATT GAGCATCGGA ATCCGAAGCC GGCGTGGTGC 
               
               
                   
               
               
                   
                 GGTGGTTTGC AATCCAAAAC AGTAATAGAA GAAGAAGTGT 
               
               
                   
               
               
                   
                 GCAGCCGTCT GTTTCTGGTC GAACACCTTC AAGATTTAGG 
               
               
                   
               
               
                   
                 AGTAGACCGC TTCTTTCAAT CAGAAATCCA ACATATTCTA 
               
               
                   
               
               
                   
                 CATCACACAT TCAGATTATG GCAGCAAAAA GATGAACAAG 
               
               
                   
               
               
                   
                 TTTTTAAAGA CGTGACATGT CGCGCCATGG CATTCAGACT 
               
               
                   
               
               
                   
                 CCTGCGTCTC GAAGGTTATC ATGTCTCGTC AGGAGAATTG 
               
               
                   
               
               
                   
                 GGGGAGTATG TTGATGAGGA AAAATTCTTT AGAACGGTAA 
               
               
                   
               
               
                   
                 GGTTAGAATG GAGAAGTACG GATACAATTC TTGAGCTGTA 
               
               
                   
               
               
                   
                 CAAAGCATCA CAGGTAAGAC TACCTGAAGA CGACAACGAC 
               
               
                   
               
               
                   
                 AATTCCAATA TCCTCAAAAA CTTGCACGAA TGGACCTTCA 
               
               
                   
               
               
                   
                 TATTTTTGAA GGAGCAGTTG CGGCGTAAAA CTATTCTTGA 
               
               
                   
               
               
                   
                 TAAAGGTTTA GAGAGAAAGG TAGAATTTTA CTTGAAGAAT 
               
               
                   
               
               
                   
                 TACCACGGCA TATTAGACGC GGTTAAGCAT AGACGAAGCC 
               
               
                   
               
               
                   
                 TCGATCACAC ACGATTCTGG AAAACTACTG CGTATAACCC 
               
               
                   
               
               
                   
                 TGCAGTGTAT GATGAGGATC TTTTCCGATT GTCGGCCCAA 
               
               
                   
               
               
                   
                 GATTTCATGG CTCGCCAAGC TCAGAGCCAG AAGGAACTTG 
               
               
                   
               
               
                   
                 AGATGTTGCT CAAGTGGTAC GATGAATGTA GACTGGACAA 
               
               
                   
               
               
                   
                 GATGGAGTAT GGGCGAAACG TGATACACGT TTCCCATTTC 
               
               
                   
               
               
                   
                 TTAAACGCAA ACAACTTCCC CGATCCTCGC CTGTCCGAAA 
               
               
                   
               
               
                   
                 CTCGTCTATC CTTTGCGAAA ACCATGACTC TCGTCACGCG 
               
               
                   
               
               
                   
                 TTTGGATGAT TTCTTCGATC ACCATGGCTC TAGAGAAGAT 
               
               
                   
               
               
                   
                 TCGGTCCTCA TCATCGAATT AATAAGGCAG TGGAATGAGC 
               
               
                   
               
               
                   
                 CTTCAACTAT TACAACAATA TTCCCCTCCG AAGAAGTGGA 
               
               
                   
               
               
                   
                 GATTCTCTAC TCTGCACTCC ACTCCACCGT AACAGATATA 
               
               
                   
               
               
                   
                 GCAGAGAAGG CTTATCCCAT CCAGGGTCGC TGCATCAAAT 
               
               
                   
               
               
                   
                 CGCTCATAAT TCATCTGTGG GTCGAGATAC TGTCGAGCTT 
               
               
                   
               
               
                   
                 CATGAGCGAA ATGGACTCGT GCACCGCGGA AACTCAGCCG 
               
               
                   
               
               
                   
                 GACTTTCACG AGTACTTAGG GTTTGCATGG ATCTCGATCG 
               
               
                   
               
               
                   
                 GCTGCAGAAT CTGCATTCTC ATAGCTATAC ATTTCTTGGG 
               
               
                   
               
               
                   
                 GGAGAAGGTA TCTCAACAAA TGGTTATGGG TGCTGAGTGC 
               
               
                   
               
               
                   
                 ACCGAGTTAT GTAGGCACGT TTCTACGATC GCACGCCTTC 
               
               
                   
               
               
                   
                 TCAACGATCT CCAAACCTTT AAGAAGGAGA GAGAAGAGAG 
               
               
                   
               
               
                   
                 GAAGGTAAAC AGCGTGATAA TCCAGCTCAA AGGGGATAAG 
               
               
                   
               
               
                   
                 ATATCGGAGG AGGTGGCCGT GTCGAATATA GAGAGAATGG 
               
               
                   
               
               
                   
                 TTGAATATCA CAGGAAAGAG CTGCTGAAGA TGGTGGTTCG 
               
               
                   
               
               
                   
                 GAGAGAAGGA AGCTTGGTTC CTAAGAGGTG TAAGGACGTG 
               
               
                   
               
               
                   
                 TTCTGGAAAT CCTGCAACAT TGCTTACTAT CTGTACGCTT 
               
               
                   
               
               
                   
                 TTACAGATGA ATTCACTTCG CCTCAACAAA TGAAGGAAGA 
               
               
                   
               
               
                   
                 TATGAAACTA CTCTTTCGTG ATCCAATCAA CTGCGTTCCT 
               
               
                   
               
               
                   
                 TCAATTCCTT CATGA 
               
            
           
         
       
     
     The  Perovskia atriplicifolia  miltiradiene synthase (PaTPS3) can have the amino acid sequence shown below (SEQ ID NO:15). 
                            MLLAFNISDV PLSQHRVILS RREHFPRHAF QEFPMIAATK                   SSVNAICSLA TPTDLMGKIK EKFKAKDGDP LAAAAIQLAA                   DIPSSLCIID TLQRLGVDRY FQSEIDSILE ETHKLWKVKD                   RDIYSEVTTH AMAFRLLRVK GYEVSSEELA PYAEQERFDL                   QTIDLATVIE LYRAAQERTC EENDNSLEKL LAWTTTFLKH                   QLLTNSIPDT KLHKQVEYYL KNYHGILDRM GVRRSLDLYD                   ISHYRPLRAR FPNLCNEDFL SFARQDFSMC QAQHQKELEQ                   LQRWYSDCRL DALLKFGRNV VRVSSFLTSA IIGEPELSEV                   RLVFAKHIIL VTLIDDLFDH GGTREESYKI LELVTEWKEK                   TAAEYGSEEV EILFTAVYNT VNELVERAHV EQGRSVKEFL                   IKLWVQILSI FKIELDTWSD ETALTLDEYL SSSWVSIGCR                   ICILMSMQFI GIKLTDEMLL SEECTDLCRH VSMVDRLLND                   VQTFEKERKE NTGNSVSLLL AANKDVTEEE AIRRAKEMAE                   CNRRQLMQIV YKTGTIFPRK CKDMFLKVCR IGCYLYASGD                   EFTSPQQMME DMKSLVYEPL YLPN            
A nucleic acid encoding the  Perovskia atriplicifolia  miltiradiene synthase (PaTPS3) with SEQ ID NO:15 is shown below as SEQ ID NO:16.
 
     
       
         
           
               
               
            
               
                   
                 ATGTTACTTG CGTTCAACAT AAGCGATGTC CCTCTCTCGC 
               
               
                   
               
               
                   
                 AGCATAGAGT AATTCTGAGC AGGAGGGAAC ATTTTCCACG 
               
               
                   
               
               
                   
                 TCATGCATTC CAGGAATTTC CGATGATCGC CGCTACTAAG 
               
               
                   
               
               
                   
                 TCATCTGTTA ATGCCATTTG CAGCCTCGCT ACTCCAACTG 
               
               
                   
               
               
                   
                 ATTTGATGGG AAAAATAAAA GAGAAGTTCA AGGCCAAGGA 
               
               
                   
               
               
                   
                 CGGCGATCCT CTTGCCGCCG CGGCTATTCA ACTCGCGGCG 
               
               
                   
               
               
                   
                 GATATACCCT CGAGTCTGTG TATAATCGAC ACCCTCCAGA 
               
               
                   
               
               
                   
                 GGTTGGGAGT CGACCGATAC TTCCAATCCG AAATCGACTC 
               
               
                   
               
               
                   
                 TATTCTAGAG GAAACACACA AGTTATGGAA AGTGAAAGAT 
               
               
                   
               
               
                   
                 AGAGATATAT ACTCTGAGGT TACTACTCAT GCAATGGCGT 
               
               
                   
               
               
                   
                 TTAGACTTCT GCGAGTGAAG GGATATGAAG TTTCATCAGA 
               
               
                   
               
               
                   
                 GGAACTAGCT CCGTATGCTG AGCAAGAGCG CTTTGACCTG 
               
               
                   
               
               
                   
                 CAAACGATTG ATCTGGCGAC GGTTATCGAG CTTTACAGAG 
               
               
                   
               
               
                   
                 CAGCACAGGA GAGAACATGC GAAGAAAACG ACAACAGTCT 
               
               
                   
               
               
                   
                 TGAGAAACTA CTTGCTTGGA CCACCACCTT TCTCAAGCAC 
               
               
                   
               
               
                   
                 CAATTGCTCA CCAACTCCAT ACCTGACACC AAATTGCACA 
               
               
                   
               
               
                   
                 AACAGGTGGA ATACTACTTG AAGAACTACC ACGGGATATT 
               
               
                   
               
               
                   
                 AGATAGAATG GGAGTTAGAC GAAGCCTCGA CCTATACGAC 
               
               
                   
               
               
                   
                 ATAAGCCATT ATCGACCTCT GAGAGCAAGA TTCCCTAATC 
               
               
                   
               
               
                   
                 TGTGTAATGA AGATTTCCTA TCATTTGCGA GGCAAGATTT 
               
               
                   
               
               
                   
                 CAGTATGTGC CAAGCCCAAC ACCAGAAGGA ACTTGAGCAA 
               
               
                   
               
               
                   
                 CTGCAAAGGT GGTATTCTGA TTGTAGGTTG GACGCGTTGT 
               
               
                   
               
               
                   
                 TGAAGTTTGG AAGAAATGTA GTGCGCGTTT CTAGCTTTCT 
               
               
                   
               
               
                   
                 GACTTCAGCA ATTATTGGTG AACCCGAATT GTCTGAAGTT 
               
               
                   
               
               
                   
                 CGACTAGTCT TTGCCAAACA TATTATTCTC GTTACACTTA 
               
               
                   
               
               
                   
                 TTGATGATTT ATTCGATCAT GGTGGAACTA GAGAAGAGTC 
               
               
                   
               
               
                   
                 ATACAAGATC CTTGAATTAG TAACAGAATG GAAAGAGAAG 
               
               
                   
               
               
                   
                 ACCGCAGCAG AATATGGTTC CGAGGAAGTT GAAATCCTTT 
               
               
                   
               
               
                   
                 TTACAGCGGT CTACAACACA GTAAATGAGT TGGTAGAGAG 
               
               
                   
               
               
                   
                 GGCTCATGTC GAACAAGGGC GCAGTGTCAA AGAATTTCTT 
               
               
                   
               
               
                   
                 ATTAAACTGT GGGTTCAAAT ACTATCAATT TTCAAGATAG 
               
               
                   
               
               
                   
                 AATTAGATAC ATGGAGCGAT GAGACTGCGC TAACCTTGGA 
               
               
                   
               
               
                   
                 TGAATACTTG TCTTCGTCGT GGGTGTCAAT TGGTTGCAGA 
               
               
                   
               
               
                   
                 ATCTGCATTC TCATGTCGAT GCAATTCATC GGTATAAAAT 
               
               
                   
               
               
                   
                 TAACTGATGA AATGCTTCTG AGTGAAGAGT GCACTGATTT 
               
               
                   
               
               
                   
                 GTGTAGGCAT GTTTCGATGG TTGACCGGCT GCTCAACGAT 
               
               
                   
               
               
                   
                 GTGCAAACCT TCGAGAAGGA ACGCAAAGAA AATACAGGAA 
               
               
                   
               
               
                   
                 ACAGTGTAAG CCTTCTGCTA GCAGCTAACA AAGATGTTAC 
               
               
                   
               
               
                   
                 TGAAGAGGAA GCAATTAGAA GAGCAAAAGA AATGGCGGAA 
               
               
                   
               
               
                   
                 TGCAACAGGA GACAACTGAT GCAGATTGTG TATAAAACAG 
               
               
                   
               
               
                   
                 GAACCATTTT CCCAAGAAAA TGCAAAGATA TGTTTCTCAA 
               
               
                   
               
               
                   
                 GGTATGCAGG ATTGGCTGTT ATTTGTATGC AAGCGGCGAC 
               
               
                   
               
               
                   
                 GAATTCACAT CTCCACAACA AATGATGGAA GATATGAAAT 
               
               
                   
               
               
                   
                 CCTTGGTTTA TGAACCCCTC TACCTACCTA ATTAA 
               
            
           
         
       
     
     A  Perovskia atriplicifolia  miltiradiene synthase (PaTPS1) can have the amino acid sequence shown below (SEQ ID NO:17). 
                            MSLTFNAGVV RFSSHRVRST KDCFTVYGFP MIANKAAFAV                   KCSLTPTDLM GRVEEKFKGK NGNSLAASTT VESADIPSNL                   CIIDTLQRLG VDRYFQTEIN AILEDTYRLW ERKDKDIYSD                   ATTHAMAFRL LRVKGYEVSS EELAPYADQE CVNVQTADVA                   TVIELYRAAQ VRISEEESSL KKLHAWTTTF LKYQLQSNSI                   PEKKLHKLVE YYLKNYHGIL DRMGVRMDLD LFDISHYRTL                   QASDRFSSLR NEDFLEFARQ DFNICQAKHQ KELQQLQRWY                   ADCRLDTLKF GRDVVRVANF LTSAIFGEPE LSDARLIFAK                   HIVLVTCIDE FFDHGGSKEE SYKILELVEE WKEKPTGEYG                   CEEVEILFTA VYSTVNELAE MAHVEQGRSV KEFLVKLWVQ                   ILSIFKIELD TWSDDTELTL DSYLNNSWVS IGCRICILMS                   MQFAGVKLSD EMLLSEECVD LCRHVSMVDR LLNDVQTFEK                   ERKENTGNSV SLLQAAAERE GRAITEEEAI TQIKELAEYH                   RRKLMQIVYK TDTIFPRKCK DMFLKVCRIG CYLYASGDEF                   TTPQQMMEDM KSLVYQPLTV DDMSAKELTS VRN            
A nucleic acid encoding the  Perovskia atriplicifolia  miltiradiene synthase (PaTPS1) with SEQ ID NO:17 is shown below as SEQ ID NO:18.
 
     
       
         
           
               
               
            
               
                   
                 ATGTCACTCA CTTTCAACGC TGGAGTCGTC CGTTTCTCCA 
               
               
                   
               
               
                   
                 GCCACCGCGT TCGGAGCACG AAAGATTGCT TTACAGTTTA 
               
               
                   
               
               
                   
                 CGGATTTCCG ATGATTGCAA ATAAGGCAGC TTTCGCAGTT 
               
               
                   
               
               
                   
                 AAATGCAGCC TTACTCCAAC CGATTTGATG GGGAGAGTAG 
               
               
                   
               
               
                   
                 AGGAGAAGTT CAAGGGCAAA AATGGTAATT CACTAGCAGC 
               
               
                   
               
               
                   
                 CTCGACGACG GTTGAATCCG CGGATATACC CTCGAACCTG 
               
               
                   
               
               
                   
                 TGTATAATCG ACACCCTCCA AAGATTGGGA GTCGACCGAT 
               
               
                   
               
               
                   
                 ACTTTCAAAC TGAAATCAAT GCCATTCTAG AGGACACTTA 
               
               
                   
               
               
                   
                 CAGATTATGG GAACGAAAAG ACAAAGACAT ATATTCCGAT 
               
               
                   
               
               
                   
                 GCCACAACTC ACGCGATGGC GTTTAGGTTA CTACGAGTGA 
               
               
                   
               
               
                   
                 AAGGATACGA AGTTTCATCA GAGGAACTGG CTCCTTACGC 
               
               
                   
               
               
                   
                 TGATCAAGAG TGCGTGAACG TGCAAACGGC TGATGTGGCA 
               
               
                   
               
               
                   
                 ACAGTTATCG AGCTTTACAG AGCAGCGCAG GTGAGAATAA 
               
               
                   
               
               
                   
                 GCGAAGAAGA GAGCAGTCTT AAGAAGCTTC ATGCTTGGAC 
               
               
                   
               
               
                   
                 CACCACCTTT CTCAAATATC AGTTGCAGAG TAACTCCATA 
               
               
                   
               
               
                   
                 CCTGAAAAGA AACTGCACAA ACTGGTGGAA TATTACTTGA 
               
               
                   
               
               
                   
                 AGAACTACCA TGGCATATTG GATAGAATGG GAGTTCGAAT 
               
               
                   
               
               
                   
                 GGACCTCGAC TTATTCGACA TCAGCCATTA TCGAACTCTA 
               
               
                   
               
               
                   
                 CAAGCTTCCG ATAGGTTCTC TAGTCTGCGT AACGAAGATT 
               
               
                   
               
               
                   
                 TTCTAGAGTT TGCAAGGCAA GATTTCAATA TCTGCCAAGC 
               
               
                   
               
               
                   
                 CAAGCACCAG AAAGAACTCC AACAACTGCA AAGGTGGTAT 
               
               
                   
               
               
                   
                 GCAGATTGCA GGCTCGACAC CTTGAAGTTC GGGAGAGACG 
               
               
                   
               
               
                   
                 TCGTACGCGT TGCTAATTTT CTGACTTCAG CAATCTTTGG 
               
               
                   
               
               
                   
                 CGAACCCGAG CTATCCGATG CTCGTCTGAT CTTTGCCAAG 
               
               
                   
               
               
                   
                 CATATCGTGC TCGTAACATG TATCGATGAA TTCTTCGATC 
               
               
                   
               
               
                   
                 ATGGTGGGTC TAAAGAAGAG TCCTACAAGA TCCTTGAATT 
               
               
                   
               
               
                   
                 AGTAGAAGAA TGGAAAGAGA AGCCAACTGG AGAATATGGG 
               
               
                   
               
               
                   
                 TGTGAGGAGG TTGAGATCCT TTTCACAGCA GTGTACAGTA 
               
               
                   
               
               
                   
                 CAGTGAATGA GTTGGCAGAG ATGGCTCATG TCGAACAAGG 
               
               
                   
               
               
                   
                 ACGTAGTGTG AAAGAGTTTC TAGTTAAACT GTGGGTGCAG 
               
               
                   
               
               
                   
                 ATACTGTCGA TTTTCAAGAT AGAACTGGAT ACATGGAGTG 
               
               
                   
               
               
                   
                 ATGACACGGA ACTGACGTTG GACAGCTACT TGAACAACTC 
               
               
                   
               
               
                   
                 GTGGGTGTCG ATCGGATGCA GAATCTGCAT TCTCATGTCG 
               
               
                   
               
               
                   
                 ATGCAGTTCG CCGGTGTAAA ACTGTCCGAC GAAATGCTTC 
               
               
                   
               
               
                   
                 TGAGTGAAGA GTGTGTTGAC TTGTGCAGGC ACGTCTCCAT 
               
               
                   
               
               
                   
                 GGTCGATCGC CTCCTGAACG ATGTGCAAAC TTTCGAGAAG 
               
               
                   
               
               
                   
                 GAACGCAAGG AAAATACAGG AAACAGTGTG AGCCTTCTGC 
               
               
                   
               
               
                   
                 AAGCAGCAGC TGAGAGAGAA GGAAGAGCCA TTACAGAAGA 
               
               
                   
               
               
                   
                 GGAAGCTATT ACACAGATCA AAGAATTGGC TGAATACCAC 
               
               
                   
               
               
                   
                 AGGAGAAAAC TGATGCAGAT TGTGTACAAA ACAGACACCA 
               
               
                   
               
               
                   
                 TTTTCCCAAG AAAATGCAAA GATATGTTCT TGAAGGTGTG 
               
               
                   
               
               
                   
                 CAGGATTGGG TGCTATCTGT ACGCAAGTGG AGACGAATTC 
               
               
                   
               
               
                   
                 ACAACTCCAC AACAAATGAT GGAAGACATG AAATCATTGG 
               
               
                   
               
               
                   
                 TTTATCAACC CCTAACAGTT GATGACATGA GTGCCAAAGA 
               
               
                   
               
               
                   
                 ATTGACTTCT GTGAGAAACT AG 
               
            
           
         
       
     
     The  Salvia officinalis  miltiradiene synthase (SoTPS1) can have the amino acid sequence shown below (SEQ ID NO:19). 
                            MSLAFNAAVA TFSGHRIRSR REILPGQGFP MITNKSSFAV                   KCNLTTTDLM GKITEKFKGR DSNFSAATAV QPAADIPSNL                   CIIDTLQRLG VDRYFQSEID TILEDTYRLW QRKEREIFSD                   ITIHAMAFRL LRVKGYVVSS EELAPYADQE RINLQRIDVA                   TVIELYRAAQ ERISEDESSL EKLHAWTATY LKQQLLTNSI                   PDKKLNKLVE CYLKNYHGIL DRMGVRQNLD LYDISHYQTL                   KAADRFSNLR NEDFLAFARQ DFNICQEQHQ KELQQLQRWY                   ADCRLDTLKY GRDVVRVANF LTSAIIGDPE LSEVRLVFAK                   HIVLVTRIDD FFDHGGSREE SYKILELLKE WKEKPAAEYG                   SKEVEILFTA VYNTVNELAE MAHIEQGRSV KEFLIKLWVQ                   IISIFKIELD TWSDETALTL DEYLSSSWVS IGCRICILMS                   MQFIGIKLSD EMLLSEECID LCRHVSMVDR LLNDVQTFEK                   ERKENTGNSV SLLLAANKDD SAFTEEEAIT KAKEMAECNR                   RQLMKIVYKT GTIFPRKCKD MFLKVCRIGC YLYASGDEFT                   SPQQMMEDMK SLVYEPLTVD PLEAKNVSGK            
A nucleic acid encoding the  Salvia officinalis  miltiradiene synthase (SoTPS1) with SEQ ID NO:19 is shown below as SEQ ID NO:20.
 
     
       
         
           
               
               
            
               
                   
                 ATGTCCCTCG CCTTCAACGC AGCAGTTGCC ACTTTCTCCG 
               
               
                   
               
               
                   
                 GCCACAGAAT TCGGAGCAGG AGAGAAATTC TTCCGGGGCA 
               
               
                   
               
               
                   
                 AGGATTTCCG ATGATCACCA ACAAGTCGTC TTTCGCCGTG 
               
               
                   
               
               
                   
                 AAATGTAACC TTACTACAAC AGATTTGATG GGCAAGATAA 
               
               
                   
               
               
                   
                 CAGAGAAATT CAAGGGAAGA GACAGTAATT TTTCAGCAGC 
               
               
                   
               
               
                   
                 AACGGCTGTT CAACCTGCGG CGGATATACC CTCTAACCTG 
               
               
                   
               
               
                   
                 TGCATAATCG ACACCCTCCA AAGGTTGGGA GTCGACCGAT 
               
               
                   
               
               
                   
                 ACTTCCAATC TGAAATCGAC ACTATTCTAG AGGACACATA 
               
               
                   
               
               
                   
                 CAGGTTATGG CAAAGGAAAG AGAGAGAGAT ATTTTCGGAT 
               
               
                   
               
               
                   
                 ATAACTATTC ATGCAATGGC ATTTAGACTT TTGCGAGTTA 
               
               
                   
               
               
                   
                 AAGGATATGT AGTTTCATCA GAGGAACTGG CTCCGTATGC 
               
               
                   
               
               
                   
                 TGACCAAGAG CGCATTAACC TGCAAAGGAT TGATGTAGCG 
               
               
                   
               
               
                   
                 ACAGTTATCG AGCTTTACAG AGCAGCACAG GAGAGAATAA 
               
               
                   
               
               
                   
                 GTGAAGACGA GAGCAGTCTT GAGAAACTAC ATGCTTGGAC 
               
               
                   
               
               
                   
                 CGCCACCTAT CTCAAGCAGC AGCTGCTCAC TAACTCCATT 
               
               
                   
               
               
                   
                 CCTGACAAGA AATTGAACAA ACTGGTGGAA TGCTACTTGA 
               
               
                   
               
               
                   
                 AGAACTATCA CGGGATATTA GATAGAATGG GAGTTAGACA 
               
               
                   
               
               
                   
                 AAACCTCGAC CTCTACGACA TAAGCCACTA TCAAACTCTA 
               
               
                   
               
               
                   
                 AAAGCTGCAG ATAGGTTCTC TAATCTACGT AATGAAGATT 
               
               
                   
               
               
                   
                 TTCTAGCATT TGCGAGGCAA GATTTTAATA TTTGCCAAGA 
               
               
                   
               
               
                   
                 ACAACACCAA AAAGAACTTC AGCAACTGCA AAGGTGGTAT 
               
               
                   
               
               
                   
                 GCAGATTGTA GGTTGGACAC ATTGAAGTAT GGAAGAGATG 
               
               
                   
               
               
                   
                 TCGTGCGGGT TGCTAATTTT CTAACATCAG CAATTATTGG 
               
               
                   
               
               
                   
                 TGATCCTGAA TTGTCTGAAG TCCGTCTAGT CTTCGCCAAA 
               
               
                   
               
               
                   
                 CATATTGTGC TTGTAACACG TATTGATGAT TTTTTCGATC 
               
               
                   
               
               
                   
                 ATGGTGGATC TAGAGAAGAG TCCTACAAGA TCCTTGAATT 
               
               
                   
               
               
                   
                 ACTAAAAGAA TGGAAAGAGA AGCCAGCTGC AGAATATGGT 
               
               
                   
               
               
                   
                 TCCAAAGAAG TTGAAATTCT TTTCACAGCA GTATACAATA 
               
               
                   
               
               
                   
                 CAGTAAACGA GTTGGCAGAG ATGGCTCACA TCGAACAAGG 
               
               
                   
               
               
                   
                 ACGTAGTGTT AAAGAATTTC TAATAAAGCT GTGGGTTCAA 
               
               
                   
               
               
                   
                 ATCATATCGA TTTTCAAGAT AGAATTAGAT ACATGGAGCG 
               
               
                   
               
               
                   
                 ATGAGACAGC GCTGACCTTG GATGAGTACT TGTCTTCGTC 
               
               
                   
               
               
                   
                 GTGGGTGTCA ATTGGGTGCA GAATCTGCAT TCTCATGTCG 
               
               
                   
               
               
                   
                 ATGCAATTCA TTGGTATAAA ATTATCTGAT GAAATGCTTC 
               
               
                   
               
               
                   
                 TGAGTGAAGA GTGTATTGAT TTGTGTCGGC ATGTCTCCAT 
               
               
                   
               
               
                   
                 GGTTGACCGG CTGCTCAACG ACGTGCAGAC TTTCGAGAAG 
               
               
                   
               
               
                   
                 GAACGCAAGG AAAATACAGG AAATAGCGTG AGCCTTCTGC 
               
               
                   
               
               
                   
                 TAGCAGCTAA CAAAGACGAC AGCGCCTTTA CTGAAGAGGA 
               
               
                   
               
               
                   
                 AGCTATTACA AAAGCAAAAG AAATGGCGGA ATGTAACAGG 
               
               
                   
               
               
                   
                 AGACAACTGA TGAAGATTGT GTATAAAACA GGAACCATTT 
               
               
                   
               
               
                   
                 TCCCAAGAAA ATGCAAAGAT ATGTTTCTGA AGGTATGCAG 
               
               
                   
               
               
                   
                 GATTGGCTGT TACTTGTATG CAAGCGGCGA TGAATTCACA 
               
               
                   
               
               
                   
                 TCTCCACAAC AAATGATGGA AGATATGAAA TCCTTGGTCT 
               
               
                   
               
               
                   
                 ATGAACCCCT AACAGTTGAT CCTCTCGAGG CCAAAAATGT 
               
               
                   
               
               
                   
                 GAGTGGCAAA TGA 
               
            
           
         
       
     
       Ajuga reptans  (+)-copalyl diphosphate synthase (ArTPS1) is a (+)-copalyl diphosphate ((+)-CPP) [31] synthase, and compound 31 is shown below. 
     
       
         
         
             
             
         
       
     
     The  Ajuga reptans  (+)-copalyl diphosphate synthase (ArTPS1) can have the amino acid sequence shown below (SEQ ID NO:21). 
                            MASLSTFHLY SSSLLHRKTL QSSPKLNLSS ECFSTRTWMN                   SSKNLSLNYQ VNQKIGKLTG TRVATVDAPQ QLEHDDSTAK                   GHDIVDIETQ DPIEYIRMLL NTTGDGRISV SPYDTAWIAL                   IKDVEGRDFP QFPSSLEWIA NHQLADGSWG DEGFFCVYDR                   LVNTIACVVA LRSWNVHHDK SQRGIQYIKE NVHQLKDGNA                   EHMMCGFEVV FPALLQKAKN MGIDDLPYEA PVIQDIYHTR                   EQKLKRIPLE MMHKVPTSLL FSLEGLENLD WDKLLKLQSA                   DGSFLTSPSS TAFAFMQTKD EKCFQFIKNT VETFNGGAPH                   TYPVDVFGRL WAVDRLQRLG ISRFFEAEIA DCLSHIHRYW                   NDKGLFSGRE SDFVDIDDTS MGFRLLRMQG YDVSPNVLRN                   FKNGDKFSCY GGQTIESSTP IYNLYRASQF RFPGEEILEE                   ADKFAHEFLS EQLGNNQLLD KWVISDRLQE EISIGLGMPF                   YATLPRVEAS YYIQHYAGAD DVWIGKTLYR MPEISNDTYL                   ELARNDFKRC QAQHQFEWIY MQEWYESCNI EEFGISRKEL                   LRVYFLACSS IFEVERTKER MAWAKSQIIS RMITSFFNKQ                   TTSSEEKETL LTEFRNINGL HKSNNTRDGD MNIVLATLHQ                   FFAGFDRYTS HQLKNAWGVW LSKLQRGAVD GGADAELITT                   TINVCAGHIA LKEDILSHDE YKTLTDLTSK ICQQLSHIQN                   EKVVEIDGGI TAKSRLKNEE LQRDMQSLVK LVLEKSVGLN                   RNIKQTFLTV AKTYYYRAYN AEETMDAHIF KVLFEPVA            
A nucleic acid encoding the  Ajuga reptans  (+)-copalyl diphosphate synthase (ArTPS1) with SEQ ID NO:21 is shown below as SEQ ID NO:22.
 
     
       
         
           
               
               
            
               
                   
                 ATGGCCTCTT TGTCCACTTT CCACCTCTAC TCTTCCTCAC 
               
               
                   
               
               
                   
                 TCCTTCACCG CAAAACACTG CAATCTTCAC CAAAGCTTAA 
               
               
                   
               
               
                   
                 CCTGTCTTCA GAATGCTTCT CCACCAGAAC TTGGATGAAC 
               
               
                   
               
               
                   
                 AGCAGCAAAA ACTTGTCGTT AAATTACCAA GTTAATCAGA 
               
               
                   
               
               
                   
                 AAATAGGAAA GCTGACAGGG ACTCGAGTTG CCACTGTGGA 
               
               
                   
               
               
                   
                 TGCGCCACAA CAACTTGAAC ACGATGATTC AACTGCTAAA 
               
               
                   
               
               
                   
                 GGCCATGATA TAGTCGATAT TGAAACTCAG GATCCAATTG 
               
               
                   
               
               
                   
                 AATATATTAG AATGCTGTTG AACACAACAG GCGATGGCAG 
               
               
                   
               
               
                   
                 AATCAGCGTT TCGCCTTACG ACACAGCATG GATTGCTCTT 
               
               
                   
               
               
                   
                 ATTAAGGACG TGGAAGGACG TGATTTTCCT CAATTTCCAT 
               
               
                   
               
               
                   
                 CCAGCCTTGA GTGGATCGCG AACCATCAAC TCGCTGATGG 
               
               
                   
               
               
                   
                 TTCATGGGGA GACGAAGGAT TTTTCTGTGT GTATGATCGG 
               
               
                   
               
               
                   
                 CTCGTAAATA CTATAGCATG TGTCGTAGCA TTGAGATCAT 
               
               
                   
               
               
                   
                 GGAATGTCCA TCACGACAAG AGCCAAAGAG GAATACAATA 
               
               
                   
               
               
                   
                 TATCAAGGAA AATGTGCATC AACTTAAGGA TGGAAATGCT 
               
               
                   
               
               
                   
                 GAGCACATGA TGTGTGGTTT CGAAGTAGTG TTTCCTGCAC 
               
               
                   
               
               
                   
                 TTCTTCAAAA AGCCAAAAAT ATGGGCATTG ATGATCTTCC 
               
               
                   
               
               
                   
                 ATATGAGGCT CCTGTCATCC AGGATATTTA CCATACAAGG 
               
               
                   
               
               
                   
                 GAGCAGAAAT TGAAAAGGAT ACCATTGGAG ATGATGCACA 
               
               
                   
               
               
                   
                 AAGTGCCTAC TTCTCTGCTG TTTAGTTTGG AAGGACTGGA 
               
               
                   
               
               
                   
                 GAATTTAGAT TGGGATAAAC TCCTTAAGTT GCAGTCAGCT 
               
               
                   
               
               
                   
                 GATGGCTCTT TCCTCACTTC TCCCTCCTCT ACTGCTTTCG 
               
               
                   
               
               
                   
                 CATTCATGCA AACAAAAGAC GAAAAATGCT TCCAGTTCAT 
               
               
                   
               
               
                   
                 CAAGAACACT GTTGAAACCT TTAATGGAGG AGCACCACAT 
               
               
                   
               
               
                   
                 ACTTATCCGG TCGATGTTTT TGGAAGACTT TGGGCGGTTG 
               
               
                   
               
               
                   
                 ATAGGCTGCA GCGCCTCGGA ATTTCTCGAT TCTTTGAGGC 
               
               
                   
               
               
                   
                 TGAGATTGCT GATTGCTTAA GTCACATTCA TAGATATTGG 
               
               
                   
               
               
                   
                 AATGATAAGG GGCTTTTCAG TGGACGTGAA TCGGACTTTG 
               
               
                   
               
               
                   
                 TCGATATTGA CGACACATCC ATGGGTTTCA GACTTCTAAG 
               
               
                   
               
               
                   
                 AATGCAAGGC TATGATGTTA GTCCAAATGT ACTGAGGAAT 
               
               
                   
               
               
                   
                 TTCAAGAATG GTGACAAGTT TTCATGTTAC GGAGGTCAAA 
               
               
                   
               
               
                   
                 CGATCGAGTC ATCAACTCCA ATATACAATC TGTACAGAGC 
               
               
                   
               
               
                   
                 TTCTCAATTC CGGTTTCCAG GAGAAGAAAT TCTTGAAGAA 
               
               
                   
               
               
                   
                 GCCGACAAGT TCGCCCATGA GTTCTTGTCC GAACAGCTTG 
               
               
                   
               
               
                   
                 GCAACAACCA ATTGCTTGAT AAATGGGTTA TATCCGACCG 
               
               
                   
               
               
                   
                 CTTGCAGGAA GAGATAAGTA TTGGATTGGG GATGCCATTT 
               
               
                   
               
               
                   
                 TATGCCACCC TTCCCAGAGT TGAAGCAAGC TACTATATAC 
               
               
                   
               
               
                   
                 AACATTACGC TGGTGCCGAC GACGTGTGGA TCGGCAAGAC 
               
               
                   
               
               
                   
                 ACTCTACAGG ATGCCGGAAA TAAGTAATGA TACATACCTG 
               
               
                   
               
               
                   
                 GAGCTAGCAA GAAATGATTT CAAGAGATGC CAAGCACAAC 
               
               
                   
               
               
                   
                 ATCAGTTCGA GTGGATCTAC ATGCAAGAAT GGTATGAGAG 
               
               
                   
               
               
                   
                 TTGCAACATT GAAGAATTCG GGATAAGCCG AAAGGAGCTC 
               
               
                   
               
               
                   
                 CTTCGCGTTT ACTTTTTGGC TTGCTCTAGC ATCTTTGAGG 
               
               
                   
               
               
                   
                 TCGAGAGGAC TAAAGAGAGA ATGGCATGGG CAAAATCTCA 
               
               
                   
               
               
                   
                 AATTATTTCT AGAATGATCA CTTCTTTCTT TAATAAACAA 
               
               
                   
               
               
                   
                 ACTACTTCAT CTGAGGAAAA AGAAACACTT TTAACCGAAT 
               
               
                   
               
               
                   
                 TCAGAAACAT CAACGGTCTG CACAAATCAA ACAATACAAG 
               
               
                   
               
               
                   
                 AGATGGAGAT ATGAACATTG TGCTTGCAAC CCTCCATCAA 
               
               
                   
               
               
                   
                 TTCTTCGCTG GATTTGACAG ATATACTAGC CATCAACTGA 
               
               
                   
               
               
                   
                 AAAATGCTTG GGGAGTATGG TTGAGCAAGC TGCAACGAGG 
               
               
                   
               
               
                   
                 AGCAGTAGAC GGTGGAGCAG ACGCAGAGCT GATAACAACC 
               
               
                   
               
               
                   
                 ACCATAAACG TATGCGCCGG TCATATAGCT CTTAAGGAAG 
               
               
                   
               
               
                   
                 ACATATTGTC CCACGATGAG TACAAGACTC TCACCGACCT 
               
               
                   
               
               
                   
                 CACCAGCAAG ATTTGTCAGC AGCTTTCTCA TATTCAAAAC 
               
               
                   
               
               
                   
                 GAAAAGGTTG TGGAAATTGA CGGTGGGATT ACAGCAAAAT 
               
               
                   
               
               
                   
                 CTAGGTTGAA GAATGAGGAA CTGCAACGTG ACATGCAATC 
               
               
                   
               
               
                   
                 ATTGGTGAAA TTAGTACTTG AGAAATCAGT TGGGCTCAAC 
               
               
                   
               
               
                   
                 CGGAATATAA AGCAAACATT TCTAACGGTT GCAAAAACAT 
               
               
                   
               
               
                   
                 ACTACTACAG AGCCTACAAT GCTGAGGAAA CTATGGATGC 
               
               
                   
               
               
                   
                 CCATATATTC AAAGTTCTTT TCGAACCAGT TGCGTGA 
               
            
           
         
       
     
       Ajuga reptans  cleroda-4(18),13E-dienyl diphosphate synthase (ArTPS2) was identified and isolated. ArTPS2 was identified as a (5R,8R,9S,10R) neo-cleroda-4(18),13E-dienyl diphosphate [38] synthase. In addition, the combination of ArTPS2 and SsSS enzymes generated neo-cleroda-4(18),14-dien-13-ol [37]. These compounds are shown below. 
     
       
         
         
             
             
         
       
     
     ArTPS2 is of particular interest for applications in agricultural biotechnology, for example, because it is useful for production of neo-clerodane diterpenoids. Neo-clerodane diterpenoids, particularly those with an epoxide moiety at the 4(18) position, have garnered significant attention for their ability to deter insect herbivores (Coll et al., Phytochem Rev 7(1):25 (2008); Klein Gebbinck et al. Phytochemistry 61(7):737-770 (2002); Li et al. Nat Prod Rep 33(10):1166-1226 (2016)). The 4(18)-desaturated products produced by ArTPS2 (e.g., compounds 37 and 38 with the ═CH 2  4(18) desaturation projecting from the A ring) the can be used in biosynthetic or semisynthetic routes to yield potent insect antifeedants. 
     The  Ajuga reptans  cleroda-4(18),13E-dienyl diphosphate synthase (ArTPS2) can have the amino acid sequence shown below (SEQ ID NO:23). 
                            MSFASQATSL LSSPNRLGHV PTPSSPARFA AGGAPFWKIL                   FTARSNGQYK AISRARNQGN VEYIDEIQKG PQVVLEAENS                   LEDDTQKDTD QIRELVENVR VKLQNIGGGG ISISAYDTAW                   VALVEDINGS GQPQFPTSLD WISNHQFPDG SWGSSKFLYY                   DRILCTLACI VALKTWNVHP DKYHKGLDFI RENIHKLADE                   EEVHMPIGFE VAFPSIIETA KKVGIEIPED FPGKKEIYAK                   RDLKLKKIPM DILHKMPTPL LFSIEGMEGL DWQKLFKFRD                   DGSFLTSPSS TAYALQQTKD ELCLKYLTDL VKKDNGGVPN                   AFPVDLFDRN YTVDRLRRLG ISRYFQPEIE ECMKYVYRFW                   DKRGISWARN TNVQDLDDTA QGFRNLRMHG YEVTLDVFKQ                   FEKCGEFFSF HGQSSDAVLG MFNLYRASQV LFPGEHMLAD                   ARKYAANYLH KRRLNNRVVD KWIINKDLEG EVAYGLDVPF                   YASLPRLEAR FYIEQYGGSD DVWIGKALYR MVNVSCDTYL                   ELAKLDYNKC QSVHQNEWKS FQKWYKSCSL GEFGFSEGSL                   LQAYYIAAST IFEPEKSGER LAWAKTAALM ETIQQLSSQQ                   KREFVDEFKH KNILKNENGE RYRSSTSLVE TLISTVNQLS                   SDILLEQGRD VHQELCHVWL KWLSTWEERG NLVEAEAELL                   LRTLHLNSGL DESSFSHPKY QQLLEVSTKV CHLLRLFQKR                   KVYDPEGCTT DIATGTTFQI EACMQELVKL VFSRSSEDLD                   SLTKLRFLDV ARSFYYTAHC DPQVVESHID KVLFEKVV            
A nucleic acid encoding the  Ajuga reptans  cleroda-4(18),13E-dienyl diphosphate synthase (ArTPS2) with SEQ ID NO:23 is shown below as SEQ ID NO:24.
 
     
       
         
           
               
               
            
               
                   
                 ATGTCATTTG CTTCCCAAGC CACCTCCCTC CTATCATCCC 
               
               
                   
               
               
                   
                 CCAACCGTCT CGGCCATGTT CCGACGCCAA GCTCGCCGGC 
               
               
                   
               
               
                   
                 TCGTTTCGCT GCCGGTGGTG CCCCATTTTG GAAGATATTA 
               
               
                   
               
               
                   
                 TTTACAGCTA GGTCTAATGG GCAGTATAAA GCTATTTCAA 
               
               
                   
               
               
                   
                 GAGCTCGTAA CCAAGGAAAT GTAGAGTACA TTGATGAGAT 
               
               
                   
               
               
                   
                 TCAGAAAGGC CCGCAAGTCG TATTGGAGGC AGAAAACAGC 
               
               
                   
               
               
                   
                 TTGGAAGATG ACACACAAAA AGATACTGAT CAGATAAGGG 
               
               
                   
               
               
                   
                 AACTAGTGGA AAATGTCCGA GTAAAGCTGC AGAATATCGG 
               
               
                   
               
               
                   
                 TGGTGGAGGG ATAAGCATAT CGGCGTACGA CACCGCATGG 
               
               
                   
               
               
                   
                 GTGGCGCTGG TGGAGGACAT CAACGGCAGT GGCCAGCCAC 
               
               
                   
               
               
                   
                 AGTTTCCGAC GAGCCTCGAT TGGATATCGA ACCATCAGTT 
               
               
                   
               
               
                   
                 CCCTGATGGG TCATGGGGCA GCAGCAAGTT TTTGTATTAT 
               
               
                   
               
               
                   
                 GATCGGATTC TATGCACATT AGCATGTATA GTTGCATTGA 
               
               
                   
               
               
                   
                 AAACCTGGAA TGTGCATCCT GATAAGTACC ACAAAGGGTT 
               
               
                   
               
               
                   
                 GGATTTCATC AGAGAGAACA TTCACAAGCT TGCGGACGAA 
               
               
                   
               
               
                   
                 GAAGAAGTGC ACATGCCAAT TGGGTTCGAA GTGGCATTCC 
               
               
                   
               
               
                   
                 CATCAATTAT TGAAACAGCT AAAAAAGTAG GAATCGAAAT 
               
               
                   
               
               
                   
                 CCCTGAGGAT TTTCCTGGCA AGAAAGAAAT TTATGCAAAA 
               
               
                   
               
               
                   
                 AGAGATTTAA AGCTAAAAAA AATACCAATG GATATACTGC 
               
               
                   
               
               
                   
                 ATAAAATGCC CACACCATTG CTCTTCAGCA TAGAAGGAAT 
               
               
                   
               
               
                   
                 GGAAGGCCTT GACTGGCAAA AGCTATTCAA ATTCCGCGAT 
               
               
                   
               
               
                   
                 GATGGCTCGT TTCTTACGTC TCCGTCCTCA ACAGCCTATG 
               
               
                   
               
               
                   
                 CACTCCAGCA AACAAAGGAT GAGCTATGCC TCAAGTATCT 
               
               
                   
               
               
                   
                 AACAGATCTT GTCAAGAAAG ACAACGGAGG AGTTCCGAAT 
               
               
                   
               
               
                   
                 GCATTTCCAG TAGACCTGTT TGATCGTAAC TATACAGTAG 
               
               
                   
               
               
                   
                 ACCGCTTGCG AAGGCTAGGA ATTTCACGGT ACTTTCAACC 
               
               
                   
               
               
                   
                 TGAAATTGAA GAATGCATGA AATATGTTTA CAGATTTTGG 
               
               
                   
               
               
                   
                 GATAAAAGAG GAATTAGCTG GGCAAGAAAT ACCAATGTTC 
               
               
                   
               
               
                   
                 AGGACCTTGA TGACACTGCA CAGGGATTCA GGAATTTAAG 
               
               
                   
               
               
                   
                 GATGCATGGT TATGAAGTCA CTCTAGATGT TTTCAAACAA 
               
               
                   
               
               
                   
                 TTTGAGAAAT GTGGAGAGTT TTTCAGTTTT CATGGGCAAT 
               
               
                   
               
               
                   
                 CCAGCGATGC TGTTTTAGGA ATGTTCAACT TGTACCGGGC 
               
               
                   
               
               
                   
                 TTCTCAGGTT TTATTTCCGG GAGAACACAT GCTTGCAGAT 
               
               
                   
               
               
                   
                 GCGAGGAAGT ATGCAGCCAA CTATTTGCAT AAACGAAGAC 
               
               
                   
               
               
                   
                 TTAATAATAG GGTGGTCGAC AAATGGATTA TCAACAAAGA 
               
               
                   
               
               
                   
                 CCTTGAAGGC GAGGTGGCAT ATGGGCTAGA TGTTCCGTTC 
               
               
                   
               
               
                   
                 TACGCCAGCC TACCTCGACT CGAAGCAAGG TTCTACATAG 
               
               
                   
               
               
                   
                 AACAATATGG GGGTAGTGAT GATGTGTGGA TTGGAAAAGC 
               
               
                   
               
               
                   
                 TTTATACAGA ATGGTAAATG TAAGCTGCGA CACTTACCTT 
               
               
                   
               
               
                   
                 GAGCTAGCAA AATTAGACTA CAACAAATGC CAATCCGTGC 
               
               
                   
               
               
                   
                 ATCAGAATGA GTGGAAAAGC TTTCAAAAAT GGTACAAAAG 
               
               
                   
               
               
                   
                 TTGCAGTCTT GGGGAGTTTG GGTTCAGTGA AGGAAGCCTA 
               
               
                   
               
               
                   
                 CTCCAAGCTT ACTACATAGC AGCCTCAACT ATATTCGAGC 
               
               
                   
               
               
                   
                 CAGAGAAATC AGGAGAACGC CTAGCTTGGG CTAAAACAGC 
               
               
                   
               
               
                   
                 AGCTCTAATG GAGACAATTC AACAACTTTC CAGCCAGCAA 
               
               
                   
               
               
                   
                 AAACGTGAAT TTGTTGATGA ATTCAAACAT AAAAACATAC 
               
               
                   
               
               
                   
                 TGAAGAATGA AAATGGAGAA AGGTATAGAT CAAGTACCAG 
               
               
                   
               
               
                   
                 TTTGGTAGAG ACTCTGATAA GCACTGTAAA TCAGCTCTCA 
               
               
                   
               
               
                   
                 TCAGACATAC TATTGGAGCA AGGCAGAGAC GTTCATCAAG 
               
               
                   
               
               
                   
                 AATTATGTCA CGTGTGGCTA AAATGGCTGA GTACATGGGA 
               
               
                   
               
               
                   
                 GGAAAGAGGA AACCTGGTGG AAGCGGAAGC CGAGCTTCTT 
               
               
                   
               
               
                   
                 CTGCGAACCT TACATCTCAA CAGCGGATTG GATGAATCAT 
               
               
                   
               
               
                   
                 CATTTTCCCA CCCTAAATAT CAACAGCTCT TGGAGGTGTC 
               
               
                   
               
               
                   
                 TACCAAAGTT TGCCACCTCC TTCGCCTATT TCAGAAACGA 
               
               
                   
               
               
                   
                 AAGGTGTATG ATCCCGAAGG GTGTACAACC GACATAGCAA 
               
               
                   
               
               
                   
                 CAGGAACAAC GTTCCAGATA GAAGCATGCA TGCAAGAACT 
               
               
                   
               
               
                   
                 AGTGAAATTA GTGTTCAGCA GATCCTCAGA AGATTTAGAT 
               
               
                   
               
               
                   
                 TCTCTTACTA AGTTGAGATT TTTGGATGTT GCTAGAAGTT 
               
               
                   
               
               
                   
                 TCTATTACAC TGCCCATTGT GATCCACAGG TGGTCGAGTC 
               
               
                   
               
               
                   
                 CCACATCGAT AAAGTATTGT TTGAGAAGGT AGTCTAG 
               
            
           
         
       
     
     The  Plectranthus barbatus  (+)-Copalyl diphosphate synthase (CfTPS16) was identified and isolated using the methods described herein, and this CfTPS116 protein can have the amino acid sequence shown below (SEQ ID NO:25). 
                            MQASMSSLNL NNAPAVCSSR SQLSAKLHPP EYSTVGAWLN                   RGNKNQRLGY RIRPKQLSKL TECRVASADV SQEIGKVGQS                   VRTPEEVNKK IEESIKYVKE LLMTSGDGRI SVAPYDTAIV                   ALIKDLEGRD APEFPSCLEW IANNQKDDGS WGDDFFCIYD                   RIVNTIASVV ALKSWNVHPD KIERGVSYIK ENAHKLKGGN                   LEHMTSGEEF VVPGCFDRAK ALGIEGLPYD DPIIKEIYAT                   KERRLSKVPK DMIYKVPTTL LFSLEGLGME DLDWQKILKL                   QSGDGSFLTS PSSTAYAFMQ TGDEKCYKFL QNAVRNCNGG                   APHTYPVDVF ARLWAVDRLQ RLGISRFFQP EIKFCLDHIK                   NVWTKNGVFS GRDSEFVDID DTSMGIRLLK MHGYDVDPNA                   LKHFKQEDGR FSCYGGQMIE SASPIYNLYR AAQLRFPGEE                   ILEEATKFAY NFLQQKLANN QIQEKWVISE HLIDEIKMGL                   KMPWYATLPR VEASYYLQYY AASGDVWIGK TFYRMPEISN                   DTYKELALLD FNRCQAQHQF EWIYMQEWYQ SNNIKEFGIS                   KKELLLAYFL AAATIFEPER SQERIVWAKT QVVSKMITSF                   LSQENALSSX QKTALFIDFG HSINGLNQIT SVEKENGLAQ                   TVLATFGQLL EEFDRYTRHQ LKNAWSQWFM KLQQGDDNGG                   ADAELLANTL NICAGHIAFN EDILSHNEYT SLSSLTNKIC                   QRLSQIRDNK ILEIEDGSIK DKELEQEMQA LVKLVLEETG                   GIDRNIKQTF LSVFKMFYYR AYHDAEAIDX HIFKVMFEPV                   V            
A nucleic acid encoding the  Plectranthus barbatus  (+)-Copalyl diphosphate synthase (CfTPS16) with SEQ ID NO:25 is shown below as SEQ ID NO:26.
 
     
       
         
           
               
               
            
               
                   
                 ATGCAGGCTT CTATGTCATC TCTGAACTTG AACAATGCAC 
               
               
                   
               
               
                   
                 CGGCCGTCTG CAGCAGCAGG TCACAGCTAT CCGCTAAACT 
               
               
                   
               
               
                   
                 TCACCCGCCG GAATATTCCA CCGTGGGTGC ATGGCTGAAT 
               
               
                   
               
               
                   
                 CGTGGCAACA AAAACCAGCG GTTGGGCTAC CGGATTCGTC 
               
               
                   
               
               
                   
                 CAAAGCAACT ATCAAAACTA ACTGAGTGTC GAGTAGCAAG 
               
               
                   
               
               
                   
                 TGCAGATGTG TCACAAGAGA TTGGAAAAGT CGGCCAATCT 
               
               
                   
               
               
                   
                 GTTCGGACTC CTGAAGAGGT AAATAAAAAG ATAGAGGAAT 
               
               
                   
               
               
                   
                 CCATCAAGTA CGTGAAGGAG CTGCTGATGA CGTCGGGCGA 
               
               
                   
               
               
                   
                 CGGGCGAATC AGTGTGGCGC CCTACGACAC GGCCATAGTT 
               
               
                   
               
               
                   
                 GCCCTTATCA AGGACTTGGA AGGGCGCGAT GCCCCGGAGT 
               
               
                   
               
               
                   
                 TTCCATCTTG CTTGGAGTGG ATTGCAAACA ATCAAAAAGA 
               
               
                   
               
               
                   
                 CGATGGTTCT TGGGGGGATG ACTTCTTCTG CATCTATGAT 
               
               
                   
               
               
                   
                 CGGATCGTTA ATACCATAGC ATCCGTCGTC GCCTTAAAAT 
               
               
                   
               
               
                   
                 CATGGAATGT GCACCCAGAC AAGATTGAGA GAGGAGTATC 
               
               
                   
               
               
                   
                 CTACATCAAG GAAAACGCGC ATAAACTAAA AGGTGGGAAT 
               
               
                   
               
               
                   
                 CTCGAACACA TGACATCAGG GTTCGAGTTC GTGGTTCCCG 
               
               
                   
               
               
                   
                 GCTGTTTTGA CAGAGCCAAA GCCTTGGGGA TCGAAGGCCT 
               
               
                   
               
               
                   
                 TCCCTATGAT GATCCCATCA TCAAGGAGAT TTATGCTACA 
               
               
                   
               
               
                   
                 AAAGAAAGGA GATTGAGCAA GGTACCGAAG GACATGATCT 
               
               
                   
               
               
                   
                 ACAAAGTTCC GACAACTCTA TTGTTTAGTT TAGAGGGACT 
               
               
                   
               
               
                   
                 GGGCATGGAG GATTTGGACT GGCAAAAGAT ACTGAAACTG 
               
               
                   
               
               
                   
                 CAGTCGGGCG ACGGCTCATT CCTCACCTCT CCGTCGTCCA 
               
               
                   
               
               
                   
                 CCGCCTACGC ATTCATGCAG ACCGGAGACG AAAAATGCTA 
               
               
                   
               
               
                   
                 CAAATTCCTC CAGAACGCCG TCAGAAATTG CAACGGCGGA 
               
               
                   
               
               
                   
                 GCGCCGCACA CTTATCCAGT CGACGTCTTT GCACGGCTCT 
               
               
                   
               
               
                   
                 GGGCGGTCGA CCGACTTCAG CGACTCGGAA TTTCTCGCTT 
               
               
                   
               
               
                   
                 CTTTCAGCCC GAGATCAAGT TTTGCCTAGA CCACATCAAA 
               
               
                   
               
               
                   
                 AATGTGTGGA CTAAGAACGG AGTTTTCAGT GGACGGGATT 
               
               
                   
               
               
                   
                 CAGAGTTTGT GGATATCGAC GACACATCCA TGGGCATCAG 
               
               
                   
               
               
                   
                 GCTTCTGAAA ATGCACGGAT ACGATGTCGA CCCAAATGCA 
               
               
                   
               
               
                   
                 CTGAAACATT TCAAGCAGGA GGATGGGAGG TTTTCATGCT 
               
               
                   
               
               
                   
                 ACGGTGGTCA AATGATCGAG TCTGCATCTC CGATTTACAA 
               
               
                   
               
               
                   
                 TCTCTACAGG GCTGCTCAGC TTCGTTTTCC AGGAGAAGAA 
               
               
                   
               
               
                   
                 ATTCTTGAAG AAGCCACTAA ATTTGCCTAC AACTTCCTGC 
               
               
                   
               
               
                   
                 AACAGAAGCT GGCCAACAAT CAAATTCAAG AAAAGTGGGT 
               
               
                   
               
               
                   
                 CATATCCGAG CACCTAATTG ATGAGATAAA AATGGGATTG 
               
               
                   
               
               
                   
                 AAGATGCCAT GGTACGCCAC CCTACCTAGA GTTGAGGCTT 
               
               
                   
               
               
                   
                 CATACTATCT CCAATATTAT GCAGCTTCTG GCGACGTATG 
               
               
                   
               
               
                   
                 GATTGGCAAG ACTTTTTACA GGATGCCAGA AATAAGTAAT 
               
               
                   
               
               
                   
                 GACACGTACA AAGAGCTTGC ACTATTGGAT TTCAACCGAT 
               
               
                   
               
               
                   
                 GCCAAGCACA ACATCAGTTC GAATGGATTT ACATGCAAGA 
               
               
                   
               
               
                   
                 GTGGTATCAA AGCAACAACA TTAAAGAATT TGGGATAAGC 
               
               
                   
               
               
                   
                 AAGAAAGAGC TTCTTCTTGC TTACTTCTTG GCTGCTGCAA 
               
               
                   
               
               
                   
                 CCATTTTTGA ACCCGAACGA TCGCAAGAGC GGATCGTGTG 
               
               
                   
               
               
                   
                 GGCTAAAACC CAAGTTGTTT CTAAGATGAT CACATCGTTT 
               
               
                   
               
               
                   
                 CTGTCTCAAG AAAACGCTTT GTCATCGGAN CAAAAGACTG 
               
               
                   
               
               
                   
                 CACTTTTCAT CGATTTTGGG CATAGTATCA ATGGCCTCAA 
               
               
                   
               
               
                   
                 TCAAATAACT AGTGTTGAGA AAGAGAATGG GCTTGCTCAG 
               
               
                   
               
               
                   
                 ACTGTGCTGG CAACCTTCGG ACAACTACTC GAGGAATTCG 
               
               
                   
               
               
                   
                 ACAGATACAC AAGGCATCAA CTGAAAAATG CTTGGAGCCA 
               
               
                   
               
               
                   
                 ATGGTTCATG AAACTGCAGC AAGGAGATGA CAATGGCGGG 
               
               
                   
               
               
                   
                 GCAGACGCAG AGCTCCTAGC AAACACATTG AACATCTGCG 
               
               
                   
               
               
                   
                 CTGGTCATAT TGCTTTTAAC GAAGACATAT TATCTCACAA 
               
               
                   
               
               
                   
                 CGAATACACC TCTCTCTCCT CCCTCACAAA CAAAATCTGT 
               
               
                   
               
               
                   
                 CAGCGGCTAA GTCAAATTCG AGATAATAAG ATACTGGAAA 
               
               
                   
               
               
                   
                 TTGAGGATGG GAGCATAAAA GATAAGGAAC TAGAACAGGA 
               
               
                   
               
               
                   
                 AATGCAGGCG CTGGTGAAGT TAGTCCTGGA AGAAACCGGT 
               
               
                   
               
               
                   
                 GGCATCGACA GGAACATCAA GCAAACATTT TTGTCAGTTT 
               
               
                   
               
               
                   
                 TCAAAATGTT TTACTACAGA GCCTACCACG ATGCTGAGGC 
               
               
                   
               
               
                   
                 TATCGATGNC CATATTTTCA AAGTAATGTT TGAACCAGTC 
               
               
                   
               
               
                   
                 GTATGA 
               
            
           
         
       
     
       Hyptis suaveolens  labda-7,13E-dienyl diphosphate synthase (HsTPS1) was identified and isolated, and is a (5S, 9S, 10S) labda-7,13E-dienyl diphosphate [21] synthase. When HsTPS1 was expressed in  N. benthamiana,  labda-7,13(16),14-triene [22] was formed. The combination of HsTPS1 with OmTPS3 produced labda-7,12E,14-triene [24]. 
     
       
         
         
             
             
         
       
     
     The  Hyptis suaveolens  labda-7,13E-dienyl diphosphate synthase (HsTPS1) can have the amino acid sequence shown below (SEQ ID NO:27). 
                            MAYMISISNL NCSSLLNTNL SAKIQLHQGL KGTWLKTSKR                   MCMDQQVHGK QIAKVIESRV TDKDVSTAQD FEVLKVNRVE                   DLISSIKSSL KTMEDGRISV SPYSTSWIAL IPSIDGRQTP                   QFPSSLEWIV KHQLSDGSWG DALFFCVYDR LVNTIACIIA                   LHTWKVHADK VKKGVSFVKE NIWKLEDANE VHMTSGFEVI                   FPILLRRARD MGIDGLPSDD TPVVRMISAA RDHKLKKIPR                   EVMHQVTTTL LYSLEGLEDL DWSRLFKLQS ADGSFLTSPS                   STAFAFMQTN NHNCLRFITS VVQTFNGGAP DNYPIDIFAR                   LWAVDRLQRL GISRFFEQEI NDCLSYVYRF WNANGVFSAG                   ATNFCDLDDT SMAFRLLRLH GYDVDPNVLR KFKEGDRFCC                   HSGEVAMSTS PTYALYRASQ IQFPGEEILD EAFSFTRDYL                   QDWLARDQVL DKWIVSKDLP DEIKVGLEVP WYASLPRVEA                   AYYMQRHYGG STDAWVAKTC YRMPDVSNDD YLELARLDFK                   RCQAQHQSEL SYMQRWYDSC NVEEFGISRK ELLVAYFVAA                   ATIFEPERAT ERIVWAKTEI VSKMIKAFFG EDSLDQKTML                   LKEFRNSINN GSHRFMKSEH RIVNILLQAL QELLHGSDDC                   RIGQLKNAWY EWLMKFEGGD EASLWGEGEL LVTTLNICTA                   HFLQHHDLLL NHDYITLSEL TNKICLKLSQ IQVGEMNEMR                   EDMQALTKLV IGESCIVNKN IKQTFLAVAK TFYYRAYFDA                   DTVDLHIFKV LFEPIV            
A nucleic acid encoding the  Hyptis suaveolens  labda-7,13E-dienyl diphosphate synthase (HsTPS1) with SEQ ID NO:27 is shown below as SEQ ID NO:28.
 
     
       
         
           
               
               
            
               
                   
                 ATGGCGTATA TGATATCTAT TTCAAATCTC AACTGTTCCT 
               
               
                   
               
               
                   
                 CGCTACTAAA CACCAATCTT TCAGCAAAGA TTCAGCTGCA 
               
               
                   
               
               
                   
                 CCAAGGTCTC AAAGGAACAT GGCTAAAAAC CAGCAAACGC 
               
               
                   
               
               
                   
                 ATGTGCATGG ATCAACAGGT TCATGGCAAG CAGATAGCAA 
               
               
                   
               
               
                   
                 AAGTGATCGA GAGCCGAGTT ACTGATAAGG ATGTTTCCAC 
               
               
                   
               
               
                   
                 TGCTCAGGAC TTTGAAGTGT TAAAGGTCAA TAGAGTGGAG 
               
               
                   
               
               
                   
                 GATCTGATAT CAAGCATTAA GAGTTCATTG AAGACAATGG 
               
               
                   
               
               
                   
                 AAGATGGAAG AATAAGCGTG TCGCCCTACA GCACATCATG 
               
               
                   
               
               
                   
                 GATCGCACTC ATTCCAAGTA TTGATGGGCG CCAGACGCCC 
               
               
                   
               
               
                   
                 CAGTTTCCAT CTTCACTGGA GTGGATCGTG AAGCATCAGC 
               
               
                   
               
               
                   
                 TATCAGATGG TTCATGGGGT GATGCCCTTT TTTTCTGCGT 
               
               
                   
               
               
                   
                 TTATGATCGT CTCGTAAATA CGATTGCATG CATCATTGCC 
               
               
                   
               
               
                   
                 CTGCACACCT GGAAGGTTCA TGCAGACAAG GTTAAAAAAG 
               
               
                   
               
               
                   
                 GAGTAAGTTT TGTGAAGGAA AATATATGGA AACTTGAAGA 
               
               
                   
               
               
                   
                 CGCCAACGAG GTCCACATGA CTAGTGGTTT CGAAGTTATA 
               
               
                   
               
               
                   
                 TTTCCCATCC TTCTTCGAAG AGCACGAGAC ATGGGAATTG 
               
               
                   
               
               
                   
                 ATGGTCTTCC TTCTGATGAT ACTCCAGTTG TTAGGATGAT 
               
               
                   
               
               
                   
                 TTCTGCTGCT AGGGATCACA AATTGAAAAA GATTCCGAGG 
               
               
                   
               
               
                   
                 GAGGTGATGC ACCAAGTGAC AACAACTCTA TTATATAGTT 
               
               
                   
               
               
                   
                 TGGAAGGGTT GGAAGATTTA GACTGGTCAA GGCTTTTCAA 
               
               
                   
               
               
                   
                 ACTTCAGTCA GCTGATGGTT CATTCTTAAC TTCTCCATCT 
               
               
                   
               
               
                   
                 TCAACTGCCT TCGCATTCAT GCAAACTAAT AACCACAATT 
               
               
                   
               
               
                   
                 GCTTGAGATT CATCACTAGC GTTGTCCAAA CATTCAATGG 
               
               
                   
               
               
                   
                 AGGAGCTCCA GATAACTATC CAATCGACAT CTTTGCGAGA 
               
               
                   
               
               
                   
                 CTGTGGGCAG TTGACAGGTT ACAGCGGTTA GGGATTTCTC 
               
               
                   
               
               
                   
                 GTTTCTTCGA GCAGGAGATA AATGATTGCC TAAGCTATGT 
               
               
                   
               
               
                   
                 ATATAGATTT TGGAATGCAA ATGGAGTTTT CAGTGCAGGA 
               
               
                   
               
               
                   
                 GCCACTAATT TTTGTGATCT TGACGACACA TCCATGGCTT 
               
               
                   
               
               
                   
                 TCCGGCTACT ACGTTTGCAT GGATATGATG TCGACCCAAA 
               
               
                   
               
               
                   
                 TGTTCTGAGG AAATTCAAAG AGGGAGACAG ATTCTGTTGC 
               
               
                   
               
               
                   
                 CACAGTGGTG AAGTGGCGAT GTCGACATCG CCAACGTACG 
               
               
                   
               
               
                   
                 CTCTCTACAG AGCTTCCCAA ATTCAGTTTC CAGGAGAAGA 
               
               
                   
               
               
                   
                 AATTCTGGAT GAAGCCTTCA GCTTCACTCG CGACTATCTA 
               
               
                   
               
               
                   
                 CAGGACTGGT TAGCAAGAGA TCAAGTTCTT GATAAGTGGA 
               
               
                   
               
               
                   
                 TTGTATCCAA GGACCTTCCA GATGAGATTA AGGTAGGACT 
               
               
                   
               
               
                   
                 AGAGGTGCCA TGGTATGCCA GCCTGCCACG GGTAGAGGCT 
               
               
                   
               
               
                   
                 GCTTATTACA TGCAACGACA TTACGGCGGG TCTACTGATG 
               
               
                   
               
               
                   
                 CGTGGGTGGC CAAGACTTGT TACAGGATGC CTGATGTGAG 
               
               
                   
               
               
                   
                 CAACGATGAT TACCTGGAGC TTGCAAGATT GGATTTCAAG 
               
               
                   
               
               
                   
                 AGATGTCAAG CCCAACATCA GAGTGAATTG AGTTACATGC 
               
               
                   
               
               
                   
                 AACGATGGTA TGACAGTTGC AATGTCGAAG AATTCGGAAT 
               
               
                   
               
               
                   
                 AAGCAGAAAA GAGTTGCTTG TAGCTTATTT TGTGGCTGCT 
               
               
                   
               
               
                   
                 GCAACTATTT TTGAACCTGA GAGAGCAACT GAGAGAATTG 
               
               
                   
               
               
                   
                 TGTGGGCAAA AACTGAAATA GTTTCTAAGA TGATCAAAGC 
               
               
                   
               
               
                   
                 ATTTTTTGGT GAAGACTCAT TAGACCAAAA AACTATGTTG 
               
               
                   
               
               
                   
                 TTAAAAGAAT TCAGAAACAG CATCAATAAT GGCTCCCACA 
               
               
                   
               
               
                   
                 GATTCATGAA GAGTGAGCAT AGAATCGTCA ACATTCTACT 
               
               
                   
               
               
                   
                 ACAAGCCTTG CAGGAGCTAT TACATGGATC TGATGATTGT 
               
               
                   
               
               
                   
                 CGTATTGGTC AACTCAAAAA TGCTTGGTAT GAGTGGCTGA 
               
               
                   
               
               
                   
                 TGAAATTCGA GGGAGGAGAT GAAGCAAGTT TGTGGGGAGA 
               
               
                   
               
               
                   
                 AGGAGAGCTT CTTGTCACCA CCTTAAACAT TTGCACAGCT 
               
               
                   
               
               
                   
                 CATTTCCTTC AACACCATGA TTTACTGTTG AATCATGACT 
               
               
                   
               
               
                   
                 ACATAACTCT TTCTGAGCTC ACAAACAAGA TCTGCCTCAA 
               
               
                   
               
               
                   
                 GCTTTCTCAG ATTCAGGTAG GAGAAATGAA TGAAATGAGA 
               
               
                   
               
               
                   
                 GAAGATATGC AGGCGTTGAC GAAATTAGTG ATTGGGGAAT 
               
               
                   
               
               
                   
                 CATGCATCGT CAACAAAAAC ATTAAGCAAA CATTTCTTGC 
               
               
                   
               
               
                   
                 AGTTGCAAAG ACTTTCTATT ACAGAGCCTA CTTCGATGCC 
               
               
                   
               
               
                   
                 GACACCGTTG ATCTCCATAT ATTTAAAGTT CTATTTGAGC 
               
               
                   
               
               
                   
                 CCATTGTCTG A 
               
            
           
         
       
     
       Leonotis leonurus  peregrinol diphosphate synthase (L1TPS1) was identified and isolated using the methods described herein. The LlTPS1 enzyme was identified as a peregrinol diphosphate (PgPP) [5] synthase, where the peregrinol diphosphate (PgPP) [5] compound is shown below. 
     
       
         
         
             
             
         
       
     
     The  Leonotis leonurus  peregrinol diphosphate synthase (L1TPS1) can have the amino acid sequence shown below (SEQ ID NO:29). 
                            MASTASTLNL TINSTPFVST KTQAKVSLPA CLWMQDRSSS                   RHVSLKHKFC RNQQLKCRAS LDVQQVRDEV FSTAQSPESV                   DKKIEERKKW VKNLLSTMDD GRINWSAYDT AWISLIKEFE                   GRDAPQFPST LMRIAENQLA DGSWGDPDYD CSYDRIINTL                   ACWALTTVVN AHPEHNKKGI KYIKENMYKL EETPVVLMTS                   AFEVVFPALL NRAKNLGIQD LPYDMPIVKE ICKIGDEKLA                   RIPKKMMEKE PTSLMYAAEG VENLDWEKLL KQRTPENGSF                   LSSPAATAVA FMHTKDENCL RYIMYLLDKF NGGAPNVYPI                   DLWSRLWATD RIQRLGISRF FKEEIKEILS YVYSYWTDIG                   VYCTRDSKYA DIDDTSMGFR LLRMHGFKMD PNVFKYFQKD                   DRFVCLGGQM NDSPTATYNL YRAAQYQFPG EKILEDARKF                   SQEFLQHCID TNNLLDKWVI SPRFPEELKF GMEMTWYSCL                   PRIEARYYVQ HYGATEDVWL GKTFFRMEEI SNENYKELAK                   LDFSKCQAQH QTEWIHMQEW YESSNAKEFG ISRKDLLFAY                   FLAAASIFET ERAKERILWA KSQIICKMVK SYLENQTASL                   EHKIAFLTGF GDNNNGLHTI NKGSGPVNNV MRTLQQLLGE                   FDGYISSQLE NAWAAWLTKL EQGEANDGEL LATTLNICSG                   RIVYNEDTLS NKEYKAFADL TNKICQNLAQ IQNKKGDEIK                   DPNEGEKDKE VEQGMQALAK LVFEESGLER SIKETFLAVV                   RTYHYGAYVA DEKIDVHMFK VLFEPVE            
A nucleic acid encoding the  Leonotis leonurus  peregrinol diphosphate synthase (L1TPS1) with SEQ ID NO:29 is shown below as SEQ ID NO:30.
 
     
       
         
           
               
               
            
               
                   
                 ATGGCCTCCA CTGCATCCAC TCTAAATTTG ACCATCAATA 
               
               
                   
               
               
                   
                 GTACACCATT TGTAAGCACC AAAACGCAAG CAAAGGTTTC 
               
               
                   
               
               
                   
                 CTTGCCCGCA TGTTTATGGA TGCAGGATAG AAGCAGCAGT 
               
               
                   
               
               
                   
                 AGACACGTGT CGTTAAAACA CAAATTCTGT CGAAATCAAC 
               
               
                   
               
               
                   
                 AACTTAAGTG TCGAGCAAGT CTGGATGTTC AGCAAGTACG 
               
               
                   
               
               
                   
                 TGATGAAGTT TTTTCCACTG CTCAATCCCC TGAATCGGTG 
               
               
                   
               
               
                   
                 GATAAAAAAA TAGAGGAACG TAAAAAATGG GTGAAGAATT 
               
               
                   
               
               
                   
                 TGTTGAGTAC AATGGACGAT GGACGAATAA ATTGGTCAGC 
               
               
                   
               
               
                   
                 CTATGACACG GCATGGATTT CACTTATTAA AGAATTTGAA 
               
               
                   
               
               
                   
                 GGACGAGATG CTCCCCAGTT TCCGTCGACT CTCATGCGCA 
               
               
                   
               
               
                   
                 TCGCGGAGAA CCAATTGGCC GACGGGTCAT GGGGCGATCC 
               
               
                   
               
               
                   
                 AGATTACGAC TGCTCCTATG ATCGGATAAT AAACACACTA 
               
               
                   
               
               
                   
                 GCGTGTGTTG TAGCCTTGAC AACATGGAAT GCTCATCCTG 
               
               
                   
               
               
                   
                 AACACAATAA AAAAGGAATA AAATACATCA AGGAAAATAT 
               
               
                   
               
               
                   
                 GTATAAACTA GAAGAGACGC CTGTTGTACT CATGACTAGT 
               
               
                   
               
               
                   
                 GCATTTGAAG TTGTGTTTCC GGCGCTTCTT AACAGAGCTA 
               
               
                   
               
               
                   
                 AAAACTTGGG CATTCAAGAT CTTCCCTATG ATATGCCCAT 
               
               
                   
               
               
                   
                 CGTGAAGGAG ATTTGTAAAA TAGGGGATGA GAAGTTGGCA 
               
               
                   
               
               
                   
                 AGGATACCAA AGAAAATGAT GGAGAAAGAG CCAACATCGC 
               
               
                   
               
               
                   
                 TGATGTATGC CGCGGAAGGA GTCGAAAACT TGGACTGGGA 
               
               
                   
               
               
                   
                 AAAGCTTCTG AAACAGCGGA CACCCGAGAA TGGCTCGTTC 
               
               
                   
               
               
                   
                 CTCTCTTCCC CGGCCGCAAC TGCCGTTGCA TTTATGCACA 
               
               
                   
               
               
                   
                 CAAAAGATGA AAATTGCTTA AGATACATCA TGTACCTTTT 
               
               
                   
               
               
                   
                 GGACAAATTT AATGGAGGAG CACCAAATGT TTATCCGATC 
               
               
                   
               
               
                   
                 GACCTCTGGT CAAGACTTTG GGCAACGGAC AGGATACAAC 
               
               
                   
               
               
                   
                 GTCTGGGAAT TTCCCGCTTC TTTAAGGAAG AGATTAAGGA 
               
               
                   
               
               
                   
                 AATCTTAAGT TATGTCTATA GCTATTGGAC AGACATTGGA 
               
               
                   
               
               
                   
                 GTCTATTGTA CACGAGATTC CAAATATGCT GACATTGACG 
               
               
                   
               
               
                   
                 ACACATCCAT GGGATTCAGG CTTCTGAGGA TGCACGGATT 
               
               
                   
               
               
                   
                 TAAAATGGAC CCAAATGTAT TTAAATACTT CCAGAAAGAC 
               
               
                   
               
               
                   
                 GACAGATTTG TTTGTCTAGG TGGTCAAATG AATGATTCTC 
               
               
                   
               
               
                   
                 CAACTGCAAC ATACAATCTT TACAGGGCTG CTCAATACCA 
               
               
                   
               
               
                   
                 ATTTCCAGGT GAAAAAATTC TAGAAGATGC TAGAAAGTTC 
               
               
                   
               
               
                   
                 TCTCAAGAGT TTCTACAACA TTGTATAGAC ACCAATAACC 
               
               
                   
               
               
                   
                 TTCTAGATAA ATGGGTGATA TCCCCGCGCT TTCCGGAAGA 
               
               
                   
               
               
                   
                 GTTGAAATTT GGAATGGAGA TGACATGGTA TTCCTGCCTA 
               
               
                   
               
               
                   
                 CCACGAATTG AGGCTAGATA CTACGTACAA CATTATGGTG 
               
               
                   
               
               
                   
                 CTACAGAGGA CGTCTGGCTT GGAAAGACTT TTTTCAGGAT 
               
               
                   
               
               
                   
                 GGAAGAAATC AGTAATGAGA ACTATAAGGA GCTTGCAAAA 
               
               
                   
               
               
                   
                 CTTGATTTCA GTAAATGCCA AGCACAACAT CAGACAGAGT 
               
               
                   
               
               
                   
                 GGATTCATAT GCAAGAGTGG TATGAAAGTA GCAATGCTAA 
               
               
                   
               
               
                   
                 GGAATTTGGG ATAAGCAGAA AAGACCTACT TTTTGCTTAC 
               
               
                   
               
               
                   
                 TTTTTGGCTG CAGCTTCCAT ATTTGAAACC GAAAGGGCAA 
               
               
                   
               
               
                   
                 AAGAGAGAAT TCTGTGGGCA AAATCTCAAA TTATTTGCAA 
               
               
                   
               
               
                   
                 GATGGTTAAG TCATATCTGG AAAACCAAAC GGCGTCGTTG 
               
               
                   
               
               
                   
                 GAGCACAAAA TCGCCTTTTT AACTGGATTC GGAGATAACA 
               
               
                   
               
               
                   
                 ACAATGGCCT GCACACAATT AATAAGGGGT CTGGACCTGT 
               
               
                   
               
               
                   
                 TAACAATGTC ATGAGAACCC TCCAACAGCT CCTTGGAGAA 
               
               
                   
               
               
                   
                 TTCGACGGAT ATATTAGTAG TCAATTGGAA AATGCTTGGG 
               
               
                   
               
               
                   
                 CAGCATGGTT GACGAAACTC GAGCAAGGCG AGGCCAACGA 
               
               
                   
               
               
                   
                 TGGCGAGCTC CTCGCAACCA CACTAAACAT TTGTTCTGGG 
               
               
                   
               
               
                   
                 CGTATTGTGT ATAACGAGGA TACATTATCG AACAAGGAGT 
               
               
                   
               
               
                   
                 ACAAGGCTTT CGCAGACCTC ACAAATAAAA TTTGTCAAAA 
               
               
                   
               
               
                   
                 TCTTGCTCAA ATCCAAAATA AAAAGGGTGA CGAAATTAAG 
               
               
                   
               
               
                   
                 GATCCGAATG AAGGCGAAAA GGACAAGGAA GTCGAGCAAG 
               
               
                   
               
               
                   
                 GCATGCAGGC ATTGGCTAAG TTAGTTTTTG AGGAATCTGG 
               
               
                   
               
               
                   
                 GCTTGAGAGG AGTATCAAAG AAACATTCTT AGCAGTGGTG 
               
               
                   
               
               
                   
                 AGAACTTATC ACTATGGGGC CTATGTTGCT GATGAGAAGA 
               
               
                   
               
               
                   
                 TTGATGTCCA CATGTTCAAG GTTTTGTTCG AACCAGTTGA 
               
               
                   
               
               
                   
                 ATGA 
               
            
           
         
       
     
       Nepeta mussinii  (+)-copalyl diphosphate synthase (NmTPS1) was identified and isolated. The NmTPS1 enzyme can synthesize compound 31, shown below. 
     
       
         
         
             
             
         
       
     
     The  Nepeta mussinii  (+)-copalyl diphosphate synthase (NmTPS1) can have the amino acid sequence shown below (SEQ ID NO:31). 
                            MTSISSLNLS NAAAARRRLQ LPANVHLPEF HSVCAWLNSS                   SKHDPFSCRI HRKQKSKVTE CRVASVDASP VSDHKMSSPV                   QTQEEANKNM EESIEYIKNL LMTSGDGRIS VSAYDTSIVA                   LIKDIEGRDA PQFPSCLEWI GQNQKADGSW GDDFFCIYDR                   FVNTLACIVA LKSWNLHPHK IQKGVTYIKK NVHKLKDGRP                   ELMTSGFEIC VPAILQRAKD LGIQDLPYDD PMIKQITDTK                   ERRLKKIPKD FIYQLPTTLL FSLEGQENLD WEKILKLQSA                   DGSFLTSPSS TAAVFMHTKD EKCLKFIENA VKNCDGGVPH                   TYPVDVFARL WAVDRLQRLG ISRFFQPEIK YFLDHIQSVW                   TENGVFSGRD SQFCDIDDTS MGIRLLKMHG YKIDPNALEH                   FKQEDGKFSC YGGQMIESAS PIYNLYRAAQ LRFPGEEILE                   EAIKFSYNFL QEKLAKDEIQ EKWVISEHLI DEIKIGLKMP                   WYATLPRVEA AYYLDYYAGS GDVWIGKTFY RMPEISNDTY                   KEMAILDFNR CQAQHQFEWI YMQEWYESSN VKEFGISKKE                   LLVAYFLAAS TIFEPERAQE RIMWAKTKIV SKMIASSLNK                   QTTLSLDQKT ALFTQLEHSL NGLDSDEKDN GVAETKNLVA                   TFQQLLDGFD KYTRHQLKNA WSQWLKQVQQ GEATGGADAE                   LEANTLNICA GHIAFNEQVL SHNEYTTLST LTNKICHRLT                   QIQDKKTLEI IDGGIRYKEL EQEMQALVKL VVEENDGGGI                   DRNIKQTFLS VFKNYYYSAY HDAHTTDVHI FKVLFGPVV            
A nucleic acid encoding the  Nepeta mussinii  (+)-copalyl diphosphate synthase (NmTPS1) with SEQ ID NO:31 is shown below as SEQ ID NO:32.
 
     
       
         
           
               
               
            
               
                   
                 ATGACTTCAA TATCCTCTCT AAATTTGAGC AATGCAGCAG 
               
               
                   
               
               
                   
                 CTGCTCGCCG CAGGTTACAA CTACCAGCAA ACGTTCACCT 
               
               
                   
               
               
                   
                 GCCGGAATTT CACTCCGTCT GTGCATGGCT GAATAGCAGC 
               
               
                   
               
               
                   
                 AGCAAACACG ATCCCTTTAG TTGCCGAATT CATCGAAAGC 
               
               
                   
               
               
                   
                 AAAAATCGAA AGTAACCGAG TGTCGAGTAG CAAGCGTGGA 
               
               
                   
               
               
                   
                 TGCATCACCA GTGAGTGATC ATAAAATGAG TTCTCCTGTT 
               
               
                   
               
               
                   
                 CAAACTCAAG AAGAGGCAAA TAAAAATATG GAGGAGTCAA 
               
               
                   
               
               
                   
                 TCGAGTACAT AAAGAATTTG TTGATGACAT CTGGAGACGG 
               
               
                   
               
               
                   
                 GCGAATAAGC GTGTCGGCAT ACGACACGTC AATAGTCGCC 
               
               
                   
               
               
                   
                 CTAATTAAGG ACATAGAAGG ACGGGACGCC CCGCAATTTC 
               
               
                   
               
               
                   
                 CATCATGCCT GGAGTGGATC GGGCAAAACC AAAAGGCCGA 
               
               
                   
               
               
                   
                 TGGCTCGTGG GGGGACGACT TCTTCTGTAT TTACGACCGC 
               
               
                   
               
               
                   
                 TTCGTAAATA CACTAGCATG TATCGTGGCC TTGAAATCAT 
               
               
                   
               
               
                   
                 GGAACCTTCA CCCTCACAAG ATTCAAAAAG GAGTGACATA 
               
               
                   
               
               
                   
                 CATCAAGAAA AACGTGCATA AGCTTAAAGA TGGGAGGCCT 
               
               
                   
               
               
                   
                 GAGCTGATGA CGTCAGGGTT CGAAATTTGT GTTCCCGCCA 
               
               
                   
               
               
                   
                 TTCTTCAAAG AGCCAAAGAC TTGGGCATCC AAGATCTTCC 
               
               
                   
               
               
                   
                 CTATGATGAT CCCATGATTA AACAGATCAC TGATACGAAA 
               
               
                   
               
               
                   
                 GAGCGACGAC TCAAAAAGAT ACCGAAGGAT TTTATATACC 
               
               
                   
               
               
                   
                 AATTGCCGAC GACTTTACTC TTCAGTTTGG AAGGGCAGGA 
               
               
                   
               
               
                   
                 GAATTTGGAC TGGGAAAAGA TACTCAAACT GCAGTCAGCT 
               
               
                   
               
               
                   
                 GACGGCTCCT TCCTTACTTC GCCGTCCTCC ACCGCCGCCG 
               
               
                   
               
               
                   
                 TCTTCATGCA TACCAAAGAT GAAAAATGCT TGAAGTTCAT 
               
               
                   
               
               
                   
                 AGAGAACGCC GTCAAAAATT GCGACGGCGG AGTGCCCCAT 
               
               
                   
               
               
                   
                 ACCTACCCAG TAGACGTGTT TGCAAGACTT TGGGCAGTTG 
               
               
                   
               
               
                   
                 ACAGACTACA ACGCCTAGGG ATTTCTCGCT TTTTTCAGCC 
               
               
                   
               
               
                   
                 TGAGATTAAA TATTTCTTAG ATCACATACA AAGCGTTTGG 
               
               
                   
               
               
                   
                 ACTGAGAACG GAGTTTTCAG TGGACGAGAT TCACAATTTT 
               
               
                   
               
               
                   
                 GCGACATTGA TGATACGTCC ATGGGGATAA GGCTTCTGAA 
               
               
                   
               
               
                   
                 AATGCATGGA TACAAAATCG ACCCAAATGC ACTTGAGCAT 
               
               
                   
               
               
                   
                 TTCAAGCAGG AGGATGGTAA ATTTTCGTGC TACGGTGGTC 
               
               
                   
               
               
                   
                 AAATGATCGA GTCTGCATCA CCGATATACA ATCTGTACCG 
               
               
                   
               
               
                   
                 AGCTGCTCAA CTCCGATTTC CAGGAGAAGA AATTCTTGAA 
               
               
                   
               
               
                   
                 GAGGCCATTA AATTTTCCTA TAACTTTTTG CAAGAAAAGC 
               
               
                   
               
               
                   
                 TAGCCAAGGA TGAAATTCAA GAAAAATGGG TCATATCGGA 
               
               
                   
               
               
                   
                 GCACTTAATT GATGAGATTA AGATCGGGCT AAAGATGCCA 
               
               
                   
               
               
                   
                 TGGTACGCCA CTCTACCCCG AGTTGAAGCT GCATATTACC 
               
               
                   
               
               
                   
                 TGGACTATTA TGCAGGATCC GGCGATGTGT GGATTGGCAA 
               
               
                   
               
               
                   
                 GACTTTCTAC AGGATGCCAG AAATCAGTAA TGATACATAC 
               
               
                   
               
               
                   
                 AAAGAAATGG CCATTTTGGA TTTCAACCGA TGCCAAGCAC 
               
               
                   
               
               
                   
                 AACATCAGTT TGAATGGATT TACATGCAAG AGTGGTATGA 
               
               
                   
               
               
                   
                 AAGTAGCAAC GTAAAGGAAT TTGGGATAAG CAAAAAAGAG 
               
               
                   
               
               
                   
                 CTACTTGTTG CTTATTTCTT GGCTGCATCA ACCATATTTG 
               
               
                   
               
               
                   
                 AACCGGAAAG AGCACAAGAG AGGATTATGT GGGCAAAAAC 
               
               
                   
               
               
                   
                 AAAAATTGTT TCCAAAATGA TCGCATCATC TCTTAACAAA 
               
               
                   
               
               
                   
                 CAAACCACTC TATCGTTAGA CCAAAAGACT GCACTTTTTA 
               
               
                   
               
               
                   
                 CCCAACTCGA ACATAGTCTC AATGGCCTCG ACAGTGATGA 
               
               
                   
               
               
                   
                 GAAAGATAAT GGAGTAGCTG AGACGAAAAA TCTAGTGGCA 
               
               
                   
               
               
                   
                 ACCTTCCAGC AGCTGCTAGA TGGATTCGAC AAATACACTC 
               
               
                   
               
               
                   
                 GCCATCAATT GAAAAATGCT TGGAGCCAGT GGTTGAAGCA 
               
               
                   
               
               
                   
                 AGTGCAGCAA GGAGAGGCGA CCGGGGGCGC AGACGCGGAG 
               
               
                   
               
               
                   
                 CTGGAAGCAA ACACGTTGAA CATCTGTGCC GGTCATATCG 
               
               
                   
               
               
                   
                 CATTCAACGA ACAAGTATTA TCGCACAACG AATACACAAC 
               
               
                   
               
               
                   
                 TCTCTCCACA CTCACAAACA AGATCTGCCA CCGGCTTACC 
               
               
                   
               
               
                   
                 CAAATTCAAG ACAAAAAGAC GCTTGAGATA ATCGACGGCG 
               
               
                   
               
               
                   
                 GCATAAGATA TAAGGAGCTG GAGCAGGAGA TGCAGGCGTT 
               
               
                   
               
               
                   
                 GGTGAAATTA GTTGTTGAAG AAAACGACGG CGGCGGCATA 
               
               
                   
               
               
                   
                 GACAGGAATA TTAAACAAAC ATTTTTATCA GTTTTCAAGA 
               
               
                   
               
               
                   
                 ATTATTACTA CAGTGCCTAC CACGATGCTC ACACAACCGA 
               
               
                   
               
               
                   
                 TGTTCATATT TTCAAAGTAT TATTTGGACC GGTCGTCTGA 
               
            
           
         
       
     
       Origanum majorana  (+)-copalyl diphosphate synthase (OmTPS1) was identified and isolated as describe herein. The OmTPS1 enzyme can synthesize compound 31. OmTPS1 can also synthesize palustradiene [29] (shown below), when combined with OmTPS5. 
     
       
         
         
             
             
         
       
     
     The  Origanum majorana  (+)-copalyl diphosphate synthase (OmTPS1) can have the amino acid sequence shown below (SEQ ID NO:33). 
                            MTDVSSLRLS NAPAAGGRLP LPGKVHLPEF RTVCAWLNNG                   CKYEPLTCRI SRRKISECRV ASLNSSQLIE KVGSPAQSLE                   EANKKIEDSI EYIKNLLMTS GDGRISVSAY DTSLVALIKD                   VKGRDAPQFP SCLEWIAQNQ MADGSWGDEF FCIYDRIVNT                   LACLVALKSW NLHPDKIEKG VTYINENVHK LKDGSTEHMT                   SGFEIVVPAT LERAKVLGIQ GLPYDHPFIK EIINTKERRL                   SKIPKDLIYK LPTTLLFSLE GQGELDWEKI LKLQSSDGSF                   LTSPSSTASV FMRTKDEKCL KFIENAVKNC GGGAPHTYPV                   DVFARLWAVD RLQRLGISRF FQHEIKYFLD HINSVWTENG                   VFSGRDSQFC DIDDTSMGVR LLKMHGYNVD PNALKHFKQE                   DGKFSCYPGQ MIESASPIYN LYRAAQLRFP GEEILEEASR                   FAFNFLQEKI ANHEIQEKWV ISEHLIDEIK LGLKMPWYAT                   LPRVEAAYYL EYYAGSGDVW IGKTFYRMPE ISNDTYKEVA                   ILDFNTCQAQ HQFEWIYMQE WYESSKVKDF GISKKDLLVA                   YFLAASTIFE PERTQERIIW AKTLILSRMI TSFLNKQATL                   SSQQKNAILT QLGESVDGLD KIYSGEKDSG LAETLLATFQ                   QLLDGFDRYT RHQLRNAWGQ WLMKVQQGEA NGGADAELIA                   NTLNICAGLI AFNEDVLLHS EYTTLSSLTN KICQRLSQIE                   DEKTLEVIEG GIKDKELEED IQALVKLALE ENGGCGVDRR                   IKQSFLSVFK TFYYRAYHDA ETTDLHIFKV LFGPVM            
A nucleic acid encoding the  Origanum majorana  (+)-copalyl diphosphate synthase (OmTPS1) with SEQ ID NO:33 is shown below as SEQ ID NO:34.
 
     
       
         
           
               
               
            
               
                   
                 ATGACCGATG TATCCTCTCT TCGTTTGAGC AATGCACCAG 
               
               
                   
               
               
                   
                 CTGCCGGCGG CAGGTTGCCG CTGCCGGGAA AGGTTCACCT 
               
               
                   
               
               
                   
                 GCCTGAATTT CGCACCGTTT GTGCATGGTT GAACAATGGC 
               
               
                   
               
               
                   
                 TGCAAATACG AGCCCTTGAC TTGTCGAATT AGTCGACGGA 
               
               
                   
               
               
                   
                 AGATATCTGA ATGTCGAGTA GCAAGTCTGA ATTCGTCGCA 
               
               
                   
               
               
                   
                 ACTAATTGAA AAGGTCGGTT CTCCTGCTCA ATCTCTAGAA 
               
               
                   
               
               
                   
                 GAGGCAAACA AAAAGATCGA GGACTCCATC GAGTACATTA 
               
               
                   
               
               
                   
                 AGAATCTATT GATGACATCT GGCGACGGGC GGATAAGTGT 
               
               
                   
               
               
                   
                 GTCGGCTTAC GACACGTCGC TAGTCGCCCT AATAAAGGAC 
               
               
                   
               
               
                   
                 GTGAAAGGAC GAGATGCCCC TCAGTTCCCG TCGTGCCTGG 
               
               
                   
               
               
                   
                 AGTGGATAGC GCAAAACCAA ATGGCCGACG GGTCGTGGGG 
               
               
                   
               
               
                   
                 GGATGAGTTC TTCTGTATTT ACGACCGGAT CGTGAATACA 
               
               
                   
               
               
                   
                 TTAGCATGCC TCGTTGCCTT GAAATCATGG AACCTTCACC 
               
               
                   
               
               
                   
                 CCGACAAGAT CGAAAAAGGA GTGACGTACA TCAACGAAAA 
               
               
                   
               
               
                   
                 TGTGCACAAA CTGAAAGACG GGAGCACCGA GCACATGACG 
               
               
                   
               
               
                   
                 TCAGGGTTCG AAATCGTGGT CCCCGCCACT CTAGAAAGAG 
               
               
                   
               
               
                   
                 CCAAAGTCTT GGGCATCCAA GGCCTCCCTT ATGATCATCC 
               
               
                   
               
               
                   
                 CTTCATTAAG GAGATTATTA ATACTAAGGA GCGAAGATTA 
               
               
                   
               
               
                   
                 AGCAAAATAC CCAAGGATTT GATATACAAA CTGCCAACGA 
               
               
                   
               
               
                   
                 CGCTGCTGTT CAGTTTAGAA GGGCAGGGAG AATTAGATTG 
               
               
                   
               
               
                   
                 GGAAAAGATA CTGAAACTGC AGTCAAGCGA TGGCTCCTTC 
               
               
                   
               
               
                   
                 CTTACTTCGC CCTCGTCGAC CGCCTCCGTC TTCATGCGGA 
               
               
                   
               
               
                   
                 CGAAAGACGA GAAATGCCTC AAGTTCATTG AGAACGCCGT 
               
               
                   
               
               
                   
                 TAAGAATTGC GGCGGGGGAG CGCCGCATAC TTACCCAGTG 
               
               
                   
               
               
                   
                 GATGTGTTTG CAAGACTTTG GGCAGTTGAC AGACTACAGC 
               
               
                   
               
               
                   
                 GATTAGGGAT TTCTCGATTC TTCCAACACG AGATTAAATA 
               
               
                   
               
               
                   
                 CTTCTTAGAT CACATTAACA GTGTATGGAC CGAGAATGGA 
               
               
                   
               
               
                   
                 GTTTTCAGTG GACGAGATTC ACAATTTTGT GATATCGACG 
               
               
                   
               
               
                   
                 ACACTTCTAT GGGAGTTAGG CTTCTAAAAA TGCATGGATA 
               
               
                   
               
               
                   
                 CAATGTTGAT CCAAATGCGC TCAAGCATTT CAAGCAGGAG 
               
               
                   
               
               
                   
                 GATGGCAAAT TCTCTTGCTA CCCTGGCCAA ATGATCGAGT 
               
               
                   
               
               
                   
                 CTGCATCTCC GATATACAAT CTCTACCGAG CCGCTCAACT 
               
               
                   
               
               
                   
                 CCGGTTCCCC GGAGAAGAAA TTCTCGAAGA AGCAAGTCGA 
               
               
                   
               
               
                   
                 TTCGCCTTCA ACTTTCTGCA GGAAAAGATA GCCAACCATG 
               
               
                   
               
               
                   
                 AAATTCAAGA AAAATGGGTC ATATCTGAGC ACTTAATTGA 
               
               
                   
               
               
                   
                 TGAGATAAAG TTGGGACTGA AGATGCCATG GTACGCGACT 
               
               
                   
               
               
                   
                 CTGCCCCGAG TTGAGGCCGC TTATTATCTA GAGTATTATG 
               
               
                   
               
               
                   
                 CTGGCTCAGG CGACGTATGG ATTGGAAAGA CTTTCTACCG 
               
               
                   
               
               
                   
                 GATGCCGGAA ATCAGTAACG ATACGTATAA AGAGGTGGCC 
               
               
                   
               
               
                   
                 ATTTTGGATT TCAACACATG CCAAGCTCAA CACCAGTTTG 
               
               
                   
               
               
                   
                 AATGGATTTA CATGCAAGAG TGGTACGAAA GTAGCAAGGT 
               
               
                   
               
               
                   
                 TAAAGATTTC GGGATAAGCA AAAAGGACCT ACTTGTTGCT 
               
               
                   
               
               
                   
                 TACTTTCTGG CGGCATCGAC TATATTTGAA CCCGAAAGAA 
               
               
                   
               
               
                   
                 CACAAGAGAG GATTATTTGG GCAAAAACCC TAATTCTTTC 
               
               
                   
               
               
                   
                 TAGGATGATC ACATCATTTC TCAACAAACA AGCTACACTT 
               
               
                   
               
               
                   
                 TCATCCCAAC AAAAGAATGC CATCTTAACA CAACTTGGAG 
               
               
                   
               
               
                   
                 AGAGTGTCGA TGGCCTCGAT AAAATATATA GTGGTGAGAA 
               
               
                   
               
               
                   
                 AGATTCTGGG CTGGCTGAGA CTCTGCTGGC TACCTTCCAG 
               
               
                   
               
               
                   
                 CAACTGCTCG ACGGATTCGA TAGATACACT CGCCATCAAC 
               
               
                   
               
               
                   
                 TGAGAAATGC TTGGGGGCAA TGGTTGATGA AAGTGCAGCA 
               
               
                   
               
               
                   
                 AGGAGAGGCC AACGGTGGCG CCGACGCTGA GCTCATAGCA 
               
               
                   
               
               
                   
                 AACACACTCA ATATCTGCGC CGGCCTTATC GCCTTCAACG 
               
               
                   
               
               
                   
                 AAGACGTATT GTTGCACAGC GAATACACGA CTCTCTCCTC 
               
               
                   
               
               
                   
                 CCTCACCAAC AAAATATGCC AGCGCCTTAG CCAGATTGAA 
               
               
                   
               
               
                   
                 GATGAAAAGA CGCTTGAAGT GATTGAAGGG GGCATAAAAG 
               
               
                   
               
               
                   
                 ATAAGGAACT GGAGGAGGAT ATTCAGGCGT TGGTGAAGCT 
               
               
                   
               
               
                   
                 AGCCCTCGAA GAAAACGGCG GCTGCGGCGT CGACAGAAGA 
               
               
                   
               
               
                   
                 ATCAAGCAGT CATTCTTATC AGTATTCAAG ACTTTTTACT 
               
               
                   
               
               
                   
                 ACAGAGCCTA CCATGATGCT GAGACCACCG ATCTTCATAT 
               
               
                   
               
               
                   
                 TTTCAAAGTA CTGTTTGGGC CGGTTATGTG A 
               
            
           
         
       
     
     A  Perovskia atriplicifolia  (+)-Copalyl diphosphate synthase (PaTPS1) enzyme was identified and isolated. This Perovskia atriplicifolia (+)-Copalyl diphosphate synthase (PaTPS1) enzyme was identified to be a (+)-copalyl diphosphate ((+)-CPP) synthase that can synthesize compound 31. The Perovskia atriplicifolia (+)-Copalyl diphosphate synthase (PaTPS1) can have the amino acid sequence shown below (SEQ ID NO:35). 
                            MTSMSSLNLS RAPATTHRLQ LQAKVHVPEF YAVCAWLNSS                   SKQAPLSCQI RCKQLSRVTE CRVASLDASQ VSEKDTSHVQ                   TPDEVNKKIE DYIEYVKNLL MTSGDGRISV SPYDTSIVAL                   IKDSKGRNIP QFPSCLEWIA QHQMADGSWG DQFFCIYDRI                   LNTLACVVAL KSWNVHGDMI EKGVTYVKEN VHKLKDGNIE                   HMTSGFEIVV PALVQRAKDL GIQGLPYDDP LIKEIADTKE                   RRLKKIPKDM IYQTPTTLLF SLEGQGDLEW EKILKLQSGD                   GSFLTSPSST AHVFVQTKDE KCLKFIENAV KNCSGGAPHT                   YPVDVFARLW AIDRLQRLGI SRFFQPEIKY FIDHINSVWT                   ENGVFSGRDS EFCDIDDTSM GIRLLKMHGY KVDPNALNHF                   KQQDGKFSCY GGQMIESASP IYNLYRAAQL RFPGEEILEE                   ASKFAFNFLQ EKIANDQFQE KWVISDHLID EVKLGLKMPW                   YATLPRVEAA YYLQYYAGSG DVWIGKVFYR MPEISNDTYK                   ELAILDFNRC QAQHQFEWIY MQEWYHRSSV SEFGISKKEL                   LRTYFLAAAT IFEPERTQER LVWAKTQIVS RMITSFVNNG                   TTLSLDQMTA LATQIGHNFD GLDQIISAMK DHGLAGTLLT                   TFQQLLDGFD RYTRHQLKNA WSQWFMKLQQ GEANGGEDAE                   LLANTLNICA GFIAFNEDVL SHDEYTTLST LTNKICKRLS                   QIQDKKALEV VDGSIKDKEL EQDMQALVKL VLEENGGGVD                   RNIKQTFLSV FKTFYYTAYH DDETTDVHIF KVLFGPVV            
A nucleic acid encoding the  Perovskia atriplicifolia  (+)-Copalyl diphosphate synthase (PaTPS1) enzyme with SEQ ID NO:35 is shown below as SEQ ID NO:36.
 
     
       
         
           
               
               
            
               
                   
                 ATGACCTCTA TGTCCTCTCT AAATTTGAGC AGAGCACCAG 
               
               
                   
               
               
                   
                 CTACCACCCA CCGGTTACAG CTACAGGCAA AGGTTCACGT 
               
               
                   
               
               
                   
                 GCCGGAATTT TATGCCGTGT GTGCATGGCT GAATAGCAGC 
               
               
                   
               
               
                   
                 AGCAAACAGG CACCCTTGAG TTGCCAAATT CGCTGCAAGC 
               
               
                   
               
               
                   
                 AACTATCAAG AGTAACTGAA TGTCGGGTAG CAAGTCTGGA 
               
               
                   
               
               
                   
                 TGCGTCGCAA GTGAGTGAAA AAGACACTTC TCATGTCCAA 
               
               
                   
               
               
                   
                 ACTCCCGATG AGGTGAACAA AAAGATCGAG GACTATATCG 
               
               
                   
               
               
                   
                 AGTACGTCAA GAATCTGTTG ATGACGTCGG GCGACGGGCG 
               
               
                   
               
               
                   
                 AATAAGCGTG TCGCCCTACG ACACGTCAAT AGTCGCCCTT 
               
               
                   
               
               
                   
                 ATTAAGGACT CGAAAGGGCG CAACATCCCG CAGTTTCCGT 
               
               
                   
               
               
                   
                 CGTGCCTCGA GTGGATAGCG CAGCACCAAA TGGCGGATGG 
               
               
                   
               
               
                   
                 CTCATGGGGG GATCAATTCT TCTGCATTTA CGACCGGATT 
               
               
                   
               
               
                   
                 CTAAATACAT TAGCATGTGT CGTAGCTTTG AAATCCTGGA 
               
               
                   
               
               
                   
                 ACGTTCACGG TGACATGATC GAAAAAGGAG TGACGTACGT 
               
               
                   
               
               
                   
                 CAAGGAAAAT GTGCATAAGC TTAAAGATGG GAATATTGAG 
               
               
                   
               
               
                   
                 CACATGACGT CGGGGTTCGA AATTGTGGTT CCCGCCCTTG 
               
               
                   
               
               
                   
                 TTCAAAGAGC CAAAGACTTG GGCATCCAAG GCCTGCCCTA 
               
               
                   
               
               
                   
                 TGATGATCCC CTCATCAAGG AGATTGCTGA TACAAAAGAA 
               
               
                   
               
               
                   
                 AGAAGATTGA AAAAGATACC CAAGGATATG ATTTACCAAA 
               
               
                   
               
               
                   
                 CGCCAACGAC ATTACTATTC AGTTTAGAAG GGCAGGGAGA 
               
               
                   
               
               
                   
                 TTTGGAGTGG GAAAAGATAC TGAAACTGCA GTCAGGCGAT 
               
               
                   
               
               
                   
                 GGCTCCTTCC TCACTTCGCC GTCATCCACC GCCCACGTGT 
               
               
                   
               
               
                   
                 TCGTGCAGAC CAAAGATGAA AAATGCTTGA AATTCATCGA 
               
               
                   
               
               
                   
                 GAACGCCGTC AAGAATTGCA GTGGAGGAGC GCCGCATACT 
               
               
                   
               
               
                   
                 TATCCAGTCG ATGTCTTCGC AAGACTTTGG GCAATTGACA 
               
               
                   
               
               
                   
                 GACTACAACG CCTAGGAATT TCTCGTTTCT TCCAGCCGGA 
               
               
                   
               
               
                   
                 AATTAAGTAT TTCATAGACC ACATCAACAG CGTTTGGACA 
               
               
                   
               
               
                   
                 GAGAACGGAG TTTTCAGTGG GCGAGATTCG GAATTTTGCG 
               
               
                   
               
               
                   
                 ATATTGATGA CACGTCCATG GGCATCAGGC TTCTCAAAAT 
               
               
                   
               
               
                   
                 GCACGGATAC AAAGTCGACC CAAATGCACT CAATCATTTC 
               
               
                   
               
               
                   
                 AAGCAGCAAG ATGGTAAATT TTCTTGCTAC GGTGGTCAAA 
               
               
                   
               
               
                   
                 TGATCGAGTC TGCATCTCCA ATATACAATC TCTACAGGGC 
               
               
                   
               
               
                   
                 TGCTCAGCTA CGATTTCCAG GAGAAGAAAT TCTTGAAGAA 
               
               
                   
               
               
                   
                 GCCAGTAAAT TTGCCTTTAA CTTTTTGCAA GAAAAAATAG 
               
               
                   
               
               
                   
                 CCAACGATCA ATTTCAAGAA AAATGGGTGA TATCCGACCA 
               
               
                   
               
               
                   
                 CTTAATCGAT GAGGTGAAGC TCGGGCTGAA GATGCCATGG 
               
               
                   
               
               
                   
                 TACGCCACTC TACCCCGGGT TGAGGCTGCA TATTATCTAC 
               
               
                   
               
               
                   
                 AATACTATGC TGGTTCTGGC GACGTATGGA TTGGCAAGGT 
               
               
                   
               
               
                   
                 TTTCTACAGG ATGCCGGAAA TCAGCAATGA TACATACAAA 
               
               
                   
               
               
                   
                 GAGCTGGCCA TATTGGATTT CAACAGATGC CAAGCACAGC 
               
               
                   
               
               
                   
                 ATCAGTTCGA ATGGATTTAT ATGCAAGAGT GGTATCACAG 
               
               
                   
               
               
                   
                 AAGCAGCGTT AGTGAATTCG GGATAAGCAA AAAAGAGCTG 
               
               
                   
               
               
                   
                 CTTCGTACTT ACTTTCTGGC TGCAGCAACC ATATTCGAAC 
               
               
                   
               
               
                   
                 CCGAGAGAAC ACAAGAGAGG CTTGTGTGGG CAAAAACCCA 
               
               
                   
               
               
                   
                 AATTGTCTCT AGGATGATCA CATCATTTGT TAACAATGGA 
               
               
                   
               
               
                   
                 ACTACACTAT CTTTGGACCA AATGACTGCA CTTGCAACAC 
               
               
                   
               
               
                   
                 AAATCGGCCA TAATTTCGAT GGCCTCGATC AAATAATTAG 
               
               
                   
               
               
                   
                 TGCTATGAAA GATCATGGAC TGGCTGGGAC TCTGCTGACA 
               
               
                   
               
               
                   
                 ACCTTCCAGC AACTTCTAGA TGGATTCGAC AGATACACTC 
               
               
                   
               
               
                   
                 GCCATCAACT CAAAAATGCT TGGAGCCAAT GGTTCATGAA 
               
               
                   
               
               
                   
                 ACTCCAGCAA GGGGAGGCGA ACGGCGGGGA AGACGCGGAG 
               
               
                   
               
               
                   
                 CTCCTAGCAA ACACGCTCAA CATCTGCGCG GGTTTCATTG 
               
               
                   
               
               
                   
                 CTTTCAACGA AGACGTATTG TCGCACGATG AATACACGAC 
               
               
                   
               
               
                   
                 TCTCTCCACC CTTACAAACA AAATCTGCAA GCGCCTTAGC 
               
               
                   
               
               
                   
                 CAAATTCAAG ATAAAAAGGC GCTGGAAGTT GTCGACGGGA 
               
               
                   
               
               
                   
                 GCATAAAGGA TAAGGAGCTC GAACAGGATA TGCAGGCGTT 
               
               
                   
               
               
                   
                 GGTGAAGTTG GTCCTTGAAG AAAATGGCGG CGGCGTCGAC 
               
               
                   
               
               
                   
                 AGGAACATCA AACAGACATT TTTGTCCGTT TTCAAGACTT 
               
               
                   
               
               
                   
                 TTTACTACAC CGCCTACCAC GATGATGAGA CCACTGATGT 
               
               
                   
               
               
                   
                 TCATATTTTC AAAGTACTGT TTGGACCGGT CGTATGA 
               
            
           
         
       
     
       Pogostemon cablin  (10R)-labda-8,13E-dienyl diphosphate synthase (PcTPS1) was identified and isolated. This  Pogostemon cablin  (10R)-labda-8,13E-dienyl diphosphate synthase (PcTPS1) enzyme was identified to be a (10R)-labda-8,13E-dienyl diphosphate synthase, which can synthesize compound 25. 
     
       
         
         
             
             
         
       
     
     The combination of PcTPS1 and SsSS, both in-vitro, and in  N. benthamiana  expression produced (10R)-labda-8,14-en-13-ol [26], shown below. 
     
       
         
         
             
             
         
       
     
     This  Pogostemon cablin  (10R)-labda-8,13E-dienyl diphosphate synthase (PcTPS1) can have the amino acid sequence shown below (SEQ ID NO:37). 
                            MSFASQSHVA FVLRRPSAVA PPPPTRIPTT AALSPLKPGD                   FSHGRSSFMP TSIKCNAIST SRVEEYKYTD DHNQSGLLEH                   DGLISDKINE LVTKIQLMLQ NMDDGEISIS PYDTAWVSLV                   EDVGGNDRPQ FPTSLEWISN NQLPDGSWGD PNAFLVHDRI                   LNTLACVVAL KSWKMHPHKC NRGVSFVREN IYRMDDEKEE                   HMPNGFEVVF PALLQKAKTL NIDIPYEFPG IQKFYAKRDL                   KFARIPMDIL HSVPTTLLFS LEGVRCGLDL DWGKLLELQA                   ADGSFLYSPS STAFALEQTK DQNCLKYLSK LVRKFDGGVP                   NVYPVDLFEH NWAVDRLQRL GISRYFTPEI NQCLDYSYRY                   WSNSKGMYSA SNSQIQDVDD TAMGFRLLRL NGYDVSTQGF                   RQFEAGGDFF CFAGQSSQAV TGMYNLYRAS QVMFPGEKLL                   EDAKKFSTNF LQQKRANNQL TDKWVIAKDV PAEVGYALDI                   PWYASLPRLE ARFFIQQYGG DDDVWIGKTL YRMGYVNNNT                   YLELAKLDYN TCQRLHQHEW ITIQRWYEIN LKITSVGLSK                   RGVLLSYYLA AANLFEPQNS THRIAWAKTS ILVSAIQLSP                   LQKRDFINQF HRSTANNGYE TSNVLVKSVI KGVHELSMDA                   MLTHNKDIHR QLFNAWRKWM SVWEEGGDGE AELLLSTLNT                   CDGVDESTFS DPKYEHLLEI TVRVTHQLHL IQNAETKRVG                   DREEIDLSMQ QLVKLVFTKS SSDLDSCIKQ RFFAIARSFY                   YVAHCDPEMV DSHIAKVLFE RVM            
A nucleic acid encoding the  Pogostemon cablin  (10R)-labda-8,13E-dienyl diphosphate synthase (PcTPS1) enzyme with SEQ ID NO:37 is shown below as SEQ ID NO:38.
 
     
       
         
           
               
               
            
               
                   
                 ATGTCATTTG CTTCTCAATC ACATGTCGCC TTTGTACTCC 
               
               
                   
               
               
                   
                 GACGGCCATC TGCCGTTGCT CCGCCACCAC CGACTAGAAT 
               
               
                   
               
               
                   
                 TCCGACAACA GCCGCTCTTT CTCCTCTCAA ACCAGGTGAT 
               
               
                   
               
               
                   
                 TTTTCCCATG GCAGATCATC ATTTATGCCC ACTTCCATTA 
               
               
                   
               
               
                   
                 AATGTAATGC AATTTCCACA TCTCGCGTCG AAGAATACAA 
               
               
                   
               
               
                   
                 GTACACGGAT GATCATAATC AGAGTGGTTT ATTGGAGCAT 
               
               
                   
               
               
                   
                 GATGGTTTGA TATCAGACAA GATAAATGAA TTGGTGACCA 
               
               
                   
               
               
                   
                 AGATACAATT GATGCTACAA AACATGGATG ACGGAGAGAT 
               
               
                   
               
               
                   
                 AAGCATCTCC CCATATGACA CCGCATGGGT GTCGTTGGTG 
               
               
                   
               
               
                   
                 GAGGATGTGG GCGGCAACGA CCGCCCACAG TTTCCTACGA 
               
               
                   
               
               
                   
                 GCCTGGAGTG GATATCGAAT AACCAGCTCC CCGACGGCTC 
               
               
                   
               
               
                   
                 GTGGGGCGAC CCGAATGCCT TTTTGGTGCA CGACCGTATC 
               
               
                   
               
               
                   
                 CTCAACACAT TGGCATGCGT CGTTGCACTC AAATCCTGGA 
               
               
                   
               
               
                   
                 AAATGCACCC CCACAAATGC AATAGAGGAG TTAGTTTCGT 
               
               
                   
               
               
                   
                 GAGAGAAAAT ATATACAGAA TGGATGATGA AAAAGAGGAA 
               
               
                   
               
               
                   
                 CACATGCCAA ATGGATTCGA AGTGGTATTT CCAGCACTCC 
               
               
                   
               
               
                   
                 TTCAAAAAGC GAAAACCCTA AACATTGATA TCCCGTACGA 
               
               
                   
               
               
                   
                 GTTTCCAGGA ATACAAAAAT TTTATGCCAA AAGAGATTTA 
               
               
                   
               
               
                   
                 AAATTCGCCA GGATTCCAAT GGATATATTG CATAGCGTTC 
               
               
                   
               
               
                   
                 CGACAACATT ACTGTTCAGC TTAGAAGGTG TAAGATGTGG 
               
               
                   
               
               
                   
                 TCTTGATCTG GATTGGGGGA AGCTTCTAGA ATTGCAAGCT 
               
               
                   
               
               
                   
                 GCTGATGGCT CATTTCTCTA CTCTCCATCC TCTACTGCCT 
               
               
                   
               
               
                   
                 TTGCACTAGA ACAAACCAAG GATCAAAACT GCCTCAAATA 
               
               
                   
               
               
                   
                 TCTATCTAAA CTTGTTCGAA AATTCGATGG CGGAGTACCC 
               
               
                   
               
               
                   
                 AACGTGTACC CGGTGGACTT GTTCGAACAT AATTGGGCAG 
               
               
                   
               
               
                   
                 TTGATCGTCT CCAAAGGCTC GGAATTTCTC GTTATTTTAC 
               
               
                   
               
               
                   
                 GCCTGAAATC AACCAATGTC TTGATTATTC TTACAGATAT 
               
               
                   
               
               
                   
                 TGGTCAAATA GTAAAGGGAT GTACTCGGCA AGCAATTCCC 
               
               
                   
               
               
                   
                 AGATTCAGGA CGTTGATGAC ACCGCCATGG GATTCAGGCT 
               
               
                   
               
               
                   
                 TTTGAGACTC AACGGCTACG ATGTCTCTAC ACAAGGGTTT 
               
               
                   
               
               
                   
                 AGGCAATTCG AGGCAGGGGG GGACTTCTTC TGCTTCGCGG 
               
               
                   
               
               
                   
                 GGCAGTCGAG CCAAGCTGTA ACCGGAATGT ACAACCTCTA 
               
               
                   
               
               
                   
                 CAGAGCTTCC CAAGTGATGT TCCCTGGAGA GAAGCTACTG 
               
               
                   
               
               
                   
                 GAAGATGCCA AGAAATTCTC CACCAACTTC TTGCAACAAA 
               
               
                   
               
               
                   
                 AACGAGCCAA TAACCAGCTC ACTGACAAGT GGGTTATTGC 
               
               
                   
               
               
                   
                 CAAAGATGTT CCAGCTGAGG TGGGATATGC CTTGGATATT 
               
               
                   
               
               
                   
                 CCCTGGTATG CCAGTCTGCC CCGACTGGAA GCAAGATTTT 
               
               
                   
               
               
                   
                 TCATACAACA ATACGGTGGA GACGACGACG TTTGGATCGG 
               
               
                   
               
               
                   
                 CAAAACCTTG TATAGAATGG GATATGTGAA CAACAACACT 
               
               
                   
               
               
                   
                 TATCTGGAAC TCGCAAAGCT AGACTACAAC ACCTGCCAAA 
               
               
                   
               
               
                   
                 GGTTGCATCA GCATGAGTGG ATAACCATTC AACGATGGTA 
               
               
                   
               
               
                   
                 CGAAATTAAT TTAAAAATTA CTAGTGTTGG GTTGAGCAAA 
               
               
                   
               
               
                   
                 AGAGGGGTCC TGTTGAGTTA TTACTTAGCC GCAGCCAATC 
               
               
                   
               
               
                   
                 TGTTTGAGCC TCAAAACTCA ACACACCGCA TCGCTTGGGC 
               
               
                   
               
               
                   
                 CAAAACTTCG ATTTTAGTAA GCGCTATTCA ACTTTCTCCC 
               
               
                   
               
               
                   
                 CTCCAAAAGC GCGACTTTAT TAACCAATTC CACCGCTCCA 
               
               
                   
               
               
                   
                 CCGCAAATAA TGGGTATGAA ACAAGTAATG TGTTGGTGAA 
               
               
                   
               
               
                   
                 GAGTGTAATC AAGGGTGTGC ATGAGCTCTC CATGGACGCT 
               
               
                   
               
               
                   
                 ATGTTGACGC ACAATAAAGA CATACATCGC CAACTTTTTA 
               
               
                   
               
               
                   
                 ATGCTTGGCG AAAGTGGATG TCAGTGTGGG AAGAGGGAGG 
               
               
                   
               
               
                   
                 TGATGGAGAA GCGGAGCTGT TATTGTCGAC GCTTAACACG 
               
               
                   
               
               
                   
                 TGCGACGGAG TAGATGAATC CACATTCAGC GATCCCAAAT 
               
               
                   
               
               
                   
                 ACGAGCACCT CTTAGAGATC ACCGTCAGAG TCACCCACCA 
               
               
                   
               
               
                   
                 GCTTCATCTC ATTCAGAATG CAGAGACGAA GCGTGTGGGT 
               
               
                   
               
               
                   
                 GACCGTGAGG AAATAGATTT GAGCATGCAA CAACTTGTTA 
               
               
                   
               
               
                   
                 AGTTGGTGTT CACTAAATCA TCATCGGATC TGGATTCTTG 
               
               
                   
               
               
                   
                 TATCAAGCAA AGATTTTTTG CGATTGCCAG AAGTTTCTAT 
               
               
                   
               
               
                   
                 TACGTGGCTC ATTGTGATCC GGAGATGGTG GACTCCCACA 
               
               
                   
               
               
                   
                 TAGCCAAAGT ATTGTTTGAG AGGGTGATGT AG 
               
            
           
         
       
     
       Prunella vulgaris  11-hydroxy vulgarisane synthase (PvHVS) was identified and isolated. The  Prunella vulgaris  11-hydroxy vulgarisane synthase (PvHVS) enzyme catalyzes the first committed step and forms the scaffold found in all Vulgarisins, a class of diterpenes with pharmaceutical applications (e.g., gout, cancer). For example, PvHVS can synthesize 11-hydroxy vulgarisane (shown below). 
     
       
         
         
             
             
         
       
     
     An example of a formula for several Vulgarisin diterpenes is shown below. 
     
       
         
         
             
             
         
       
     
     Vulgarisins B (1) and C (2) exhibit modest cytotoxicity activity against human lung carcinoma A549 cell line (Lou et al. Tetrahedron Letters 58: 401-404 (2017)). 
     The  Prunella vulgaris  11-hydroxy vulgarisane synthase (PvHVS) can have the amino acid sequence shown below (SEQ ID NO:39). 
     
       
         
           
               
               
            
               
                   
                 MSSLSIPFSS AICTSSIPKI STGHHRRTAR MPAHDTSRLV 
               
               
                   
               
               
                   
                 FRPSAVMVEG SPMTTSSNGK EVQRLITTFK PSMWKDIFST 
               
               
                   
               
               
                   
                 FSFDNQVQEK YLKEIEELKK EVRSTLMSAT HRKLFDLIDN 
               
               
                   
               
               
                   
                 LERMGIAYHF ETEIEDKLKQ AHASLEEEDD YDLFTTALRF 
               
               
                   
               
               
                   
                 RLLRQHRYHV SCDPFAKFVD QDNKLKESLS SDVEGLLSLF 
               
               
                   
               
               
                   
                 EASHLRIHNE DVLDEAIVFT THHLNRMMPQ LESPLKEEVK 
               
               
                   
               
               
                   
                 HALRYPLHKC LGILSLRFHI DRYENDKSRD EVVLRLGQVN 
               
               
                   
               
               
                   
                 FNYMQNIYMN ELYEITTWWN KLQMTSKVPY FRDRLVECYM 
               
               
                   
               
               
                   
                 WGLAYHFEPE YAPVRVLITK YYMTATTVDD TYDNYATLEE 
               
               
                   
               
               
                   
                 IELFTQAIDR WSEDEIDQLP DEYLKIVYKG LMNFTEEFRR 
               
               
                   
               
               
                   
                 DAEERGKGYV IPYFIEETKR ATQGYANEQR WIMKREMPSF 
               
               
                   
               
               
                   
                 EEYMVNSRVT SLMYVTYVAV VAVIESATKE TVDWALSDSD 
               
               
                   
               
               
                   
                 IFVYTNDIGR LIDDLATHRR ERKDGTMLTS MDYYMKEYGG 
               
               
                   
               
               
                   
                 TMEEGEAAFR KLMEEKWKLL NAAWVDTING KESKEIVVQV 
               
               
                   
               
               
                   
                 LDLARICGTL YGDEEDGFTY PEKNFAPLVA ALLMNPIHI 
               
            
           
         
       
     
     A nucleic acid encoding the i Prunella vulgaris 11-hydroxy vulgarisane synthase (PvHVS) enzyme with SEQ ID NO:39 is shown below as SEQ ID NO:40. 
     
       
         
           
               
               
            
               
                   
                 ATGAGCTCTC TCTCAATTCC CTTTTCTTCC GCCATTTGCA 
               
               
                   
                   
               
               
                   
                 CTTCATCAAT CCCAAAGATC AGTACTGGGC ATCATCGCCG 
               
               
                   
                   
               
               
                   
                 CACCGCGAGG ATGCCCGCGC ACGACACATC GCGTCTCGTC 
               
               
                   
                   
               
               
                   
                 TTTCGCCCTT CAGCTGTGAT GGTGGAAGGA AGTCCGATGA 
               
               
                   
                   
               
               
                   
                 CTACTTCAAG CAACGGGAAG GAAGTCCAAC GACTTATAAC 
               
               
                   
                   
               
               
                   
                 CACTTTCAAG CCTAGCATGT GGAAAGATAT TTTTTCTACC 
               
               
                   
                   
               
               
                   
                 TTCTCTTTCG ATAATCAGGT GCAAGAAAAG TATTTGAAAG 
               
               
                   
                   
               
               
                   
                 AAATTGAGGA ATTGAAGAAA GAAGTAAGAA GCACACTAAT 
               
               
                   
                   
               
               
                   
                 GAGTGCTACG CATAGGAAAT TGTTTGACTT GATCGACAAT 
               
               
                   
                   
               
               
                   
                 CTCGAGCGTA TGGGAATCGC CTATCATTTC GAGACAGAAA 
               
               
                   
                   
               
               
                   
                 TCGAAGACAA GCTCAAACAA GCTCATGCTT CTCTAGAGGA 
               
               
                   
                   
               
               
                   
                 GGAAGATGAC TACGACTTGT TCACTACTGC ACTTCGCTTT 
               
               
                   
                   
               
               
                   
                 CGTCTGCTCA GACAACATCG CTATCATGTT TCTTGCGATC 
               
               
                   
                   
               
               
                   
                 CCTTTGCGAA ATTTGTTGAC CAAGACAACA AATTGAAAGA 
               
               
                   
                   
               
               
                   
                 GAGTCTTAGT AGCGACGTCG AGGGGCTATT AAGCTTGTTC 
               
               
                   
                   
               
               
                   
                 GAGGCATCCC ATCTTCGGAT CCACAACGAG GATGTTCTAG 
               
               
                   
                   
               
               
                   
                 ATGAAGCTAT AGTGTTCACA ACCCATCACT TGAATCGAAT 
               
               
                   
                   
               
               
                   
                 GATGCCACAA TTGGAATCGC CCCTTAAAGA AGAAGTGAAG 
               
               
                   
                   
               
               
                   
                 CATGCTCTTC GATACCCCCT TCACAAGTGT CTTGGAATCC 
               
               
                   
                   
               
               
                   
                 TTAGCCTTCG TTTTCATATC GACAGATATG AGAATGATAA 
               
               
                   
                   
               
               
                   
                 GTCGAGGGAT GAAGTTGTTC TCAGACTAGG CCAAGTTAAT 
               
               
                   
                   
               
               
                   
                 TTCAATTACA TGCAGAACAT TTACATGAAC GAGCTCTATG 
               
               
                   
                   
               
               
                   
                 AAATCACCAC GTGGTGGAAC AAGTTGCAGA TGACTTCAAA 
               
               
                   
                   
               
               
                   
                 AGTACCTTAC TTTAGAGATA GATTGGTAGA GTGCTATATG 
               
               
                   
                   
               
               
                   
                 TGGGGTTTGG CATATCATTT CGAACCAGAA TACGCTCCCG 
               
               
                   
                   
               
               
                   
                 TTCGAGTCCT CATTACCAAG TACTATATGA CCGCCACAAC 
               
               
                   
                   
               
               
                   
                 TGTCGACGAT ACCTATGATA ATTATGCTAC ACTCGAAGAA 
               
               
                   
                   
               
               
                   
                 ATCGAACTCT TCACTCAGGC CATTGACAGG TGGAGCGAGG 
               
               
                   
                   
               
               
                   
                 ATGAGATTGA TCAGCTACCT GATGAATACC TAAAAATAGT 
               
               
                   
                   
               
               
                   
                 GTACAAAGGT CTAATGAACT TCACTGAAGA GTTTAGACGT 
               
               
                   
                   
               
               
                   
                 GACGCAGAAG AGCGAGGGAA AGGCTATGTG ATTCCTTACT 
               
               
                   
                   
               
               
                   
                 TTATTGAAGA AACGAAGAGA GCAACACAGG GTTATGCAAA 
               
               
                   
                   
               
               
                   
                 CGAGCAGAGG TGGATAATGA AGAGAGAAAT GCCGAGTTTT 
               
               
                   
                   
               
               
                   
                 GAAGAGTATA TGGTGAACTC AAGGGTAACA TCACTTATGT 
               
               
                   
                   
               
               
                   
                 ATGTGACCTA CGTTGCTGTT GTGGCAGTCA TAGAATCAGC 
               
               
                   
                   
               
               
                   
                 TACCAAAGAA ACCGTAGATT GGGCGCTAAG TGACTCCGAT 
               
               
                   
                   
               
               
                   
                 ATCTTTGTCT ACACTAACGA TATCGGCCGA CTTATCGACG 
               
               
                   
                   
               
               
                   
                 ACCTTGCCAC TCATCGACGC GAGAGGAAAG ACGGGACAAT 
               
               
                   
                   
               
               
                   
                 GCTTACATCG ATGGATTATT ACATGAAGGA ATATGGCGGT 
               
               
                   
                   
               
               
                   
                 ACGATGGAAG AGGGGGAAGC TGCATTTAGG AAATTGATGG 
               
               
                   
                   
               
               
                   
                 AGGAGAAATG GAAACTTTTG AATGCAGCAT GGGTAGATAC 
               
               
                   
                   
               
               
                   
                 TATTAATGGA AAAGAGTCGA AGGAAATAGT TGTGCAAGTT 
               
               
                   
                   
               
               
                   
                 CTCGACCTCG CCAGGATATG CGGAACGCTC TATGGGGACG 
               
               
                   
                   
               
               
                   
                 AAGAAGATGG CTTCACCTAC CCAGAGAAGA ATTTTGCACC 
               
               
                   
                   
               
               
                   
                 ACTCGTTGCT GCTCTATTGA TGAATCCTAT ACATATTTGA 
               
            
           
         
       
     
     A  Chiococca alba  ent-CPP synthase (CaTPS1) was identified and isolated. This CaTPS1 enzyme was identified that converts GGPP to ent-CPP [16]. 
     
       
         
         
             
             
         
       
     
     The  Chiococca alba  ent-CPP synthase (CaTPS1) has the amino acid sequence shown below (SEQ ID NO:41). 
     
       
         
           
               
               
            
               
                 1 
                 MSSSTSAAAT LLGLSPASRR FVSFPPANGP IETITGIWSP 
               
               
                   
               
               
                 41 
                 GKALHHFNFR LRCSTVSSPR TQELGQVSQN GMSGIKWHDI 
               
               
                   
               
               
                 81 
                 VEEGVTEKGT LEANTSSWIK ESIEAIRWML RTMDDGDISI 
               
               
                   
               
               
                 121 
                 SAYDTAWVAL VEDINGSGGP QFPSSLEWIA NNQLPDGSWG 
               
               
                   
               
               
                 161 
                 DSDIFSAHDR ILNTLGCVVA LKSWNMHPEK SEKGLLYLRD 
               
               
                   
               
               
                 201 
                 NIHKLEDENV EHMPIGFEVA FPSLIEIAKK LSIDIPDDSA 
               
               
                   
               
               
                 241 
                 ILQEIYARRN LKLTRIPKDI MHTVPTTLLH SLEGMPELDW 
               
               
                   
               
               
                 281 
                 KRLISLKCED GSFLFSPSST AFALTQTKDA DCLRYLTKTV 
               
               
                   
               
               
                 321 
                 QKFNGGVPNV YPVDLFEHIW AVDRLQRLGI SRYFQSEIRE 
               
               
                   
               
               
                 361 
                 CIDYVHRYWT DKGICWARNT HVYDIDDTAM GFRLLRLHGY 
               
               
                   
               
               
                 401 
                 DVSADVFRYY EKDGEFVCFA GQSNQAVTGM YNLYRASQVM 
               
               
                   
               
               
                 441 
                 FPGENILSDA RKFSSEFLHD KRANNELLDK WIITKDLPGE 
               
               
                   
               
               
                 481 
                 VAYALDVPWY ASLPRLETRL YLEQYGGEDD VWIGKTLYRM 
               
               
                   
               
               
                 521 
                 QKVNNNIYLE LGKLDYNNCQ ALHQLEWRSI QKWYNECGLG 
               
               
                   
               
               
                 561 
                 EYGLSERSLL LSYYLAAASI FEPERSKERL AWAKTTMLIR 
               
               
                   
               
               
                 601 
                 TIESYLSSEQ MVEDHNGAFV SEFQYYCSNL DYVNGGRHKP 
               
               
                   
               
               
                 641 
                 TQRLVRTLLG TLNQISLDAV LVHGRDIHQY LRQAWEKWLI 
               
               
                   
               
               
                 681 
                 ALQEGDDSDM GQEEAELLVR TLNLCAGRYA SEELLLSHPK 
               
               
                   
               
               
                 721 
                 YQQLLHITTR VCNQIRHFQH KKVQDGENGR ANMGDGITSI 
               
               
                   
               
               
                 761 
                 SSIESDMQEL TKLVVGNTQN DLDADTKQTF LTVAKSFYYT 
               
               
                   
               
               
                 801 
                 AHCNPGTINC HIAKVLFERV L 
               
            
           
         
       
     
     A nucleic acid encoding the  Chiococca alba  ent-CPP synthase (CaTPS1) with SEQ ID NO:41 is shown below as SEQ ID NO:42. 
     
       
         
           
               
               
            
               
                 1 
                 ATGTCTTCTT CTACCTCAGC AGCAGCAACC CTTCTCGGAT 
               
               
                   
               
               
                 41 
                 TATCGCCGGC AAGCCGCCGG TTTGTATCAT TTCCTCCGGC 
               
               
                   
               
               
                 81 
                 AAATGGACCT ATAGAAACTA TTACCGGTAT TTGGTCGCCC 
               
               
                   
               
               
                 121 
                 GGCAAAGCTC TTCATCACTT TAATTTCCGT CTGCGTTGTA 
               
               
                   
               
               
                 161 
                 GCACGGTGTC CAGTCCTCGC ACCCAAGAAT TGGGCCAGGT 
               
               
                   
               
               
                 201 
                 GTCACAAAAT GGCATGTCTG GTATAAAGTG GCATGACATA 
               
               
                   
               
               
                 241 
                 GTGGAAGAAG GAGTCACAGA AAAAGGAACT CTTGAGGCGA 
               
               
                   
               
               
                 281 
                 ACACATCAAG CTGGATAAAA GAAAGCATAG AAGCCATTCG 
               
               
                   
               
               
                 321 
                 TTGGATGCTG CGTACCATGG ATGACGGGGA TATCAGCATA 
               
               
                   
               
               
                 361 
                 TCTGCTTATG ATACTGCATG GGTTGCCCTT GTGGAAGATA 
               
               
                   
               
               
                 401 
                 TCAACGGAAG TGGCGGTCCT CAATTTCCTT CAAGCCTCGA 
               
               
                   
               
               
                 441 
                 GTGGATTGCC AACAATCAGC TTCCTGATGG TTCATGGGGC 
               
               
                   
               
               
                 481 
                 GACAGCGACA TCTTTTCAGC TCACGATCGG ATTCTCAACA 
               
               
                   
               
               
                 521 
                 CTTTGGGATG CGTTGTTGCA TTAAAATCTT GGAACATGCA 
               
               
                   
               
               
                 561 
                 CCCTGAAAAG AGTGAAAAAG GATTATTATA TTTAAGGGAT 
               
               
                   
               
               
                 601 
                 AACATTCACA AGCTTGAGGA TGAAAATGTC GAGCACATGC 
               
               
                   
               
               
                 641 
                 CTATCGGTTT TGAAGTGGCA TTTCCTTCAC TAATTGAGAT 
               
               
                   
               
               
                 681 
                 AGCCAAAAAG TTGAGCATTG ATATTCCGGA TGATTCTGCA 
               
               
                   
               
               
                 721 
                 ATCTTGCAGG AGATATATGC CAGAAGAAAT CTAAAGCTAA 
               
               
                   
               
               
                 761 
                 CAAGGATACC GAAGGACATT ATGCACACAG TGCCCACAAC 
               
               
                   
               
               
                 801 
                 ATTGCTCCAC AGCTTGGAAG GCATGCCAGA ACTAGACTGG 
               
               
                   
               
               
                 841 
                 AAAAGGCTAA TATCTCTAAA GTGTGAGGAT GGTTCCTTTC 
               
               
                   
               
               
                 881 
                 TGTTTTCTCC ATCCTCCACT GCTTTTGCCC TCACGCAAAC 
               
               
                   
               
               
                 921 
                 TAAAGATGCT GATTGCCTCA GATATTTAAC TAAAACCGTA 
               
               
                   
               
               
                 961 
                 CAAAAATTCA ATGGAGGAGT TCCCAATGTT TACCCCGTGG 
               
               
                   
               
               
                 1001 
                 ACTTATTCGA ACACATCTGG GCTGTTGATC GACTTCAAAG 
               
               
                   
               
               
                 1041 
                 ACTAGGAATT TCTCGATACT TCCAGTCAGA AATCCGCGAG 
               
               
                   
               
               
                 1081 
                 TGCATCGATT ATGTTCACCG ATATTGGACG GATAAAGGTA 
               
               
                   
               
               
                 1121 
                 TCTGTTGGGC TAGAAATACC CACGTTTATG ACATTGATGA 
               
               
                   
               
               
                 1161 
                 TACAGCTATG GGTTTTAGAC TTCTAAGGTT GCATGGCTAC 
               
               
                   
               
               
                 1201 
                 GATGTTTCTG CAGATGTTTT CAGATACTAT GAGAAGGATG 
               
               
                   
               
               
                 1241 
                 GCGAATTCGT TTGCTTTGCC GGACAGTCAA ACCAGGCGGT 
               
               
                   
               
               
                 1281 
                 GACCGGAATG TATAACCTGT ATAGAGCTTC TCAAGTGATG 
               
               
                   
               
               
                 1321 
                 TTTCCAGGGG AGAATATACT TTCGGATGCT AGGAAATTCT 
               
               
                   
               
               
                 1361 
                 CGTCCGAATT CTTGCATGAT AAGCGAGCCA ACAATGAGCT 
               
               
                   
               
               
                 1401 
                 CCTAGATAAA TGGATCATAA CCAAAGATTT GCCTGGGGAG 
               
               
                   
               
               
                 1441 
                 GTAGCATATG CTTTAGATGT TCCATGGTAT GCCAGTTTAC 
               
               
                   
               
               
                 1481 
                 CTCGTTTAGA AACCAGATTG TATTTGGAAC AATATGGCGG 
               
               
                   
               
               
                 1521 
                 CGAAGATGAT GTCTGGATTG GCAAGACATT GTACAGGATG 
               
               
                   
               
               
                 1561 
                 CAAAAAGTTA ACAACAACAT CTATCTTGAA CTTGGCAAAT 
               
               
                   
               
               
                 1601 
                 TAGATTACAA CAACTGTCAG GCATTGCATC AGCTTGAGTG 
               
               
                   
               
               
                 1641 
                 GAGAAGCATC CAAAAATGGT ACAATGAATG CGGTCTTGGA 
               
               
                   
               
               
                 1681 
                 GAGTACGGAT TAAGCGAGAG AAGCCTCCTT CTTTCGTATT 
               
               
                   
               
               
                 1721 
                 ATTTGGCCGC AGCCAGTATA TTTGAACCGG AGAGGTCAAA 
               
               
                   
               
               
                 1761 
                 GGAACGGCTT GCCTGGGCCA AAACTACTAT GCTAATCCGC 
               
               
                   
               
               
                 1801 
                 ACAATTGAAT CTTATTTGAG TAGTGAACAA ATGGTTGAGG 
               
               
                   
               
               
                 1841 
                 ATCACAATGG AGCCTTTGTT AGCGAGTTCC AATACTATTG 
               
               
                   
               
               
                 1881 
                 CAGTAACCTT GACTACGTAA ATGGTGGAAG GCATAAGCCA 
               
               
                   
               
               
                 1921 
                 ACACAAAGGC TAGTGAGGAC TCTACTCGGA ACTTTAAATC 
               
               
                   
               
               
                 1961 
                 AGATTTCTTT GGACGCAGTG TTAGTCCACG GCAGAGATAT 
               
               
                   
               
               
                 2001 
                 CCATCAATAT TTGCGTCAAG CCTGGGAAAA GTGGTTGATA 
               
               
                   
               
               
                 2041 
                 GCTTTGCAAG AGGGAGATGA TAGTGACATG GGTCAAGAGG 
               
               
                   
               
               
                 2081 
                 AAGCAGAACT TTTAGTGCGC ACACTAAACC TATGCGCCGG 
               
               
                   
               
               
                 2121 
                 TCGCTACGCA TCGGAGGAGC TATTGTTGTC CCATCCCAAG 
               
               
                   
               
               
                 2161 
                 TATCAACAAC TTTTGCACAT CACTACTAGA GTCTGTAACC 
               
               
                   
               
               
                 2201 
                 AAATTCGTCA TTTCCAACAC AAAAAGGTGC AAGATGGGGA 
               
               
                   
               
               
                 2241 
                 AAATGGAAGA GCAAACATGG GTGATGGCAT CACAAGCATC 
               
               
                   
               
               
                 2281 
                 AGCTCAATAG AGTCGGACAT GCAAGAACTA ACGAAATTAG 
               
               
                   
               
               
                 2321 
                 TTGTCGGCAA TACCCAAAAC GATCTAGATG CTGATACGAA 
               
               
                   
               
               
                 2361 
                 GCAAACATTT CTCACGGTGG CAAAAAGCTT CTACTACACC 
               
               
                   
               
               
                 2401 
                 GCCCACTGCA ATCCCGGAAC AATCAATTGC CATATTGCTA 
               
               
                   
               
               
                 2441 
                 AAGTATTATT TGAGAGAGTA CTTTGA 
               
            
           
         
       
     
     A  Chiococca alba  (5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) synthase (CaTPS2) was identified and isolated. This CaTPS2 enzyme was identified as an 5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) synthase, which converts GGPP to 5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP, [7]). 
     
       
         
         
             
             
         
       
     
     The  Chiococca alba  (5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) synthase (CaTPS2) has the amino acid sequence shown below (SEQ ID NO:43). 
     
       
         
           
               
               
            
               
                 1 
                 MPVIKSHEFI EEVGPEKGTL KLSRSSRINE LVESIQTMLQ 
               
               
                   
               
               
                 41 
                 SMDDGEISMS AYDTAWVALV EDINGSSYPQ FPMSLEWIAN 
               
               
                   
               
               
                 81 
                 NQLPDGSWGD GSIFSVHDRI ISTLGCVLAL KSWNMHPDKS 
               
               
                   
               
               
                 121 
                 EKGLLFIRDN IHKVGDESAE HMPIGFEVVF PSLIERAKNL 
               
               
                   
               
               
                 161 
                 DIDIPDISAI LQEIYARRNL KLARIPKDIL YTVPTTLLHS 
               
               
                   
               
               
                 201 
                 LEGMPELDWQ KLLPLKCEDG SFLFSPSCTA FALMQTKDGD 
               
               
                   
               
               
                 241 
                 CLRYLTNTIE KFNGGVPGVY PVDLFEHIWA VDRLQRLGIS 
               
               
                   
               
               
                 281 
                 RYFQTEIEEC MSYVYRYWTD KGICWARNSK VEDIDDTAMG 
               
               
                   
               
               
                 321 
                 FRLLRLHGYM VSADVFAQFE KGGEFVCFAG QSNQALTGMF 
               
               
                   
               
               
                 361 
                 NLYRASQVMF PGEKILADAK KFSSNFLHEK RANNELLDKW 
               
               
                   
               
               
                 401 
                 IITKDLPGEV TYALDVPWYA SLPRVETRLY LEQYGGEDDV 
               
               
                   
               
               
                 441 
                 WIAKTLYRMR KVNNKIYLEL GILDYNNCQA LHQLEWRSIQ 
               
               
                   
               
               
                 481 
                 KWYKDSGLEE YGLSERNLLL AYYLATACIF EPERLVERLS 
               
               
                   
               
               
                 521 
                 WAKTTALIYT TKSYFRTECN SGEQRKAFLH EFQQYCNDLD 
               
               
                   
               
               
                 561 
                 YVSGARHKPT IRLIEALLGT LEQVSLDAIL DHGRYIHQDL 
               
               
                   
               
               
                 601 
                 RNAWEKWLIA LQEGVDMDQE EAELTVLTLH LCAGSYTSEE 
               
               
                   
               
               
                 641 
                 LLLSHPKYQQ LLNITSRVCH QIRQFQREKA QDTDNGRENL 
               
               
                   
               
               
                 681 
                 VAITSIKAIE SDMQELAKLV LTKSTGDLAA KIKQTFLIVA 
               
               
                   
               
               
                 721 
                 KSFYYTAHCL PGIISTHIAK VLFEKVF 
               
            
           
         
       
     
     A nucleic acid encoding the  Chiococca alba  (5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) synthase (CaTPS2) with SEQ ID NO:43 is shown below as SEQ ID NO:44. 
     
       
         
           
               
               
            
               
                 1 
                 ATGCCAGTAA TAAAGTCGCA TGAGTTTATT GAAGAGGTCG 
               
               
                   
               
               
                 41 
                 GCCCGGAAAA AGGAACTCTG AAGCTGAGCA GATCAAGTAG 
               
               
                   
               
               
                 81 
                 GATAAACGAA CTTGTAGAAT CAATTCAAAC GATGCTTCAA 
               
               
                   
               
               
                 121 
                 TCGATGGATG ATGGGGAAAT AAGCATGTCT GCTTATGACA 
               
               
                   
               
               
                 161 
                 CCGCGTGGGT TGCCCTTGTG GAAGATATTA ATGGAAGCAG 
               
               
                   
               
               
                 201 
                 CTACCCTCAA TTCCCTATGA GCCTCGAGTG GATTGCCAAC 
               
               
                   
               
               
                 241 
                 AATCAGCTTC CTGATGGTTC ATGGGGTGAC GGCAGTATCT 
               
               
                   
               
               
                 281 
                 TTTCGGTTCA TGATCGGATA ATCAGCACAT TAGGATGTGT 
               
               
                   
               
               
                 321 
                 TCTTGCATTA AAATCATGGA ACATGCACCC GGACAAAAGC 
               
               
                   
               
               
                 361 
                 GAAAAAGGAC TGTTATTTAT AAGGGACAAT ATTCACAAGG 
               
               
                   
               
               
                 401 
                 TTGGAGATGA GAGCGCTGAG CACATGCCTA TTGGTTTTGA 
               
               
                   
               
               
                 441 
                 GGTGGTATTT CCTTCGCTTA TTGAGAGAGC CAAAAACTTG 
               
               
                   
               
               
                 481 
                 GACATTGATA TTCCAGATAT TTCTGCTATC TTGCAAGAGA 
               
               
                   
               
               
                 521 
                 TTTATGCACG AAGAAATCTA AAGCTCGCAA GGATTCCAAA 
               
               
                   
               
               
                 561 
                 GGATATACTG TATACCGTGC CCACGACATT ACTTCATAGC 
               
               
                   
               
               
                 601 
                 TTAGAAGGAA TGCCAGAACT GGACTGGCAA AAGCTACTGC 
               
               
                   
               
               
                 641 
                 CATTAAAATG TGAGGATGGT TCATTTCTAT TTTCTCCATC 
               
               
                   
               
               
                 681 
                 GTGCACTGCT TTTGCCCTCA TGCAGACTAA GGATGGTGAT 
               
               
                   
               
               
                 721 
                 TGCCTCAGAT ATCTAACTAA TACCATAGAA AAATTCAATG 
               
               
                   
               
               
                 761 
                 GGGGAGTTCC CGGTGTATAC CCTGTGGACT TGTTCGAACA 
               
               
                   
               
               
                 801 
                 CATTTGGGCT GTTGATCGCT TGCAAAGACT AGGAATTTCC 
               
               
                   
               
               
                 841 
                 CGGTATTTTC AGACAGAAAT TGAAGAATGT ATGAGTTATG 
               
               
                   
               
               
                 881 
                 TTTACCGATA TTGGACGGAT AAAGGTATCT GTTGGGCTAG 
               
               
                   
               
               
                 921 
                 AAACTCCAAA GTTGAAGACA TCGATGACAC AGCCATGGGT 
               
               
                   
               
               
                 961 
                 TTTAGACTTC TAAGGTTGCA TGGTTACATG GTTTCTGCAG 
               
               
                   
               
               
                 1001 
                 ATGTGTTTGC ACAGTTTGAG AAAGGGGGTG AATTCGTTTG 
               
               
                   
               
               
                 1041 
                 CTTTGCTGGA CAGTCGAACC AGGCGCTGAC TGGAATGTTT 
               
               
                   
               
               
                 1081 
                 AACCTGTATA GAGCTTCTCA AGTAATGTTT CCAGGGGAGA 
               
               
                   
               
               
                 1121 
                 AGATACTTGC TGATGCCAAG AAATTCTCAT CGAACTTCTT 
               
               
                   
               
               
                 1161 
                 ACATGAAAAG CGTGCAAACA ACGAGCTTCT AGATAAATGG 
               
               
                   
               
               
                 1201 
                 ATCATAACTA AAGATTTGCC TGGAGAGGTG ACGTATGCGC 
               
               
                   
               
               
                 1241 
                 TAGATGTTCC ATGGTACGCC AGTTTACCTC GTGTAGAAAC 
               
               
                   
               
               
                 1281 
                 GAGATTATAT CTGGAACAAT ATGGAGGAGA GGATGATGTC 
               
               
                   
               
               
                 1321 
                 TGGATTGCCA AGACATTGTA CAGGATGAGA AAAGTTAACA 
               
               
                   
               
               
                 1361 
                 ACAAAATTTA CCTTGAACTT GGCATATTAG ATTACAATAA 
               
               
                   
               
               
                 1401 
                 CTGTCAAGCA TTGCATCAGC TGGAGTGGAG AAGCATCCAA 
               
               
                   
               
               
                 1441 
                 AAATGGTATA AGGATTCTGG CCTTGAAGAG TACGGGTTGA 
               
               
                   
               
               
                 1481 
                 GCGAGAGGAA CCTTCTCCTG GCATATTATC TGGCCACAGC 
               
               
                   
               
               
                 1521 
                 TTGTATATTT GAACCCGAAA GGTTGGTGGA GCGCCTTTCC 
               
               
                   
               
               
                 1561 
                 TGGGCGAAAA CAACCGCCTT AATCTACACA ACAAAATCTT 
               
               
                   
               
               
                 1601 
                 ATTTCAGAAC TGAATGCAAC TCTGGGGAAC AGAGAAAAGC 
               
               
                   
               
               
                 1641 
                 TTTTCTTCAT GAGTTCCAAC AGTACTGCAA TGACCTGGAC 
               
               
                   
               
               
                 1681 
                 TACGTTAGTG GCGCAAGGCA CAAGCCAACA ATAAGATTGA 
               
               
                   
               
               
                 1721 
                 TCGAAGCTCT ACTTGGAACC CTAGAGCAGG TCTCTTTGGA 
               
               
                   
               
               
                 1761 
                 TGCAATATTA GATCATGGCC GATATATCCA TCAAGATTTG 
               
               
                   
               
               
                 1801 
                 CGTAATGCTT GGGAGAAATG GTTGATAGCT TTGCAAGAGG 
               
               
                   
               
               
                 1841 
                 GAGTTGACAT GGACCAAGAA GAAGCAGAAC TTACAGTGCT 
               
               
                   
               
               
                 1881 
                 CACACTACAC CTGTGTGCCG GCAGCTACAC ATCGGAGGAG 
               
               
                   
               
               
                 1921 
                 TTACTGTTAT CTCATCCCAA GTATCAACAA CTTTTAAATA 
               
               
                   
               
               
                 1961 
                 TCACTAGTAG AGTCTGCCAC CAAATTCGTC AATTCCAGCG 
               
               
                   
               
               
                 2001 
                 CGAAAAGGCA CAGGATACGG ATAATGGAAG AGAAAACTTG 
               
               
                   
               
               
                 2041 
                 GTTGCCATCA CAAGCATCAA GGCGATAGAA TCAGACATGC 
               
               
                   
               
               
                 2081 
                 AAGAACTTGC GAAATTAGTT CTGACCAAAT CCACTGGCGA 
               
               
                   
               
               
                 2121 
                 TTTAGCTGCT AAAATCAAGC AAACATTTCT TATAGTGGCA 
               
               
                   
               
               
                 2161 
                 AAGAGCTTCT ACTACACCGC ACATTGCCTT CCTGGAATTA 
               
               
                   
               
               
                 2201 
                 TCAGTACCCA CATTGCCAAA GTACTATTTG AGAAAGTTTT 
               
               
                   
               
               
                 2241 
                 CTGA 
               
            
           
         
       
     
     A  Chiococca alba  CaTPS3 and CaTPS4 were identified and isolated. CaTPS3 and CaTPS4 were identified as an ent-kaurene synthase, converting ent-CPP [16] into ent-kaurene [19]. 
     
       
         
         
             
             
         
       
     
     The  Chiococca alba  ent-kaurene synthase (CaTPS3) has the amino acid sequence shown below (SEQ ID NO:45). 
                        1   MMMMMVVMNT APAHSYHPFP FAGPKSSATL FSNYYCSSRK               41   KSSPPRISAS VSLLTGVEST TAINSSDPEI KERIRKLFHD               81   VDISLSSYDT AWVAMVPAPH SSQSPLFPQC INWLLDNQLP               121   DGSWSLPPPH HHPLLLKDAL SSTLACVLAL RRWGIGQEQV               161   DKGIRFVELN FASASDQNQH LPVGFDIIFP GMLEYARDLN               201   LNLQLESATV NALLLKRDQE LTRFFKSYSD ESKAYLAYVS               241   EGIVKLQNWD TVMKFQRKNG SLFNSPSATA AAVMHVHNPG               281   CLDYLHSVLE KHGNAVPTVY PLDIYPRLCL VDNLERLGIC               321   GHFRKEILSV LDDTYRCWMQ GDEEIFAEKS TCAIAFTLLR               361   KHGYNISADP LTPFLKEECF SNSLGGCLKD TSAVLELYRA               401   LEMIISQNES ALVKKSLWSR SFLKEHISGG CDLKGFSNQI               441   SILVDDILNF PSHATLQRVA NRRSIEQYNL DSTKILKTSY               481   CSSNFSNKDL LILAVKDFNH CQLIHREELK ELERWVTDNR               521   LDKLKFARQK SAYCYFSAAA TIFSPELSDA RMSWAKNGVL               561   ATLVDDFFDV GGSLEELKKL IELVEKWDIN VSDGCCSEPV               601   QILFSALHST IQEIGDKAFK WQARSVTNHI FKIWLDLLNS               641   MLREAEWARN ATVPTVEEYM TNGYVSFALG PIILPALYLV               681   GPKLSEEVVK DSEFHSLFKL VSTCGRLLND VHSFERESKS               721   GQLNALSLRL IHGGVGITEA AAVAEMKSSI ENLRRELLRL               761   VLRKEGSVVP RACKDLFWNM SKVLHQFYNK DDGFTSEEMI               801   QLVKSIIYEP IAVNEFLNSC HT            
A nucleic acid encoding the  Chiococca alba  ent-kaurene synthase (CaTPS3) with SEQ ID NO:45 is shown below as SEQ ID NO:46.
 
     
       
         
           
               
               
            
               
                 1 
                 ATGATGATGA TGATGGTGGT GATGAACACA GCTCCCGCCC 
               
               
                   
               
               
                 41 
                 ACTCTTACCA TCCTTTCCCC TTTGCCGGCC CAAAATCCTC 
               
               
                   
               
               
                 81 
                 AGCCACACTT TTTTCCAATT ATTATTGTTC CAGTAGGAAG 
               
               
                   
               
               
                 121 
                 AAATCATCGC CACCTCGCAT CTCTGCCTCA GTTTCTTTGC 
               
               
                   
               
               
                 241 
                 TAACTGGAGT TGAAAGCACA ACTGCAATTA ATTCTTCAGA 
               
               
                   
               
               
                 281 
                 CCCGGAGATC AAAGAAAGAA TAAGGAAACT ATTTCATGAT 
               
               
                   
               
               
                 321 
                 GTTGATATCT CGCTTTCTTC ATATGACACT GCATGGGTGG 
               
               
                   
               
               
                 361 
                 CAATGGTCCC TGCTCCACAT TCTTCCCAGT CTCCCCTTTT 
               
               
                   
               
               
                 401 
                 TCCCCAGTGC ATTAATTGGT TATTGGACAA TCAGCTTCCT 
               
               
                   
               
               
                 441 
                 GATGGCTCAT GGAGTCTTCC TCCTCCTCAT CATCATCCTC 
               
               
                   
               
               
                 481 
                 TATTACTTAA AGATGCATTA TCCTCTACCC TTGCATGTGT 
               
               
                   
               
               
                 521 
                 TCTTGCGCTC AGGAGATGGG GAATTGGTCA AGAACAAGTT 
               
               
                   
               
               
                 561 
                 GACAAGGGTA TTCGTTTTGT TGAGTTAAAT TTTGCTTCAG 
               
               
                   
               
               
                 601 
                 CATCTGACCA GAACCAGCAT TTGCCAGTTG GATTTGACAT 
               
               
                   
               
               
                 641 
                 TATATTCCCT GGCATGCTCG AATATGCTAG AGATTTAAAT 
               
               
                   
               
               
                 681 
                 TTAAATCTTC AACTAGAATC TGCAACAGTA AATGCCTTAC 
               
               
                   
               
               
                 721 
                 TTCTTAAAAG AGATCAGGAG CTTACAAGAT TCTTTAAAAG 
               
               
                   
               
               
                 761 
                 CTACTCAGAC GAGAGTAAAG CATACCTTGC ATATGTATCA 
               
               
                   
               
               
                 801 
                 GAAGGTATAG TAAAGTTACA GAACTGGGAT ACAGTTATGA 
               
               
                   
               
               
                 841 
                 AGTTCCAAAG AAAGAACGGG TCACTATTCA ATTCACCTTC 
               
               
                   
               
               
                 881 
                 AGCTACAGCA GCTGCTGTTA TGCATGTCCA CAATCCTGGT 
               
               
                   
               
               
                 921 
                 TGCCTCGATT ACCTTCACTC AGTGTTGGAG AAGCATGGAA 
               
               
                   
               
               
                 961 
                 ATGCTGTTCC AACAGTTTAC CCTTTGGATA TATATCCACG 
               
               
                   
               
               
                 1001 
                 CCTCTGCTTG GTTGACAACC TTGAGAGACT GGGTATTTGT 
               
               
                   
               
               
                 1041 
                 GGTCATTTTA GGAAGGAAAT TCTGAGTGTA TTGGATGATA 
               
               
                   
               
               
                 1081 
                 CATACAGATG CTGGATGCAG GGGGATGAAG AGATATTTGC 
               
               
                   
               
               
                 1121 
                 AGAAAAATCA ACTTGTGCCA TAGCATTTAC ATTATTGCGA 
               
               
                   
               
               
                 1161 
                 AAGCATGGGT ACAACATCTC TGCAGATCCA TTGACCCCAT 
               
               
                   
               
               
                 1201 
                 TCTTAAAGGA AGAGTGTTTT TCCAATTCTT TGGGTGGATG 
               
               
                   
               
               
                 1241 
                 TTTGAAAGAT ACTAGTGCTG TACTTGAATT ATACCGGGCA 
               
               
                   
               
               
                 1281 
                 TTAGAGATGA TTATTAGCCA GAATGAATCA GCTCTGGTGA 
               
               
                   
               
               
                 1321 
                 AAAAAAGCTT GTGGTCCAGA AGCTTCCTGA AAGAGCATAT 
               
               
                   
               
               
                 1361 
                 TTCTGGTGGT TGTGATTTAA AGGGATTCAG CAATCAAATT 
               
               
                   
               
               
                 1401 
                 TCCATACTGG TGGATGATAT CCTCAACTTT CCATCGCATG 
               
               
                   
               
               
                 1481 
                 CTACTTTGCA ACGGGTTGCT AACAGGAGAA GCATAGAGCA 
               
               
                   
               
               
                 1521 
                 ATACAACTTA GACAGTACAA AAATTTTAAA AACTTCATAT 
               
               
                   
               
               
                 1561 
                 TGCTCGTCGA ATTTTAGCAA CAAAGATTTA TTGATCCTGG 
               
               
                   
               
               
                 1601 
                 CAGTCAAAGA TTTTAATCAT TGCCAACTCA TACACCGTGA 
               
               
                   
               
               
                 1641 
                 AGAACTGAAA GAACTAGAAA GGTGGGTCAC AGACAATAGA 
               
               
                   
               
               
                 1681 
                 TTGGACAAGT TAAAGTTTGC TAGGCAGAAG TCTGCATACT 
               
               
                   
               
               
                 1721 
                 GTTACTTTTC TGCTGCAGCA ACCATATTCT CACCTGAACT 
               
               
                   
               
               
                 1761 
                 TTCTGATGCC CGCATGTCAT GGGCCAAGAA TGGTGTACTT 
               
               
                   
               
               
                 1801 
                 GCTACTTTGG TTGATGACTT CTTTGACGTG GGAGGTTCTC 
               
               
                   
               
               
                 1841 
                 TAGAGGAATT AAAGAAACTG ATTGAGTTGG TTGAAAAGTG 
               
               
                   
               
               
                 1881 
                 GGATATAAAT GTCAGTGATG GTTGTTGCTC TGAACCAGTG 
               
               
                   
               
               
                 1921 
                 CAAATCCTCT TCTCAGCACT ACATAGTACA ATCCAGGAGA 
               
               
                   
               
               
                 1961 
                 TTGGAGATAA AGCATTCAAA TGGCAAGCAC GCAGTGTAAC 
               
               
                   
               
               
                 2001 
                 AAACCACATA TTTAAGATAT GGTTAGATTT GCTTAATTCT 
               
               
                   
               
               
                 2041 
                 ATGTTGAGGG AAGCTGAGTG GGCTAGAAAT GCAACAGTGC 
               
               
                   
               
               
                 2081 
                 CTACAGTTGA AGAATATATG ACAAATGGTT ATGTATCATT 
               
               
                   
               
               
                 2121 
                 TGCTTTGGGG CCAATTATCC TCCCTGCTCT TTATCTTGTT 
               
               
                   
               
               
                 2161 
                 GGACCTAAGC TGTCAGAGGA AGTAGTTAAG GATTCTGAAT 
               
               
                   
               
               
                 2201 
                 TCCACTCCCT TTTTAAGCTA GTGAGTACCT GTGGGCGGCT 
               
               
                   
               
               
                 2241 
                 TCTGAATGAT GTCCACAGCT TCGAGAGGGA ATCAAAGTCC 
               
               
                   
               
               
                 2281 
                 GGCCAACTAA ATGCTCTGTC TCTGCGCCTG ATTCATGGTG 
               
               
                   
               
               
                 2321 
                 GTGTTGGCAT TACTGAAGCA GCTGCTGTTG CAGAGATGAA 
               
               
                   
               
               
                 2361 
                 GAGTTCAATT GAGAATCTAA GGAGAGAACT GCTGAGACTA 
               
               
                   
               
               
                 2401 
                 GTCTTGCGCA AAGAGGGTAG TGTAGTTCCA AGAGCTTGCA 
               
               
                   
               
               
                 2441 
                 AGGATTTGTT TTGGAATATG AGTAAAGTGC TACATCAATT 
               
               
                   
               
               
                 2481 
                 TTACAACAAA GATGATGGAT TTACTTCAGA GGAGATGATT 
               
               
                   
               
               
                 2521 
                 CAGCTTGTGA AGTCGATCAT TTATGAGCCA ATTGCGGTCA 
               
               
                   
               
               
                 2561 
                 ATGAATTTTT GAATAGTTGC CATACATGA 
               
            
           
         
       
     
     The  Chiococca alba  ent-kaurene synthase (CaTPS4) has the amino acid sequence shown below (SEQ ID NO:47). 
                        1   MMIMVMNTAP VHAYHALPIP TQKSSTTLFP NYNCSSRKKS               41   SPPRISAASV SLQTGVERTT AIHSSDLEIK ERIRKLFHDV               81   DISLSSYDTA WVAMVPAPHS SQSPLFPQCI NWLLDNQLPD               121   GSWSLPPHHH HHHPLLLKDA LSSTLACVLA LRRWGIGQEQ               161   VDKGIRFVEL NFASASDQNQ HLPVGFDIIF PGMLEYARDL               201   NLNLQLESAT VDALLLKRDQ ELIRFFKSYS DESKAYLAYV               241   SEGIIKLQNW DTVMKFQRKN GSLFNSPSAT AAAVMHVHNP               281   GCLDYLHSVL EKHGNAVPTV YPLDIYPRLC LVDNLERLGI               321   CGHFRKEILS VLDDTYRCWM QGDEEIFAEK STCAIAFTLL               361   RKHGYNISAD PLTPFLKEEC FSNSLGGCLK DTSAVLELYR               401   ALEMIISQNE SALVKKSLWS RSFLKEHISG GCDLKGFSNQ               441   ISKQVDDILN FPSHATLQRV ANRRSIEQYN LDSTKILKTS               481   YCSSNFSNKD LLILAVKDFN HCQLIHREEL KELERWVADN               521   RLDKLKFARQ KSAYCYFSAA ATIFSPELSD ARISWARNGV               561   LTTLVDDFFD VGGSLEELKK LIELVEKWDI NVSDGCCSEP               601   VQILFSALHS TIQEIGDKAF KWQARSVTNH IIKIWLDLLN               641   SMLREAEWAR NATVPTVEEY MTNGYVSFAL GPIILPALYL               681   VGPKLSEELV KDSEFHSLFK LVSTCGRLLN DVHSFERESK               721   AGQLNALSLR LIHGGVGITE AAAVAEMKSS IEKQRRELLR               761   LVLRKEGSVV PRACKDLFWN MSRVLHQFYV KDDGFTSEEM               801   IELVKSIIYE PIAVNEF            
A nucleic acid encoding the  Chiococca alba  ent-kaurene synthase (CaTPS4) with SEQ ID NO:47 is shown below as SEQ ID NO:48.
 
     
       
         
           
               
               
            
               
                 1 
                 ATGATGATAA TGGTGATGAA CACAGCTCCC GTCCACGCTT 
               
               
                   
               
               
                 41 
                 ACCACGCTTT ACCCATTCCC ACCCAAAAAT CCTCAACCAC 
               
               
                   
               
               
                 81 
                 ACTTTTTCCC AATTATAACT GTTCCAGTAG GAAGAAATCA 
               
               
                   
               
               
                 121 
                 TCGCCACCTC GCATCTCTGC CGCCTCAGTT TCTTTGCAAA 
               
               
                   
               
               
                 161 
                 CTGGAGTTGA AAGAACGACG GCAATTCATT CTTCAGACCT 
               
               
                   
               
               
                 201 
                 AGAGATCAAA GAAAGAATAA GGAAACTATT TCATGATGTT 
               
               
                   
               
               
                 241 
                 GATATCTCGC TTTCTTCATA TGACACTGCA TGGGTGGCAA 
               
               
                   
               
               
                 281 
                 TGGTCCCTGC TCCACATTCT TCCCAGTCTC CCCTTTTTCC 
               
               
                   
               
               
                 321 
                 CCAGTGCATT AATTGGTTAT TGGACAATCA GCTTCCTGAT 
               
               
                   
               
               
                 361 
                 GGCTCATGGA GTCTTCCTCC TCATCATCAT CATCATCATC 
               
               
                   
               
               
                 401 
                 CCCTATTACT TAAAGATGCA TTATCCTCTA CGCTTGCATG 
               
               
                   
               
               
                 441 
                 TGTTCTTGCG CTCAGGAGAT GGGGAATTGG TCAAGAACAA 
               
               
                   
               
               
                 481 
                 GTTGACAAGG GTATTCGTTT TGTTGAGTTA AATTTTGCTT 
               
               
                   
               
               
                 521 
                 CTGCATCTGA CCAGAACCAG CATTTGCCAG TTGGATTTGA 
               
               
                   
               
               
                 561 
                 CATTATATTC CCTGGCATGC TCGAATATGC TAGAGATTTA 
               
               
                   
               
               
                 601 
                 AATTTAAATC TTCAACTAGA ATCCGCAACT GTAGATGCCT 
               
               
                   
               
               
                 641 
                 TACTTCTCAA AAGAGATCAG GAGCTTATAA GATTCTTTAA 
               
               
                   
               
               
                 681 
                 AAGCTACTCA GACGAGAGTA AAGCATACCT TGCATATGTA 
               
               
                   
               
               
                 721 
                 TCAGAAGGTA TCATAAAGTT ACAGAACTGG GATACAGTTA 
               
               
                   
               
               
                 761 
                 TGAAGTTCCA AAGAAAGAAC GGGTCACTGT TCAATTCACC 
               
               
                   
               
               
                 801 
                 TTCAGCTACA GCAGCTGCTG TTATGCATGT CCACAATCCT 
               
               
                   
               
               
                 841 
                 GGCTGCCTCG ATTACCTTCA CTCAGTGTTG GAGAAGCATG 
               
               
                   
               
               
                 881 
                 GCAATGCTGT TCCAACAGTT TACCCTTTGG ATATATATCC 
               
               
                   
               
               
                 921 
                 ACGCCTCTGC TTGGTTGACA ACCTTGAGAG ACTGGGTATT 
               
               
                   
               
               
                 961 
                 TGTGGTCATT TTAGGAAGGA AATTCTGAGT GTATTGGATG 
               
               
                   
               
               
                 1001 
                 ATACATACAG ATGCTGGATG CAGGGGGATG AAGAGATATT 
               
               
                   
               
               
                 1041 
                 TGCAGAAAAA TCAACTTGTG CCATAGCATT TACATTATTG 
               
               
                   
               
               
                 1081 
                 CGAAAGCATG GGTACAACAT CTCTGCAGAT CCATTGACCC 
               
               
                   
               
               
                 1121 
                 CATTCTTAAA GGAAGAGTGT TTTTCCAATT CTTTGGGTGG 
               
               
                   
               
               
                 1161 
                 ATGTTTGAAA GATACTAGTG CTGTACTTGA ATTATACCGG 
               
               
                   
               
               
                 1201 
                 GCATTAGAGA TGATTATTAG CCAGAATGAA TCAGCTCTGG 
               
               
                   
               
               
                 1241 
                 TGAAAAAAAG CTTGTGGTCC AGAAGCTTCC TGAAAGAGCA 
               
               
                   
               
               
                 1281 
                 TATTTCTGGT GGTTGTGATT TAAAGGGATT CAGCAATCAA 
               
               
                   
               
               
                 1321 
                 ATTTCCAAAC AGGTGGATGA TATCCTCAAC TTTCCATCGC 
               
               
                   
               
               
                 1361 
                 ATGCTACTTT GCAACGGGTT GCTAACAGGA GAAGCATAGA 
               
               
                   
               
               
                 1401 
                 GCAATACAAC TTAGACAGTA CAAAAATTTT AAAAACTTCA 
               
               
                   
               
               
                 1441 
                 TATTGCTCGT CGAATTTTAG TAACAAAGAT TTATTGATCC 
               
               
                   
               
               
                 1481 
                 TGGCAGTCAA AGATTTTAAT CATTGCCAAC TCATACACCG 
               
               
                   
               
               
                 1521 
                 TGAAGAACTG AAAGAACTAG AAAGGTGGGT CGCAGACAAT 
               
               
                   
               
               
                 1561 
                 AGATTGGACA AGTTAAAGTT TGCTAGGCAG AAGTCTGCAT 
               
               
                   
               
               
                 1601 
                 ACTGTTACTT TTCTGCTGCA GCAACCATAT TCTCACCTGA 
               
               
                   
               
               
                 1641 
                 ACTTTCTGAT GCCCGCATCT CATGGGCCAA AAATGGTGTA 
               
               
                   
               
               
                 1681 
                 CTTACTACTT TGGTTGATGA CTTCTTTGAC GTGGGAGGTT 
               
               
                   
               
               
                 1721 
                 CTCTAGAGGA ATTAAAGAAA CTGATTGAGT TGGTTGAAAA 
               
               
                   
               
               
                 1761 
                 GTGGGATATA AATGTCAGTG ATGGTTGTTG CTCTGAACCA 
               
               
                   
               
               
                 1801 
                 GTGCAAATCC TCTTCTCAGC ACTACATAGT ACAATCCAGG 
               
               
                   
               
               
                 1841 
                 AGATTGGAGA TAAAGCATTC AAATGGCAAG CACGCAGTGT 
               
               
                   
               
               
                 1881 
                 AACAAACCAC ATAATTAAGA TATGGTTAGA TTTGCTTAAT 
               
               
                   
               
               
                 1921 
                 TCTATGTTGA GGGAAGCTGA GTGGGCTAGA AATGCAACAG 
               
               
                   
               
               
                 1961 
                 TGCCTACAGT TGAAGAATAT ATGACAAATG GTTATGTATC 
               
               
                   
               
               
                 2001 
                 ATTTGCCTTG GGGCCAATTA TCCTCCCTGC TCTTTATCTT 
               
               
                   
               
               
                 2041 
                 GTTGGACCTA AGCTGTCAGA GGAATTAGTT AAGGATTCTG 
               
               
                   
               
               
                 2081 
                 AATTCCACTC CCTTTTTAAG CTAGTGAGTA CCTGTGGGCG 
               
               
                   
               
               
                 2121 
                 GCTTCTGAAT GATGTCCACA GCTTCGAGAG GGAATCAAAG 
               
               
                   
               
               
                 2161 
                 GCCGGCCAAC TAAATGCTCT TTCTCTGCGC CTGATTCATG 
               
               
                   
               
               
                 2201 
                 GTGGAGTTGG CATTACTGAA GCAGCTGCTG TTGCAGAGAT 
               
               
                   
               
               
                 2241 
                 GAAGAGTTCA ATTGAGAAGC AAAGGAGAGA ACTGCTGAGA 
               
               
                   
               
               
                 2281 
                 CTAGTCTTGC GCAAAGAGGG TAGTGTAGTT CCAAGAGCTT 
               
               
                   
               
               
                 2321 
                 GCAAGGATTT GTTTTGGAAT ATGAGTAGGG TGCTACATCA 
               
               
                   
               
               
                 2361 
                 ATTTTACGTC AAAGATGATG GATTTACTTC AGAGGAGATG 
               
               
                   
               
               
                 2401 
                 ATTGAGCTTG TGAAGTCGAT CATTTATGAG CCAATTGCGG 
               
               
                   
               
               
                 2441 
                 TCAATGAATT TTGA 
               
            
           
         
       
     
     A  Chiococca alba  13(R)-epi-dolabradiene synthase (CaTPS5) was identified and isolated. This CaTPS5 enzyme was identified as an 13(R)-epi-dolabradiene synthase, which converts ent-CPP [16] to 13(R)-epi-dolabradiene. 
     
       
         
         
             
             
         
       
     
     The  Chiococca alba  13(R)-epi-dolabradiene synthase (CaTPS5) has the amino acid sequence shown below (SEQ ID NO:49). 
     
       
         
           
               
               
            
               
                 1 
                 MIHTLPHGGQ AHFISHKTQP YYSSRPRFSS AASLDTRVRR 
               
               
                   
               
               
                 41 
                 TSPSNSSVLD FNETKERITK LFHNVDYSIS SYDTAWVAMV 
               
               
                   
               
               
                 81 
                 PDPHSSQAPL FPECINWLLD NQFHDGSWSL PHHNSLLLKD 
               
               
                   
               
               
                 121 
                 VLSSTLACVL ALKRWGIGGR QIDKGVRFIE MNFGSASDNC 
               
               
                   
               
               
                 161 
                 QHTPIGFDII FPGMLENARD LDLNLRLEPR IVTDMQRKRD 
               
               
                   
               
               
                 201 
                 MQLTRLHESD LKGDQAYLAY VSEGMQKLQN WDLAMKFQRK 
               
               
                   
               
               
                 241 
                 NGSLFNSPSA TAAAVMHVQN PASLNYLHSV VDKFGHAVPA 
               
               
                   
               
               
                 281 
                 VYPLDLYARL CLVDNLERLG ICRHFTNEIE IVMEDTYRCW 
               
               
                   
               
               
                 321 
                 LQDDEDIFAE ISTCALAFRL LRKHGYVVSP DPLTKIIEEE 
               
               
                   
               
               
                 401 
                 DVSNSSGNGY WNDIHAVMEV HRASEVVIHE NESDLKNQNT 
               
               
                   
               
               
                 441 
                 ISKHLLRHHL FNGSDVKPFP NPIYKQVDYA LKFPTPLILQ 
               
               
                   
               
               
                 481 
                 RVENKTLIQN YDVDSTRLLK TSYRSSNFCN EDLLRLAVKD 
               
               
                   
               
               
                 521 
                 FNDCQLLHRK ELKELERWSA DNRLHELKFA RQKAIYCSFS 
               
               
                   
               
               
                 561 
                 AAATIFIPEW YEARMSLAKN SVLATVVDDF FDVGGSMEEL 
               
               
                   
               
               
                 601 
                 KKLIEFVEKW DIDITKESCS EPLKIIFSAL HSTISEIGEQ 
               
               
                   
               
               
                 641 
                 AVKWQGRNVT SHIIEIWLDL LNSMLRESEW TTDVHMPTLD 
               
               
                   
               
               
                 681 
                 EYMEAAYVSF AMGPIIIPAL YFVGPKLSDE IVRDPEIRSL 
               
               
                   
               
               
                 721 
                 HKLVSICGRL LNDMQGFERE KKAGKPNAVS IRISQNGDGI 
               
               
                   
               
               
                 761 
                 TESAAFEEVK MELEDARREL LRLVVQKDGS VVPRACKDAF 
               
               
                   
               
               
                 801 
                 WSVSRMLHHF YFNNDGYTSE VEMVELVNSI IHEPLK 
               
            
           
         
       
     
     A nucleic acid encoding the  Chiococca alba  13(R)-epi-dolabradiene synthase (CaTPS5) with SEQ ID NO:49 is shown below as SEQ ID NO:50. 
     
       
         
           
               
               
            
               
                 1 
                 ATGATTCATA CTCTCCCTCA TGGCGGCCAG GCTCACTTCA 
               
               
                   
               
               
                 41 
                 TTTCCCACAA AACACAGCCT TATTATTCCA GTAGACCTCG 
               
               
                   
               
               
                 81 
                 CTTTTCTTCA GCAGCTTCTT TGGACACACG AGTCCGGAGA 
               
               
                   
               
               
                 121 
                 ACATCGCCCT CTAATTCCTC TGTCCTAGAC TTCAACGAGA 
               
               
                   
               
               
                 161 
                 CCAAAGAAAG AATCACAAAA TTATTTCATA ATGTTGATTA 
               
               
                   
               
               
                 201 
                 TTCAATTTCT TCATATGATA CAGCATGGGT TGCTATGGTC 
               
               
                   
               
               
                 241 
                 CCGGACCCAC ATTCTTCTCA GGCTCCCCTT TTCCCAGAGT 
               
               
                   
               
               
                 281 
                 GCATAAATTG GTTGCTAGAT AATCAATTTC ATGATGGCTC 
               
               
                   
               
               
                 321 
                 CTGGAGTCTT CCTCATCACA ATTCTCTATT GCTTAAGGAT 
               
               
                   
               
               
                 361 
                 GTTTTATCCT CTACGCTTGC GTGTGTTCTT GCTCTTAAGA 
               
               
                   
               
               
                 401 
                 GATGGGGAAT AGGAGGAAGG CAGATTGACA AAGGTGTTCG 
               
               
                   
               
               
                 441 
                 CTTTATTGAG ATGAATTTTG GCTCAGCATC TGACAATTGC 
               
               
                   
               
               
                 481 
                 CAGCATACTC CAATAGGATT TGACATAATA TTTCCAGGAA 
               
               
                   
               
               
                 521 
                 TGCTTGAAAA TGCCAGAGAT TTGGATCTAA ATCTTAGACT 
               
               
                   
               
               
                 561 
                 AGAACCCAGA ATTGTAACTG ACATGCAACG TAAAAGAGAC 
               
               
                   
               
               
                 601 
                 ATGCAGCTTA CAAGACTCCA TGAAAGCGAT CTAAAGGGGG 
               
               
                   
               
               
                 641 
                 ACCAAGCATA CTTGGCATAT GTATCCGAAG GGATGCAAAA 
               
               
                   
               
               
                 681 
                 GTTACAGAAT TGGGATTTGG CGATGAAGTT TCAAAGGAAG 
               
               
                   
               
               
                 721 
                 AATGGATCGC TCTTCAACTC ACCATCAGCT ACAGCAGCCG 
               
               
                   
               
               
                 801 
                 CTGTTATGCA TGTCCAAAAT CCTGCTTCCC TCAATTATCT 
               
               
                   
               
               
                 841 
                 TCATTCAGTC GTCGACAAAT TCGGCCATGC AGTTCCGGCT 
               
               
                   
               
               
                 881 
                 GTTTACCCTT TGGATCTCTA TGCGCGCCTT TGCTTGGTTG 
               
               
                   
               
               
                 921 
                 ACAATCTTGA GAGGCTGGGT ATCTGTCGAC ATTTTACTAA 
               
               
                   
               
               
                 961 
                 TGAAATTGAA ATTGTAATGG AGGACACGTA CAGGTGCTGG 
               
               
                   
               
               
                 1001 
                 CTGCAGGATG ATGAAGATAT ATTTGCCGAA ATATCAACTT 
               
               
                   
               
               
                 1041 
                 GTGCCTTAGC TTTTCGGTTA TTGAGAAAAC ATGGCTATGT 
               
               
                   
               
               
                 1081 
                 TGTCTCCCCA GATCCACTGA CAAAAATCAT AGAAGAAGAA 
               
               
                   
               
               
                 1121 
                 GATGTTTCCA ATTCTTCTGG TAATGGATAT TGGAATGATA 
               
               
                   
               
               
                 1161 
                 TACATGCTGT AATGGAAGTG CATCGGGCAT CAGAGGTGGT 
               
               
                   
               
               
                 1201 
                 TATACATGAA AATGAATCAG ATTTAAAGAA TCAAAATACC 
               
               
                   
               
               
                 1241 
                 ATATCAAAAC ACCTTCTCAG ACACCATCTT TTCAATGGTT 
               
               
                   
               
               
                 1281 
                 CTGATGTGAA GCCCTTTCCT AATCCAATAT ACAAGCAGGT 
               
               
                   
               
               
                 1321 
                 GGACTATGCT CTCAAGTTTC CAACCCCCTT AATTCTACAA 
               
               
                   
               
               
                 1361 
                 CGTGTTGAAA ACAAGACCCT CATACAGAAC TACGACGTAG 
               
               
                   
               
               
                 1401 
                 ACAGTACAAG ACTTCTTAAA ACTTCATATC GATCATCAAA 
               
               
                   
               
               
                 1441 
                 TTTCTGCAAT GAAGATTTAC TGAGGTTAGC AGTGAAAGAT 
               
               
                   
               
               
                 1481 
                 TTTAATGACT GTCAACTCCT GCACCGGAAA GAACTAAAAG 
               
               
                   
               
               
                 1521 
                 AACTAGAAAG ATGGTCCGCA GATAACAGAC TGCACGAACT 
               
               
                   
               
               
                 1601 
                 AAAATTTGCT CGGCAGAAAG CTATATACTG CTCCTTTTCT 
               
               
                   
               
               
                 1641 
                 GCTGCAGCAA CGATTTTCAT ACCTGAATGG TACGAAGCCC 
               
               
                   
               
               
                 1681 
                 GCATGTCATT GGCCAAAAAT AGTGTACTTG CTACTGTGGT 
               
               
                   
               
               
                 1721 
                 TGATGACTTC TTTGATGTGG GTGGTTCGAT GGAGGAATTA 
               
               
                   
               
               
                 1761 
                 AAGAAGCTAA TTGAATTTGT TGAAAAGTGG GATATTGACA 
               
               
                   
               
               
                 1801 
                 TCACCAAGGA ATCCTGCTCT GAGCCACTCA AAATCATATT 
               
               
                   
               
               
                 1841 
                 TTCAGCACTG CACAGTACAA TCTCTGAGAT TGGAGAGCAA 
               
               
                   
               
               
                 1881 
                 GCAGTTAAAT GGCAAGGACG CAATGTAACA AGCCACATAA 
               
               
                   
               
               
                 1921 
                 TTGAGATCTG GTTGGATTTG CTCAATTCGA TGTTGAGGGA 
               
               
                   
               
               
                 1961 
                 GTCTGAATGG ACTACAGATG TGCACATGCC AACATTGGAT 
               
               
                   
               
               
                 2001 
                 GAATATATGG AAGCTGCTTA TGTATCATTC GCCATGGGGC 
               
               
                   
               
               
                 2041 
                 CAATTATCAT CCCTGCTCTG TATTTTGTTG GGCCTAAGCT 
               
               
                   
               
               
                 2081 
                 ATCTGATGAA ATTGTTCGGG ATCCTGAAAT ACGATCCCTC 
               
               
                   
               
               
                 2121 
                 CATAAGCTTG TGAGCATTTG TGGGCGGCTT CTAAATGATA 
               
               
                   
               
               
                 2161 
                 TGCAAGGGTT CGAGAGGGAA AAGAAGGCTG GTAAACCAAA 
               
               
                   
               
               
                 2201 
                 TGCCGTGTCT ATACGCATTA GTCAAAATGG TGATGGCATT 
               
               
                   
               
               
                 2241 
                 ACCGAATCAG CAGCTTTCGA AGAAGTGAAG ATGGAATTAG 
               
               
                   
               
               
                 2281 
                 AGGATGCAAG GAGAGAATTG CTAAGATTAG TTGTGCAAAA 
               
               
                   
               
               
                 2321 
                 AGATGGTAGT GTAGTTCCAA GAGCTTGCAA GGATGCGTTT 
               
               
                   
               
               
                 2361 
                 TGGAGCGTAA GCAGAATGTT GCATCATTTC TACTTCAATA 
               
               
                   
               
               
                 2401 
                 ATGATGGATA CACGTCAGAG GTGGAGATGG TTGAGCTCGT 
               
               
                   
               
               
                 2441 
                 GAATTCAATT ATTCATGAAC CACTAAAATA A 
               
            
           
         
       
     
     A  Salvia hispanica  (−)-kolavenyl diphosphate synthase (ShTPS1) was identified and isolated. This ShTPS1 enzyme was identified as an (−)-kolavenyl 35 diphosphate synthase, which converts GGPP to (−)-kolavenyl diphosphate [36]. 
     
       
         
         
             
             
         
       
     
     The  Salvia hispanica  (−)-kolavenyl diphosphate synthase (ShTPS1) has, for example, an amino acid sequence shown below (SEQ ID NO:51). 
     
       
         
           
               
               
            
               
                 1 
                 MSIQANMSFA TSLHRSTTPG VGLPLKPCIS PSPSLSFSPN 
               
               
                   
               
               
                 41 
                 FGTFNNTSLR LKPEAGSKSY EGIRRSHQLA ASTILEGQTP 
               
               
                   
               
               
                 81 
                 ITPEVESEKT RLIERIRSML QDMDNDGQIS VSPYDTAWVA 
               
               
                   
               
               
                 121 
                 LVEDIGGSGG PQFPTSLEWI SNHQYDDGSW GDRKFVLYDR 
               
               
                   
               
               
                 161 
                 ILNTLACVVA LTNWKMHPNK CEKGLRFIHE NIKKLADEDE 
               
               
                   
               
               
                 201 
                 ELMPVGFEIA LPSVIDLAKR LGIEIPENSA SIKRIYELRD 
               
               
                   
               
               
                 241 
                 SKLKKIPMDL VHKRPTSLLF SLEGMEGLNW DKLMNFLAEG 
               
               
                   
               
               
                 281 
                 SFLSSPSSTA YALQHTKNEL CLEYLLKAVK RFNGGVPNAY 
               
               
                   
               
               
                 321 
                 PVDMFEHLWS VDRLQRLGIS RYFQAEIEEN MAYAYRYWTN 
               
               
                   
               
               
                 361 
                 KGITWARNMV VQDSDDSAQG FRLLRLYGYD IPIDVFKHFE 
               
               
                   
               
               
                 401 
                 QGGQFCSIPG QMTHAITGMY NLYRASELLF PGEHILSDAR 
               
               
                   
               
               
                 441 
                 KYTGNFLHQR RITNTWDKVV IITKDLHGEV AYALDVPFYA 
               
               
                   
               
               
                 481 
                 SLPRLEARFF IEQYGGDEDV WIGKTLYRMF KVNSDTYLEM 
               
               
                   
               
               
                 521 
                 AKLDYKQCQS VHQLEWNSMQ RLYRDCNLGE FGLSERSLLL 
               
               
                   
               
               
                 561 
                 AYYIAASTTF EPEKSSERLA WAITTILVEI IASQKLSDEQ 
               
               
                   
               
               
                 601 
                 KREFVDEFVK GSIVNNQNGG RHKPGNRLVE VLINNITLMA 
               
               
                   
               
               
                 641 
                 EGRGTYQQLS NAWKKWLKTW EEGGDLGEAE ARLLLHTIHL 
               
               
                   
               
               
                 681 
                 SSGLDDSSFS HPKYQQLLEA TSKVCHQLRV FQSVKVYDDQ 
               
               
                   
               
               
                 721 
                 ESTSQLVTRT TFQIEAGMQE LVKLVFTKTL EDLPSTTKQS 
               
               
                   
               
               
                 761 
                 FFSVARSFYY TACIHADTID SHINKVLFEK IV 
               
            
           
         
       
     
     A nucleic acid encoding the Salvia hispanica (−)-kolavenyl diphosphate synthase (ShTPS1) with SEQ ID NO:51 is shown below as SEQ ID NO:52. 
     
       
         
           
               
               
            
               
                 1 
                 ATGAGTATTC AAGCAAACAT GTCATTTGCC ACCTCCCTCC 
               
               
                   
               
               
                 41 
                 ACCGATCAAC CACCCCCGGA GTTGGCCTTC CGCTAAAACC 
               
               
                   
               
               
                 81 
                 ATGTATCTCT CCCTCTCCCT CTCTTTCCTT TTCCCCAAAC 
               
               
                   
               
               
                 121 
                 TTTGGCACTT TTAACAACAC AAGTTTGAGA CTCAAACCAG 
               
               
                   
               
               
                 161 
                 AGGCTGGGAG CAAAAGTTAT GAGGGGATTC GAAGAAGTCA 
               
               
                   
               
               
                 201 
                 TCAATTAGCA GCATCAACAA TTTTGGAGGG TCAAACTCCG 
               
               
                   
               
               
                 241 
                 ATTACTCCGG AGGTTGAATC GGAGAAAACA CGCCTGATTG 
               
               
                   
               
               
                 281 
                 AAAGGATTCG TTCGATGTTA CAAGACATGG ACAACGATGG 
               
               
                   
               
               
                 321 
                 CCAGATAAGT GTGTCACCAT ACGACACAGC ATGGGTGGCG 
               
               
                   
               
               
                 361 
                 CTCGTGGAAG ATATTGGTGG CAGCGGAGGG CCACAGTTTC 
               
               
                   
               
               
                 401 
                 CAACGAGCCT AGAGTGGATT TCTAACCACC AGTACGACGA 
               
               
                   
               
               
                 441 
                 TGGATCGTGG GGGGATCGCA AATTTGTTCT CTATGACCGG 
               
               
                   
               
               
                 481 
                 ATACTCAATA CATTAGCATG TGTTGTCGCA CTCACGAATT 
               
               
                   
               
               
                 521 
                 GGAAAATGCA TCCTAACAAA TGCGAAAAAG GGTTGAGGTT 
               
               
                   
               
               
                 561 
                 TATTCATGAG AATATTAAGA AACTCGCGGA TGAAGATGAA 
               
               
                   
               
               
                 601 
                 GAGCTCATGC CCGTAGGATT CGAAATCGCA CTGCCATCAG 
               
               
                   
               
               
                 641 
                 TCATTGATTT AGCTAAAAGA CTGGGTATAG AAATCCCAGA 
               
               
                   
               
               
                 681 
                 AAATTCTGCA AGCATAAAAA GAATTTATGA ATTGAGAGAT 
               
               
                   
               
               
                 721 
                 TCAAAACTTA AAAAAATACC AATGGATTTA GTGCACAAAA 
               
               
                   
               
               
                 761 
                 GGCCCACATC ACTACTCTTC AGCTTGGAAG GCATGGAAGG 
               
               
                   
               
               
                 801 
                 CCTTAACTGG GACAAACTAA TGAATTTTCT AGCCGAGGGT 
               
               
                   
               
               
                 841 
                 TCGTTTCTTT CATCGCCATC GTCCACTGCC TACGCTCTCC 
               
               
                   
               
               
                 881 
                 AACACACCAA GAATGAGTTA TGCCTAGAGT ATTTACTCAA 
               
               
                   
               
               
                 921 
                 GGCAGTCAAG AGATTCAATG GTGGAGTTCC AAATGCATAC 
               
               
                   
               
               
                 961 
                 CCTGTCGACA TGTTTGAGCA TCTGTGGTCC GTGGATCGCT 
               
               
                   
               
               
                 1001 
                 TACAGAGATT AGGAATTTCT CGGTATTTTC AAGCTGAAAT 
               
               
                   
               
               
                 1041 
                 TGAAGAAAAC ATGGCCTATG CTTACAGATA CTGGACAAAT 
               
               
                   
               
               
                 1081 
                 AAAGGAATCA CCTGGGCAAG AAATATGGTT GTCCAAGACA 
               
               
                   
               
               
                 1121 
                 GTGACGACAG CGCACAGGGA TTCAGGCTCT TAAGGTTGTA 
               
               
                   
               
               
                 1161 
                 CGGATACGAT ATTCCTATAG ATGTTTTCAA ACATTTCGAG 
               
               
                   
               
               
                 1201 
                 CAAGGTGGAC AATTCTGCAG CATACCAGGA CAGATGACAC 
               
               
                   
               
               
                 1241 
                 ACGCTATTAC AGGAATGTAC AACTTGTATA GAGCTTCTGA 
               
               
                   
               
               
                 1281 
                 ACTTCTGTTC CCTGGAGAAC ACATACTTTC TGATGCTAGA 
               
               
                   
               
               
                 1321 
                 AAATACACAG GTAACTTCTT GCATCAAAGA AGAATTACTA 
               
               
                   
               
               
                 1361 
                 ACACGGTAGT AGACAAGTGG ATCATTACCA AAGACCTTCA 
               
               
                   
               
               
                 1401 
                 CGGCGAGGTG GCTTATGCAT TGGATGTGCC ATTCTACGCC 
               
               
                   
               
               
                 1441 
                 AGTCTGCCAC GACTGGAAGC ACGATTCTTC ATAGAACAAT 
               
               
                   
               
               
                 1481 
                 ATGGGGGTGA TGAAGATGTT TGGATTGGGA AAACATTGTA 
               
               
                   
               
               
                 1521 
                 CAGGATGTTT AAAGTAAACT CCGACACATA CCTTGAGATG 
               
               
                   
               
               
                 1561 
                 GCAAAATTAG ATTACAAACA ATGCCAGTCT GTGCATCAGT 
               
               
                   
               
               
                 1601 
                 TAGAGTGGAA TAGCATGCAA AGATTGTATA GAGATTGCAA 
               
               
                   
               
               
                 1641 
                 TCTAGGAGAG TTTGGGTTGA GCGAAAGAAG CCTTCTCCTA 
               
               
                   
               
               
                 1681 
                 GCTTACTACA TAGCAGCCTC AACTACATTT GAGCCGGAAA 
               
               
                   
               
               
                 1721 
                 AATCAAGTGA AAGACTGGCT TGGGCTATAA CAACAATTTT 
               
               
                   
               
               
                 1761 
                 AGTCGAAATA ATCGCATCCC AAAAACTCTC TGATGAGCAA 
               
               
                   
               
               
                 1801 
                 AAGAGAGAGT TTGTTGATGA ATTTGTAAAA GGAAGCATCG 
               
               
                   
               
               
                 1841 
                 TCAATAACCA AAATGGAGGA AGACATAAAC CGGGAAACAG 
               
               
                   
               
               
                 1881 
                 ATTGGTTGAA GTTTTGATCA ACAATATAAC ACTGATGGCA 
               
               
                   
               
               
                 1921 
                 GAAGGCAGAG GCACATATCA GCAGTTGTCT AATGCGTGGA 
               
               
                   
               
               
                 1961 
                 AAAAATGGCT AAAGACATGG GAAGAGGGAG GTGACCTGGG 
               
               
                   
               
               
                 2001 
                 GGAAGCAGAA GCACGGCTTC TCCTGCACAC GATACATTTG 
               
               
                   
               
               
                 2041 
                 AGCTCCGGAT TGGATGATTC ATCATTTTCC CATCCAAAAT 
               
               
                   
               
               
                 2081 
                 ATCAGCAGCT CTTGGAGGCA ACCAGCAAAG TCTGCCACCA 
               
               
                   
               
               
                 2121 
                 ACTTCGCGTA TTCCAGAGTG TAAAGGTGTA TGATGACCAA 
               
               
                   
               
               
                 2161 
                 GAGTCTACAA GCCAACTGGT AACTAGGACA ACTTTCCAAA 
               
               
                   
               
               
                 2201 
                 TAGAAGCAGG CATGCAAGAA CTAGTGAAAT TAGTTTTCAC 
               
               
                   
               
               
                 2241 
                 AAAAACCTTG GAAGATTTGC CTTCTACTAC CAAGCAAAGC 
               
               
                   
               
               
                 2281 
                 TTTTTTAGTG TTGCTAGAAG TTTCTATTAC ACTGCCTGTA 
               
               
                   
               
               
                 2321 
                 TTCATGCAGA CACTATAGAC TCCCACATAA ACAAAGTATT 
               
               
                   
               
               
                 2361 
                 GTTTGAAAAA ATTGTCTAG 
               
            
           
         
       
     
     A  Teucrium canadense  cleroda-4(18),13E-dienyl diphosphate synthase (TcTPS1) was identified and isolated. This TcTPS1 enzyme was identified as a cleroda-4(18),13E-dienyl diphosphate synthase, which converts GGPP to cleroda-4(18),13E-dienyl diphosphate [38]. In addition, the combination of TcTPS1 and SsSS enzymes generated neo-cleroda-4(18),14-dien-13-ol [37]. These compounds are shown below. 
     
       
         
         
             
             
         
       
     
     5 The  Teucrium canadense  cleroda-4(18),13E-dienyl diphosphate synthase (TcTPS1) amino acid sequence is shown below as SEQ ID NO:53. 
     
       
         
           
               
               
            
               
                 1 
                 MSFASQATSL LLSSHNATAL PPLSAARLPP LTAGAAPFGR 
               
               
                   
               
               
                 41 
                 ISFTTTSLRQ YKLVSRAQSQ EVDEIEKVTQ WLEAEKDID 
               
               
                   
               
               
                 81 
                 QEAKVRELVE NVRVKLQNIG EGGISISPYD TAWVALVEDV 
               
               
                   
               
               
                 121 
                 GGSGRPQFPE SLDWISNHQF PDGSWGSHKF LYYDRVLCTL 
               
               
                   
               
               
                 161 
                 ACIVALKTWN LHPHKFDKGL KFVRENIGKL ADEEDVHMPI 
               
               
                   
               
               
                 201 
                 GFEVAFPSLI ETAKRKGIDI PEDFPGKKEI YAKRDLKLKK 
               
               
                   
               
               
                 241 
                 IPMDILHKIP TPLLFSIEGI EGLDWQKLFK FRDHGSFLTS 
               
               
                   
               
               
                 281 
                 PSSTAHALQQ TKDELCLKYL TNLVKKNNGG VPNAFPVDLF 
               
               
                   
               
               
                 321 
                 DRNYTVDRLR RLGILRYFQP EIEECMKYVY RFWDKRGISW 
               
               
                   
               
               
                 361 
                 ARNTHVQDLD DTVQGFRNLR MHGYDVTLDV FKQFERCGEF 
               
               
                   
               
               
                 401 
                 FSFHGQSSDA VLGMFNLYRA SQVLFPGEDM LADARKYAAN 
               
               
                   
               
               
                 441 
                 YLHKRRVSNR VVDKWIINKD LPGEVAYGLD VPFYASLPRL 
               
               
                   
               
               
                 481 
                 EARFYVEQYG GNDDVWIGKA LYRMLNVSCD TYLELAKLDY 
               
               
                   
               
               
                 521 
                 NICQAVHQKE WKSFQKWHRD GEFGLDEKSL LLAYYIAAST 
               
               
                   
               
               
                 561 
                 VFEPEKSLER LAWAKTAVLM EAILSQQLPS TKKHELVDEF 
               
               
                   
               
               
                 601 
                 KHASILNNQN GGSYKTRTPL VETLVNAISE LSTTILLEQD 
               
               
                   
               
               
                 641 
                 RDIHLQLSNA WLKWLSRWEA RGNLVEAEAE LLLQTLHLSN 
               
               
                   
               
               
                 681 
                 GLEESSFSHP KYQQLLQVTS KVCHLLRLFQ KRKVHDPEGC 
               
               
                   
               
               
                 721 
                 TTDIATGTTF QIEACMQQVV KLVFTKSSHD LDSWKQRFL 
               
               
                   
               
               
                 761 
                 DVARSFYYTA HCDPQVIQSH INKVLFEKW 
               
            
           
         
       
     
     A nucleic acid encoding the  Teucrium canadense  Cleroda-4(18),13E-dienyl diphosphate synthase (TcTPS1) has with SEQ ID NO:53 is shown below as SEQ ID NO:54. 
     
       
         
           
               
               
            
               
                 1 
                 ATGTCATTTG CTTCCCAAGC CACCTCCCTC CTCCTTTCTT 
               
               
                   
               
               
                 41 
                 CCCACAACGC CACCGCTCTT CCGCCTCTCT CTGCCGCCCG 
               
               
                   
               
               
                 81 
                 CCTTCCGCCT CTCACTGCCG GTGCTGCTCC ATTCGGAAGA 
               
               
                   
               
               
                 121 
                 ATATCATTTA CTACTACCTC TCTTCGGCAG TATAAACTGG 
               
               
                   
               
               
                 161 
                 TGTCAAGAGC TCAAAGCCAA GAGGTGGATG AGATTGAAAA 
               
               
                   
               
               
                 201 
                 AGTGACACAA GTGGTATTGG AGGCAGAAAA AGACATCGAT 
               
               
                   
               
               
                 241 
                 CAAGAGGCGA AGGTAAGGGA GCTGGTGGAA AATGTCCGAG 
               
               
                   
               
               
                 281 
                 TGAAGCTGCA AAATATCGGG GAAGGAGGGA TAAGCATATC 
               
               
                   
               
               
                 321 
                 GCCGTACGAC ACCGCATGGG TGGCGCTGGT GGAGGATGTC 
               
               
                   
               
               
                 361 
                 GGCGGCAGCG GCAGACCGCA GTTCCCGGAG AGCCTGGATT 
               
               
                   
               
               
                 401 
                 GGATATCAAA CCACCAGTTC CCGGACGGGT CGTGGGGCAG 
               
               
                   
               
               
                 441 
                 CCACAAATTC TTGTACTATG ACCGGGTTTT GTGCACGTTA 
               
               
                   
               
               
                 481 
                 GCATGTATAG TTGCATTGAA AACTTGGAAT CTGCATCCTC 
               
               
                   
               
               
                 521 
                 ACAAATTCGA CAAAGGGTTG AAATTCGTCA GAGAGAACAT 
               
               
                   
               
               
                 561 
                 TGGAAAGCTC GCGGATGAAG AAGACGTGCA CATGCCGATT 
               
               
                   
               
               
                 601 
                 GGGTTCGAAG TGGCATTCCC ATCACTTATA GAGACTGCAA 
               
               
                   
               
               
                 641 
                 AGAGAAAAGG AATTGACATC CCGGAAGATT TCCCTGGCAA 
               
               
                   
               
               
                 681 
                 GAAAGAAATC TATGCAAAAA GAGACCTAAA GCTGAAAAAG 
               
               
                   
               
               
                 721 
                 ATACCTATGG ATATACTGCA CAAAATCCCC ACACCATTAC 
               
               
                   
               
               
                 761 
                 TGTTCAGCAT AGAAGGGATA GAAGGCCTTG ATTGGCAGAA 
               
               
                   
               
               
                 801 
                 GCTATTCAAA TTCCGCGATC ACGGCTCCTT CCTCACGTCC 
               
               
                   
               
               
                 841 
                 CCGTCCTCAA CGGCCCACGC TCTCCAGCAA ACAAAGGACG 
               
               
                   
               
               
                 881 
                 AGTTATGCCT CAAATATCTG ACCAATCTTG TCAAAAAGAA 
               
               
                   
               
               
                 921 
                 CAATGGGGGA GTTCCAAATG CATTTCCGGT GGACCTATTT 
               
               
                   
               
               
                 961 
                 GATCGTAACT ATACAGTAGA TCGCCTGAGG AGGCTGGGAA 
               
               
                   
               
               
                 1001 
                 TTTTGCGCTA TTTTCAACCT GAAATCGAGG AATGCATGAA 
               
               
                   
               
               
                 1041 
                 ATATGTATAC AGATTCTGGG ATAAAAGAGG AATCAGCTGG 
               
               
                   
               
               
                 1081 
                 GCAAGAAATA CCCATGTTCA GGACCTTGAT GATACCGTAC 
               
               
                   
               
               
                 1121 
                 AGGGATTCAG GAACTTAAGG ATGCATGGTT ATGATGTCAC 
               
               
                   
               
               
                 1161 
                 CTTAGATGTT TTCAAACAGT TCGAGAGATG TGGAGAATTC 
               
               
                   
               
               
                 1201 
                 TTTAGCTTCC ACGGGCAATC AAGTGATGCT GTCTTAGGAA 
               
               
                   
               
               
                 1241 
                 TGTTCAACTT GTACCGAGCT TCTCAGGTTC TGTTTCCAGG 
               
               
                   
               
               
                 1281 
                 AGAAGACATG CTTGCAGATG CAAGGAAGTA CGCGGCCAAC 
               
               
                   
               
               
                 1321 
                 TATTTGCATA AAAGAAGAGT TAGTAATAGG GTCGTGGACA 
               
               
                   
               
               
                 1401 
                 AATGGATTAT TAACAAAGAT CTTCCAGGCG AGGTGGCGTA 
               
               
                   
               
               
                 1441 
                 TGGGCTAGAT GTTCCGTTCT ACGCCAGTCT ACCTCGACTG 
               
               
                   
               
               
                 1481 
                 GAAGCAAGAT TCTACGTCGA ACAATATGGG GGTAACGATG 
               
               
                   
               
               
                 1521 
                 ATGTCTGGAT TGGAAAAGCT TTATATAGAA TGTTGAATGT 
               
               
                   
               
               
                 1601 
                 GAGCTGTGAT ACTTACCTTG AGCTAGCAAA ATTAGACTAC 
               
               
                   
               
               
                 1641 
                 AATATTTGCC AGGCTGTGCA TCAGAAAGAG TGGAAAAGCT 
               
               
                   
               
               
                 1681 
                 TTCAAAAATG GCACAGGGAT GGGGAGTTTG GATTGGATGA 
               
               
                   
               
               
                 1721 
                 AAAAAGCTTA CTTTTAGCTT ACTACATAGC AGCCTCGACT 
               
               
                   
               
               
                 1761 
                 GTTTTCGAGC CTGAAAAATC TCTAGAGCGA CTGGCTTGGG 
               
               
                   
               
               
                 1801 
                 CTAAAACCGC AGTTCTAATG GAGGCAATTT TGTCCCAACA 
               
               
                   
               
               
                 1841 
                 ACTTCCTAGC ACAAAAAAAC ATGAGCTTGT TGACGAATTT 
               
               
                   
               
               
                 1881 
                 AAACATGCAA GCATCCTCAA CAACCAAAAT GGAGGAAGCT 
               
               
                   
               
               
                 1921 
                 ATAAAACAAG AACTCCTTTG GTAGAGACTC TAGTAAACGC 
               
               
                   
               
               
                 1961 
                 CATAAGTGAG CTCTCAACTA CCATACTATT GGAGCAAGAC 
               
               
                   
               
               
                 2001 
                 AGAGACATTC ATCTGCAATT ATCTAATGCG TGGCTGAAGT 
               
               
                   
               
               
                 2041 
                 GGCTAAGTAG ATGGGAGGCA AGAGGCAACC TAGTGGAAGC 
               
               
                   
               
               
                 2081 
                 AGAAGCAGAG CTTCTTCTGC AAACCTTACA TCTGAGCAAT 
               
               
                   
               
               
                 2121 
                 GGATTAGAAG AATCATCATT TTCTCATCCA AAATATCAAC 
               
               
                   
               
               
                 2161 
                 AACTCTTACA GGTTACCAGC AAAGTCTGTC ACCTACTTCG 
               
               
                   
               
               
                 2201 
                 GCTATTCCAG AAACGAAAGG TGCATGATCC GGAAGGGTGT 
               
               
                   
               
               
                 2241 
                 ACAACAGACA TTGCAACAGG GACAACTTTC CAAATAGAAG 
               
               
                   
               
               
                 2281 
                 CATGCATGCA ACAAGTAGTG AAATTAGTGT TCACCAAATC 
               
               
                   
               
               
                 2321 
                 CTCACATGAT TTAGATTCTG TTGTTAAGCA GAGATTTTTG 
               
               
                   
               
               
                 2361 
                 GATGTTGCCA GAAGTTTCTA TTACACAGCC CACTGTGATC 
               
               
                   
               
               
                 2401 
                 CACAAGTGAT CCAGTCCCAC ATTAATAAAG TGTTGTTTGA 
               
               
                   
               
               
                 2441 
                 AAAAGTAGTC TAG 
               
            
           
         
       
     
       Salvia officinalis  (SoTPS2),  Scutellaria baicalensis  SbTPS1, and SbTPS2 enzymes were identified and isolated. These SoTPS2, SbTPS1, SbTPS2, CfTPS18a and CfTPS18b enzymes were all identified as ent-CPP synthases, which convert GGPP to ent-CPP. 
     
       
         
         
             
             
         
       
     
     The  Salvia officinalis  (SoTPS2) enzyme can have the amino acid sequence shown below (SEQ ID NO:55). 
                        1   MSFASTTSLL RPSVTGFGVS PRVTSTSILS RSYGQILKGK               41   TKYITDNRRN RQLAVKFEGQ IALDLEDGVA KQTNQEAESE               81   KIRQLKGKIR WILQNMEDGE MSVSPYDTAW VALVEDISGG               121   GGPQFPTSLE WISKNQLADG SWGDPNYFLL YDRILNTLAC               161   VVALTTWNMH PHKCDQGLRF IRDNIEKLED EDEELILVGF               201   EIALPSLIDY AQNLGIQIQY DSPFIKKICA KRDLKLRKIP               241   MDLMHRKPTS LLYSLEGMEG LEWEKLMNLR SEGSFLSSPS               281   STAYALQHTK DELCLDYLVK AVNKFNGGVP NVYPVDMYEH               321   LWCVDRLQRL GISRYFQLEI QQCLDYVYRY WTNEGISWAR               361   YTNIRDSDDT AMGFRLLRLY GYDVSIDAFK PFEESGEFYS               401   MAGQMNHAVT GMYNLYRASQ LMFPQEHILS DARNFSAKFL               441   HQKRRTNALV DKWIITKDLP GEVGYALDVP FYASLPRLEA               481   RFFLEQYGGD DDVWIGKTLY RMPYVNSNTY LELAKVDYKN               521   CQSVHQLEWK SMQKWYRECN IGEFGLSERS LLLAYYIAAS               561   TTFEPEKSGE RLAWATTAIL IETIASQQLS DEQKREFVDE               601   FENSIIIKNQ NGGRYKARNR LVKVLINTVT LVAEGRGINQ               641   QLFNAWQKWL KTWEEGGDMG EAEAQLLLRT LHLSSGFDQS               681   SFSHPKYEQL LEATSKVCHQ LRLFQNRKVD DGQGCISRLV               721   IGTTSQIEAG MQEVVKLVFT KTSQDLTSAT KQSFFNIARS               761   FYYTAYFHAD TIDSHIYKVL FQTIV            
A nucleic acid encoding the  Salvia officinalis  (SoTPS2) has with SEQ ID NO:55 is shown below as SEQ ID NO:56.
 
     
       
         
           
               
               
            
               
                 1 
                 ATGTCATTTG CTTCCACCAC CTCCCTCCTC CGACCAAGCG 
               
               
                   
               
               
                 41 
                 TCACTGGGTT CGGTGTTTCT CCAAGGGTTA CTTCCACCTC 
               
               
                   
               
               
                 81 
                 CATTCTTAGC CGAAGTTATG GTCAAATATT AAAAGGAAAA 
               
               
                   
               
               
                 121 
                 ACAAAATACA TAACTGATAA CCGTAGAAAT CGACAATTGG 
               
               
                   
               
               
                 161 
                 CGGTAAAATT TGAGGGCCAA ATTGCTTTGG ATTTGGAGGA 
               
               
                   
               
               
                 201 
                 TGGCGTAGCA AAGCAGACGA ATCAAGAGGC GGAATCTGAG 
               
               
                   
               
               
                 241 
                 AAGATAAGGC AACTGAAGGG AAAGATCCGA TGGATTCTGC 
               
               
                   
               
               
                 281 
                 AAAACATGGA GGACGGCGAG ATGAGCGTGT CGCCGTACGA 
               
               
                   
               
               
                 321 
                 CACCGCATGG GTGGCGCTGG TGGAAGATAT CAGCGGCGGC 
               
               
                   
               
               
                 361 
                 GGCGGGCCGC AGTTCCCGAC GAGCCTCGAG TGGATTTCCA 
               
               
                   
               
               
                 401 
                 AGAATCAGTT GGCGGATGGG TCATGGGGGG ATCCTAATTA 
               
               
                   
               
               
                 441 
                 TTTCCTTCTC TACGACAGAA TACTCAATAC TTTAGCATGT 
               
               
                   
               
               
                 481 
                 GTAGTCGCAC TCACGACTTG GAATATGCAT CCTCACAAAT 
               
               
                   
               
               
                 521 
                 GCGATCAAGG GTTGAGGTTT ATAAGAGACA ACATTGAGAA 
               
               
                   
               
               
                 561 
                 ACTTGAGGAT GAAGATGAGG AGCTAATTCT CGTAGGATTC 
               
               
                   
               
               
                 601 
                 GAGATCGCAC TGCCTTCACT CATTGATTAT GCTCAAAACC 
               
               
                   
               
               
                 641 
                 TTGGGATACA AATCCAATAT GATTCTCCAT TCATTAAAAA 
               
               
                   
               
               
                 681 
                 AATTTGTGCA AAGAGAGATC TAAAACTCAG AAAAATACCA 
               
               
                   
               
               
                 721 
                 ATGGATTTAA TGCACAGAAA GCCAACATCA TTGCTCTACA 
               
               
                   
               
               
                 761 
                 GCTTGGAAGG CATGGAAGGC CTTGAGTGGG AAAAGCTAAT 
               
               
                   
               
               
                 801 
                 GAATTTGCGA TCGGAGGGTT CGTTTCTGTC ATCGCCGTCG 
               
               
                   
               
               
                 841 
                 TCCACGGCCT ACGCTCTCCA ACACACCAAG GATGAGTTAT 
               
               
                   
               
               
                 881 
                 GCCTTGACTA TCTGGTCAAG GCGGTCAACA AATTCAATGG 
               
               
                   
               
               
                 921 
                 TGGAGTTCCC AACGTGTACC CTGTCGACAT GTATGAGCAT 
               
               
                   
               
               
                 961 
                 CTATGGTGCG TAGACCGCTT GCAGAGGTTG GGAATTTCTC 
               
               
                   
               
               
                 1001 
                 GCTATTTTCA ACTTGAAATT CAACAATGCC TCGACTATGT 
               
               
                   
               
               
                 1041 
                 TTACAGATAC TGGACAAATG AAGGAATTTC GTGGGCAAGA 
               
               
                   
               
               
                 1081 
                 TATACTAATA TCCGGGATAG TGACGACACC GCAATGGGAT 
               
               
                   
               
               
                 1121 
                 TCAGGCTTCT AAGGTTGTAC GGCTATGATG TCTCTATAGA 
               
               
                   
               
               
                 1161 
                 TGCTTTTAAA CCATTCGAGG AAAGCGGAGA ATTCTATAGC 
               
               
                   
               
               
                 1201 
                 ATGGCAGGGC AGATGAACCA CGCTGTTACA GGAATGTACA 
               
               
                   
               
               
                 1241 
                 ACTTGTACAG AGCTTCTCAA CTTATGTTCC CTCAAGAACA 
               
               
                   
               
               
                 1281 
                 CATACTTTCC GATGCCAGAA ACTTCTCTGC CAAATTCTTG 
               
               
                   
               
               
                 1321 
                 CATCAAAAGA GGCGTACTAA TGCACTAGTA GACAAGTGGA 
               
               
                   
               
               
                 1361 
                 TCATTACCAA AGACCTTCCC GGCGAGGTTG GATATGCATT 
               
               
                   
               
               
                 1401 
                 GGATGTGCCG TTCTACGCCA GTCTGCCTCG ACTGGAAGCA 
               
               
                   
               
               
                 1441 
                 CGATTCTTCT TAGAACAATA TGGGGGTGAT GATGATGTTT 
               
               
                   
               
               
                 1481 
                 GGATTGGAAA AACTTTGTAC AGGATGCCAT ATGTGAACTC 
               
               
                   
               
               
                 1521 
                 CAACACATAC CTTGAGCTTG CAAAAGTAGA CTACAAAAAC 
               
               
                   
               
               
                 1561 
                 TGCCAGTCCG TGCATCAGTT GGAGTGGAAG AGCATGCAAA 
               
               
                   
               
               
                 1601 
                 AATGGTACAG AGAATGCAAT ATAGGTGAGT TTGGGTTGAG 
               
               
                   
               
               
                 1641 
                 CGAAAGAAGC CTTCTCCTAG CTTACTACAT AGCAGCCTCA 
               
               
                   
               
               
                 1681 
                 ACTACATTCG AGCCAGAAAA ATCAGGTGAG CGGCTCGCTT 
               
               
                   
               
               
                 1721 
                 GGGCTACAAC AGCAATTTTA ATCGAGACAA TCGCGTCCCA 
               
               
                   
               
               
                 1761 
                 ACAACTCTCC GATGAACAAA AGAGAGAGTT CGTTGATGAA 
               
               
                   
               
               
                 1801 
                 TTTGAAAACA GCATCATTAT CAAGAATCAA AATGGAGGGA 
               
               
                   
               
               
                 1841 
                 GATATAAAGC AAGAAACAGA TTGGTCAAGG TTTTGATCAA 
               
               
                   
               
               
                 1881 
                 CACTGTAACA CTGGTAGCAG AAGGCAGAGG CATAAATCAG 
               
               
                   
               
               
                 1921 
                 CAGTTGTTTA ATGCGTGGCA AAAATGGCTA AAGACATGGG 
               
               
                   
               
               
                 1961 
                 AAGAAGGAGG TGACATGGGG GAAGCAGAAG CCCAGCTTCT 
               
               
                   
               
               
                 2001 
                 TCTGCGCACG CTACATTTGA GCTCCGGATT CGATCAATCA 
               
               
                   
               
               
                 2041 
                 TCATTTTCCC ATCCAAAATA TGAGCAGCTC TTGGAGGCGA 
               
               
                   
               
               
                 2081 
                 CCAGCAAAGT TTGCCACCAA CTTCGCCTAT TCCAGAATCG 
               
               
                   
               
               
                 2121 
                 AAAGGTGGAT GATGGCCAAG GGTGTATAAG TCGATTGGTA 
               
               
                   
               
               
                 2161 
                 ATTGGGACAA CTTCCCAAAT AGAAGCAGGC ATGCAAGAAG 
               
               
                   
               
               
                 2201 
                 TAGTGAAATT AGTTTTCACC AAAACCTCAC AAGACTTGAC 
               
               
                   
               
               
                 2241 
                 TTCTGCTACC AAGCAAAGCT TTTTCAATAT TGCTAGAAGT 
               
               
                   
               
               
                 2281 
                 TTCTATTATA CTGCCTACTT TCATGCAGAC ACTATAGACT 
               
               
                   
               
               
                 2321 
                 CCCACATATA CAAAGTATTG TTTCAAACAA TAGTATAG 
               
            
           
         
       
     
     A  Scutellaria baicalensis  SbTPS1 amino acid sequence shown below (SEQ ID NO:57). 
     
       
         
           
               
               
            
               
                 1 
                 MPFLLPSSAT SSPAFYTPAA PLAGHHVFPS FKPLIISRSS 
               
               
                   
               
               
                 41 
                 LQCNAISRPR TQEYIDVIQN GLPVIKWHEA VEEDETDKDS 
               
               
                   
               
               
                 81 
                 LNKEATSDKI RELVNLIRSM LQSMGDGEIS SSPYDAAWVA 
               
               
                   
               
               
                 121 
                 LVPDVGGSGG PQFPSSLEWI SKNQLPDGSW GDTCTFSIYD 
               
               
                   
               
               
                 161 
                 RIINTLACVV ALKSWNIHPH KTYQGISFIK ANMDKLEDEN 
               
               
                   
               
               
                 201 
                 EEHMPIGFEV ALPSLIEIAK RLDIDISSDS RGLQEIYTRR 
               
               
                   
               
               
                 241 
                 EVKLKRIPKE IMHQVPTTLL HSLEGMAELT WHKLLKLQCQ 
               
               
                   
               
               
                 281 
                 DGSFLFSPSS TAFALHQTKD HNCLHYLTKY VHKFHGGVPN 
               
               
                   
               
               
                 321 
                 VYPVDLFEHL WAVDRIQRLG ISRHFKPQVD ECIAYVYRYW 
               
               
                   
               
               
                 361 
                 TDKGICWARN SVVQDLDDTA MGFRLLRLHG YDVSADVFKH 
               
               
                   
               
               
                 401 
                 FENGGEFFCF KGQSTQAVTG MYNLYRASQL MFPGESILED 
               
               
                   
               
               
                 441 
                 AKTFSSKFLQ RKRANNELLD KWIITKDLPG EVGYALDVPW 
               
               
                   
               
               
                 481 
                 YASLPRVETR FYLEQYGGED DVWIGKTLYR MPYVNNNKYL 
               
               
                   
               
               
                 521 
                 ELAKLDYSNC QSLHQQEWKN IQKWYESCNL GEFGLSERRV 
               
               
                   
               
               
                 561 
                 LLAYYVAAAC IYEPEKSNQR LAWAKTVILM ETITSYFEHQ 
               
               
                   
               
               
                 601 
                 QLSAEQRRAF VNEFEHGSIL KYANGGRYKR RSVLGTLLKT 
               
               
                   
               
               
                 641 
                 LNQLSLDILL THGRNVHQPF KNAWHKWLKT WEEGGDIEEG 
               
               
                   
               
               
                 681 
                 EAEVLVRTLN LSGEGRHDSY VLEQSLLSQP IYEQLLKATM 
               
               
                   
               
               
                 721 
                 SVCKKLRLFQ HRKDENGCMT KMRGITTLEI ESEMQELVKL 
               
               
                   
               
               
                 761 
                 VFTKSSDDLD CEIKQNFFTI ARSFYYVAYC NQGTINFHIA 
               
               
                   
               
               
                 801 
                 KVLFERVL 
               
            
           
         
       
     
     A nucleic acid encoding the  Scutellaria baicalensis  SbTPS1 with SEQ ID NO:57 is shown below as SEQ ID NO:58. 
     
       
         
           
               
               
            
               
                 1 
                 ATGCCTTTCC TCCTCCCTTC CTCCGCCACC AGCTCCCCCG 
               
               
                   
               
               
                 41 
                 CGTTCTATAC TCCGGCCGCG CCTCTCGCCG GTCATCATGT 
               
               
                   
               
               
                 81 
                 TTTTCCATCT TTCAAGCCAC TCATTATTTC CCGTTCTTCA 
               
               
                   
               
               
                 121 
                 CTCCAATGCA ATGCAATCTC TCGACCTCGT ACCCAAGAAT 
               
               
                   
               
               
                 161 
                 ACATAGATGT GATTCAGAAT GGATTGCCAG TAATAAAGTG 
               
               
                   
               
               
                 201 
                 GCACGAAGCT GTGGAAGAAG ATGAGACAGA TAAAGATTCT 
               
               
                   
               
               
                 241 
                 CTTAATAAGG AGGCCACGTC AGACAAGATA AGAGAGTTGG 
               
               
                   
               
               
                 281 
                 TAAATCTGAT CCGTTCGATG CTCCAATCAA TGGGCGACGG 
               
               
                   
               
               
                 521 
                 AGAGATAAGC TCGTCGCCGT ACGACGCCGC ATGGGTGGCG 
               
               
                   
               
               
                 561 
                 CTGGTGCCGG ACGTCGGCGG CTCCGGCGGG CCCCAGTTCC 
               
               
                   
               
               
                 601 
                 CCTCCAGCCT CGAATGGATA TCCAAAAACC AACTCCCCGA 
               
               
                   
               
               
                 641 
                 CGGCTCCTGG GGCGACACGT GTACCTTTTC CATTTATGAT 
               
               
                   
               
               
                 681 
                 CGAATCATCA ACACACTGGC TTGCGTTGTT GCTTTGAAAT 
               
               
                   
               
               
                 721 
                 CTTGGAACAT ACATCCCCAC AAAACTTATC AAGGGATTTC 
               
               
                   
               
               
                 761 
                 ATTCATAAAG GCAAATATGG ACAAACTTGA AGACGAGAAC 
               
               
                   
               
               
                 801 
                 GAGGAGCACA TGCCGATCGG ATTTGAAGTG GCACTCCCGT 
               
               
                   
               
               
                 841 
                 CGCTAATCGA GATAGCGAAA AGGCTCGATA TCGATATTTC 
               
               
                   
               
               
                 881 
                 CAGCGATTCG AGAGGGCTGC AAGAGATATA CACGAGGAGG 
               
               
                   
               
               
                 921 
                 GAGGTAAAGC TGAAAAGGAT ACCGAAAGAG ATAATGCACC 
               
               
                   
               
               
                 961 
                 AAGTGCCCAC AACACTGCTT CATAGCTTGG AGGGTATGGC 
               
               
                   
               
               
                 1041 
                 CGAGCTGACG TGGCACAAGC TTTTGAAATT ACAGTGCCAA 
               
               
                   
               
               
                 1081 
                 GATGGCTCCT TTCTTTTCTC TCCATCTTCA ACTGCCTTTG 
               
               
                   
               
               
                 1121 
                 CTCTTCACCA AACTAAGGAC CATAATTGTC TCCATTATTT 
               
               
                   
               
               
                 1161 
                 GACCAAATAT GTTCACAAAT TTCATGGTGG AGTGCCAAAT 
               
               
                   
               
               
                 1201 
                 GTGTATCCGG TGGACTTGTT CGAGCATCTA TGGGCAGTTG 
               
               
                   
               
               
                 1241 
                 ATCGGATCCA ACGGCTGGGG ATTTCCCGGC ATTTCAAGCC 
               
               
                   
               
               
                 1281 
                 CCAAGTTGAT GAATGTATTG CCTATGTTTA TAGATATTGG 
               
               
                   
               
               
                 1321 
                 ACAGATAAAG GAATATGCTG GGCAAGAAAT TCAGTAGTTC 
               
               
                   
               
               
                 1361 
                 AAGATCTTGA TGACACAGCC ATGGGATTCA GGCTTCTTAG 
               
               
                   
               
               
                 1401 
                 GTTGCATGGC TACGATGTTT CAGCAGATGT TTTCAAACAT 
               
               
                   
               
               
                 1441 
                 TTTGAAAATG GTGGAGAGTT CTTCTGCTTC AAAGGGCAAA 
               
               
                   
               
               
                 1481 
                 GCACGCAGGC AGTGACTGGA ATGTACAATC TGTACAGAGC 
               
               
                   
               
               
                 1521 
                 TTCTCAGTTG ATGTTTCCTG GAGAAAGCAT ACTGGAAGAT 
               
               
                   
               
               
                 1601 
                 GCTAAGACCT TCTCATCTAA GTTTTTGCAA CGAAAACGAG 
               
               
                   
               
               
                 1641 
                 CCAATAACGA GTTGTTAGAT AAGTGGATTA TTACCAAGGA 
               
               
                   
               
               
                 1681 
                 TCTTCCTGGA GAGGTGGGAT ATGCTCTAGA TGTACCATGG 
               
               
                   
               
               
                 1721 
                 TATGCTAGCT TACCTAGAGT TGAAACTAGA TTCTACTTGG 
               
               
                   
               
               
                 1801 
                 AACAATATGG TGGTGAAGAT GATGTTTGGA TTGGCAAAAC 
               
               
                   
               
               
                 1841 
                 TTTATACAGG ATGCCATATG TTAACAATAA TAAATATCTA 
               
               
                   
               
               
                 1881 
                 GAACTGGCAA AATTAGACTA TAGTAACTGC CAGTCATTAC 
               
               
                   
               
               
                 1921 
                 ATCAACAAGA GTGGAAAAAC ATTCAAAAAT GGTATGAGAG 
               
               
                   
               
               
                 1961 
                 TTGCAATCTG GGAGAATTTG GTTTGAGTGA AAGAAGGGTT 
               
               
                   
               
               
                 2001 
                 CTACTAGCCT ACTACGTAGC TGCTGCGTGT ATATATGAGC 
               
               
                   
               
               
                 2041 
                 CCGAAAAGTC AAACCAGCGC TTGGCTTGGG CCAAAACCGT 
               
               
                   
               
               
                 2081 
                 AATTTTAATG GAGACTATTA CTTCCTATTT TGAGCACCAA 
               
               
                   
               
               
                 2121 
                 CAACTCTCCG CAGAACAGAG ACGCGCCTTT GTTAATGAAT 
               
               
                   
               
               
                 2161 
                 TTGAACATGG GAGTATCCTC AAATATGCAA ATGGAGGAAG 
               
               
                   
               
               
                 2201 
                 ATACAAAAGG AGGAGTGTTT TGGGGACTTT GCTCAAAACA 
               
               
                   
               
               
                 2241 
                 CTAAATCAGC TTTCATTGGA TATATTATTG ACACACGGTC 
               
               
                   
               
               
                 2281 
                 GAAACGTCCA TCAGCCTTTC AAAAATGCGT GGCACAAGTG 
               
               
                   
               
               
                 2321 
                 GCTAAAAACG TGGGAAGAAG GAGGTGACAT TGAAGAAGGC 
               
               
                   
               
               
                 2361 
                 GAAGCAGAGG TATTGGTCCG AACCCTAAAC CTAAGCGGCG 
               
               
                   
               
               
                 2401 
                 AAGGGAGGCA CGACTCCTAT GTATTGGAGC AATCATTATT 
               
               
                   
               
               
                 2441 
                 GTCACAACCT ATATATGAAC AACTTTTGAA AGCCACCATG 
               
               
                   
               
               
                 2481 
                 AGTGTTTGCA AGAAGCTTCG ATTGTTCCAA CATCGAAAGG 
               
               
                   
               
               
                 2521 
                 ATGAGAATGG ATGTATGACG AAGATGAGAG GCATTACAAC 
               
               
                   
               
               
                 2561 
                 GTTAGAGATA GAATCGGAGA TGCAAGAATT AGTGAAATTA 
               
               
                   
               
               
                 2601 
                 GTATTTACTA AATCCTCAGA TGATTTAGAT TGTGAAATTA 
               
               
                   
               
               
                 2641 
                 AACAAAACTT TTTTACAATT GCTAGGAGTT TCTATTATGT 
               
               
                   
               
               
                 2681 
                 GGCTTATTGT AACCAAGGAA CTATCAACTT TCACATTGCT 
               
               
                   
               
               
                 2721 
                 AAGGTGCTCT TTGAAAGAGT TCTTTAG 
               
            
           
         
       
     
     A  Scutellaria baicalensis  SbTPS2 amino acid sequence is shown below (SEQ ID NO:59)
         1 MASLSTLSLN FSPAIHRKIQ QSSAKLQFQG HCFTISSCMN       

                        41   NSKRLSLNHQ SNHKRTSNVS ELQVATLDAP QIREKEDYST               81   AQGYEKVDEV EDPIEYIRML LNTTGDGRIS VSPYDTAWIA               121   LIKDVEGRDA PQFPSSLEWI ANNQLSDGSW GDEKFFCVYD               161   RLVNTLACVV ALRSWNIDAE KSEKGIRYIK ENVDKLKDGN               201   PEHMTCGFEV VFPSLLQRAQ SMGIHDLPYD APVIQDIYNT               241   RESKLKRIPM EVMHKVPTSL LFSLEGLENL EWDKLLKLQS               281   SDGSFLTSPS STAYAFMHTK DPKCFEFIKN TVETFNGGAP               321   HTYPVDVFGR LWAIDRLQRL GISRFFESEI ADCLDHIYKY               361   WTDKGVFSGR ESDFVDVDDT SMGVRLLRMH GYQVDPNVLR               401   NFKQGDKFSC YGGQMIESSS PIYNLYRASQ LRFPGEDILE               441   DANKFAYEFL QEQLSNNQLL DKWVISKHLP DEIKLGLQMP               481   WYATLPRVEA KYYLQYYAGA DDVWIGKTLY RMPEISNDTY               521   LELARMDFKR CQAQHQFEWI SMQEWYESCN IEEFGISRKE               561   LLQAYFLAGS SVFELERTTE RIGWAKSQII SRMIASFFNN               601   ETTTADEKDA LLTRFRNING PNKTKSGQRE SEAVNMLVAT               641   LQQYLAGFDR YTRHQLKDAW SVWFRKVQEE EAIYGAEAEL               681   LTTTLNICAG HIAFDENIMA NKDYTTLSSL TSKICQKLSE               721   IRNEKVEEME SGIKAKSSIK DKEVEHDMQS LVKLVLERCE               761   GINNRKLKQT FLSVAKTYYY RAYNADETMD IHMFKVLFEP               801   VM            
A nucleic acid encoding the  Scutellaria baicalensis  SbTPS2 with SEQ ID NO:59 is shown below as SEQ ID NO:60.
 
     
       
         
           
               
               
            
               
                 1 
                 ATGGCCTCTC TATCAACTCT GAGCCTCAAC TTTTCCCCAG 
               
               
                   
               
               
                 41 
                 CAATTCACCG CAAAATACAG CAATCATCTG CAAAACTTCA 
               
               
                   
               
               
                 81 
                 GTTCCAGGGA CATTGTTTCA CCATAAGTTC ATGCATGAAC 
               
               
                   
               
               
                 121 
                 AACAGTAAAA GACTGTCTTT GAACCACCAA TCTAATCACA 
               
               
                   
               
               
                 161 
                 AAAGAACGTC AAACGTATCT GAGCTGCAAG TTGCCACTTT 
               
               
                   
               
               
                 201 
                 GGATGCGCCC CAAATACGTG AAAAAGAAGA CTACTCCACT 
               
               
                   
               
               
                 241 
                 GCTCAAGGCT ATGAGAAGGT GGATGAAGTA GAGGATCCTA 
               
               
                   
               
               
                 281 
                 TCGAATATAT TAGAATGCTG TTGAACACAA CAGGTGATGG 
               
               
                   
               
               
                 321 
                 GCGAATAAGT GTGTCGCCAT ACGACACAGC CTGGATCGCT 
               
               
                   
               
               
                 361 
                 CTTATTAAAG ACGTGGAAGG ACGTGATGCT CCCCAGTTCC 
               
               
                   
               
               
                 401 
                 CATCTAGTCT CGAATGGATT GCCAATAATC AACTGAGTGA 
               
               
                   
               
               
                 441 
                 TGGGTCGTGG GGCGATGAGA AGTTTTTCTG TGTGTATGAT 
               
               
                   
               
               
                 481 
                 CGCCTTGTTA ATACACTTGC ATGTGTCGTG GCATTGAGAT 
               
               
                   
               
               
                 521 
                 CATGGAATAT TGATGCTGAA AAGAGCGAGA AAGGAATAAG 
               
               
                   
               
               
                 561 
                 ATACATAAAA GAAAACGTGG ATAAACTGAA AGATGGGAAT 
               
               
                   
               
               
                 601 
                 CCAGAGCACA TGACCTGTGG TTTTGAGGTG GTGTTTCCTT 
               
               
                   
               
               
                 641 
                 CCCTTCTTCA GAGAGCCCAA AGTATGGGAA TTCATGATCT 
               
               
                   
               
               
                 681 
                 TCCCTATGAT GCTCCTGTCA TCCAAGACAT TTACAATACC 
               
               
                   
               
               
                 721 
                 AGGGAGAGTA AATTGAAAAG GATTCCAATG GAGGTTATGC 
               
               
                   
               
               
                 761 
                 ACAAGGTGCC AACATCTCTA TTGTTCAGCT TGGAAGGATT 
               
               
                   
               
               
                 801 
                 GGAGAATTTG GAGTGGGATA AGCTCCTCAA ACTTCAGTCT 
               
               
                   
               
               
                 841 
                 TCTGATGGTT CATTCCTCAC TTCTCCATCC TCAACTGCCT 
               
               
                   
               
               
                 881 
                 ATGCTTTCAT GCACACTAAG GACCCTAAAT GCTTCGAATT 
               
               
                   
               
               
                 921 
                 CATCAAAAAC ACCGTCGAAA CATTTAATGG AGGAGCACCT 
               
               
                   
               
               
                 961 
                 CATACTTATC CGGTGGATGT TTTTGGAAGA CTGTGGGCCA 
               
               
                   
               
               
                 1001 
                 TTGACAGGCT GCAGCGCCTC GGAATCTCTC GCTTCTTTGA 
               
               
                   
               
               
                 1041 
                 GTCCGAGATT GCTGATTGCT TAGATCACAT CTATAAATAT 
               
               
                   
               
               
                 1081 
                 TGGACAGACA AAGGAGTGTT CAGTGGAAGA GAATCAGATT 
               
               
                   
               
               
                 1121 
                 TTGTGGATGT GGATGACACA TCCATGGGTG TTAGGCTTCT 
               
               
                   
               
               
                 1161 
                 AAGGATGCAC GGATATCAAG TTGATCCAAA TGTATTGAGG 
               
               
                   
               
               
                 1201 
                 AACTTCAAGC AGGGTGACAA ATTTTCATGC TATGGTGGTC 
               
               
                   
               
               
                 1241 
                 AAATGATAGA GTCATCATCT CCGATATACA ATCTCTATAG 
               
               
                   
               
               
                 1281 
                 GGCTTCTCAA CTCCGATTTC CAGGAGAAGA CATTCTTGAA 
               
               
                   
               
               
                 1321 
                 GATGCCAACA AATTCGCATA CGAGTTCTTG CAAGAACAGC 
               
               
                   
               
               
                 1361 
                 TATCCAACAA TCAACTTTTG GACAAATGGG TTATATCCAA 
               
               
                   
               
               
                 1401 
                 GCACTTGCCT GATGAGATAA AGCTTGGATT GCAGATGCCA 
               
               
                   
               
               
                 1441 
                 TGGTATGCCA CCCTACCCCG AGTGGAGGCT AAATACTACC 
               
               
                   
               
               
                 1481 
                 TACAGTATTA TGCTGGTGCT GATGATGTCT GGATCGGCAA 
               
               
                   
               
               
                 1521 
                 GACTCTCTAC AGAATGCCAG AAATCAGTAA TGATACATAT 
               
               
                   
               
               
                 1561 
                 CTGGAGTTAG CAAGAATGGA TTTCAAGAGA TGCCAAGCAC 
               
               
                   
               
               
                 1601 
                 AGCATCAATT TGAGTGGATT TCCATGCAAG AATGGTATGA 
               
               
                   
               
               
                 1641 
                 AAGTTGCAAC ATTGAAGAAT TTGGGATAAG CAGAAAAGAG 
               
               
                   
               
               
                 1681 
                 CTTCTTCAGG CTTACTTTTT GGCCTGCTCA AGTGTATTTG 
               
               
                   
               
               
                 1721 
                 AACTCGAGAG GACAACAGAG AGAATAGGAT GGGCCAAATC 
               
               
                   
               
               
                 1761 
                 CCAAATTATT TCAAGGATGA TAGCTTCTTT CTTCAACAAT 
               
               
                   
               
               
                 1801 
                 GAAACTACAA CAGCCGATGA AAAAGATGCA CTTTTAACCA 
               
               
                   
               
               
                 1841 
                 GATTCAGAAA CATCAATGGC CCAAACAAAA CAAAAAGTGG 
               
               
                   
               
               
                 1881 
                 TCAGAGAGAG AGTGAAGCTG TGAACATGTT GGTAGCAACG 
               
               
                   
               
               
                 1921 
                 CTCCAACAAT ACCTGGCAGG ATTTGATAGA TATACCAGAC 
               
               
                   
               
               
                 1961 
                 ATCAATTGAA AGATGCTTGG AGTGTGTGGT TCAGAAAAGT 
               
               
                   
               
               
                 2001 
                 GCAAGAAGAA GAGGCCATCT ACGGGGCAGA AGCGGAGCTT 
               
               
                   
               
               
                 2041 
                 CTAACAACCA CCTTAAACAT CTGTGCTGGT CATATTGCTT 
               
               
                   
               
               
                 2081 
                 TCGACGAAAA CATAATGGCC AACAAAGATT ACACCACTCT 
               
               
                   
               
               
                 2121 
                 TTCCAGCCTT ACAAGCAAAA TTTGCCAGAA GCTTTCTGAA 
               
               
                   
               
               
                 2161 
                 ATTCGAAATG AAAAGGTTGA GGAAATGGAG AGTGGAATTA 
               
               
                   
               
               
                 2201 
                 AAGCAAAATC AAGCATCAAA GACAAGGAAG TGGAACATGA 
               
               
                   
               
               
                 2241 
                 TATGCAGTCA CTGGTGAAAT TAGTCCTGGA GAGATGTGAA 
               
               
                   
               
               
                 2281 
                 GGCATAAACA ACAGAAAACT GAAGCAAACA TTTCTATCGG 
               
               
                   
               
               
                 2321 
                 TTGCAAAAAC ATATTACTAC AGAGCCTATA ATGCTGATGA 
               
               
                   
               
               
                 2361 
                 AACCATGGAC ATCCATATGT TCAAAGTACT TTTCGAACCA 
               
               
                   
               
               
                 2401 
                 GTCATGTGA 
               
            
           
         
       
     
     An example of a  Salvia sclarea  sclareol synthase amino acid sequence is shown below (SEQ ID NO:61; NCBI accession no. AET21246.1). 
                        1   MSLAFNVGVT PFSGQRVGSR KEKFPVQGFP VTTPNRSRLI               41   VNCSLTTIDF MAKMKENFKR EDDKFPTTTT LRSEDIPSNL               81   CIIDTLQRLG VDQFFQYEIN TILDNTFRLW QEKHKVIYGN               121   VTTHAMAFRL LRVKGYEVSS EELAPYGNQE AVSQQTNDLP               161   MIIELYRAAN ERIYEEERSL EKILAWTTIF LNKQVQDNSI               201   PDKKLHKLVE FYLRNYKGIT IRLGARRNLE LYDMTYYQAL               241   KSTNRFSNLC NEDFLVFAKQ DFDIHEAQNQ KGLQQLQRWY               281   ADCRLDTLNF GRDVVIIANY LASLIIGDHA FDYVRLAFAK               321   TSVLVTIMDD FFDCHGSSQE CDKIIELVKE WKENPDAEYG               361   SEELEILFMA LYNTVNELAE RARVEQGRSV KEFLVKLWVE               401   ILSAFKIELD TWSNGTQQSF DEYISSSWLS NGSRLTGLLT               441   MQFVGVKLSD EMLMSEECTD LARHVCMVGR LLNDVCSSER               481   EREENIAGKS YSILLATEKD GRKVSEDEAI AEINEMVEYH               521   WRKVLQIVYK KESILPRRCK DVFLEMAKGT FYAYGINDEL               561   TSPQQSKEDM KSFVF            
A nucleic acid encoding the  Salvia sclarea  sclareol synthase with SEQ ID NO:61 is shown below as SEQ ID NO:62.
 
     
       
         
           
               
               
            
               
                 1 
                 ATGTCGCTCG CCTTCAACGT CGGAGTTACG CCTTTCTCCG 
               
               
                   
               
               
                 41 
                 GCCAAAGAGT TGGGAGCAGG AAAGAAAAAT TTCCAGTCCA 
               
               
                   
               
               
                 81 
                 AGGATTTCCT GTGACCACCC CCAATAGGTC ACGTCTCATC 
               
               
                   
               
               
                 121 
                 GTTAACTGCA GCCTTACTAC AATAGATTTC ATGGCGAAAA 
               
               
                   
               
               
                 161 
                 TGAAAGAGAA TTTCAAGAGG GAAGACGATA AATTTCCAAC 
               
               
                   
               
               
                 201 
                 GACAACGACT CTTCGATCCG AAGATATACC CTCTAATTTG 
               
               
                   
               
               
                 241 
                 TGTATAATCG ACACCCTTCA AAGGTTGGGG GTCGATCAAT 
               
               
                   
               
               
                 281 
                 TCTTCCAATA TGAAATCAAC ACTATTCTAG ATAACACATT 
               
               
                   
               
               
                 321 
                 CAGGTTGTGG CAAGAAAAAC ACAAAGTTAT ATATGGCAAT 
               
               
                   
               
               
                 361 
                 GTTACTACTC ATGCAATGGC ATTTAGGCTT TTGCGAGTGA 
               
               
                   
               
               
                 401 
                 AAGGATACGA AGTTTCATCA GAGGAGTTGG CTCCATATGG 
               
               
                   
               
               
                 441 
                 TAACCAAGAG GCTGTTAGCC AGCAAACAAA TGACCTGCCG 
               
               
                   
               
               
                 481 
                 ATGATTATTG AGCTTTATAG AGCAGCAAAT GAGAGAATAT 
               
               
                   
               
               
                 521 
                 ATGAAGAAGA GAGGAGTCTT GAAAAAATTC TTGCTTGGAC 
               
               
                   
               
               
                 561 
                 TACCATCTTT CTCAATAAGC AAGTGCAAGA TAACTCAATT 
               
               
                   
               
               
                 601 
                 CCCGACAAAA AACTGCACAA ACTGGTGGAA TTCTACTTGA 
               
               
                   
               
               
                 641 
                 GGAATTACAA AGGCATAACC ATAAGATTGG GAGCTAGACG 
               
               
                   
               
               
                 681 
                 AAACCTCGAG CTATATGACA TGACCTACTA TCAAGCTCTG 
               
               
                   
               
               
                 721 
                 AAATCTACAA ACAGGTTCTC TAATTTATGC AACGAAGATT 
               
               
                   
               
               
                 761 
                 TTCTAGTTTT CGCAAAGCAA GATTTCGATA TACATGAAGC 
               
               
                   
               
               
                 801 
                 CCAGAACCAG AAAGGACTTC AACAACTGCA AAGGTGGTAT 
               
               
                   
               
               
                 841 
                 GCAGATTGTA GGTTGGACAC CTTAAACTTT GGAAGAGATG 
               
               
                   
               
               
                 881 
                 TAGTTATTAT TGCTAATTAT TTGGCTTCAT TAATTATTGG 
               
               
                   
               
               
                 921 
                 TGATCATGCG TTTGACTATG TTCGTCTCGC ATTTGCCAAA 
               
               
                   
               
               
                 961 
                 ACATCTGTGC TTGTAACAAT TATGGATGAT TTTTTCGACT 
               
               
                   
               
               
                 1001 
                 GTCATGGCTC TAGTCAAGAG TGTGACAAGA TCATTGAATT 
               
               
                   
               
               
                 1041 
                 AGTAAAAGAA TGGAAGGAGA ATCCGGATGC AGAGTACGGA 
               
               
                   
               
               
                 1081 
                 TCTGAGGAGC TTGAGATCCT TTTTATGGCG TTGTACAATA 
               
               
                   
               
               
                 1121 
                 CAGTAAATGA GTTGGCGGAG AGGGCTCGTG TTGAACAGGG 
               
               
                   
               
               
                 1161 
                 GCGTAGTGTC AAAGAGTTTC TAGTCAAACT GTGGGTTGAA 
               
               
                   
               
               
                 1201 
                 ATACTCTCAG CTTTCAAGAT AGAATTAGAT ACATGGAGCA 
               
               
                   
               
               
                 1241 
                 ATGGCACGCA GCAAAGCTTC GATGAATACA TTTCTTCGTC 
               
               
                   
               
               
                 1281 
                 GTGGTTGTCG AACGGTTCCC GGCTGACAGG TCTCCTGACG 
               
               
                   
               
               
                 1321 
                 ATGCAATTCG TCGGAGTAAA ATTGTCCGAT GAAATGCTTA 
               
               
                   
               
               
                 1361 
                 TGAGTGAAGA GTGCACTGAT TTGGCTAGGC ATGTCTGTAT 
               
               
                   
               
               
                 1401 
                 GGTCGGCCGG CTGCTCAACG ACGTGTGCAG TTCTGAGAGG 
               
               
                   
               
               
                 1441 
                 GAGCGCGAGG AAAATATTGC AGGAAAAAGT TATAGCATTC 
               
               
                   
               
               
                 1481 
                 TACTAGCAAC TGAGAAAGAT GGAAGAAAAG TTAGTGAAGA 
               
               
                   
               
               
                 1521 
                 TGAAGCCATT GCAGAGATCA ATGAAATGGT TGAATATCAC 
               
               
                   
               
               
                 1561 
                 TGGAGAAAAG TGTTGCAGAT TGTGTATAAA AAAGAAAGCA 
               
               
                   
               
               
                 1601 
                 TTTTGCCAAG AAGATGCAAA GATGTATTTT TGGAGATGGC 
               
               
                   
               
               
                 1641 
                 TAAGGGTACG TTTTATGCTT ATGGGATCAA CGATGAATTG 
               
               
                   
               
               
                 1681 
                 ACTTCTCCTC AGCAATCCAA GGAAGATATG AAATCCTTTG 
               
               
                   
               
               
                 1721 
                 TCTTTTGA 
               
            
           
         
       
     
     An example of a  Marrubium vulgare  (Mv) CPS1 amino acid sequence is shown below (SEQ ID NO:63). 
     
       
         
           
               
               
            
               
                 1 
                 MASTPTLNLS ITTPFVRTKI PAKISLPACS WLDRSSSRHV 
               
               
                   
               
               
                 41 
                 ELNHKFCRKL ELKVAMCRAS LDVQQVRDEV YSNAQPHELV 
               
               
                   
               
               
                 81 
                 DKKIEERVKY VKNLLSTMDD GRINWSAYDT AWISLIKDFE 
               
               
                   
               
               
                 121 
                 GRDCPQFPST LERIAENQLP DGSWGDKDFD CSYDRIINTL 
               
               
                   
               
               
                 161 
                 ACVVALTTWN VHPEINQKGI RYLKENMRKL EETPTVLMTC 
               
               
                   
               
               
                 201 
                 AFEVVFPALL KKARNLGIHD LPYDMPIVKE ICKIGDEKLA 
               
               
                   
               
               
                 241 
                 RIPKKMMEKE TTSLMYAAEG VENLDWERLL KLRTPENGSF 
               
               
                   
               
               
                 281 
                 LSSPAATVVA FMHTKDEDCL RYIKYLLNKF NGGAPNVYPV 
               
               
                   
               
               
                 321 
                 DLWSRLWATD RLQRLGISRY FESEIKDLLS YVHSYWTDIG 
               
               
                   
               
               
                 361 
                 VYCTRDSKYA DIDDTSMGFR LLRVQGYNMD ANVFKYFQKD 
               
               
                   
               
               
                 401 
                 DKFVCLGGQM NGSATATYNL YRAAQYQFPG EQILEDARKF 
               
               
                   
               
               
                 441 
                 SQQFLQESID TNNLLDKWVI SPHIPEEMRF GMEMTWYSCL 
               
               
                   
               
               
                 481 
                 PRIEASYYLQ HYGATEDVWL GKTFFRMEEI SNENYRELAI 
               
               
                   
               
               
                 521 
                 LDFSKCQAQH QTEWIHMQEW YESNNVKEFG ISRKDLLFAY 
               
               
                   
               
               
                 561 
                 FLAAASIFET ERAKERILWA RSKIICKMVK SFLEKETGSL 
               
               
                   
               
               
                 601 
                 EHKIAFLTGS GDKGNGPVNN AMATLHQLLG EFDGYISIQL 
               
               
                   
               
               
                 641 
                 ENAWAAWLTK LEQGEANDGE LLATTINICG GRVNQDTLSH 
               
               
                   
               
               
                 681 
                 NEYKALSDLT NKICHNLAQI QNDKGDEIKD SKRSERDKEV 
               
               
                   
               
               
                 721 
                 EQDMQALAKL VFEESDLERS IKQTFLAVVR TYYYGAYIAA 
               
               
                   
               
               
                 761 
                 EKIDVHMFKV LFKPVG 
               
            
           
         
       
     
     An example of a  Marrubium vulgare  (Mv) TPS5 (syn. MvELS) amino acid sequence is shown below (SEQ ID NO:64). 
     
       
         
           
               
               
            
               
                 1 
                 MSITFNLKIA PFSGPGIQRS KETFPATEIQ ITASTKSTMT 
               
               
                   
               
               
                 41 
                 TKCSFNASTD FMGKLREKVG GKADKPPVVI HPVDISSNLC 
               
               
                   
               
               
                 81 
                 MIDTLQSLGV DRYFQSEINT LLEHTYRLWK EKKKNIIFKD 
               
               
                   
               
               
                 121 
                 VSCCAIAFRL LREKGYQVSS DKLAPFADYR IRDVATILEL 
               
               
                   
               
               
                 161 
                 YRASQARLYE DEHTLEKLHD WSSNLLKQHL LNGSIPDHKL 
               
               
                   
               
               
                 201 
                 HKQVEYFLKN YHGILDRVAV RRSLDLYNIN HHHRIPDVAD 
               
               
                   
               
               
                 241 
                 GFPKEDFLEY SMQDFNICQA QQQEELHQLQ RWYADCRLDT 
               
               
                   
               
               
                 281 
                 LNYGRDVVRI ANFLTSAIFG EPEFSDARLA FAKHIILVTR 
               
               
                   
               
               
                 321 
                 IDDFFDHGGS REESYKILDL VQEWKEKPAE EYGSKEVEIL 
               
               
                   
               
               
                 361 
                 FTAVYNTVND LAEKAHIEQG RCVKPLLIKL WVEILTSFKK 
               
               
                   
               
               
                 401 
                 ELDSWTEETA LTLDEYLSSS WVSIGCRICI LNSLQYLGIK 
               
               
                   
               
               
                 441 
                 LSEEMLSSQE CTDLCRHVSS VDRLLNDVQT FKKERLENTI 
               
               
                   
               
               
                 481 
                 NSVGLQLAAH KGERAMTEED AMSKIKEMAD YHRRKLMQIV 
               
               
                   
               
               
                 521 
                 YKEGTVFPRE CKDVFLRVCR IGYYLYSSGD EFTSPQQMKE 
               
               
                   
               
               
                 561 
                 DMKSLVYQPV KIHPLEAINV 
               
            
           
         
       
     
     An example of a  Kitasatospora griseola  TPS2 (KgTPS2) amino acid sequence is shown below (SEQ ID NO:65). 
     
       
         
           
               
               
            
               
                 1 
                 MPDAIEFEHE GRRNPNSAEA ESAYSSIIAA LDLQESDYAV 
               
               
                   
               
               
                 41 
                 ISGHSRIVGA AALVYPDADA ETLLAASLWT ACLIVNDDRW 
               
               
                   
               
               
                 81 
                 DYVQEDGGRL APGEWFDGVT EVVDTWRTAG PRLPDPFFEL 
               
               
                   
               
               
                 121 
                 VRTTMSRLDA ALGAEAADEI GHEIKRAITA MKWEGVWNEY 
               
               
                   
               
               
                 161 
                 TKKTSLATYL SFRRGYCTMD VQVVLDKWIN GGRSFAALRD 
               
               
                   
               
               
                 201 
                 DPVRRAIDDV VVRFGCLSND YYSWGREKKA VDKSNAVRIL 
               
               
                   
               
               
                 241 
                 MDHAGYDEST ALAHVRDDCV QAITDLDCIE ESIKRSGHLG 
               
               
                   
               
               
                 281 
                 SHAQELLDYL ACHRPLIYAA ATWPTETNRY R 
               
            
           
         
       
     
     An example of an  Origanum majorana  TPS1 (0mTPS1) amino acid sequence is shown below (SEQ ID NO:66). 
     
       
         
           
               
               
            
               
                 1 
                 MTDVSSLRLS NAPAAGGRLP LPGKVHLPEF RTVCAWLNNG 
               
               
                   
               
               
                 41 
                 CKYEPLTCRI SRRKISECRV ASLNSSQLIE KVGSPAQSLE 
               
               
                   
               
               
                 81 
                 EANKKIEDSI EYIKNLLMTS GDGRISVSAY DTSLVALIKD 
               
               
                   
               
               
                 121 
                 VKGRDAPQFP SCLEWIAQNQ MADGSWGDEF FCIYDRIVNT 
               
               
                   
               
               
                 161 
                 LACLVALKSW NLHPDKIEKG VTYINENVHK LKDGSTEHMT 
               
               
                   
               
               
                 201 
                 SGFEIVVPAT LERAKVLGIQ GLPYDHPFIK EIINTKERRL 
               
               
                   
               
               
                 241 
                 SKIPKDLIYK LPTTLLFSLE GQGELDWEKI LKLQSSDGSF 
               
               
                   
               
               
                 281 
                 LTSPSSTASV FMRTKDEKCL KFIENAVKNC GGGAPHTYPV 
               
               
                   
               
               
                 321 
                 DVFARLWAVD RLQRLGISRF FQHEIKYFLD HINSVWTENG 
               
               
                   
               
               
                 361 
                 VFSGRDSQFC DIDDTSMGVR LLKMHGYNVD PNALKHFKQE 
               
               
                   
               
               
                 401 
                 DGKFSCYPGQ MIESASPIYN LYRAAQLRFP GEEILEEASR 
               
               
                   
               
               
                 441 
                 FAFNFLQEKI ANHEIQEKWV ISEHLIDEIK LGLKMPWYAT 
               
               
                   
               
               
                 481 
                 LPRVEAAYYL EYYAGSGDVW IGKTFYRMPE ISNDTYKEVA 
               
               
                   
               
               
                 521 
                 ILDFNTCQAQ HQFEWIYMQE WYESSKVKDF GISKKDLLVA 
               
               
                   
               
               
                 561 
                 YFLAASTIFE PERTQERIIW AKTLILSRMI TSFLNKQATL 
               
               
                   
               
               
                 601 
                 SSQQKNAILT QLGESVDGLD KIYSGEKDSG LAETLLATFQ 
               
               
                   
               
               
                 641 
                 QLLDGFDRYT RHQLRNAWGQ WLMKVQQGEA NGGADAELIA 
               
               
                   
               
               
                 681 
                 NTLNICAGLI AFNEDVLLHS EYTTLSSLTN KICQRLSQIE 
               
               
                   
               
               
                 721 
                 DEKTLEVIEG GIKDKELEED IQALVKLALE ENGGCGVDRR 
               
               
                   
               
               
                 741 
                 IKQSFLSVFK TFYYRAYHDA ETTDLHIFKV LFGPVM 
               
            
           
         
       
     
     An example of an  Origanum majorana  TPS4 (OmTPS4) amino acid sequence is shown below (SEQ ID NO:67). 
     
       
         
           
               
               
            
               
                 1 
                 MSLAFSHVST FFSGQRVVGS RREIIPVNGV PTTANKPSFA 
               
               
                   
               
               
                 41 
                 VKCNLTTKDL MVKMKEKLKG QDGNLTVGVA DMPSSLCVID 
               
               
                   
               
               
                 81 
                 TLERLGVDRY FRSEIHVILH DTYRLWQQKD KDICSNVTTH 
               
               
                   
               
               
                 121 
                 AMAFRLLRVN GYEVSSEELA PYANLEHFSQ QKVDTAMAIE 
               
               
                   
               
               
                 161 
                 LYRAAQERIH EDESGLDKIL AWTTTFLEQQ LLTNSILDNK 
               
               
                   
               
               
                 201 
                 LHKLVEYYLN NYHGQTNRVG ARRHLDLYEM SHYQNLKPSH 
               
               
                   
               
               
                 241 
                 SLCNEDLLAF AKQGFRDFQI QQQKEFEQLQ RWYEDCRLDK 
               
               
                   
               
               
                 281 
                 LSYGRDVVKI SSFMASILMD DPELADVRLS IAKQMVLVTR 
               
               
                   
               
               
                 321 
                 IDDFFDHGGS REDSYKIIEL VKEWKEKAEY DSEEVKILFT 
               
               
                   
               
               
                 361 
                 AVYTTVNELA EACVQQGRNS TTVKEFLVQL WIEILSAFKV 
               
               
                   
               
               
                 401 
                 ELDTWSDGTE VSLDEYLSWS WISNGCRVSI VTTMHLLPTK 
               
               
                   
               
               
                 441 
                 LCSDEMLRSE ECKDLCRHVS MVGRLLNDIH SFEKEHEENT 
               
               
                   
               
               
                 481 
                 GNSVSILVAG EDTEEEAIGK IKEIVEYERR KLMQIVYKRG 
               
               
                   
               
               
                 521 
                 TILPRECKDI FLKACRATFY VYSSTDEFTS PRQVMEDMKT 
               
               
                   
               
               
                 561 
                 LSS 
               
            
           
         
       
     
     The inventors have described a CYP71D381 from  C. forskohlii,  which resulted in oxidized derivatives at alternative positions outside the known forskolin chemistry (Pateraki et al. Elife 6 (2017)). The sequence for the CYP71D381 from  Plectranthus barbatus  is shown below (SEQ ID NO:68). 
     
       
         
           
               
               
            
               
                 1 
                 MEFDFPSALI FPAVSLLLLL WLTKTRKPKS DLDRIPGPRR 
               
               
                   
               
               
                 41 
                 LPLIGNLHHL ISLTPPPRLF REMAAKYGPL MRLQLGGVPF 
               
               
                   
               
               
                 81 
                 LIVSSVDVAK HVVKTNDVPF ANRPPMHAAR AITYNYTDIG 
               
               
                   
               
               
                 121 
                 FAPYGEYWRN LRKICTLELL SARRVRSFRH IREEENAGVA 
               
               
                   
               
               
                 161 
                 KWIASKEGSP ANLSERVYLS SFDITSRASI GKATEEKQTL 
               
               
                   
               
               
                 201 
                 TSSIKDAMKL GGFNVADLYP SSKLLLLITG LNFRIQRVFR 
               
               
                   
               
               
                 241 
                 KTDRILDDLL SQHRSTSATT ERPEDLVDVL LKYQKEETEV 
               
               
                   
               
               
                 281 
                 HLNNDKIKAV IMDMFLAGGE TSATAVDWAM AEMIRNPTTL 
               
               
                   
               
               
                 321 
                 KKAQEEVRRV FDGKGYVDEE EFHELKYLKL VIKEMLRMHP 
               
               
                   
               
               
                 361 
                 PLPFLVPRMN SERCEINGYE IPANTRLLIN AWAIGRDPKY 
               
               
                   
               
               
                 401 
                 WNDAEKFIPE RFENSSIDFK GNNLEYIPFG AGRRMCPGMT 
               
               
                   
               
               
                 441 
                 FGLASVEFTL AMLLYHFDWK MPQGIKLDMT ESFGASLKRK 
               
               
                   
               
               
                 481 
                 HDLLMIPTLK RPLRLAP 
               
            
           
         
       
     
     Mining of nearly 50 transcriptomes of related members of the mint family (Lamiaceae; Johnson et al.,  J. Biol. Chem.  294: 1349-1362 (2019)) indicates that the mint family provides rich repository of members of the CYP71D and CYP76AH enzymes (over 200 candidates, functional characterization, preliminary results by the inventors). Any of these enzymes can be used for additional/alternative oxidation chemistries to produce useful products. 
     The cyclization of diterpenes is among the most complex reactions found in nature. Typically, more than half of the carbons of GGPP undergo changes in connection, hybridization (sp-status), and stereochemistry during the carbocationic cascade. Stabilized in the active site of diterpene synthases, those carbocation intermediates undergo electron delocalization, hydride and alkyl-shifts, and can be quenched by access to water. For example, predicted cyclization reactions for conversion of GGPP to hydroxy-vulgarisane are shown below. 
     
       
         
         
             
             
         
       
     
     The inventors have recently discovered the PvHVS enzyme (SEQ ID NO:40), which can generate the irregular diterpene founding the class of bioactive vulgarisane compounds in  Prunella vulgaris  (see Pelot et al. Plant Physiol. 178: 54 LP-71 (2018)). 
     Mutated variants of diTPS can be deployed for diversification of the enzymes to increase the range of products produced, for example, by controlling the stereochemistry of the product outcome. Previously, generation of individual compounds from GGPP remained limited to the natural C20 chemical space of diterpenes (Schulte et al. Biochemistry 57: 3473-3479 (2018)). Terpene cyclization has been investigated through crystallography, structural modelling and mutagenesis studies including unreactive fluorinated, azaisoprenyl or thioloisoprenyl diphosphate analogs, by quantum-chemical calculations of intermediates, and by isotopically labelled natural precursors. With exceptions, the majority of approaches are static (non-dynamic) and have not yet been applied to terpenoid synthases, where reports are limited to single-enzyme-analog tests. Crystal structures for plant diTPS are similarly restricted to three enzymes only, the grand fir bifunctional class II/I abietadiene synthase, the class II Arabidopsis thaliana ent-CPS30, and the class I Taxus taxadiene synthase. Cyclization of rationally designed substrates with both altered spatial and electronic properties will provide a unique and dynamic facet by evaluation of the previously unrecognized substrate tolerance: steric constraints, stabilization of transition states and kinetics of the enzymes. With that, the proposed technology complements current tools exploring the mechanism of the cationic cascade of terpene cyclization. Structure-guided mutational studies for identified optimal modules, combined with the substrate tolerance described herein can broaden the accessible range of enzymes and products produced. 
     Enzymes that exhibit the following characteristics are generally preferred for use in methods of producing desirable products: (i) terpenoid synthases with high natural substrate tolerance, (ii) those generating a set of intermediates with maximized chemical diversity, and (iii) enzymes that provide intermediates in the pathways to forskolin and jolkinol C (P450s, ADHs, ACTs). In some cases, the enzymes can be active as recombinant enzymes in E. coli and/or the enzymes have demonstrated functionally in yeast. 
     As one example of an enzyme that can accept multiple unnatural substrates is CfTps2, which the inventors have demonstrated has such useful activity. The CfTps2 enzyme can provide the first step in synthesis of the cardiac stimulant and cognition enhancer forskolin which is derived from  Coleus forskohiii.  The CfTps2 enzyme can also serve as the first step in production of sclareol, which is an industrial precursor for ambroxoid fragrance substances. The ability to modify, in a targeted manner, these biological active or industrially significant natural products would facilitate the design, testing, and production of novel materials and biologically active agents. 
     Enzymes described herein can therefore have one or more deletions, insertions, replacements, or substitutions in a part of the enzyme. The enzyme(s) described herein can have, for example, at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 93%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to a sequence described herein. 
     In some cases, enzymes can have conservative changes such as one or more deletions, insertions, replacements, or substitutions that have no significant effect on the activities of the enzymes. Examples of conservative substitutions are provided below in Table IA. 
     
       
         
           
               
             
               
                 TABLE 1A 
               
             
            
               
                   
               
               
                 Conservative Substitutions 
               
            
           
           
               
               
               
            
               
                   
                 Type of  
                 Substitutable  
               
               
                   
                 Amino Acid 
                 Amino Acids 
               
               
                   
                   
               
               
                   
                 Hydrophilic 
                 Ala, Pro, Gly, Glu, Asp,  
               
               
                   
                   
                 Gln, Asn, Ser, Thr 
               
               
                   
                 Sulfhydryl 
                 Cys 
               
               
                   
                 Aliphatic 
                 Val, Ile, Leu, Met 
               
               
                   
                 Basic 
                 Lys, Arg, His 
               
               
                   
                 Aromatic 
                 Phe, Tyr, Trp 
               
               
                   
                   
               
            
           
         
       
     
     The enzymes can also include a tag, for example, as a label or to facilitate purification of the enzyme. Examples of such tags include histidine tags, streptavidin tags, biotin tags, antibody fragments, and the like. 
     Hosts 
     Terpenes, including diterpenes and terpenoids, can be made in a variety of host organisms in vivo. In some cases, the enzymes described herein can be made in host cells, and those enzymes can be extracted from the host cells for use in vitro. As used herein, a “host” means a cell, tissue or organism capable of replication. The host can have an expression cassette or expression vector that can include a nucleic acid segment encoding an enzyme that is involved in the biosynthesis of terpenes. 
     The term “host cell”, as used herein, refers to any prokaryotic or eukaryotic cell that can be transformed with an expression cassettes or vector carrying the nucleic acid segment encoding an enzyme that is involved in the biosynthesis of one or more terpenes or terpenoids. The host cells can, for example, be a plant, bacterial, insect, or yeast cell. Expression cassettes encoding biosynthetic enzymes can be incorporated or transferred into a host cell to facilitate manufacture of the enzymes described herein or the terpene, diterpene, or terpenoid products of those enzymes. The host cells can be present in an organism. For example, the host cells can be present in a host such as a microorganism, fungus, or plant. 
     Expression of Enzymes 
     Also described herein are expression systems that include at least one expression cassette (e.g., expression vectors or transgenes) that encode one or more of the enzyme(s) described herein. For example, the expression systems can also include one or more expression cassettes any of the monoterpene synthase, diterpene synthase, sesquiterpene synthase, sesterterpene synthase, triterpene synthase, tetraterpene synthase, polyterpene synthase, transcription factor, cytochrome P450, cytochrome P450 reductase, 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate-reducto-isomerase, cytidine 5′-diphosphate-methylerythritol (CDP-ME) synthetase (IspD), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF), HMG-CoA synthase, HMG-CoA reductase (HMGR), mevalonic acid kinase (MVK), phosphomevalonate kinase (PMK), mevalonate-5-diphosphate decarboxylase (MPD), isopentenyl diphosphate isomerase, abietadiene synthase (ABS), farnesylpyrophosphate synthase (FPPS), or squalene synthase (SQS), LDSP-protein fusions, or enzymes that facilitate production of terpenoids, terpene precursors, terpene building blocks, or products derived from terpenoids. 
     Nucleic acids encoding the enzymes can have sequence modifications. For example, nucleic acid sequences described herein can be modified to more optimally express the enzymes. Hence, the nucleic acid segment encoding the enzymes can be optimized to improve expression in different host cells. Most amino acids can be encoded by more than one codon, but when an amino acid is encoded by more than one codon, the codons are referred to as degenerate codons. A listing of degenerate codons is provided in Table 1B below. 
                     TABLE 1B                  Degenerate Amino Acid Codons                             Amino               Acid   Three Nucleotide Codon                       Ala/A   GCT, GCC, GCA, GCG                       Arg/R   CGT, CGC, CGA, CGG, AGA, AGG                       Asn/N   AAT, AAC                       Asp/D   GAT, GAC                       Cys/C   TGT, TGC                       Gln/Q   CAA, CAG                       Glu/E   GAA, GAG                       Gly/G   GGT, GGC, GGA, GGG                       His/H   CAT, CAC                       Ile/I   ATT, ATC, ATA                       Leu/L   TTA, TTG, CTT, CTC, CTA, CTG                       Lys/K   AAA, AAG                       Met/M   ATG                       Phe/F   TTT, TTC                       Pro/P   CCT, CCC, CCA, CCG                       Ser/S   TCT, TCC, TCA, TCG, AGT, AGC                       Thr/T   ACT, ACC, ACA, ACG                       Trp/W   TGG                       Tyr/Y   TAT, TAC                       Val/V   GTT, GTC, GTA, GTG                       START   ATG                       STOP   TAG, TGA, TAA                        
Different organisms may translate different codons more or less efficiently (e.g., because they have different ratios of tRNAs) than other organisms. Hence, when some amino acids can be encoded by several codons, a nucleic acid segment can be designed to optimize the efficiency of expression of an enzyme by using codons that are preferred by an organism of interest. For example, the nucleotide coding regions of the enzymes described herein can be codon optimized for expression in various microorganisms, fungi, or plant species.
 
     An optimized nucleic acid can have less than 100%, less than 99%, less than 98%, less than 97%, less than 95%, or less than 94%, or less than 93%, or less than 92%, or less than 91%, or less than 90%, or less than 89%, or less than 88%, or less than 85%, or less than 83%, or less than 80%, or less than 75% nucleic acid sequence identity to a corresponding non-optimized (e.g., a non-optimized parental or wild type enzyme nucleic acid) sequence. Nucleic acid segment(s) encoding one or more enzyme(s) can therefore have one or more nucleotide deletions, insertions, replacements, or substitutions. 
     The nucleic acid segments encoding one or more enzyme can be operably linked to a promoter, which provides for expression of mRNA from the nucleic acid segments. The promoter is typically a promoter functional in a microorganism, fungus or plant. A nucleic acid segment encoding one or more enzyme is operably linked to the promoter, for example, when it is located downstream from the promoter. The combination of a coding region for an enzyme operably linked to a promoter forms an expression cassette, which can include other elements and regulatory sequences as well. 
     Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both the prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences can also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous DNAs, that is a DNA different from the native or homologous DNA. 
     Promoter sequences are also known to be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, a bacterial promoter such as the P tac  promoter can be induced to varying levels of gene expression depending on the level of isopropyl-beta-D-thiogalactoside added to the transformed cells. Promoters can also provide for tissue specific or developmental regulation. An isolated promoter sequence that is a strong promoter for heterologous DNAs is often advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired. 
     Examples of prokaryotic promoters that can be used include, but are not limited to, SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters. Examples of eukaryotic promoters that can be used include, but are not limited to, constitutive promoters, e.g., viral promoters such as CMV, SV40 and RSV promoters, as well as regulatable promoters, e.g., an inducible or repressible promoter such as the tet promoter, the hsp70 promoter and a synthetic promoter regulated by CRE. 
     Examples of plant promoters include the CaMV 35S promoter (Odell et al.,  Nature.  313:810-812 (1985)), or others such as CaMV 19S (Lawton et al.,  Plant Molecular Biology.  9:315-324 (1987)), nos (Ebert et al.,  Proc. Natl. Acad. Sci. USA.  84:5745-5749 (1987)),  Adhl  (Walker et al.,  Proc. Natl. Acad. Sci. USA.  84:6624-6628 (1987)), sucrose synthase (Yang et al.,  Proc. Natl. Acad. Sci. USA.  87:4144-4148 (1990)), α-tubulin, ubiquitin, actin (Wang et al.,  Mol. Cell. Biol.  12:3399 (1992)), cab (Sullivan et al.,  Mol. Gen. Genet.  215:431 (1989)), PEPCase (Hudspeth et al.,  Plant Molecular Biology.  12:579-589 (1989)) or those associated with the R gene complex (Chandler et al.,  The Plant Cell.  1:1175-1183 (1989)). Further suitable promoters include a CYP71D16 trichome-specific promoter and the CBTS (cembratrienol synthase) promotor, cauliflower mosaic virus promoter, the Z10 promoter from a gene encoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zein protein, the plastid rRNA-operon (rrn) promoter, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene (Coruzzi et al.,  EMBO J.  3:1671 (1971)), RUBISCO-SSU light inducible promoter (SSU) from tobacco and the actin promoter from rice (McElroy et al.,  The Plant Cell.  2:163-171 (1990)). Other promoters that are useful can also be employed. 
     Examples of leaf-specific promoters include the promoter from the  Populus  ribulose-1,5-bisphosphate carboxylase small subunit gene (Wang et al.  Plant Molec Biol Reporter  31 (1): 120-127 (2013)), the promoter from the  Brachypodium distachyon  sedoheptulose-1,7-bisphosphatase (SBPase-p) gene (Alotaibi et al.  Plants  7(2): 27 (2018)), the fructose-1,6-bisphosphate aldolase (FBPA-p) gene from  Brachypodium distachyon  (Alotaibi et al.  Plants  7(2): 27 (2018)), and the photosystem-II promoter (CAB2-p) of the rice ( Oryza sativa  L.) light-harvest chlorophyll a/b binding protein (CAB) (Song et al.  J Am Soc Hort Sci  132(4): 551-556 (2007)). Additional promoters that can be used include those available in expression databases, see for example, website bar.utoronto.ca/eplant/ which includes poplar or heterologous promoters from Arabidopsis (for example from AT2G26020/PDF1.2b or AT5G44420 / LCR77). 
     Alternatively, novel tissue specific promoter sequences may be employed. cDNA clones from a particular tissue can be isolated and those clones which are expressed specifically in that tissue can be identified, for example, using Northern blotting. Preferably, the gene isolated is not present in a high copy number but is relatively abundant in specific tissues. The promoter and control elements of corresponding genomic clones can then be localized using techniques well known to those of skill in the art. 
     Plant plastid originated promoters can also be used, for example, to improve expression in plastids, for example, a rice clp promoter, or tobacco rrn promoter. Chloroplast-specific promoters can also be utilized for targeting the foreign protein expression into chloroplasts. For example, the 16S ribosomal RNA promoter (Prrn) like psbA and atpA gene promoters can be used for chloroplast transformation. 
     A nucleic acid encoding one or more enzyme can be combined with the promoter by standard methods to yield an expression cassette, for example, as described in Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL. Second Edition (Cold Spring Harbor, NY: Cold Spring Harbor Press (1989); MOLECULAR CLONING: A LABORATORY MANUAL. Third Edition (Cold Spring Harbor, NY: Cold Spring Harbor Press (2000)). Briefly, a plasmid containing a promoter such as the 35S CaMV promoter or the CYP71D16 trichome-specific promoter can be constructed as described in Jefferson ( Plant Molecular Biology Reporter  5:387-405 (1987)) or obtained from Clontech Lab in Palo Alto, California (e.g., pBI121 or pBI221). Typically, these plasmids are constructed to have multiple cloning sites having specificity for different restriction enzymes downstream from the promoter. 
     The expression cassette or vector can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Marker genes can include the  E. coli  lacZ gene which encodes β-galactosidase, and green fluorescent protein. In some embodiments the marker can be a selectable marker. When such selectable markers are successfully transferred into a host cell, the transformed host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell&#39;s metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). 
     The expression cassettes can be within vectors such as plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or artificial chromosomes. 
     Transfer of the expression cassettes or vectors into host cells can be by methods available in the art and readily adaptable for use in the method described herein. Expression cassettes and vectors can be incorporated into host cells, for example, calcium-mediated transformation, electroporation, microinjection, lipofection, particle bombardment, chemical transfectants, physico-mechanical methods such as electroporation, or direct diffusion of DNA. 
     In some cases, one or more enzyme cassettes can be introduced into a single host cell. Such transformed host cells can then be used either for producing one or more enzymes or for chemical conversion of an unnatural substrate into a useful terpene product. 
     After expression in a suitable host, in some cases the enzymes can be purified or semi-purified for use within in vitro enzyme catalyzed reactions to generate terpenes. For example, the host cells can be lysed, and the enzymes purified or semi-purified to the extent needed to reduce side reactions. Purification of the enzymes also removes cellular debris that can complicate purification of the terpene products of enzymatic reactions. Purification of the enzymes can include lysis of host cells, removal of cellular debris by centrifugation or precipitation, solubilization of proteins, column chromatography (e.g., size selection chromatography, ion exchange chromatography), retrieval of tagged enzymes using affinity chromatography, and combinations thereof. For example, in some cases the enzymes can be histidine-tagged and purified or semi-purified by Ni-NTA agarose or Ni-NTA columns. 
     Methods 
     Methods are described herein that are useful for synthesizing terpenoids and products made from terpenoids. The methods can involve contacting one or more of the substrates described herein with one or more enzymes capable of synthesizing at least one terpene to produce a terpenoid product. In some cases, the methods can involve incubating one or more of the substrates described herein with a population of host cells having a at least one heterologous expression cassette or expression vector that can express one or more enzymes capable of synthesizing at least one terpenoid product. The enzymes capable of synthesizing at least one terpenoid product can be referred to as a primary enzyme. The methods can also involve contacting the terpenoid product with a secondary enzyme that can modify the terpenoid product into another useful product. 
     For example, one method can involve contacting one or more of the substrates described herein with one or more enzymes capable of synthesizing at least one terpene to produce a terpenoid product. 
     For example, another method can involve (a) incubating a population of host cells or host tissue that includes one or more expression cassettes (or vectors) that have a promoter operably linked to a nucleic acid segment encoding an enzyme capable of synthesizing at least one terpene; and (b) isolating at least one terpenoid product from the population of host cells or the host tissue. 
     The enzymes can be any of the enzymes described herein. For example, the enzymes can be a monoterpene synthase, diterpene synthase, sesquiterpene synthase, sesterterpene synthase, triterpene synthase, tetraterpene synthase, or polyterpene synthase. Enzymes used for modifying a terpenoid product (e.g., secondary enzymes) can include one or more transcription factor, cytochrome P450, cytochrome P450 reductase, 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate-reducto-isomerase, cytidine 5′-diphosphate-methylerythritol (CDP-ME) synthetase (IspD), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF), geranylgeranyl diphosphate synthase (GGDPS), HMG-CoA synthase, HMG-CoA reductase (HMGR), mevalonic acid kinase (MVK), phosphomevalonate kinase (PMK), mevalonate-5-diphosphate decarboxylase (MPD), isopentenyl diphosphate isomerase (IDI), abietadiene synthase (ABS), farnesylpyrophosphate synthase (FPPS), ribulose bisphosphate carboxylase, squalene synthase (SQS), patchoulol synthase, or WRI1 protein; and (b) isolating lipids from the population of host cells, the host plant&#39;s cells, or the host tissue. In some cases, a combination of enzymes, transcription factors, and lipid droplet proteins can be expressed in host cells, host plant, or host tissues. 
     Definitions 
     As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also, as used herein, “and/or” refers to, and encompasses, any and all possible combinations of one or more of the associated listed items. Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. 
     The term “about”, as used herein, can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. 
     The term “enzyme” or “enzymes”, as used herein, refers to a protein catalyst capable of catalyzing a reaction. Herein, the term does not mean only an isolated enzyme, but also includes a host cell expressing that enzyme. Accordingly, the conversion of A to B by enzyme C should also be construed to encompass the conversion of A to B by a host cell expressing enzyme C. However, in some cases, purified or semi-purified enzymes are used to catalyze formation of terpenes within in vitro reactions. 
     The term “heterologous” when used in reference to a nucleic acid refers to a nucleic acid that has been manipulated in some way. For example, a heterologous nucleic acid includes a nucleic acid from one species introduced into another species. A heterologous nucleic acid also includes a nucleic acid native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous nucleic acids can include cDNA forms of a nucleic acid; the cDNA may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). For example, heterologous nucleic acids can be distinguished from endogenous plant nucleic acids in that the heterologous nucleic acids are typically joined to nucleic acids comprising regulatory elements such as promoters that are not found naturally associated with the natural gene for the protein encoded by the heterologous gene. Heterologous nucleic acids can also be distinguished from endogenous plant nucleic acids in that the heterologous nucleic acids are in an unnatural chromosomal location or are associated with portions of the chromosome not found in nature (e.g., the heterologous nucleic acids are expressed in tissues where the gene is not normally expressed). 
     The terms “identical” or percent “identity”, as used herein, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 97% identity, 98% identity, 99% identity, or 100% identity in pairwise comparison). Sequence identity can be determined by comparison and/or alignment of sequences for maximum correspondence over a comparison window, or over a designated region as measured using a sequence comparison algorithm, or by manual alignment and visual inspection. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence. 
     As used herein, a “native” nucleic acid or polypeptide means a DNA, RNA, or amino acid sequence or segment thereof that has not been manipulated in vitro, i.e., has not been isolated, purified, amplified and/or modified. 
     The terms “in operable combination,” “in operable order,” and “operably linked” refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a coding region (e.g., gene) and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. 
     As used herein the term “terpene” includes any type of terpene or terpenoid, including for example any monoterpene, diterpene, sesquiterpene, sesterterpene, triterpene, tetraterpene, polyterpene, and any mixture thereof. 
     As used herein, the term “wild-type” when made in reference to a gene refers to a functional gene common throughout an outbred population. As used herein, the term “wild-type” when made in reference to a gene product refers to a functional gene product common throughout an outbred population. A functional wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. 
     The following Examples illustrate some of the experimental work involved in development of the invention. 
     EXAMPLE 1 
     Method Overview 
     This Example summarizes methods that include synthesizing and screening an inventory of unnatural substrates producing novel decalin-core diphosphate intermediates and irregular terpene-like products. Preliminary results indicate that novel, structurally diverse unnatural diterpene substrates, mimicking the natural precursor, are accessible and can be processed to produce unnatural terpenes. 
     Initially, a panel of substrates with altered carbon number, inserted heteroatoms and rearranged linear and branched structures will be prepared. Class II diterpene synthases (diTPS) produce a characteristic decalin core intermediate. Screens are performed of functionally distinct class II diTPS enzymes with a panel of substrates ( FIG.  1 A ) to identify enzymes with substrate tolerance and capacity. As illustrated in  FIG.  1 B , class I diTPS directly produce irregular scaffolds, such as polycyclic diterpenes. Various enzymes can be used for bioprocessing of unnatural forskolin and jolkinol c compounds. Hence, sets of diTPS enzymes selected for functional diversity will be probed for their substrate tolerance to generate unnatural diterpene scaffolds. 
     Many class I diTPS of the decalin-core diterpene biosynthesis accept a range of class II intermediates, producing diverse products. The inventors have selected promiscuous class I diTPS and will examine their substrate tolerance against the unnatural class II intermediates. Products formed, for example as illustrated in  FIG.  1 B- 1 C , constitute a pilot library of diverse unnatural diterpene scaffolds. 
     EXAMPLE 2 
     Methods for Development of Substrates for Making Terpenoids 
     Modular pairs of diterpene synthases forming decalin core scaffolds were assembled through combinatorial biochemistry into new-to-nature pathways, yielding regioselective and stereoselective access to a panel of over 50 diterpene scaffolds, including novel compounds and those previously inaccessible. P450 enzymes were found to catalyze oxygenations of multiple substrates not native to the pathways and could also be substituted by enzymes from other species. Hence, our current repertoire of diTPS gives access to an estimated 75 scaffolds, while P450s, ACTs and ADHs can further modify each scaffold leading to an at least ten-fold diversification of possible diterpene pathways (see  FIG.  3   ). 
     A prototype pipeline was developed to generate chemically diversified and naturally inspired small molecules of the diterpene class at unprecedented chemical diversity (see  FIG.  3   ). Specifically, this required (i) establishment of a routine scheme for chemical synthesis of novel unnatural substrate derivatives of GGPP; (ii) combinatorial bioprocessing through a set of enzymes selected for their promiscuity; and (iii) iterative refinement of identified combinations of enzymes with their respective substrates. This process therefore involves test-learn-design cycles. 
     Over a dozen unnatural GGPP substrates were developed. The test-learn-design cycle informs further structural refinement of substrates, bringing the anticipated number to approximately 100 compounds. With its inherent building block principle, this strategy will be invaluable for high-throughput development of similar substrates for other classes of terpenoids and the resulting library of substrates will serve as a screening tool for future studies against an ever-expanding number of isolated enzymes. 
     EXAMPLE 3 
     Library of Unnatural Isoprenyl-Diphosphate Derivatized Substrates 
     This Example describes preparation of a library of unnatural isoprenyl-diphosphate derivatized substrates and screening a panel of class II labdane-type and class I macrocyclic, irregular-type diterpene synthases to advance mechanistic and structural understanding of the cationic cyclization cascades of these enzymes and to produce a collection of novel unnatural small molecules. 
     The diversity of substrates is synthetically explored that can be tolerated by the inventors&#39; expansive toolbox of class II and I diterpene synthases (diTPS). Initial findings will provide key data guiding more extensive investigation of features that influence the cationic cyclization cascade and an understanding of substrate features that are tolerated, to generate a wide diversity of previously unknown products. The goal is to prepare unnatural products using a diversity of structural motifs that would, upon cyclization generate novel structures. These cyclization precursors will then be tested/fed to both class I and class II enzymes and the products isolated and characterized. 
     A broad spectrum of GGPP unnatural substrates are initially be prepared, exploring both spatial and electronic considerations. Altered backbones manipulating carbon numbers, insertion of heteroatoms and shifting double bonds are of interest. These substrates will also be functionalized with halogens, oxygen, nitrogen, and sulfur. More than a dozen compounds have been synthesized. The test-learn-design cycle is used to identify subgroups of acceptable substrates for further subtle structural refinement will be applied. Substrates are prepared according to Scheme 1, shown below. 
     
       
         
         
             
             
         
       
     
     Recognizing that Scheme 1 generally shows activation of an allylic center and formation of the pyrophosphate, those of skill in the art should recognize that compounds such as those of the Formulae (III) and (IV) described herein can be accessed via the general methodology described in Scheme 1. 
     The substrate with a terminal allylic alcohol, substituted as described above, is prepared using methods described by Oberhauser et al. Angew. Chemie Int. Ed. 57, 11802-11806 (2018); Hoshino et al. Chem.—A Eur. J. 18: 13108-13116 (2012); Isaka et al. Biosci. Biotechnol. Biochem. 75: 2213-2222 (2011). The substrate with a terminal allylic alcohol is then converted via a simple two-step process (Davisson et al.  J. Org. Chem.  51, 4768-4779 (1986)) to generate the non-natural substrates. 
     EXAMPLE 4 
     Analysis of an Unnatural Methyl-Derivative of GGPP 
     A methyl-derivative of GGPP (‘unGGPP’) was synthesized as described in the previous Example. A comparison of the structures of GGPP and this methyl-derivative of GGPP (‘unGGPP’) is shown below. 
     
       
         
         
             
             
         
       
     
     DgTPS1 (casbene synthase) was reacted with the unGGPP substrate to yield a novel product with a shifted retention time as detected by gas chromatography (see  FIG.  5 A- 5 B ). The product had a mass consistent with a methyl-derivative of casbene ( FIG.  5 C- 5 D ). A conserved irregular, macrocyclic structure is consistent with the fragmentation pattern of the major fragments of casbene ( FIG.  5 C- 5 D ). 
     Systematic testing against five additional irregular-type diTPS indicated successful bioprocessing of this first substrate by three enzymes. The molecular mass and the fragmentation pattern of the products were consistent with unnatural diterpene-analogs. 
     A dozen modified unnatural substrates were synthesized and tested for conversion to unnatural products. The results indicated that the enzymes employed had broad substrate tolerance. With only two exceptions, chain and sidechain substituted derivatives were readily accepted and converted by select enzymes of both the class I irregular type and class II labdane-type diTPS. The conversion pattern across all enzymes indicated astounding levels of activity. A total of fifty-six products (possibly with some structural redundancy) were identified in 159 assays ( FIG.  6 A- 6 B ). 
     EXAMPLE 5 
     Screen of Unnatural Substrates Against 25 Class II Diterpene Synthases 
     Class II diTPS forming the decalin core labdanoid-type products catalyze cyclizations initiated by cation formation at carbon C 15  of the linear achiral isoprenyl diphosphate, retaining the diphosphate moiety, for example as shown below. 
     
       
         
         
             
             
         
       
     
     Shown is a typical cyclo-isomerization of GGPP into (4,13)-CLPP and ent-(8,13)-LPP by class II diTPS, where Ar refers to  Ajuga reptans,  and Pc refers to  Pogostemon cablin.    
     To assess substrate tolerance of class II diTPS, substrates are screened against a panel of twenty-five enzymes. Recombinant diTPS were expressed heterologously in  E. coli,  purified, and reconstituted in in vitro assays with the substrate to be tested. The products from the enzymatic action on the substrate were analyzed structural elucidation and downstream functionalization. 
     In particular, pET28b+plasmids containing N-terminally truncated diTPS variants (having the plastidial targeting signal removed to generate pseudomature enzymes) are transformed into  E. coli  BL-21DE3-C41 OverExpress cells. Cultures are grown at 37° C. and 180 rpm until the optical density at 600 nm reached 0.3 to 0.4. Cultures are cooled to 16° C., and expression is induced at an optical density at 600 nm of approximately 0.6 with 0.2 mM isopropylthiogalactoside. Cells are collected and lysed before purification of the His6-tagged enzymes with Ni-NTA columns. 
     A typical high-throughput in vitro diTPS assay in lml contained 5 μg substrate, 200 μg purified enzyme (class II plus class I for labdanoid-type diterpenes, or class I for irregular diterpenes), and 10mM buffer with magnesium. Reactions are carried out for 1 hour at 16° C., followed by vortexing with an equal volume of hexane to extract the products into the organic phase, prior to removal for GC/MS analysis. 
     Active enzyme/substrate combinations are validated by GC/MS analysis of the extract and products compared against references and authentic standards. Structural elucidation of novel products can be by NMR in some cases. The diphosphate intermediate can be converted by lysis to an alcohol for analysis, and the universally acting class I diTPS sclareol synthase from Salvia sclarea can be used for this purpose. 
     Reactions leading to novel compounds can be scaled up for structural elucidation. The scale-up procedure involved the same composition. However, in coupled assays of pairs of diTPS, the class II enzyme may be pre-incubated with substrate for two hours, before adding the class I diTPS. The diTPS enzymes exhibit excellent stability. Hence, the assays can be extended to overnight reactions to increase product yields, before extraction with hexane. 
     Results 
     Thirteen labdane-type diphosphate intermediates (partially redundant with intermediates made from substrate GGPP) were made by the twenty-five plant class II enzymes (see  FIG.  6 A- 6 B ): 
     ent-8,13-copalyl diphosphate (ent-CPP) 
     normal-(+)-copalyl diphosphate ((+)-CPP) 
     syn-copalyl diphosphate (syn-CPP) 
     (+)-8,13-copalyl diphosphate ((8,13)-CPP) 
     (5S,9S,10S)-labda-7,13Edienyl diphosphate((7,13)-LPP) 
     ent-(10R)-labda-8,13E-dienyl diphosphate (ent-(8,13)-LPP) 
     normal-H-labda-13-en-8-ol diphosphate ((+)-8-LPP) 
     peregrinol (labda-13-en-9-ol diphosphate (PGPP) 
     (−)-kolavenyl diphosphate (KPP) 
     (5R,8S,9S,10S)-labda-13-en-8-ol diphosphate (ent-8-LPP) 
     ent-neo-cis-transclerodienyl diphosphate (CT-CLPP) 
     (5R,8R,9S,10R)-neo-cleroda-4(18),13E-dienyl diphosphate ((4,13)-CLPP) 
     (+)-labden-9-ol diphosphate ((+)-9-LPP). 
     Approximately 100 substrate analogs will be generated. Based on preliminary results ( FIGS.  5  and  6   ), a significant number of these substrates will, upon testing provide diversified chemistries, novel structures and structural motifs not previously seen with known diTPS (products in the range of 100-200 compounds). Insights associated with the mechanistic details of how these enzymes operate in relation to the unnatural steric and electronic properties of the substrate, and structural information of which substrates are tolerated will guide the test-learn-design cycle. Specifically, after identification of well-accepted substrates, individual unnatural chemical features will be combined, and further subtle modifications will permit refining the substrates for iterative testing against a subset of diTPS identified as active and highly tolerant. 
     EXAMPLE 6 
     Screening of Unnatural Substrates Against 15 class I Irregular-Type Diterpene Synthases, Including 5 Macrocyclase- and Vulgarisane-Type Enzymes 
     Class I diTPS use a different chemical strategy for the initial carbocation formation. The diTPS initiate the cascade of cyclization into irregular, macrocyclic or polycyclic compounds by lysis of the isoprenoid diphosphate to yield an allylic cation at the opposite end of the substrate, carbon Ci and inorganic pyrophosphate, for example, as shown below. 
     
       
         
         
             
             
         
       
     
     Analogously to class II diTPS, the resulting carbocation intermediate further undergoes cyclo-isomerizations including hydride shifts, alkyl migrations and double bond rearrangements before termination of the reaction by proton abstraction or addition of a water molecule. In contrast to the paired modules of class II and I diTPS involved in formation of the labdanoid-type chemistry, irregular diterpenes are formed by the class I diTPS directly (Mau et al. Proc. Natl. Acad. Sci. 91: 8497 LP-8501 (1994)). 
     To explore unnatural substrate tolerance of the irregular diterpene formation, the inventors are screening all substrates produced in the library against a panel of six plant diTPS, including four macrocyclase-type and two polycyclic—type enzymes, followed by analysis of the products. 
     The products include the entry-step into the formation of jolkinol C, casbene and the closely related neo-cembrene, next to the structurally more complex taxadiene and hydroxyvulgarisane. 
     EXAMPLE 7 
     Screen of Class I Enzymes Against Substrates 
     The general function of class I enzymes of labdane-type diterpene metabolism is shared with those yielding irregular polycyclic diterpenes, i.e., generation of the initial carbocation at carbon C 1  by metal-dependent ionization. 
     Instead of accepting the acyclic GGPP, class I enzymes can use structurally diverse decalin-core diphosphate intermediates generated by class II enzymes. At this stage, additional cyclizations, double-bond-, hydride and alkyl shifts can occur, followed by either proton abstraction or quenching of the final carbocation through a water molecule. A panel of eight (seven plant and one microbial) class I labdane-type diTPS was selected for their demonstrated substrate promiscuity (Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Class I labdane-type diTPS and tested substrates converted 
               
            
           
           
               
               
            
               
                 Class I diTPS 
                 Substrate 
               
               
                   
               
               
                 SsSCS 
                 ent-, syn-, (+)-CPP, (+)-8-LPP, ent-8-LPP, KPP, 9-LPP 
               
               
                 CfTPS3 
                 syn-, (+)-CPP, (+)-8-LPP, ent-8-LPP, KPP, 9-LPP 
               
               
                 EpTPS8 
                 ent-, (+)-CPP, 9-LPP, KPP 
               
               
                 ArTPS3 
                 PgPP, (+)-CPP, (+)-8-LPP, ent-CPP 
               
               
                 OmTPS4 
                 PgPP, (+)-CPP, (+)-8-LPP, ent-CPP 
               
               
                 MvTPS5 
                 syn-, (+)-CPP, (+)-8-LPP, KPP, 9-LPP 
               
               
                 EpTPS1 
                 ent-CPP, ent-8-LPP 
               
               
                 KgTPS2 
                 ent-, syn-, (+)-CPP, (+)-8-LPP, ent-8-LPP, KPP, 9-LPP 
               
               
                   
               
               
                 Ss  Salvia sclarea ; 
               
               
                 Cf  Coleus forskohlii ; 
               
               
                 Ep  Euphorbia   peplus ; 
               
               
                 Ar  Ajuga reptans;   
               
               
                 Om  Origanum majoranum ; 
               
               
                 Mv  Marrubium vulgare ; 
               
               
                 Kg  Kitasatospora griseola . 
               
            
           
         
       
     
     All enzymes in Table 2 were functionally expressed. Microbial sequences were expressed as synthetic variants, expression optimized for  E. coli.  See also  FIG.  7   . 
     One example of an enzyme that can accept multiple unnatural substrates is CfTps2, which the inventors have demonstrated can provide the first step in synthesis of the cardiac stimulant and cognition enhancer forskolin. CfTps2 derived from  Coleus forskohiii  (also referred to as  Plectranthus barbatus ), an is shown below as SEQ ID NO:69. 
     
       
         
           
               
               
               
               
               
            
               
                 1 
                 MKMLMIKSQE 
                 RVHSIVSAWA 
                 NNSNKRQSLG 
                 HQIRRKQRSQ 
               
               
                   
               
               
                 41 
                 VTECRVASLD 
                 ALNGIQKVGP 
                 ATIGTPEEEN 
                 KKIEDSIEYV 
               
               
                   
               
               
                 81 
                 KELLKTMGDG 
                 RISVSPYDTA 
                 IVALIKDLEG 
                 GDGPEFPSCL 
               
               
                   
               
               
                 121 
                 EWIAQNQLAD 
                 GSWGDHFFCI 
                 YDRVVNTAAC 
                 VVALKSWNVH 
               
               
                   
               
               
                 161 
                 ADKIEKGAVY 
                 LKENVHKLKD 
                 GKIEHMPAGF 
                 EFVVPATLER 
               
               
                   
               
               
                 201 
                 AKALGIKGLP 
                 YDDPFIREIY 
                 SAKQTRLTKI 
                 PKGMIYESPT 
               
               
                   
               
               
                 241 
                 SLLYSLDGLE 
                 GLEWDKILKL 
                 QSADGSFITS 
                 VSSTAFVFMH 
               
               
                   
               
               
                 281 
                 TNDLKCHAFI 
                 KNALTNCNGG 
                 VPHTYPVDIF 
                 ARLWAVDRLQ 
               
               
                   
               
               
                 321 
                 RLGISRFFEP 
                 EIKYLMDHIN 
                 NVWREKGVFS 
                 SRHSQFADID 
               
               
                   
               
               
                 361 
                 DTSMGIRLLK 
                 MHGYNVNPNA 
                 LEHFKQKDGK 
                 FTCYADQHIE 
               
               
                   
               
               
                 401 
                 SPSPMYNLYR 
                 AAQLRFPGEE 
                 ILQQALQFAY 
                 NFLHENLASN 
               
               
                   
               
               
                 441 
                 HFQEKWVISD 
                 HLIDEVRIGL 
                 KMPWYATLPR 
                 VEASYYLQHY 
               
               
                   
               
               
                 481 
                 GGSSDVWIGK 
                 TLYRMPEISN 
                 DTYKILAQLD 
                 FNKCQAQHQL 
               
               
                   
               
               
                 521 
                 EWMSMKEWYQ 
                 SNNVKEFGIS 
                 KKELLLAYFL 
                 AAATMFEPER 
               
               
                   
               
               
                 561 
                 TQERIMWAKT 
                 QVVSRMITSF 
                 LNKENTMSFD 
                 LKIALLTQPQ 
               
               
                   
               
               
                 601 
                 HQINGSEMKN 
                 GLAQTLPAAF 
                 RQLLKEFDKY 
                 TRHQLRNTWN 
               
               
                   
               
               
                 641 
                 KWLMKLKQGD 
                 DNGGADAELL 
                 ANTLNICAGH 
                 NEDILSHYEY 
               
               
                   
               
               
                 681 
                 TALSSLTNKI 
                 CQRLSQIQDK 
                 KMLEIEEGSI 
                 KDKEMELEIQ 
               
               
                   
               
               
                 721 
                 TLVKLVLQET 
                 SGGIDRNIKQ 
                 TFLSVFKTFY 
                 YRAYHDAKTI 
               
               
                   
               
               
                 761 
                 DAHIFQVLFE 
                 PW 
                   
                   
               
            
           
         
       
     
     The CfTps2 enzyme can also serve as the first step in production of sclareol, manoyl oxide and structurally related compounds which are industrial precursors for ambroxoid fragrance substances. 
     Similarly, the neo-cleroda-4(18),13E-dienyl diphosphate synthase, which affords entry into a class of insect-antifeedants ArTPS2 is of particular interest for applications in agricultural biotechnology. Neo-clerodane diterpenoids, particularly those with an epoxide moiety at the 4(18) position, such as clerodin, the ajugarins, and the jodrellins have garnered significant attention for their ability to deter insect herbivores. The 4(18) desaturated product of ArTPS2 could be used in biosynthetic or semisynthetic routes to these potent insect antifeedants (BRH: compound 38, below). 
     
       
         
         
             
             
         
       
     
     The ability to modify, in a targeted manner, these biological active or industrially significant natural products would facilitate the design, testing, and production of novel materials and biologically active agents. 
     EXAMPLE 8 
     Enzymatic Pathway to Jolkinol C 
     Genetic information was used to reconstruct the pathways to the pharmacologically active cyclic AMP booster forskolin, and jolkinol C ( FIG.  8   ), which are precursors of phorbol esters drugs with unique anti-cancer, anti-HIV and analgesic activities. 
     For example, the inventors have described a CYP726A27 from  Euphorbia lathyris,  which has the following sequence (SEQ ID NO:70). 
     
       
         
           
               
               
               
               
               
            
               
                 1 
                 MDLQLQIPSY 
                 PIIFSFFIFI 
                 FMLIKIWKKQ 
                 TQTSIFPPGP 
               
               
                   
               
               
                 41 
                 FKFPIVGNIP 
                 QLATGGTLPH 
                 HRLRDLAKIY 
                 GPIMTIQLGQ 
               
               
                   
               
               
                 81 
                 VKSVVISSPE 
                 TAKEVLKTQD 
                 IQFADRPLLL 
                 AGEMVLYNRK 
               
               
                   
               
               
                 121 
                 DILYGTYGDQ 
                 WRQMRKICTL 
                 ELLSAKRIQS 
                 FKSVREKEVE 
               
               
                   
               
               
                 161 
                 SFIKTLRSKS 
                 GIPVNLTNAV 
                 FELTNTIMMI 
                 TTIGQKCKNQ 
               
               
                   
               
               
                 201 
                 EAVMSVIDRV 
                 SEAAAGFSVA 
                 DVFPSLKFLH 
                 YLSGEKTKLQ 
               
               
                   
               
               
                 241 
                 KLHKETDQIL 
                 EEIISEHKAN 
                 AKVGAQADNL 
                 LDVLLDLQKN 
               
               
                   
               
               
                 281 
                 GNLQVPLTND 
                 NIKAATLEMF 
                 GAGSDTSSKT 
                 TDWAMAOMMR 
               
               
                   
               
               
                 321 
                 KPTTMKKAQE 
                 EVRRVFGENG 
                 KVEESRIQEL 
                 KYLKLVVKET 
               
               
                   
               
               
                 361 
                 LRLHPAVALI 
                 PRECREKTKI 
                 DGFDIYPKTK 
                 ILVNPWAIGR 
               
               
                   
               
               
                 401 
                 DPKVWNEPES 
                 FNPERFQDSP 
                 IDYKGTNFEL 
                 IPFGAGKRIC 
               
               
                   
               
               
                 441 
                 PGMTLGITNL 
                 ELFLANLLYH 
                 FDWKFPDGIT 
                 SENLDMTEAI 
               
               
                   
               
               
                 481 
                 GGAIKRKLDL 
                 ELISIPYTSS 
                   
                   
               
            
           
         
       
     
     The inventors have also described a CYP71D445 from  Euphorbia lathyris,  which has the following sequence (SEQ ID NO:71). 
     
       
         
           
               
               
               
               
               
            
               
                 1 
                 MELEFRSPSS 
                 PSEWAITSTI 
                 TLLFLILLRK 
                 ILKPKTPTPN 
               
               
                   
               
               
                 41 
                 LPPGPKKLPL 
                 IGNIHQLIGG 
                 IPHQKMRDLS 
                 QIHGPIMHLK 
               
               
                   
               
               
                 81 
                 LGELENVIIS 
                 SKEAAEKILK 
                 THDVLFAQRP 
                 QMIVAKSVTY 
               
               
                   
               
               
                 121 
                 DEHDITFSPY 
                 GDYWRQLRKI 
                 TMIELLAAKR 
                 VLSFRAIREE 
               
               
                   
               
               
                 161 
                 ETTKLVELIR 
                 GFQSGESINF 
                 TRMIDSTTYG 
                 ITSRAACGKI 
               
               
                   
               
               
                 201 
                 WEGENLFISS 
                 LEKIMFEVGS 
                 GISFADAYPS 
                 VKLLKVFSGI 
               
               
                   
               
               
                 241 
                 RIRVDRLQKN 
                 IDKIFESIIE 
                 EHREERKGRK 
                 KGEDDLDLVD 
               
               
                   
               
               
                 281 
                 VLLNLQESGT 
                 LEIPLSDVTI 
                 KAVIMDMFVA 
                 GVDTSAATTE 
               
               
                   
               
               
                 321 
                 WLMSELIKNP 
                 EVMKKAQAEI 
                 REKFKGKASI 
                 DEADLQDLHY 
               
               
                   
               
               
                 361 
                 LKLVIKETFR 
                 LHPSVPLLVP 
                 RECRESCVIE 
                 GYDIPVKTKI 
               
               
                   
               
               
                 401 
                 MVNAWAMGRD 
                 TKYWGEDAEK 
                 FKPERFIDSP 
                 IDFKGHNFEY 
               
               
                   
               
               
                 441 
                 LPFGSGRRSC 
                 PGMAFGVANV 
                 EIAVAKLLYH 
                 FDWRLGDGMV 
               
               
                   
               
               
                 481 
                 PENLDMTEKI 
                 GGTTRRLSEL 
                 YIIPTPYVPQ 
                 NSA 
               
            
           
         
       
     
     
       
         
         
             
             
         
       
     
     As illustrated, GGPP is cyclized to the irregular diterpene scaffold Casbene, which is subsequently oxidized and further re-arranged by P450 enzymes and an ADH1. All the functionalization enzymes involved are inherently promiscuous. 
     EXAMPLE 9 
     Selective Exploration of Substrate Tolerance of Two Model Pathways Functionalizing Bioactive Labdane-Type and Irregular, Macrocyclic Diterpenes 
     The inventors have earlier established the metabolic pathway for oxidative functionalization of casbene to jolkinol C within  Euphorbia  ( FIG.  8   ) and they have established functional yeast ( S. cerevisiae ) lines expressing the complete pathways from sugar to the labdane-type diterpene forskolin (40 mg/L), as illustrated below. 
     
       
         
         
             
             
         
       
     
     Yeast lines expressing the corresponding characterized functionalization pathways only, i.e., P450s, ACTs and ADHs, can be supplemented with natural untested, and unnatural diterpenes synthetic analogs. Products and intermediates can be purified through the procedures described herein. The structurally elucidated products so generated can include rationally designed derivatives that are not accessible through formal synthesis. Analogs of forskolin are of high interest for their specificity to interact with the specific subgroups of adenylate cyclase, while jolkinol C analogs, not being an immediate pharmaceutical candidate, based on current knowledge, can serve as lead compounds for further chemical diversification. 
     EXAMPLE 10 
     Use of Natural and Unnatural Diterpene Scaffolds in Biosynthetic Routes for the Labdane-Type Forskolin and Non-Labdane Type Ingenol Therapeutics 
     The inventors have shown highly efficient conversion of labdane-type, synthetic diterpenes, by yeast cell lines expressing P450 enzymes (Hamberger et al. Plant Physiol. 157: 1677-1695 (2011)). See  FIG.  9 A- 9 C . The enzymes also showed conversion of non-native (yet natural) diterpenes into the corresponding oxidized forms, in the limited range where tested. Analogously, an acyl transferase was identified, which indiscriminately converted accessible alcohols into the corresponding acetyl-esters of forskolin (Pateraki et al. Elife 6 (2017). 
     Yeast cell lines are generated in the industrial strain CEN.PK (CEN.PK2-1C, MATa; his3D1; leu2-3_112; ura3-52; trpl-289; MAL2-8c, SUC2; Entian et al. Methods in Microbiology 36: 629-666 (2007)) that exhibited several advantages, including improved transformability and high tolerance for functionalized terpenoids. Also, the EasyClone 2.0 set of integrative vectors can be used as appropriate for over-expression of heterologous genes in industrial yeast strains. The vectors allow for selection in auxotrophic yeast strains (four different selection markers) and can carry two genes each, which allows for generation of multigene pathways. As the compounds are supplemented to the cultures, this project will not require engineering of the diterpene scaffold biosynthesis, significantly simplifying the generation of yeast strains. P450s, the corresponding cytochrome P450 reductase and enzymes encoding downstream functionalization steps can be stably, chromosomally integrated and driven by various promoters, including constitutive promoters. Isolation of products and analysis can be adapted to the physicochemical properties of the molecules. LC/MS can be used for analysis to offset problems with increasing oxygenation and the increased polarity of products. 
     EXAMPLE 11 
     Bioactivity of Unnatural Forskolin and Related Intermediate Labdane-Type Products with Adenylyl Cyclase 
     Forskolin derivatives are tested for their activity at a representative of each of the three families of membrane adenylyl cyclase (AC1, AC2, and AC5; Dessauer et al.  Pharmacol. Rev.  69: 93 LP-139 (2017)). Activation of AC1 could be a potential cognition enhancing target while inhibition may be beneficial in Fragile X syndrome—a genetic autism syndrome. Counter-screens can be done to assess selectivity against AC2 and AC5. Activation of AC5 would be expected to mediate cardiovascular side effects and inhibition may be beneficial. Forskolin itself activates all subtypes of AC so identifying novel derivatives that show selective activation of AC1 without stimulating AC2 or AC5 would be of significant interest. A full exploration of AC drug discovery is beyond the scope of this technology development grant application, but this section will provide initial proof-of-concept results to show potential value of our synthetic biology compound library approach in rationally designing specificity into a known terpenoid AC modulator, forskolin. 
     AC activity can be tested, as described by Feng et al. Neurology 89: 762 LP-770 (2017) with enhancements made possible by a novel ACA3/6 HEK cell line (Doyle et al. Biochem. Pharmacol. 163: 169-177 (2019)). The inventors can perform subtype-enriched cell-based assays using HEK293 cells transfected with AC1, AC2, and AC5. Cells with vector control plasmid or with plasmids for AC1, 2, or 5 can be stimulated with various concentrations of forskolin analogs (100 nM-30 μM) in the presence of the general PDE inhibitor IBMX. cAMP production can be assessed using the LANCE Ultra cAMP kit (Perkin Elmer; Waltham, Mass.) which is based on a TR-FRET detection method as described by Feng et al. (Neurology 89: 762 LP-770 (2017), see supplement). ACD73/6 HEK-293 cells transfected as indicated above are dissociated from dishes using Versene on the day of experiment. Two thousand cells cells/well in 5 μlin white 384-well microplate (Perkin Elmer) are incubated with various concentrations of forskolin or analogs for 30 min at room temperature. DMSO (0.1%) will be included in all samples for control and forskolin analogs. A cAMP standard curve was generated in triplicate according to the manual. Finally, europium (Eu)-cAMP tracer (54) and ULight™-anti-cAMP (54) were added to each well and incubated for lh at room temperature. Plates will be read on a TR-FRET microplate reader (Synergy NEO; Biotek, Winooski, Vt.) in the MSU Assay Development and Drug Repurposing Core. 
     Data analysis for forskolin-analog concentration-response curves can include background subtraction of activity in mock-transfected cells to estimate AC1, 2, or 5 specific activity. The resulting curves will be analyzed by non-linear least squares regression analysis to a 4-parameter logistic equation (R min , R max , —logEC 50  e.g. pEC50, and n H ) using GraphPad Prism, as described by Feng et al. (Neurology 89: 
     762 LP-770 (2017). Where curves are well-defined, the pEC 50  values for AC1, AC2, and AC5 as well as Rmax values are compared. Where curves may not provide a clear pEC50 value, major differences in R max  can be noted. Significant selectivity can be defined as a 5-fold or greater differential potency (based on pEC50 values) or 5-fold or greater R max  value for the chosen AC subtype. In addition to testing for AC activation, the inventors can also test for AC inhibition. Cells will be activated by a forskolin concentration that produces approximately 30% activation (ca. 1 μM) in the presence of increasing concentrations of the forskolin analogs. Any identified selective activators or any derivatives that significantly inhibit AC subtype activity can be tagged for future follow-up studies in receptor-regulated AC activity in HEK or native cells and in WT and AC-subtype KO animals (beyond the scope of the present application). 
     The catalytic capacities can be determined through gas chromatography and LC-MS analysis of products, i.e., substrate tolerance of entire assembled pathways, which will provide unique mechanistic insights (flux through the pathway, intermediates will indicate the order of conversion, potential steric/electronic hindrance). Hence, novel bioactive labdane and non-labdane type diterpenes can be identified. Structural elucidation of the products of biological interest can be performed using the procedures detailed herein. Analysis of their biological activity against a representative of adenylyl cyclases, either activation, or inhibition is expected to provide valuable data for structural refinement and is of pharmacological relevance. 
     EXAMPLE 12 
     {[(2E,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2Z,6E, 10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     Ethyl (2E,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraenoate and ethyl (2Z,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraenoate. A 25.0 mL 14/20 round bottom flask was charged with sodium hydride (0.344 g, 8.60 mmol), tetrahydrofuran (4.00 mL) was, under an argon atmosphere at 0° C., was treated with triethyl-2-phosphonopropionate (2.05 g, 8.60 mmol) dissolved in tetrahydrofuran (1.00 mL). Once gas evolution ceased, farnesyl acetone (2.25 g, 8.60 mmol) was added, dissolved in tetrahydrofuran (1.00 mL), and the mixture heated to 45° C. for 24 hours. The mixture was cooled to 0° C., quenched with water, and partitioned into ethyl acetate. The organic layer was then washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was dissolved in ethanol (20.0 mL) and cooled to 0° C. Sodium borohydride, to reduce any remaining ketone to ease purification, was added (0.312 g, 8.30 mmol) and the mixture was stirred for 1 hour at room temperature, cooled to 0° C. and quenched with 1.00 N hydrochloric acid. The reaction mixture was concentrated in vacuo and partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The product was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield a mixture of ethyl (2E,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraenoate and ethyl (2Z,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraenoate (1.50 g, 50%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.18-5.04 (m, 3H), 4.22-4.13 (m, 2H), 2.35 (dd, J=9.6, 6.4 Hz, 1H), 2.20-2.02 (m, 9H), 2.02-1.94 (m, 5H), 1.89-1.82 (m, 3H), 1.78 (d, J=3.3 Hz, 1H), 1.68 (s, 5H), 1.60 (d, J=6.5 Hz, 7H), 1.29 (td, J=7.2, 4.0 Hz, 3H). 
     (2E,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraen-1-ol and (2Z,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraen-1-ol. A 50.0 mL 24/40 round bottom flask was charged with a mixture of ethyl (2E,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraenoate and ethyl (2Z,6E,10E)-2,3 ,7,11,15 -pentamethylhexadeca-2,6,10,14-tetraenoate (1.10 g, 3.17 mmol), dichloromethane (15.0 mL) and on cooling to 0° C. (argon atmosphere) was treated with a 1.00 M solution of diisobutylaluminum hydride (12.7 mL, 12.7 mmol) in heptanes. The reaction was stirred for 24 hours, the mixture allowed to warm to room temperature then quenched with ethanol (2.00 mL). A solution of sodium potassium tartrate was added (4.50 g, 15.9 mmol in 20.0 mL water) and the biphasic mixture stirred vigorously for 24 hours. The product was then extracted with dichloromethane, the organic layers combined, dried over sodium sulfate, filtered, and concentrated in vacuo to an oil used without further purification (0.722 g, 75%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.18-5.06 (m, 3H), 4.15-4.05 (m, 2H), 2.20-1.91 (m, 12H), 1.76 (ddd, J=11.4, 3.0, 1.5 Hz, 4H), 1.71-1.66 (m, 6H), 1.64-1.58 (m, 8H). HRMS ESI (+) calc&#39;d for [M+Na]=327.2664, found=327.2662. 
     {[(2E,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2Z,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate. A 100 mL 24/40 round bottom flask was charged with a mixture of (2E,6E,10E)-2,3,7,11,15-pentamethylhexadeca-2,6,10,14-tetraen-1-ol and (2Z,6E,10E)-2,3 ,7,11,15 -pentamethylhexadeca-2,6,10,14-tetraen-1-ol (0.300 g, 1.00 mmol), diethyl ether (5.00 mL) and, under an argon atmosphere, at 0° C. phosphorus tribromide (0.0500 mL, 0.500 mmol) added as a solution in diethyl ether (1.00 mL). After 15 minutes the mixture was diluted with hexanes, washed with brine, sodium bicarbonate and brine, dried over sodium sulfate, filtered, and concentrated in vacuo to dryness as an oil. The residue was redissolved in acetonitrile (5.00 mL), under an argon atmosphere, and treated with tetrabutylammonium pyrophosphate (2.10 g, 2.30 mmol). After 2 hours the reaction mixture was concentrated in vacuo to a viscous liquid and purified on a DOWEX50 column prepared by first stirring the resin (8.70 g) in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL), suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate mixture) and poured into a column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude product applied to the column (dissolved in 3.00 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.550 g, 100%).  31 P NMR (202 MHz, Deuterium Oxide) δ −8.57, −10.48 (d, J=20.2 Hz). HRMS ESI [M—H] calcd=463.2020, observed=463.2038. 
     EXAMPLE 13 
     {[(2Z,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2E,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate. 
     
       
         
         
             
             
         
       
     
     Ethyl (2Z,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2E,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate. A 50.0 mL 14/20 round bottom flask was charged with sodium hydride (0.705 g, 21.0 mmol), tetrahydrofuran (20.0 mL) and at 0° C. (argon atmosphere) was added triethyl-2-fluoro-phosphonoacetate (4.84 g, 20.0 mmol) dissolved in tetrahydrofuran (5.00 mL) via syringe. Once gas evolution ceased, farnesyl acetone (2.62 g, 10 0 mmol) was added as a solution in tetrahydrofuran (1.00 mL). The mixture was heated to 45° C. for 22 hours, then concentrated and partitioned between ethyl acetate and 1.00 N hydrochloric acid. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield the pure product as a mixture of cis and trans isomers ethyl (2Z,6E,10E)-2-fluoro-3,7,11,15 -tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2E,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate (3.43 g, 98%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.18-5.04 (m, 3H), 4.38-4.21 (m, 3H), 4.11 (p, J=7.2 Hz, 1H), 2.58-2.48 (m, 1H), 2.25 (tt, J=8.8, 4.6 Hz, 1H), 2.16 (dq, J=14.1, 7.0 Hz, 2H), 2.11-2.00 (m, 7H), 1.97 (q, J=7.9 Hz, 3H), 1.86 (d, J=4.3 Hz, 2H), 1.68 (d, J=3.7 Hz, 5H), 1.60 (t, J=4.6 Hz, 7H).  19 F NMR (470 MHz, CDCl 3 ) δ −126.96 (dd, J=14.3, 4.8 Hz), −128.66-−128.97 (m). 
     (2Z,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. A 50.0 mL 24/40 round bottom flask was charged with a mixture of ethyl (2Z,6E,10E)-2-fluoro-3,7,11,15 -tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2E,6E,10E)-2-fluoro-3,7,11,15 -tetramethylhexadeca-2,6,10,14-tetraenoate (2.31 g, 6.50 mmol), dichloromethane (20.0 mL) and under an argon atmosphere at 0° C. was added diisobutylaluminum hydride (27.0 mL, 27.0 mmol, 1.00 M in heptanes). The reaction was stirred for 18 hours, warming to room temperature, then quenched with ethanol (5.00 mL) and a solution of sodium potassium tartrate was added (7.00 g, 24.8 mmol in 50.0 mL water). The biphasic mixture was stirred vigorously for 24 hours. The mixture was partitioned in a separatory funnel and the aqueous layer washed with dichloromethane. The organic layers were combined, dried over sodium sulfate, filtered, and concentrated in vacuo to an oil. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield the products (2Z,6E,10E)-2-fluoro-3,7,11,15 -tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol as a mixture of cis and trans isomers (0.859 g, 43%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.11 (dt, J=14.5, 7.5 Hz, 3H), 4.22 (dd, J=22.4, 16.6 Hz, 2H), 4.11 (p, J=7.2 Hz, 1H), 2.05 (tdd, J=33.1, 30.9, 10.8, 4.5 Hz, 12H), 1.69 (q, J=3.6, 3.1 Hz, 7H), 1.60 (t, J=3.2 Hz, 8H).  19 F NMR (470 MHz, CDCl 3 ) δ −119.15 -−120.01 (m), −121.10-−121.57 (m). HRMS ESI (+) calc&#39;d for [M+Na]=331.2412, found=331.2442. 
     {[(2Z,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2E,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with a mixture of (2Z,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-fluoro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol (0.200 g, 0.640 mmol), diethyl ether (5.00 mL) and at 0° C., under an argon atmosphere, was added phosphorus tribromide (0.270 g, 1.00 mmol) dissolved in diethyl ether (1.00 mL). After 15 minutes, the reaction was partitioned between hexanes and brine. The organic layer was washed with sodium bicarbonate, brine, dried over sodium sulfate, filtered, and concentrated in vacuo to an oil. This crude mixture of isomers was dissolved in acetonitrile (2.00 mL), under an argon atmosphere, and treated with tetrabutylammonium pyrophosphate (0.904 g, 1.00 mmol). The reaction mixture was stirred for 2 hours, concentrated in vacuo to a viscous liquid and purified over DOWEX50 (9.40 g) resin. The resin was prepared by first stirring the DOWEX50 in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was then filtered and washed four times with water (100 mL), then suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude product material applied to the top of the column (dissolved in 3.00 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.250 g, 84%).  31 P NMR (202 MHz, Deuterium Oxide) δ −9.34, − 11 . 34 .  19 F NMR (470 MHz, D 2 O) δ −117.52 (d, J=133.4 Hz), −118.74 (d, J=144.3 Hz). HRMS ESI [M−H] calcd=467.1769, observed=467.1786. 
     EXAMPLE 14 
     {[(2E,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2Z,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate. 
     
       
         
         
             
             
         
       
     
     Ethyl (2E,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2Z,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate. A 25.0 mL 14/20 round bottom flask was charged with sodium hydride (0.839 g, 34.9 mmol), tetrahydrofuran (20.0 mL) was, under argon atmosphere at 0° C., charged with triethyl phosphonobutyrate (4.89 g, 19.4 mmol) dissolved in tetrahydrofuran (2.00 mL). Once gas evolution ceased farnesyl acetone (1.05 g, 4.00 mmol) was added as a solution in tetrahydrofuran (2.00 mL). The reaction mixture was heated to 45° C. for 170 hours, cooled to 0° C., quenched with water and partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to provide a mixture of ethyl (2E,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2Z,6E,10E)-2-ethyl-3,7,11,15 -tetramethylhexadeca-2,6,10,14-tetraenoate, in a 1 to 1 mixture, and unreacted farnesyl acetone. The crude mixture was dissolved in ethanol (20.0 mL), cooled to 0° C. and unreacted farnesyl acetone was reduced with sodium borohydride (0.230 g, 6.20 mmol) to ease purification. The reaction mixture was stirred for 1 hour, allowed to warm to room temperature, cooled to 0° C. and quenched with 1.00 N hydrochloric acid. The reaction mixture was concentrated in vacuo, partitioned between ethyl acetate and water, the organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The mixture was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to yield a mixture of cis and trans isomers, ethyl (2E,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2Z,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate (0.770 g, 31%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.12 (dt, J=13.8, 6.0 Hz, 3H), 4.24-4.07 (m, 2H), 2.35-2.21 (m, 3H), 2.19-1.88 (m, 12H), 1.78 (s, 1H), 1.68 (s, 5H), 1.60 (d, J=5.0 Hz, 8H), 1.29 (td, J=7.1, 5.4 Hz, 3H), 1.04-0.80 (m, 3H). 
     (2E,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2Z,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. A 50.0 mL 24/40 round bottom flask was charged with a mixture ethyl (2E,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2Z,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate (0.720 g, 2.00 mmol), dichloromethane (20.0 mL) and at 0° C., under an argon atmosphere, treated with diisobutylaluminum hydride (10.0 mL, 10.0 mmol, 1.00 M in heptanes). The mixture was stirred for 18 hours, warming to room temperature. The mixture was again cooled to 0° C., quenched with ethanol (2.00 mL), and a solution of sodium potassium tartrate added (7.10 g, 24.8 mmol in 50.0 mL water) and the biphasic mixture vigorously stirred for 24 hours. The reaction mixture was partitioned, and the aqueous layer washed with dichloromethane. The organic layers were combined, dried over sodium sulfate, filtered, and concentrated in vacuo to an oil. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield an oil (mixture of cis and trans isomers, 0.622 g, 98%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.12 (dttd, J=12.5, 5.5, 2.8, 1.4 Hz, 3H), 4.17-4.06 (m, 2H), 2.22-2.12 (m, 3H), 2.08 (tq, J=10.7, 6.2, 5.1 Hz, 8H), 2.02-1.94 (m, 3H), 1.77 (s, 1H), 1.73-1.67 (m, 6H), 1.65-1.57 (m, 8H), 1.01 (qd, J=7.9, 5.5 Hz, 3H), 0.94-0.80 (m, 2H). HRMS ESI (+) calc&#39;d for [M+Na]=341.2820, found=341.2816. 
     {[(2E,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2Z,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with a mixture of (2E,6E,10E)-2-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2Z,6E,10E)-2-ethyl-3,7,11,15 -tetramethylhexadeca-2,6,10,14-tetraen-1-ol (0.311 g, 1.00 mmol) and anhydrous diethyl ether (5.00 mL). The reaction vessel was sealed, flushed with argon, cooled to 0° C. and phosphorus tribromide (0.405 g, 1.50 mmol) was added dissolved in diethyl ether (1.00 mL). After 15 minutes, the reaction was partitioned between hexanes and brine. The organic layer was then washed with sodium bicarbonate, brine, dried over sodium sulfate, and concentrated in vacuo to dryness as an oil. To this material was added acetonitrile (2.00 mL) and tetrabutylammonium pyrophosphate (0.585 g, 0.645 mmol). The reaction vessel was sealed and stirred, under an argon atmosphere, for 2 hours, then concentrated to a viscous liquid and purified over DOWEX50 resin column. The column was prepared by stirring DOWEX50 resin (8.50 g) in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.230 g, 48%).  31 P NMR (202 MHz, Methanol-d 4 ) δ −5.96, −9.82 (d, J=19.2 Hz). HRMS ESI [M−H] calcd.=477.2177, observed=477.2190. 
     EXAMPLE 15 
     {[(5E,9E)-6,10,14-trimethyl-2-oxopentadeca-5,9,13-trien-1-yl phosphonato]oxy}phosphonate. 
     
       
         
         
             
             
         
       
     
     {[(5E,9E)-6,10,14-trimethyl-2-oxopentadeca-5,9,13-trien-1-yl phosphonato]oxy} phosphonate. Using a reported procedure (Hu, T.; Corey, E. J.;  Org. Lett.,  2002, 4, 2441) a 25.0 mL 14/20 round bottom flask was charged with farnesyl acetone (0.524 g, 2.00 mmol), dichloromethane (32.0 mL), and cooled to 0° C. (argon atmosphere). Diisopropylethylamine (1.55 g, 12.0 mmol) was added, followed by trimethylsilyl triflate (1.77 g, 6.00 mmol) and the mixture stirred at 0° C. for 1.5 hours and then quenched by the addition of sodium bicarbonate. The mixture was extracted with hexanes, the organic layers combined, dried over sodium sulfate, filtered, and concentrated to yield an oil (0.720 g). The crude material was dissolved in tetrahydrofuran (40.0 mL) and solid sodium bicarbonate (0.189 g, 2.25 mmol) was added. The mixture was cooled to −78° C. and, under an argon atmosphere, n-bromosuccinimide (0.371 g, 2.10 mmol) added. The reaction mixture was stirred for 2 hours at −78° C., warmed to room temperature, filtered, and concentrated to yield the crude as a 1:4 mixture of starting material and bromide product (0.572 g, 55%). The crude product was dissolved in acetonitrile (3.00 mL) and tetrabutylammonium pyrophosphate (1.23 g, 1.30 mmol) added. The reaction was stirred at room temperature, under an argon atmosphere for 2 hours, concentrated and purified over DOWEX50 resin according to the following method. DOWEX50 resin (11.8 g) was stirred in concentrated ammonium hydroxide (40.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 millimolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25.0 millimolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25.0 millimolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL of the 1:49 2-propanol: 25.0 millimolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.692 g, 68%).  31 P NMR (202 MHz, Deuterium Oxide) δ −5.91, −10.60. HRMS ESI [M−H] calcd=437.1500, observed=437.1511. 
     EXAMPLE 16 
     {[2-({[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}methyl)prop-2-en-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     2-({ [(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}methyl)prop en-1-ol. A 25.0 mL 14/20 round bottom flask was charged with trans, trans-farnesol (0.889 g, 4.00 mmol), diethyl ether (8.00 mL) and at 0° C. was, under an argon atmosphere, added phosphorus tribromide (1.35 g, 5.00 mmol) dissolved in diethyl ether (1.00 mL). After 1 hour the reaction mixture was diluted with hexanes, washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield trans, trans-farnesyl bromide. A separate 25.0 mL 14/20 round bottom flask was charged with sodium hydride (0.336 g, 10.0 mmol), tetrahydrofuran (8.00 mL) and at to 0° C., under an argon atmosphere, 2-methylidenepropane-1,3-diol (0.704 g, 8.00 mmol) was added in a dropwise fashion. Once gas evolution had ceased, the trans, trans-farnesyl bromide was added (dissolved in 3.00 mL tetrahydrofuran). The reaction was heated to 45° C. for 19 hours, quenched with saturated aqueous ammonium chloride (10.0 mL) and partitioned with ethyl acetate. The crude material was purified by silica gel chromatography (10-100% ethyl acetate in hexanes) to yield the pure product as an oil (0.900 g, 77%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.82 (dddd, J=12.6, 7.7, 4.6, 1.4 Hz, 1H), 5.72 (dtd, J=11.1, 6.1, 1.3 Hz, 1H), 5.35 (ddt, J=6.9, 5.5, 1.3 Hz, 1H), 5.14-5.05 (m, 2H), 4.20 (d, J=6.3 Hz, 2H), 4.06-4.03 (m, 2H), 4.01 (d, J=6.9 Hz, 2H), 2.10 (dd, J=14.5, 6.9 Hz, 3H), 2.07-2.01 (m, 5H), 1.97 (dd, J=9.1, 6.2 Hz, 3H), 1.67 (s, 6H), 1.59 (s, 6H). HRMS ESI (+) calc&#39;d for [M+Na]=315.2300, found=315.2314. 
     {[2-({[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}methyl)prop-2-en-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with 2-({[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}methyl)prop-2-en-1-ol (0.200 g, 0.680 mmol), ether (3.00 mL) and treated, under an argon atmosphere at 0° C., with phosphorus tribromide (0.270, 1.00 mmol) dissolved in diethyl ether (1.00 mL). The reaction mixture was stirred for 30 minutes at 0° C. The organic layer was dried over sodium sulfate, filtered, and concentrated to yield crude (6E,10E)-12-{[2-(bromomethyl)prop-2-en-1-yl]oxy}-2,6,10-trimethyldodeca-2,6,10-triene (0.182 g, 72%). The crude material was dissolved in acetonitrile (2.00 mL), tetrabutylammonium pyrophosphate (0.634 g, 0.7 mmol) was added, the reaction mixture was stirred under argon for 3 hours, at which time it was concentrated and purified over DOWEX50 resin according to the following method. DOWEX50 resin (11.8 g) was stirred in concentrated ammonium hydroxide (40.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.0 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.259 g, 90%).  31 P NMR (202 MHz, Deuterium Oxide) δ −5.98 (t, J=21.2 Hz), −9.92 (d, J=20.2 Hz). HRMS ESI [M−H] calc&#39;d=451.1656, observed=451.1666. 
     EXAMPLE 17 
     {[(2E)-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     (2E)-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-ol. A 25.0 mL 14/20 round bottom flask was charged with trans, trans-farnesol (0.222 g, 1.00 mmol), diethyl ether (5.00 mL) and at 0° C., under an argon atmosphere, phosphorus tribromide (0.475 mL, 5.00 mmol) dissolved in diethyl ether (1.00 mL) was added. The mixture was stirred for 30 minutes, diluted with hexanes, washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield trans, trans-farnesyl bromide. A separate 25.0 mL 14/20 round bottom flask was charged with sodium hydride (0.134 g, 4.00 mmol), tetrahydrofuran (6.00 mL) and, under an argon atmosphere at 0° C., but-2-ene-1,4-diol (purchased commercially) was added (0.178 g, 2.00 mmol) in a dropwise fashion. Once gas evolution had ceased, the trans, trans-farnesyl bromide previously prepared was added as a solution in tetrahydrofuran (3.00 mL). The reaction was heated to 45° C. for 19 hours, quenched with saturated ammonium chloride (10.0 mL) and partitioned with ethyl acetate. The crude material was purified by silica gel chromatography (10-100% ethyl acetate in hexanes) to yield the pure product as a clear oil (0.252 g, 86%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.89-5.58 (m, 3H), 5.38-5.30 (m, 1H), 5.13-5.04 (m, 2H), 4.70-4.62 (m, 2H), 4.25 (dd, J=6.8, 1.4 Hz, 1H), 4.20 (d, J=6.4 Hz, 2H), 4.04 (d, J=6.2 Hz, 2H), 4.00 (d, J=7.0 Hz, 2H), 2.17-1.90 (m, 8H), 1.67 (s, 6H), 1.59 (s, 6H). HRMS ESI (+) calc&#39;d for [M+Na]=315.2300, found=315.2300. 
     {[(2E)-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with (2E)-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-ol (0.314 g, 1.10 mmol), diethyl ether (3.00 mL) and at 0° C., under an argon atmosphere, was added phosphorus tribromide (0.324 g, 1.20 mmol). The mixture was stirred for 20 minutes, diluted with hexanes, washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield crude (6E,10E)-12-{[(2E)-4-bromobut-2-en-1-yl]oxy}-2,6,10-trimethyldodeca-2,6,10-triene (0.262 g, 62%). The crude material was dissolved in acetonitrile (2.00 mL), stirred and treated with tetrabutylammonium pyrophosphate (0.604 g, 0.660 mmol). The reaction mixture was stirred, under an argon atmosphere, for 2 hours, at which time it was concentrated in vacuo and purified over DOWEX50 resin column. The column was prepared by stirring DOWEX50 resin (8.50 g) in concentrated ammonium hydroxide (25.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25.0 aqueous mmolar ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of 1:49 2-propanol: 25 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.196 g, 44%).  31 P NMR (202 MHz, Deuterium Oxide) δ −5.70-−6.25 (m), −9.92 (dd, J=66.8, 21.3 Hz). HRMS ESI [M−H] calcd=451.1656, observed=451.1662. 
     EXAMPLE 18 
     {[(2E)-3-methyl-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     tert-butyl({[2E)-3-methyl-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-yl]oxy})diphenylsilane. A 25.0 mL 14/20 round bottom flask was charged with a stir bar, trans, trans-farnesol (0.444 g, 2.00 mmol), diethyl ether (10.0 mL) and phosphorus tribromide (0.812 g, 3.00 mmol dissolved in 1.00 mL diethyl ether) at 0° C. under an argon atmosphere. After 30 minutes the mixture was diluted with hexanes, washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield trans, trans-farnesyl bromide. A separate 25.0 mL 14/20 round bottom flask was charged with sodium hydride (0.087 g, 2.60 mmol), tetrahydrofuran (5.00 mL), and under argon at 0° C. (2E)-4-[(tert-butyldiphenylsilyl)oxy]-2-methylbut-2-en-1-ol (0.749 g, 2.20 mmol, prepared according to the method described in Oberhauser, C.; Harms, V.; Seidel, K.; Schrçder, B.; Ekramzadeh, K.; Beutel, S,; Winkler, S.; Lauterbach, L.; Dickschat, J. S.; and Kirschning, A.;  Angew. Chemie. Int. Ed.,  2018, 57, 11802.) was added. Once gas evolution ceased, the trans, trans-farnesyl bromide previously prepared was added as a solution dissolved in tetrahydrofuran (2.00 mL). The reaction was heated to 45° C. for 21 hours, quenched with saturated ammonium chloride (10.0 mL) and partitioned with ethyl acetate. The crude material was purified by silica gel chromatography (hexanes) to yield the pure product as an oil (0.390 g, 37%).  1 H NMR (500 MHz, CDCl 3 ) δ 7.75-7.67 (m, 4H), 7.47-7.36 (m, 6H), 5.66 (ddt, J=7.5, 4.9, 1.4 Hz, 1H), 5.37 (dddd, J=8.1, 5.5, 2.6, 1.3 Hz, 1H), 5.16-5.07 (m, 2H), 4.28 (dq, J=6.0, 0.9 Hz, 2H), 3.93 (d, J=6.6 Hz, 2H), 3.84 (d, J=1.2 Hz, 2H), 2.17-2.03 (m, 7H), 1.99 (dd, J=9.2, 5.9 Hz, 3H), 1.69 (q, J=1.3 Hz, 3H), 1.67 (d, J=1.4 Hz, 3H), 1.61 (dd, J=2.2, 1.2 Hz, 6H), 1.50 (t, J=1.1 Hz, 3H), 1.06 (d, J=2.8 Hz, 9H). 
     (2E)-3-methyl-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-ol. A 50.0 mL 24/40 round bottom flask was charged with tert-butyl({[(2E)-3-methyl-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-yl]oxy})diphenylsilane (1.01 g, 1.90 mmol) and a stir bar. Under an argon atmosphere, tetrabutylammonium fluoride was added (15.0 mL, 15.0 mmol). The reaction mixture was heated to 45° C. for 16 hours, diluted with ethyl acetate, washed with 1.00 N HCl (20.0 mL), brine, and concentrated to an oil. The crude material was purified by silica gel chromatography (0-100% ethyl acetate in hexanes) to yield the product as an oil (0.263 g, 45%,).  1 H NMR (500 MHz, CDCl 3 ) δ 5.67 (tq, J=6.8, 1.3 Hz, 1H), 5.37 (tq, J=6.8, 1.3 Hz, 1H), 5.11 (ddddd, J=11.4, 7.0, 5.6, 2.8, 1.4 Hz, 2H), 4.22 (d, J=6.7 Hz, 2H), 3.97 (d, J=6.8 Hz, 2H), 3.87 (d, J=1.3 Hz, 2H), 2.16-2.03 (m, 7H), 1.98 (dd, J=9.1, 6.1 Hz, 2H), 1.72 (d, J=1.4 Hz, 3H), 1.69 (q, J=1.3 Hz, 3H), 1.67 (d, J=1.3 Hz, 3H), 1.61 (s, 6H). HRMS ESI (+) calc&#39;d for [M+Na]=329.2457, found=329.2475. 
     {[(2E)-3-methyl-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien yl]oxy}but-2-en-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with (2E)-3-methyl-4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-en-1-ol (0.263 g, 0.850 mmol), diethyl ether (4.00 mL) and at 0° C., under an argon atmosphere, phosphorus tribromide (0.270 g, 1.00 mmol) was added. The mixture stirred for 30 minutes, diluted with hexanes, washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to yield the crude (6E,10E)-12-{[(2E)-4-bromo-2-methylbut-2-en-1-yl]oxy}-2,6,10-trimethyldodeca-2,6,10-triene (0.0720 g). The crude material was then dissolved in acetonitrile (1.00 mL), stirred and tetrabutylammonium pyrophosphate (0.497 g, 0.540 mmol) added and stirred under an argon atmosphere for 3 hours. The mixture was then concentrated in vacuo and purified over DOWEX50 resin column. The resin (6.80 g) was prepared by stirring in concentrated ammonium hydroxide (25.0 mL) for 20 minutes, then filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.156 g, 40%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.01 (d, J=21.2 Hz), −9.88 (t, J=25.2 Hz). HRMS ESI [M−H] calcd=465.1813, observed=465.1814. 
     EXAMPLE 19 
     {[(2E,6E,10E)-3,7,11-trimethyl-12-[(3-methylbut-2-en-1-yl)oxy]dodeca-2,6,10-trien-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     (2E,6E,10E)-12-[(tert-butyldiphenylsilyl)oxy]-2,6,10-trimethyldodeca-2,6,10-trien-1-ol. A 100 mL 24/40 round bottom flask was charged with trans,trans-farnesol (4.50 g, 20.2 mmol), imidazole (2.99 g, 44.4 mmol) and dimethylformamide (25.0 mL). The reaction mixture was stirred, under an argon atmosphere, and tert-butyldiphenylsilyl chloride added (5.70 mL, 22.0 mmol) dropwise. The mixture was stirred for 19 hours at room temperature, then partitioned between 1.00 N HCl (30.0 mL) and ethyl acetate. The organic layer was washed with sodium bicarbonate, twice with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to provide crude tert-butyldiphenyl {[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}silane (8.71 g, 95%). 
     In a separate 100 mL 24/40 round bottom flask was added selenium(IV) dioxide (0.103 g, 0.94 mmol), salicylic acid (0.259 g, 1.88 mmol) and dichloromethane (40.0 mL). The mixture was stirred at room temperature and tent-butylhydroperoxide added (9.00 mL, 65.8 mmol, 70% solution in water), followed by tert-butyldiphenyl {[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}silane (8.71 g, 18.8 mmol, dissolved in 5.00 mL dichloromethane). The mixture was stirred at room temperature for 50 hours, washed with saturated sodium thiosulfate and concentrated in vacuo. The material was then dissolved in ethanol, cooled to 0° C., and treated with sodium borohydride (0.720 g, 19.0 mmol). After gas evolution ceased the reaction was warmed to room temperature and stirred for 30 minutes. The reaction was quenched with 1.00 N HCl (10.0 mL), partitioned between ethyl acetate and sodium bicarbonate, washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to yield the crude product as a red oil. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield the product as a clear oil (1.2 g, 13% yield).  1 H NMR (500 MHz,CDCl 3 ) δ 7.74-7.66 (m, 4H), 7.46-7.34 (m, 6H), 5.40 (dddt, J=6.3, 5.0, 2.6, 1.3 Hz, 2H), 5.14 (tq, J=6.9, 1.4 Hz, 1H), 4.29-4.19 (m, 2H), 4.02 (d, J=19.9 Hz, 2H), 2.20-1.95 (m, 8H), 1.69 (dd, J=14.6, 1.4 Hz, 3H), 1.62 (s, 3H), 1.45 (d, J=1.2 Hz, 3H), 1.05 (s, 9H). 
     (2E,6E,10E)-3,7,11-trimethyl-12-[(3-methylbut-2-en-1-yl)oxy]dodeca-2,6,10-trien-1-ol. A 25.0 mL 14/20 round bottom flask was charged with (2E,6E,10E)-12-[(tert-butyldiphenylsilyl) oxy]-2,6,10-trimethyldodeca-2,6,10-trien-1-ol (0.478 g, 1.00 mmol) and tetrahydrofuran (5.00 mL). The mixture was cooled to 0° C. and sodium hydride added (0.170 g, 7.00 mmol) under an argon atmosphere, followed by the addition of prenyl bromide (1.00 g, 6.20 mmol). The mixture was stirred at 40° C. for 22 hours and quenched with saturated ammonium chloride, partitioned into ethyl acetate and the organic layer washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (100% hexanes) to yield the product as an oil. This material was dissolved in tetrabutylammonium fluoride (10.0 mL, 10.0 mmol) and heated to 40° C. for 19 hours under argon. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield the product (0.171 g, 58%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.43-5.31 (m, 3H), 5.10 (tq, J=6.9, 1.4 Hz, 1H), 4.12 (dd, J=13.9, 7.1 Hz, 2H), 3.90-3.84 (m, 2H), 3.82 (d, J=1.1 Hz, 2H), 2.17-1.97 (m, 8H), 1.73 (d, J=1.4 Hz, 3H), 1.66 (d, J=1.4 Hz, 3H), 1.65 (d, J=1.4 Hz, 3H), 1.64 (d, J=1.4 Hz, 3H), 1.59 (d, J=1.4 Hz, 3H). 
     {[(2E,6E,10E)-3,7,11-trimethyl-12-[(3-methylbut-2-en-1-yl)oxy]dodeca-2,6,10-trien-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with (2E,6E,10E)-3,7,11-trimethyl-12-[(3-methylbut-2-en-1-yl)oxy]dodeca-2,6,10-trien-1-ol (0.171 g, 0.580 mmol), diethyl ether (4.00 mL) and at 0° C. under an argon atmosphere, treated phosphorus tribromide (0.094 mL, 1.00 mmol). The reaction mixture was stirred for 30 minutes, diluted with hexanes, the organic layer was then washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield crude (2E,6E,10E)-12-bromo-2,6,10-trimethyl-1-[(3 -methylbut-2-en-1-yl)oxy]dodeca-2,6,10-triene (0.122 g). The crude material was then dissolved in acetonitrile (1.00 mL), stirred and treated with tetrabutylammonium pyrophosphate (0.500 g, 0.540 mmol). The reaction mixture was stirred, under an argon atmosphere, for 2 hours, then concentrated in vacuo and purified over DOWEX50 resin (6.89 g) column prepared by stirring in concentrated ammonium hydroxide (30.0 mL) for 20 minutes, then filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL of the 1:49 2-propanol: 25 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.138 g, 60%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.03 (d, J=21.2 Hz), -9.99 (d, J=20.6 Hz). HRMS ESI [M−H] calcd=465.1813, observed=465.1810. 
     EXAMPLE 20 
     {[2-({[(5E)-6,10-dimethylundeca-5,9-dien-2-yl]oxy}methyl)prop-2-en-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     Tert-butyl({[2-({[(5E)-6,10-dimethylundeca-5,9-dien-2-yl]oxy}methyl)prop-2-en-1-yl]oxy})diphenylsilane. A 100 mL 24/40 round bottom flask was charged with geranyl acetone (1.94 g, 10.0 mmol) and ethanol (30.0 mL). The reaction mixture was cooled to 0° C. and sodium borohydride added (0.529 g, 14.0 mmol) and stirred for 1.0 hour, quenched with 1.00 N HCl (10.0 mL) and partitioned with ethyl acetate. The organic layer was filtered through a plug of silica gel (eluted with 100% ethyl acetate) and concentrated to yield crude (5E)-6,10-dimethylundeca-5,9-dien-2-ol (1.80 g, 91%), which was used without further purification. 
     Using the method of Vita and coworkers (Vita, M. V.; Caramenti. P.; Waser, J.  Org. Lett.,  2015, 17, 5832.), a separate 250 mL 24/40 round bottom flask was charged prop-2-ene-1,3-diol (8.40 mL, 102 mmol) and tetrahydrofuran (50.0 mL) under an argon atmosphere. The flask was cooled to 0° C. and sodium hydride added (3.69 g, 110 mmol), followed by tent-butyldiphenylsilyl chloride (25.9 mL, 100 mmol). The reaction was stirred for 20 hours, partitioned into ethyl acetate, which was washed with a saturated ammonium chloride solution, concentrated in vacuo and purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield 2-{[(tert-butyldiphenylsilyl)oxy]methyl}prop-2-en-1-ol (8.00 g, 23%).  1 H NMR (500 MHz, Chloroform-d) δ 7.71-7.68 (m, 4H), 7.48-7.38 (m, 6H), 5.72 (dtt, J=11.5, 5.7, 1.3 Hz, 1H), 5.65 (dtt, J=11.2, 6.4, 1.4 Hz, 1H), 4.31-4.25 (m, 2H), 4.02 (d, J=6.2 Hz, 2H), 1.06 (s, 9H). 
     2-{[(tert-butyldiphenylsilyl)oxy]methyl}prop-2-en-1-ol (1.31 g, 4.00 mmol, Heidelbrecht, R. W. Jr.; Gulledge, B.; Martin, S.,  Org. Lett.,  2010, 12, 2492.) was dissolved in dichloromethane (15.0 mL), cooled to 0° C. and triphenylphosphine added (1.25 g, 4.80 mmol), followed by n-bromosuccinimide (0.782 g, 4.80 mmol). The mixture was stirred under an argon atmosphere for 2 hours at 0° C. and treated with hexanes (200 mL). The solid was filtered and the filtrate concentrated to yield {[2-(bromomethyl)prop-2-en-1-yl]oxy}(tert-butyl)diphenylsilane (1.10 g, 71%). The product was used without further purification. 1H NMR (500 MHz, Chloroform-d) δ 7.71-7.67 (m, 4H), 7.46-7.39 (m, 6H), 5.78-5.72 (m, 2H), 4.34-4.32 (m, 2H), 3.87-3.84 (m, 2H), 1.06 (s, 9H). 
     A 14/20 25.0 mL round bottom flask was charged with crude {[2-(bromomethyl)prop-2-en-1-yl]oxy }(tert-butyl)diphenylsilane (0.960 g, 2.30 mmol), crude (5E)-6,10-dimethylundeca-5,9-dien-2-ol (0.976 g, 5.00 mmol), tetrahydrofuran (5.00 mL), cooled to 0° C., and treated with sodium hydride (0.235 g, 7.00 mmol). After gas evolution was complete, the mixture was heated to 45° C. for 19 hours under an argon atmosphere. The reaction was partitioned between ethyl acetate and ammonium chloride, washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield an oil (0.500 g, 1.00 mmol).  1 H NMR (500 MHz, Chloroform-d) δ 7.72-7.67 (m, 4H), 7.48-7.36 (m, 6H), 5.79-5.71 (m, 2H), 5.64-5.55 (m, 1H), 5.10 (ttq, J=7.2, 4.4, 1.3 Hz, 1H), 4.33 (dd, J=3.3, 2.2 Hz, 1H), 4.29-4.25 (m, 1H), 3.97-3.76 (m, 4H), 2.10-1.95 (m, 4H), 1.69 (t, J=1.3 Hz, 3H), 1.61 (t, J=1.7 Hz, 3H), 1.59-1.54 (m, 3H), 1.09-1.03 (m, 9H), 1.00 (s, 3H). 
     A 25.0 mL 14/20 round bottom flask was charged with tert-butyl({[2-({[(5E)-6,10-dimethylundeca-5,9-dien-2-yl]oxy}methyl)prop-2-en-1-yl]oxy})diphenylsilane (0.500 g, 1.00 mmol) and, under an argon atmosphere, tetrabutylammonium fluoride (5.00 mL, 5.00 mmol) added. The reaction mixture was stirred at 45° C. for 15 hours then partitioned between ethyl acetate and 1.00 N HCl (15.0 mL). The organic layer was washed with brine and purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield 2-({[(5E)-6,10-dimethylundeca-5,9-dien-2-yl]oxy}methyl)prop-2-en-1-ol (0.100 g, 38%).  1 H NMR (500 MHz, Chloroform-d) δ 5.83-5.75 (m, 1H), 5.75-5.66 (m, 1H), 5.09 (dddddd, J=12.7, 7.0, 5.7, 4.3, 2.8, 1.4 Hz, 2H), 4.18 (dt, J=6.4, 1.2 Hz, 2H), 4.14-4.06 (m, 1H), 3.97 (dddd, J=12.4, 6.2, 2.3, 1.4 Hz, 1H), 3.44 (hept, J=6.4 Hz, 1H), 2.09-1.94 (m, 7H), 1.68 (dq, J=4.2, 1.3 Hz, 3H), 1.64-1.50 (m, 6H), 1.47-1.36 (m, 1H), 1.15 (dd, J=6.2, 2.3 Hz, 3H). 
     {[2-({[(5E)-6,10-dimethylundeca-5,9-dien-2-yl]oxy}methyl)prop-2-en-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with 2-({[(5E)-6,10-dimethylundeca-5,9-dien-2-yl]oxy}methyl)prop-2-en-1-ol (0.100 g, 0.380 mmol), diethyl ether (4.00 mL), cooled to 0° C., and phosphorus tribromide was added (0.270 g, 1.00 mmol). He reaction mixture was stirred for 30 minutes at 0° C., diluted with hexanes, washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield the crude (6E)-10-{[2-(bromomethyl)prop-2-en-1-yl]oxy}-2,6-dimethylundeca-2,6-diene (0.0400 g, 32%). The crude material was dissolved in acetonitrile (2.00 mL), stirred, and tetrabutylammonium pyrophosphate (0.604 g, 0.670 mmol) was added. The reaction mixture was stirred under an argon atmosphere for 2 hours, at which time it was concentrated in vacuo and purified over DOWEX50 resin column. The column was prepared by stirring DOWEX50 resin (7.00 g) in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.128 g, 82%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.03 (d, J=22.8 Hz), −10.05 (d, J=22.0 Hz). HRMS ESI [M−H] calcd=425.1500, observed=425.1502. 
     EXAMPLE 21 
     {[(2E,6E)-8-{[(2Z)-3,7-dimethylocta-2,6-dien-lyl]oxy}-3,7-dimethylocta-2,6-dien-1-yl phosphonato]oxy}phosphonate. 
     
       
         
         
             
             
         
       
     
     Tert-butyl({[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy})diphenylsilane. A 250 mL 24/40 round bottom flask was charged with trans,trans-geraniol (4.62 g, 30.0 mmol), imidazole (2.72 g, 40.0 mmol) and dimethylformamide (90.0 mL). The reaction mixture was stirred under an argon atmosphere and tert-butyldiphenylsilyl chloride added (8.55 g, 31.0 mmol) in a dropwise fashion. The reaction was stirred for 19 hours at room temperature, portioned between 1.00 N HCl (30.0 mL) and ethyl acetate. The organic layer was washed with a saturated sodium bicarbonate solution, twice with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to a clear oil (8.41 g, 70%). 1H NMR (500 MHz, Chloroform-d) δ 7.74-7.69 (m, 4H), 7.47-7.35 (m, 6H), 5.40 (dddt, J=7.6, 6.2, 3.3, 1.4 Hz, 2H), 4.23 (dq, J=6.3, 0.9 Hz, 2H), 4.00 (d, J=1.3 Hz, 2H), 2.21-2.10 (m, 2H), 2.03 (dd, J=9.1, 6.3 Hz, 2H), 1.68 (d, J=1.3 Hz, 3H), 1.46 (d, J=1.3 Hz, 4H), 1.05 (s, 9H). 
     (2E,6E)-8-[(tert-butyldiphenylsilyl)oxy]-2,6-dimethylocta-2,6-dien-1-ol. In a 100 mL 24/40 round bottom flask was added selenium(IV) dioxide (0.118 g, 1.07 mmol), salicylic acid (0.295 g, 2.14 mmol) and dichloromethane (40.0 mL). The reaction mixture was stirred at room temperature and tent-butylhydroperoxide was added (10.0 mL, 73.1 mmol, 70% solution in water), followed by tert-butyl({[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy})diphenylsilane (8.71 g, 18.8 mmol dissolved in 5.00 mL dichloromethane). The reaction mixture was stirred at room temperature for 75 hours, washed with a saturated sodium thiosulfate solution and concentrated in vacuo. This material was dissolved in ethanol, cooled to 0° C., and treated with sodium borohydride (0.832 g, 22.0 mmol). After gas evolution ceased, the reaction was warmed to room temperature and stirred for 1 hour, quenched with 1.00 N hydrochloric acid (20.0 mL), partitioned between ethyl acetate and sodium bicarbonate, the organic layer washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to yield the crude product as an oil. The crude material was purified by silica gel chromatography (0-100% ethyl acetate in hexanes) to yield the product as an oil (1.20 g, 13% yield).  1 H NMR (500 MHz, CDCl 3 ) δ 7.74-7.66 (m, 4H), 7.47-7.34 (m, 6H), 5.40 (dddt, J=7.6, 6.3, 3.3, 1.4 Hz, 2H), 4.23 (dq, J=6.3, 0.9 Hz, 2H), 4.00 (d, J=1.1 Hz, 2H), 2.19-2.09 (m, 2H), 2.03 (dd, J=9.1, 6.3 Hz, 2H), 1.68 (d, J=1.4 Hz, 3H), 1.46 (d, J=1.3 Hz, 3H), 1.05 (s, 9H). HRMS ESI (+) calc&#39;d for [M+Na]=329.2457, found=329.2448. 
     Tert-butyl({[(2E,6E)-8-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}-3,7-dimethylocta-2,6-dien-1-yl]oxy})diphenylsilane. A 25.0 mL 14/20 round bottom flask was charged with (2E,6E)-8-[(tert-butyldiphenylsilyl)oxy]-2,6-dimethylocta-2,6-dien-1-ol (0.752 g, 2.0 mmol) and tetrahydrofuran (5.00 mL). The reaction mixture was cooled to 0° C. and sodium hydride was added (0.134 g, 4.00 mmol). Under an argon atmosphere, geranyl bromide (0.650 g, 3.00 mmol, Brundel, B., J., J., M.; Steen, H.; Heeres, A.; Seerden, J. P. G., WO2013157926) was added and the mixture stirred at 40° C. for 20 hours. The reaction was quenched with ammonium chloride, partitioned with ethyl acetate, washed with brine, dried over sodium sulfate, filtered and the solvent removed in vacuo. The crude material was purified by silica gel chromatography (100% hexanes) to yield the product as an oil (0.470 g, 43%).  1 H NMR (500 MHz, CDCl 3 ) δ 7.73-7.67 (m, 4H), 7.46-7.34 (m, 6H), 5.44-5.33 (m, 3H), 5.15-5.02 (m, 1H), 4.23 (d, J=6.0 Hz, 2H), 3.95-3.90 (m, 2H), 3.87-3.80 (m, 2H), 2.16-1.99 (m, 8H), 1.72-1.65 (m, 6H), 1.59 (s, 6H), 1.45 (d, J=1.2 Hz, 3H), 1.05 (s, 9H). 
     (2E,6E)-8-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}-3,7-dimethylocta-2,6-dien-1-ol. Tert-butyl({[(2E,6E)-8-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}-3,7-dimethylocta-2,6-dien-1-yl]oxy})diphenylsilane was dissolved in tetrahydrofuran (2.00 mL), treated with a 1.00 M tetrabutylammonium fluoride (10.0 mL, 10.0 mmol) solution (in tetrahydrofuran) and heated to 40° C. for 19 hours under an argon atmosphere. The mixture was treated with water then extracted with ethyl acetate. The organic layers were combined, dried with sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield the product (0.100 g, 42% yield).  1 H NMR (500 MHz, CDCl 3 ) δ 5.46-5.30 (m, 3H), 5.10 (ddp, J=7.1, 5.8, 1.5 Hz, 1H), 4.15 (d, J=6.9 Hz, 2H), 3.93 (d, J=6.8 Hz, 2H), 3.83 (d, J=1.2 Hz, 2H), 2.24-1.97 (m, 8H), 1.69 (d, J=1.3 Hz, 6H), 1.66 (d, J=1.3 Hz, 5H), 1.62-1.59 (m, 3H). {[(2E,6E)-8-{[(2Z)-3,7-dimethylocta-2,6-dien-1yl]oxy}-3,7-dimethylocta-2,6-dien-1-yl phosphonato]oxy}phosphonate. The alcohol (2E,6E)-8-{[(2Z)-3,7-dimethylocta-2,6-dien-1-yl]oxy}-3,7-dimethylocta-2,6-dien-1-ol (0.100 g, 0.340 mmol) was dissolved in diethyl ether (2.00 mL) and at 0° C., under an argon atmosphere, was added phosphorus tribromide (0.270 mL, 1.00 mmol). The reaction mixture was stirred for 30 minutes, diluted with hexanes, washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield (2E,6E)-8-bromo-1-{[(2Z)-3,7-dimethylocta-2,6-dien-1-yl]oxy}-2,6-dimethylocta-2,6-diene (0.121 g, 96%). The crude material was dissolved in acetonitrile (2.00 mL) and tetrabutylammonium pyrophosphate (0.255 g, 0.280 mmol) added. The reaction mixture was stirred under an argon atmosphere for 2 hours, at which time it was concentrated in vacuo and purified over a DOWEX50 resin column. The column was prepared by treating DOWEX50 resin (7.20 g) with concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.066 g, 60% yield by mass, 95% yield by  31 P NMR integration).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.43 (d, J=97.1 Hz), −9.83-−11.41 (m). HRMS ESI [M−H] calcd=465.1813, observed=465.1814. 
     EXAMPLE 22 
     {[(2E,6E,10E)-13-(3,3-dimethyloxiran-2-yl)-3,7,11-trimethyltrideca-2,6,10-trien-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     (2E,6E,10E)-13-(3,3-dimethyloxiran-2-yl)-3,7,11-trimethyltrideca-2,6,10-trien-1-ol and (2Z,6E,10E)-13-(3,3-dimethyloxiran-2-yl)-3,7,11-trimethyltrideca-2,6,10-trien-1-ol. A 50.0 mL 24/40 round bottom flask was charged with geranyl geraniol (1.00 g, 3.50 mmol, Look, G. C., WO2015006614) as a mixture of (2E,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2Z,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol), dichloromethane (10.0 mL), triethylamine (0.696 mL, 5.00 mmol), stirred and cooled to 0° C. To the mixture was added acetic anhydride (0.378 mL, 4.00 mmol) and dimethylaminopyridine (0.0240 g, 0.200 mmol). The reaction was stirred for 1 hour at 0° C. and quenched with brine, dried over sodium sulfate, and concentrated to yield the product as a mixture of (2E,6E,10E)-13-(3,3-dimethyloxiran-2-yl)-3,7,11-trimethyltrideca-2,6,10-trien-1-yl acetate and (2Z,6E,10E)-13-(3,3-dimethyloxiran-2-yl)-3,7,11-trimethyltrideca-2,6,10-trien-1-yl acetate (0.980 g, 84%). The oil was dissolved in a mixture of tetrahydrofuran (25.0 mL) and water (10.0 mL), cooled to 0° C., and n-bromosuccinimide (0.623 g, 3.50 mmol). The reaction was stirred at 0° C. for 2 hours, concentrated and extracted with hexanes. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to yield the intermediate as an oil (1.21 g). This material was dissolved in methanol (15.0 mL), potassium carbonate was added (0.720 g, 5.20 mmol) and the mixture was stirred for 17 hours. The reaction mixture was partitioned with ethyl acetate, filtered through a plug of silica (100% ethyl acetate) and concentrated to yield the product 3-[(3E,7E,11E)-13-bromo-3,7,11-trimethyltrideca-3,7,11-trien-1-yl]-2,2-dimethyloxirane and 3-[(3E,7E,11Z)-13-bromo-3,7,11-trimethyltrideca-3,7,11-trien-1-yl]-2,2-dimethyloxirane (0.823 g, 96%). 1H NMR (500 MHz, Chloroform-d) δ 5.39-5.31 (m, 1H), 5.10 (dddqd, J=8.4, 6.9, 4.1, 2.7, 1.9, 1.4 Hz, 3H), 4.62-4.54 (m, 2H), 2.16-2.01 (m, 14H), 1.97 (dd, J=9.1, 6.3 Hz, 2H), 1.74-1.66 (m, 8H), 1.63-1.57 (m, 6H). HRMS ESI (+) calc&#39;d for [M+Na]=329.2457, found=329.2455. 
     {[(2E,6E,10E)-13-(3,3-dimethyloxiran-2-yl)-3,7,11-trimethyltrideca-2,6,10-trien-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with dichloromethane (10.0 mL) and n-chlorosuccinimide (0.267 g, 2.00 mmol). The mixture was stirred under argon, cooled to −30° C., and dimethyl sulfide was added (0.146 mL, 2.00 mmol). The reaction was warmed to 0° C. for 5 minutes, again cooled to −30° C. and (2E,6E,10E)-13-(3,3-dimethyloxiran-2-yl)-3,7,11-trimethyltrideca-2,6,10-trien-1-ol added (0.306 g, 1.00 mmol, dissolved in 1.0 mL dichloromethane). The mixture was stirred for 5 minutes at −30° C. then warmed to 0° C. for 2 hours. The mixture was then washed with brine and concentrated to dryness to yield the crude 3-[(3E,7E,11E)-13-chloro-3,7,11-trimethyltrideca-3,7,11-trien-1-yl]-2,2-dimethyloxirane (0.301 g, 0.920 mmol). This material was dissolved in acetonitrile (2.00 mL), stirred (argon atmosphere), then treated with tetrabutylammonium pyrophosphate (0.525 g, 0.570 mmol). The reaction mixture was stirred for 2 hours, then concentrated in vacuo and purified on a DOWEX50 resin column. The column was prepared by treating the resin (7.20 g) with concentrated ammonium hydroxide (30 mL) for 20 minutes then filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20 mL of 1:49 2-propanol: 25.0 mmolar ammonium bicarbonate) and poured into a column. The excess buffer was drained from the column and the crude product was applied to the column (dissolved in 3.00 mL of the same buffer). The material was eluted with 30 mL buffer and lyophilized to a waxy solid (0.083 g, 18%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.24 (d, J=21.8 Hz), −10.09 (d, J=22.4 Hz). HRMS ESI [M−H] calc&#39;d=465.1813, observed=465.1816. 
     EXAMPLE 23 
     {[(3-{[(2E,6E)-3,6,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)methyl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     3-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}benzaldehyde. A 25.0 mL 14/20 round bottom flask was charged with trans,trans-farnesol (0.889 g, 4.00 mmol), diethyl ether, (10.0 mL), and at 0° C. under argon was added phosphorus tribromide (1.35 g, 5.00 mmol). The reaction mixture was stirred for 30 minutes, diluted with hexanes, washed with brine, a saturated sodium bicarbonate solution, and brine. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to yield crude trans, trans-farnesyl bromide (0.937 g, 83%). This material was diluted with tetrahydrofuran (10.0 mL) and 3-hydroxybenzaldehyde (0.463 g, 3.80 mmol) was added. The reaction mixture was cooled to 0° C. and sodium hydride added (0.151 g, 4.50 mmol). Once gas evolution ceased, the reaction mixture was heated to 45° C. for 23 hours under an argon atmosphere. The mixture was then partitioned between ethyl acetate and saturated ammonium chloride. The organic layer washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by silica gel chromatography (1:9:0.1 ethyl acetate: hexanes: triethylamine) to yield the product as an oil (0.490 g, 46%).  1 H NMR (500 MHz, CDCl 3 ) δ 9.97 (s, 1H), 7.49-7.37 (m, 3H), 7.19 (dt, J=6.7, 2.5 Hz, 1H), 5.50 (tq, J=6.6, 1.3 Hz, 1H), 5.09 (ddddt, J=11.3, 5.7, 4.3, 2.9, 1.4 Hz, 2H), 4.60 (dd, J=6.6, 1.0 Hz, 2H), 2.18-2.02 (m, 6H), 2.01-1.94 (m, 2H), 1.76 (d, J=1.3 Hz, 3H), 1.68 (q, J=1.3 Hz, 3H), 1.60 (dd, J=2.3, 1.3 Hz, 6H). 
     (3-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)methanol. A 50.0 mL 14/20 round bottom flask was charged with 3-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}benzaldehyde (0.470 g 1.50 mmol), ethanol (6.00 mL), cooled to 0° C., and sodium borohydride (0.0750 g, 2.00 mmol). After 10 minutes, the reaction mixture was partitioned between ethyl acetate and ammonium chloride, the organic layer washed with brine, dried over sodium sulfate, filtered, and concentrated to an oil. The crude material was purified by silica gel chromatography (0-50% ethyl acetate in hexanes) to yield the product as an oil (0.251 g, 51%).  1 H NMR (500 MHz, CDCl 3 ) δ 7.27 (t, J=7.8 Hz, 1H), 6.98-6.90 (m, 2H), 6.86 (ddd, J=8.2, 2.7, 1.0 Hz, 1H), 5.51 (tq, J=6.6, 1.3 Hz, 1H), 5.17-5.06 (m, 2H), 4.68 (s, 2H), 4.56 (d, J=6.6 Hz, 2H), 2.21-2.03 (m, 6H), 1.98 (dd, J=9.1, 6.2 Hz, 2H), 1.75 (d, J=1.3 Hz, 3H), 1.69 (d, J=1.4 Hz, 3H), 1.62 (d, J=1.4 Hz, 6H). HRMS ESI (+) calc&#39;d for [M+Na]=351.2300, found=351.2315. 
     {[(3-{[(2E,6E)-3,6,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)methyl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with the alcohol from the prior step, (3-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)methanol (0.212 g, 0.640 mmol), diethyl ether (3.00 mL), cooled to 0° C., and under argon was added phosphorus tribromide (0.270 g, 1.00 mmol). The reaction was stirred at 0° C. for 1 hour, diluted with hexanes, washed with brine, sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield the crude bromide as an oil (0.135 g). This material was dissolved in acetonitrile (2.00 mL) and tetrabutylammonium pyrophosphate added (0.409 g, 0.450 mmol) and stirred under an argon atmosphere for 2 hours, at which time it was concentrated in vacuo and purified over DOWEX50 resin column. The column was prepared by stirring DOWEX50 resin (7.20 g) in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propano1:25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propano1:25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of the 1:49 2-propano1:25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol:25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.163 g, 53%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.45 (d, J=21.9 Hz), −10.37 (d, J=22.6 Hz). HRMS ESI [M−H] calc&#39;d=487.1656, observed=487.1653. 
     EXAMPLE 24 
     {[(2-{[(2E,6E)-3,6,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)methyl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     (2-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)methanol. A 25.0 mL 14/20 round bottom flask was charged with trans, trans-farnesol (0.889 g, 4.00 mmol), diethyl ether, (10.0 mL), and at 0° C., under an argon atmosphere, was added phosphorus tribromide (1.35 g, 5.00 mmol) and the mixture stirred for 1 hour. The mixture was then diluted with hexanes and the organic layer washed with brine, a saturated sodium bicarbonate solution, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield crude trans, trans-farnesyl bromide (0.769 g, 2.70 mmol). This material was diluted with tetrahydrofuran (10.0 mL) and treated with 2-hydroxy-benzyl alcohol (0.369 g, 3.00 mmol). The mixture was cooled to 0° C. and sodium hydride added (0.100 g, 3.00 mmol). After gas evolution ceased the reaction mixture was heated to 45° C. for 19 hours under an argon atmosphere, partitioned between ethyl acetate and a saturated ammonium chloride solution, washed with brine and concentrated in vacuo as an oil. The crude material was purified by silica gel chromatography (1:9:0.1 ethyl acetate: hexanes: triethylamine) to yield the product as an oil (0.258 g, 29%).  1 H NMR (500 MHz, CDCl 3 ) δ 7.26 (ddd, J=7.8, 6.5, 2.0 Hz, 2H), 6.97-6.86 (m, 2H), 5.49 (tq, J=6.4, 1.3 Hz, 1H), 5.10 (dtp, J=8.4, 4.3, 1.4 Hz, 2H), 4.69 (s, 2H), 4.60 (d, J=6.5 Hz, 2H), 2.47 (s, 1H), 2.22-2.03 (m, 6H), 1.98 (dd, J=9.1, 6.0 Hz, 2H), 1.74 (d, J=1.3 Hz, 3H), 1.68 (p, J=1.6 Hz, 3H), 1.61 (dd, J=2.9, 1.4 Hz, 6H). HRMS ESI (+) calc&#39;d for [M+Na]=351.2300, found=351.2340. 
     {[(2-{[(2E,6E)-3,6,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)methyl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with (2-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)methanol (0.218 g, 0.660 mmol), diethyl ether (3.00 mL), cooled to 0° C., and under argon atmosphere, treated with phosphorus tribromide (0.270 g, 1.00 mmol). The mixture was stirred at 0° C. for 1 hour, diluted with hexanes, washed with brine, aqueous saturated sodium bicarbonate solution and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to yield crude 1-(bromomethyl)-2-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}benzene as an oil (0.132 g). This material was dissolved in acetonitrile (2.00 mL) and tetrabutylammonium pyrophosphate (0.292 g, 0.450 mmol) added. The reaction mixture was stirred under an argon atmosphere for 2 hours, then concentrated in vacuo and purified over a DOWEX50 resin column. The DOWEX50 column was prepared by first stirring the DOWEX50 (7.55 g) in concentrated ammonium hydroxide (30.0 mL) for 20 minutes and then filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propano1:25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propano1:25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.0 mL of the same buffer). The material was eluted with 30.0 mL 1:49 2-propano1:25.0 mmolar aqueous ammonium bicarbonate and lyophilized to a waxy solid (0.075 g, 46%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.22 (d, J=21.9 Hz), −10.05 (d, J=21.9 Hz). HRMS ESI [M−H] calc&#39;d=487.1656, observed=487.1644. 
     EXAMPLE 25 
     {[(2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2E,6E,10E) ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     Ethyl (2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate. A 100 mL 14/20 round bottom flask was charged with ethyl 2-(diethoxyphosphoryl)-2-ethoxyacetate (2.36 g, 8.80 mmol. Prepared according to the method described in Bach, K.; Hesham, R., E.-S.; Jensen, H. M.; Nielsen, H. B.; Thomson, I.; Torssell, K. B. G;  Tetrahedron,  1994, 50, 7543), tetrahydrofuran (15.0 mL), cooled to 0° C., and sodium hydride (0.336 g, 10.0 mmol) added under an argon atmosphere. Farnesyl acetone (1.57 g, 6.00 mmol) dissolved in tetrahydrofuran (5.00 mL) was added in dropwise fashion. The stirring mixture was heated to 45° C. for 26 hours. The reaction was partitioned between ethyl acetate and a saturated ammonium chloride solution, the organic layer washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was dissolved in ethanol (30.0 mL), cooled to 0° C., and treated with sodium borohydride (0.226 g, 6.00 mmol). After 1 hour, the reaction was partitioned between ethyl acetate and a saturated ammonium chloride solution, washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to yield the product as a clear oil (0.953 g, 41%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.18-5.04 (m, 3H), 4.23 (ttd, J=7.1, 5.1, 2.6 Hz, 2H), 3.74-3.64 (m, 2H), 2.48-2.40 (m, 1H), 2.25 (ddd, J=8.7, 6.0, 1.5 Hz, 1H), 2.21-2.09 (m, 2H), 2.10-2.00 (m, 8H), 1.97 (dd, J=8.8, 5.5 Hz, 2H), 1.88-1.81 (m, 2H), 1.68 (dt, J=4.2, 1.4 Hz, 6H), 1.60 (dd, J=4.8, 2.6 Hz, 6H), 1.32 (td, J=7.1, 5.3 Hz, 3H), 1.28 (tdd, J=7.1, 2.7, 2.1 Hz, 3H). 
     (2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. A mixture of ethyl (2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate was dissolved in dichloromethane (5.00 mL), cooled to 0° C., and treated, under an argon atmosphere, with diisobutylaluminum hydride (8.00 mL, 8.00 mmol, 1.00 M in heptanes). The reaction was warmed to room temperature and stirred for 22 hours. The reaction was cooled to 0° C., quenched with ethanol (2.00 mL) and stirred vigorously for 24 hours with a solution of sodium potassium tartrate (10.0 g, 35.0 mmol in 50.0 mL water). The mixture was partitioned, and the aqueous layer washed with dichloromethane. The organic layers were combined, dried over sodium sulfate, filtered and concentrated in vacuo to yield the crude product, which was purified by silica gel (1:4 ethyl acetate: hexanes) chromatography to yield (2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol as a mixture of isomers (0.665 g, 80%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.17-5.04 (m, 3H), 4.34-4.07 (m, 2H), 3.81-3.69 (m, 2H), 2.19-2.12 (m, 1H), 2.12-2.00 (m, 9H), 1.98 (q, J=7.5, 7.0 Hz, 3H), 1.72-1.65 (m, 9H), 1.63-1.55 (m, 6H), 1.29-1.21 (m, 3H). 
     {[(2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with a mixture of (2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol (0.270 g, 0.830 mmol), dichloromethane (4.00 mL), triethylamine (0.223 mL, 1.60 mmol), cooled to 0° C., and treated with methane sulfonyl chloride (0.0770 mL, 1.00 mmol). The mixture was stirred for 1 hour then quenched with brine and partitioned. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to provide crude mixture of (2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl methanesulfonate and of (2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl methanesulfonate (0.374 g). This material was dissolved in acetonitrile (2.00 mL), stirred, and tetrabutylammonium pyrophosphate was added (0.454 g, 0.500 mmol). The reaction mixture was stirred under argon for 2 hours, concentrated and purified over DOWEX50 resin column. The column was prepared with DOWEX50 resin (7.55 g) stirred in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess buffer 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate was drained from the column and the crude product material applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.141 g, 35%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.52, −10.38 (d, J=22.0 Hz). HRMS ESI [M−H] calc&#39;d=493.2126, observed=493.2130. 
     EXAMPLE 26 
     {[(2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2E,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     Ethyl (2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2E,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate. A 50.0 mL 14/20 round bottom flask was charged with sodium hydride (0.151 g, 4.50 mmol). Under an argon atmosphere at 0° C. was added anhydrous tetrahydrofuran (10.0 mL), followed by triethyl-2-chloro-phosphonoacetate (1.00 g, 3.86 mmol) dissolved in tetrahydrofuran (2.00 mL). Once gas evolution ceased, farnesyl acetone (1.04 g, 4.00 mmol) was added dissolved in tetrahydrofuran (1.00 mL). The mixture was heated to 45° C. for 19 hours, concentrated in vacuo and partitioned between ethyl acetate and a saturated ammonium chloride solution. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to provide an oil. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield a mixture of ethyl (2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2E,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate (0.880 g, 62%). NMR (500 MHz, CDCl 3 ) δ 5.19-5.05 (m, 3H), 4.33-4.09 (m, 2H), 2.59-2.51 (m, 1H), 2.49-2.43 (m, 1H), 2.40 (dt, J=8.7, 7.2 Hz, 1H), 2.32-2.24 (m, 1H), 2.22-2.12 (m, 3H), 2.12-1.94 (m, 8H), 1.69 (dt, J=2.7, 1.3 Hz, 6H), 1.66-1.60 (m, 6H), 1.34-1.23 (td, J=7.1, 4.1 Hz, 3H). 
     (2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. A 50.0 mL 24/40 round bottom flask was charged with a mixture of ethyl (2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate and ethyl (2E,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenoate (0.840 g, 2.30 mmol). The material was dissolved in dichloromethane (5.00 mL) and, under an argon atmosphere at 0° C., treated with diisobutylaluminum hydride (7.00 mL, 7.00 mmol, 1.00 M in heptanes). The mixture was warmed to room temperature and stirred for 18 hours, then quenched with ethanol (5.00 mL). A solution of sodium potassium tartrate (7.00 g, 24.8 mmol in 50.0 mL water) was added and the biphasic mixture vigorously stirred for 24 hours. The reaction mixture partitioned, and the aqueous layer twice washed with dichloromethane (20.0 mL per wash). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield a mixture of (2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol (0.220 g, 30%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.18-5.06 (m, 3H), 4.29 (d, J=14.7 Hz, 1H), 4.19-3.76 (m, 1H), 2.31-2.19 (m, 1H), 2.17-2.01 (m, 9H), 1.98 (dd, J=9.5, 6.1 Hz, 2H), 1.69 (ddd, J=5.5, 2.6, 1.3 Hz, 9H), 1.64-1.58 (m, 6H). HRMS ESI (+) calc&#39;d for [M+Na]=347.2118, found=347.2159. 
     {[(2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate and {[(2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with a mixture of (2Z,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-chloro-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. (0.227 g, 0.670 mmol) and anhydrous ether (3.00 mL). At 0° C., under an argon atmosphere, phosphorus tribromide (0.270 g, 1.00 mmol) dissolved in ether (1.00 mL) was added. The mixture was stirred for 2 hours, diluted with hexanes, washed with brine, a saturated sodium bicarbonate solution and brine, then dried over sodium sulfate, filtered, and concentrated in vacuo. To this material dissolved in acetonitrile (1.50 mL) and treated with tetrabutylammonium pyrophosphate (0.504 g, 0.550 mmol). The reaction vessel was sealed and stirred under an argon atmosphere for 2 hours, then concentrated in vacuo to a viscous liquid and purified on DOWEX50 resin column. The resin column was prepared by stirring DOWEX50 resin (7.70 g) in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was then filtered and washed four times with water (100 mL) and subsequently suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate), then poured into a column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude product material applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.177 g, 55%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.17, −10.51. HRMS ESI [M−H] calc&#39;d=483.1474, observed=483.1471. 
     EXAMPLE 27 
     [(4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-yn-1-yl phosphonato)oxy]phosphonate 
     
       
         
         
             
             
         
       
     
     4-{[dimethyl(phenyl)silyl]oxy}but-2-yn-1-ol. A 250 mL 24/40 round bottom flask was charged with 2-butyne-1,4-diol (2.15 g, 25.0 mmol), tetrahydrofuran (75.0 mL), cooled to 0° C., and treated with sodium hydride (0.840 g, 25.0 mmol). Under an argon atmosphere, phenyldimethylchlorosilane (1.65 mL, 10.0 mmol) was added and the mixture stirred at room temperature for 19 hours, concentrated to a solid, partitioned between ethyl acetate and a saturated ammonium chloride solution, then washed with brine, dried over sodium sulfate, filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield the product (0.320 g, 14%).  1 H NMR (500 MHz, CDCl 3 ) δ 7.65-7.57 (m, 2H), 7.46-7.36 (m, 3H), 4.32 (t, J=1.8 Hz, 2H), 4.24 (t, J=2.0 Hz, 2H), 0.46 (s, 6H). 
     4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-yn-1-ol. A 50 mL 14/20 round bottom flask was charged with trans, trans-farnesol (0.889 g, 4.00 mmol), diethyl ether, (10.0 mL), and at 0° C. (argon atmosphere), treated with phosphorus tribromide (1.35 g, 5.00 mmol) and stirred for 30 minutes. The mixture was then diluted with hexanes, washed with brine, a saturated solution of sodium bicarbonate, and brine. The organic layer was then dried over sodium sulfate, filtered, and concentrated in vacuo to provide crude (6E,10E)-12-bromo-2,6,10-trimethyldodeca-2,6,10-triene (0.523 g, 1.80 mmol). The crude was diluted with tetrahydrofuran (10.0 mL) and charged with 4-{[dimethyl(phenyl)silyl]oxy}but-2-yn-1-ol (0.320 g, 1.30 mmol) and the mixture cooled to 0° C. and treated with sodium hydride (0.122 g, 5.00 mmol). After gas evolution ceased, the mixture (argon atmosphere) was heated to 45° C. for 19 hours. The reaction mixture was partitioned between ethyl acetate and a saturated ammonium chloride solution, the organic layer washed with brine, dried with sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to provide crude dimethyl(phenyl)[(4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-yn-1-yl)oxy]silane (0.394 g, 71%). A 25.0 mL 14/20 round bottom flask was charged with this material and, under an argon atmosphere, tetrabutylammonium fluoride (5.00 mL, 5.00 mmol, 1.00 M solution in tetrahydrofuran) was added and the mixture stirred at 45° C. for 20 hours. The mixture was partitioned between ethyl acetate and 1.00 N HCl (10.0 mL), the organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield the product (0.050 g, 18%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.45-5.28 (m, 1H), 5.09 (dddt, J=8.4, 7.0, 5.6, 1.4 Hz, 2H), 4.30 (t, J=1.8 Hz, 2H), 4.19-4.11 (m, 2H), 4.06 (d, J=6.9 Hz, 2H), 2.15-2.00 (m, 6H), 2.00-1.91 (m, 2H), 1.73-1.65 (m, 6H), 1.63-1.56 (m, 6H). 
     [(4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-yn-1-yl phosphonato)oxy]phosphonate. A 10.0 mL 14/20 round bottom flask was charged with 4-{[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}but-2-yn-1-ol (0.050, 0.170 mmol), diethyl ether (1.00 mL), cooled to 0° C., and treated with phosphorus tribromide (0.0280 mL, 0.300 mmol). The mixture was stirred at 0° C. for 15 minutes, diluted with hexanes, washed with brine, a saturated sodium bicarbonate solution, and brine. The organic layer was dried over sodium sulfate and concentrated to provide crude (6E,10E)-12-[(4-bromobut-2-yn-1-yl)oxy]-2,6,10-trimethyldodeca-2,6,10-triene (0.0600 g, 100%). This material was dissolved in acetonitrile (2.00 mL) and, under an argon atmosphere, treated with tetrabutylammonium pyrophosphate (0.251 g, 0.270 mmol), then stirred for 2 hours. The mixture was concentrated to a viscous liquid and purified over DOWEX50 resin column, the column prepared by dissolving DOWEX50 resin (5.98 g) in concentrated ammonium hydroxide (30.0 mL) for 20 minutes, then filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20 mL of 1:49 2-propanol: 25 mmolar ammonium bicarbonate) and poured into the column. The excess buffer was drained from the column and the crude material applied to the column (dissolved in 3.00 mL of the same buffer). The material was eluted with 30.0 mL buffer and lyophilized to a waxy solid (0.076 g, 100%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.46 (d, J=21.5 Hz), -10.18--11.03 (m). HRMS ESI [M−H] calc&#39;d=449.1497, observed=449.1494. 
     EXAMPLE 28 
     {[(6E,10E)-3,7,11,15-tetramethyl-2-oxohexadeca-6,10,14-trien-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     (2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. A 100 mL 14/20 round bottom flask was charged with ethyl 2-(diethoxyphosphoryl)-2-ethoxyacetate (2.36 g, 8.80 mmol. Prepared according to the method described in Bach, K.; Hesham, R., E.-S.; Jensen, H. M.; Nielsen, H. B.; Thomson, I.; Torssell, K. B. G.,  Tetrahedron,  1994, 50, 7543), tetrahydrofuran (15.0 mL) then cooled to 0° C., and treated with sodium hydride (0.336 g, 10.0 mmol) under an argon atmosphere. Farnesyl acetone (1.57 g, 6.00 mmol), dissolved in tetrahydrofuran (5.00 mL), was added in dropwise fashion. The mixture was then heated to 45° C. for 26 hours and quenched with a saturated ammonium chloride solution. The reaction was partitioned with ethyl acetate, washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was dissolved in ethanol (30.0 mL) then cooled to 0° C. and treated with sodium borohydride (0.226 g, 6.00 mmol) and the mixture stirred for 1 hour. The mixture was partitioned between ethyl acetate and saturated ammonium chloride solution, washed with brine, dried over sodium sulfate, and concentrated in vacuo to dryness. The crude material was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to yield an oil (0.953 g, 41%). The crude was dissolved in dichloromethane (5.00 mL), cooled to 0° C., under an argon atmosphere, and treated with diisobutylaluminum hydride (8.00 mL, 8.00 mmol, 1.00 M in heptanes). The reaction was warmed to room temperature, stirred for 22 hours, then cooled to 0° C. and quenched with ethanol. The mixture was then treated with a solution of sodium potassium tartrate (10.0 g, 35.0 mmol in 50.0 mL water) and vigorously stirred for 24 hours. The mixture was partitioned, and the aqueous layer washed with dichloromethane. The organic layers were combined, dried over sodium sulfate, filtered, concentrated in vacuo and purified by silica gel chromatography to yield the product (0.665 g, 80%).  1 H NMR (500 MHz, CDCl 3 ) δ 5.17-5.04 (m, 3H), 4.34-4.07 (m, 2H), 3.81-3.69 (m, 2H), 2.19-2.12 (m, 1H), 2.12-2.00 (m, 9H), 1.98 (q, J=7.5, 7.0 Hz, 3H), 1.72-1.65 (m, 9H), 1.63-1.55 (m, 6H), 1.29-1.21 (m, 3H). 
     {[(6E,10E)-3,7,11,15-tetramethyl-2-oxohexadeca-6,10,14-trien-1-yl phosphonato]oxy} phosphonate. A 25.0 mL 14/20 round bottom flask was charged with (2Z,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol and (2E,6E,10E)-2-ethoxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol (0.385 g, 1.15 mmol), dichloromethane (5.00 mL) and triphenylphosphine (0.314 mL, 1.20 mmol). The mixture was cooled to 0° C. and treated with carbon tetrabromide (0.379 mL, 1.20 mmol dissolved in 1.00 mL dichloromethane). The mixture was stirred for 10 minutes at 0° C. and 20 minutes at room temperature. The mixture was concentrated to ˜1.00 mL in vacuo and diluted with hexanes (15.0 mL). A solid precipitated and the mixture filtered through Celite, this process was repeated twice to yield crude (6E,10E)-1-bromo-3,7,11,15-tetramethylhexadeca-6,10,14-trien-2-one (0.397 g) as an oil. This material was dissolved in acetonitrile (2.00 mL) and treated with tetrabutylammonium pyrophosphate (0.775 g, 0.850 mmol). The reaction mixture was stirred under an argon atmosphere for 2 hours, concentrated in vacuo and purified on a DOWEX50 resin column, the column prepared by first suspending the DOWEX50 resin (11.7 g) in concentrated ammonium hydroxide (45.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and loaded into the DOWEX50 column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material on the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.291 g, 55%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.10, −10.74. HRMS ESI [M−H] calc&#39;d=465.1813, observed=465.1812. 
     EXAMPLE 29 
     {[(2E)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-en-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}benzaldehyde. A 25.0 mL 14/20 round bottom flask was charged with geranyl bromide (0.975 g, 4.50 mmol, Brundel, B., J., J., M.; Steen, H.; Heeres, A.; Seerden, J. P. G., WO2013157926), acetone (15.0 mL), 4-hydroxybenzaldehyde (0.732 g, 6.00 mmol) and potassium carbonate (1.10 g, 8.00 mmol). The mixture was stirred overnight, then partitioned between ethyl acetate and a saturated aqueous ammonium chloride solution. The organic layer was then washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by filtration through a triethylamine-deactivated silica (100% ethyl acetate) to yield the pure product (0.980 g, 84%).  1 H NMR (500 MHz, CDCl 3 ) δ 9.95-9.82 (m, 1H), 7.90-7.72 (m, 2H), 7.05-6.94 (my, 2H), 5.55-5.42 (m, 1H), 5.13-5.03 (m, 1H), 4.70-4.52 (m, 2H), 2.24-1.99 (m, 4H), 1.79-1.75 (m, 3H), 1.68 (d, J=1.4 Hz, 3H), 1.61 (d, J=1.3 Hz, 3H). 
     Ethyl (2E)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-enoate and ethyl (2Z)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-enoate. A 50.0 mL 14/20 round bottom flask was charged with 4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}benzaldehyde (0.800 g, 3.10 mmol), tetrahydrofuran (15.0 mL), cooled to 0° C., and treated with sodium hydride (0.201 g, 6.00 mmol) under an argon atmosphere. Triethyl-2-phosphonopropionate (0.952 g, 4.00 mmol), dissolved in tetrahydrofuran (2.00 mL), was added dropwise. After gas evolution ceased, the mixture was heated to 45° C. for 70 hours and quenched with methanol. The mixture was partitioned between ethyl acetate and a saturated ammonium chloride solution, the organic layer washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was dissolved in ethanol (20.0 mL), cooled to 0° C., and sodium borohydride added (0.082 g, 2.10 mmol). After 10 minutes, the reaction was quenched with a saturated ammonium chloride solution (30.0 mL) and partitioned with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to yield an oil (0.43 g, 41%).  1 H NMR (500 MHz, CDCl 3 ) δ 7.65 (d, J=1.8 Hz, 1H), 7.44-7.34 (m, 2H), 7.00-6.86 (m, 2H), 5.50 (tp, J=6.6, 1.4 Hz, 1H), 5.10 (ddp, J=6.8, 5.4, 1.4 Hz, 1H), 4.62-4.54 (m, 2H), 4.27 (q, J=7.1 Hz, 2H), 2.20-2.01 (m, 7H), 1.76 (t, J=1.3 Hz, 3H), 1.69 (q, J=1.3 Hz, 3H), 1.62 (d, J=1.3 Hz, 3H), 1.36 (t, J=7.1 Hz, 3H). 
     (2E)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-en-1-ol and (2Z)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-en-1-ol. A mixture of ethyl (2E)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-enoate and ethyl (2Z)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-enoate (0.430, 1.20 mmol) was dissolved in dichloromethane (5.00 mL), cooled to 0° C. under an argon atmosphere, and treated with diisobutylaluminum hydride (3.00 mL, 3.00 mmol, 1.00 M in heptanes). The reaction was allowed to warm to room temperature and stirred for 22 hours, cooled to 0° C., quenched with ethanol (2.00 mL) and stirred vigorously for 24 hours with a solution of sodium potassium tartrate (10.0 g, 35.0 mmol in 40.0 mL water). The mixture was partitioned, the aqueous layer washed with dichloromethane and the organic layers combined, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude was purified by silica gel chromatography to yield an oil (0.240 g, 80%).  1 H NMR (500 MHz, CDCl 3 ) δ 7.25-7.19 (m, 2H), 6.93-6.85 (m, 2H), 6.49-6.41 (m, 1H), 5.50 (tq, J=6.6, 1.3 Hz, 1H), 5.10 (ddp, J=7.0, 5.8, 1.4 Hz, 1H), 4.55 (d, J=6.5 Hz, 2H), 4.17 (d, J=1.3 Hz, 2H), 2.20-2.02 (m, 4H), 1.91 (d, J=1.4 Hz, 3H), 1.74 (d, J=1.4 Hz, 3H), 1.69 (d, J=1.4 Hz, 3H), 1.61 (d, J=1.4 Hz, 3H). 
     {[(2E)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-en-1-yl phosphonato]oxy}phosphonate and {[(2Z)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-en-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with a mixture of (2E)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-en-1-ol and (2Z)-3-(4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-2-methylprop-2-en-1-ol (0.212 g, 0.570 mmol), diluted in dichloromethane (3.00 mL), cooled to 0° C., and, under an argon atmosphere, treated with phosphorus tribromide (0.0940 mL, 1.00 mmol). The reaction was stirred at 0° C. for 1 hour then diluted with hexanes, quenched with brine, washed with sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to provide crude mixture of 1-[{(1E)-3-bromo-2-methylprop-1-en-1-yl]-4-[(2E)-3,7-dimethylocta-2,6-dien-1 -yl]oxy}benzene and 1-[(1Z)-3-bromo-2-methylprop-1-en-1-yl]-4-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}benzene (0.216 g, 100%). This material was dissolved in acetonitrile (2.00 mL), then treated with tetrabutylammonium pyrophosphate (0.394 g, 0.430 mmol). The mixture was stirred under an argon atmosphere for 2 hours, at which time it was concentrated in vacuo and purified over DOWEX50 resin column, which was prepared according to the following method. DOWEX50 resin (6.70 g) was stirred in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a waxy solid (0.170 g, 65%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.49 (d, J=21.8 Hz), −10.26 (d, J=21.4 Hz). HRMS ESI [M−H] calc&#39;d=459.1343, observed=459.1305. 
     EXAMPLE 30 
     {[(2E,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl phosphonato]oxy}phosphonate, {[(2Z,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl phosphonato]oxy}phosphonate, {[(2E,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl phosphonato]oxy}phosphonate, {[(2Z,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl phosphonato]oxy}phosphonate 
     
       
         
         
             
             
         
       
     
     Ethyl (2E,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaenoate, ethyl (2Z,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaenoate, ethyl (2E,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaenoate, and ethyl (2Z,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaenoate. A 100 mL 14/20 round bottom flask was charged with triethyl-4-phosphonocrotonate (3.00 g, 12.0 mmol), tetrahydrofuran (30.0 mL), cooled to 0° C., and sodium hydride (0.504 g, 15.0 mmol). After gas evolution ceased, a mixture of (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienal and (2Z,6E)-3,7,11-trimethyldodeca-2,6,10-trienal (2.04 g, 9.20 mmol, Hu, H.; Harrison, T. J.; Wilson, P. D.,  J. Org. Chem.,  2004, 69, 3782.) was added as a solution in tetrahydrofuran (2.00 mL). After 2 hours the mixture was quenched with a saturated ammonium chloride solution and partitioned into ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered, concentrated in vacuo, and purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to provide an oil (1.64 g, 56%).  1 H NMR (500 MHz, Chloroform-d) δ 7.35 (dddd, J=15.2, 11.3, 6.9, 0.8 Hz, 1H), 6.85-6.76 (m, 1H), 6.21 (dt, J=14.7, 10.7 Hz, 1H), 5.96 (dd, J=11.3, 2.0 Hz, 1H), 5.82 (dd, J=15.2, 2.0 Hz, 1H), 5.18-5.04 (m, 2H), 4.20 (q, J=7.1 Hz, 2H), 2.28-2.10 (m, 4H), 2.09-2.03 (m, 2H), 2.01-1.93 (m, 2H), 1.85 (dd, J=9.4, 1.3 Hz, 3H), 1.68 (d, J=1.6 Hz, 3H), 1.60 (dd, J=4.4, 2.8 Hz, 6H), 1.30 (t, J=7.1 Hz, 3H). 
     (2E,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-ol, (2Z,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-ol, (2E,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-ol, and (2Z,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-ol. A mixture of ethyl (2E,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaenoate, ethyl (2Z,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaenoate, ethyl (2E,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaenoate, and ethyl (2Z,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaenoate (1.64 g, 5.1 mmol) was dissolved in dichloromethane (10.0 mL), cooled to 0° C. under an argon atmosphere, and diisobutylaluminum hydride added (15.0 mL, 15.0 mmol, 1.00 M in heptanes). The mixture was warmed to room temperature and stirred for 22 hours. The reaction was cooled to 0° C., quenched with ethanol (2.00 mL) and stirred vigorously for 24 hours with a solution of sodium potassium tartrate (10.0 g, 35.0 mmol in 40.0 mL water). The reaction mixture was partitioned, and the aqueous layer washed with dichloromethane. The organic layers were combined, dried over sodium sulfate and concentrated in vacuo to provide the crude product mixture, which was purified by silica gel chromatography (1:9 ethyl acetate: hexanes) to yield a mixture of the products (0.610 g, 44%).  1 H NMR (500 MHz, CDCl 3 ) δ 6.50-6.40 (m, 1H), 6.36-6.26 (m, 1H), 6.13 (dt, J=14.8, 10.7 Hz, 1H), 5.91-5.85 (m, 1H), 5.80 (ddd, J=15.2, 7.3, 5.0 Hz, 1H), 5.10 (dtdq, J=9.9, 7.0, 2.9, 1.4 Hz, 2H), 4.20 (dd, J=6.0, 1.4 Hz, 2H), 2.20-2.09 (m, 6H), 1.98 (dd, J=9.2, 6.1 Hz, 2H), 1.80 (dd, J=14.2, 1.3 Hz, 3H), 1.68 (t, J=1.4 Hz, 3H), 1.60 (d, J=1.3 Hz, 6H). HRMS ESI (+) calc&#39;d for [M+Na]=297.2195, found=297.2242. 
     {[(2E,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl phosphonato]oxy}phosphonate, {[(2Z,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl phosphonato]oxy}phosphonate, {[(2E,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl phosphonato]oxy}phosphonate, {[(2Z,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl phosphonato]oxy}phosphonate. A 25.0 mL 14/20 round bottom flask was charged with a mixture of (2E,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-ol, (2Z,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-ol, (2E,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-ol, and (2Z,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-ol (0.164 g, 0.590 mmol) was dissolved in diethyl ether (3.00 mL) and under an argon atmosphere at 0° C., treated with triethylamine (0.208 mL, 1.50 mmol) followed by methanesulfonyl chloride (0.0770 mL, 1.00 mmol). A precipitate formed upon the addition and after 30 minutes at 0° C., the reaction was diluted with hexanes and washed with brine (three times), the organic layer dried over sodium sulfate, and concentrated in vacuo to yield the crude (2E,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl methanesulfonate, (2Z,4E,6E,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl methanesulfonate, (2E,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl methanesulfonate, and (2Z,4E,6Z,10E)-7,11,15-trimethylhexadeca-2,4,6,10,14-pentaen-1-yl methanesulfonate (0.211 g, 100%). This mixture was dissolved in acetonitrile (2.00 mL) and treated with tetrabutylammonium pyrophosphate (0.332 g, 0.360 mmol). The reaction mixture was stirred under argon for 2 hours, at which time it was concentrated in vacuo and purified over DOWEX50 resin column. The column was prepared via the following method. DOWEX50 resin (6.7 g) was stirred in concentrated ammonium hydroxide (30.0 mL) for 20 minutes. The resin was filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propanol: 25.0 mmolar ammonium bicarbonate) and poured into a column. The excess buffer was drained from the column and the crude material was applied to the column (dissolved in 3.0 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a solid (0.127 g, 50%).  31 P NMR (202 MHz, Deuterium Oxide) δ −5.88-−6.54 (m), −21.17--21.93 (m). HRMS ESI [M−H] calc&#39;d=433.1550, observed=433.1552. 
     EXAMPLE 31 
     ({[2-({[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1 yl]oxy}methyl) cyclopropyl]methyl phosphonato}oxy)phosphonate 
     
       
         
         
             
             
         
       
     
     [2-({[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}methyl)cyclopropyl]methanol. A 50.0 mL 14/20 round bottom flask was charged with trans, trans-farnesol (0.889 g, 4.00 mmol), diethyl ether (10.0 mL), and cooled to 0° C. (argon atmosphere). Phosphorus tribromide (1.35 g, 5.00 mmol) was added and the mixture allowed to stir for 1 hour, then diluted with hexanes, washed with brine, a saturated sodium bicarbonate solution, and then a second time with brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to yield crude trans, trans-farnesyl bromide (1.06 g, 3.7 mmol).  1 H NMR (500 MHz, Chloroform-d) δ 4.13-4.04 (m, 2H), 3.40 (s, 2H), 3.29-3.19 (m, 2H), 1.37-1.25 (m, 2H), 0.79 (td, J=8.2, 5.1 Hz, 1H), 0.20 (q, J=5.3 Hz, 1H). A separate flask was charged with [2-(hydroxymethyl)cyclopropyl]methanol (0.612 g, 6.0 mmol, (Ito, M.; Osaku, A.; Shiibashi, A.; Ikariya, T., Org. Lett, 2007, 9, 1821. and tetrahydrofuran (15.0 mL). The mixture was cooled to 0° C. and sodium hydride added (0.268 g, 8.0 mmol). After gas evolution ceased, a solution of crude trans, trans-farnesyl bromide dissolved in tetrahydrofuran (5.0 mL) was added and the mixture heated to 45° C. for 19 hours under an argon atmosphere. The mixture was partitioned between ethyl acetate and a saturated ammonium chloride solution and the organic layer washed with brine and concentrated to dryness to provide an oil. The crude material was purified by silica gel chromatography (1:4 ethyl acetate: hexanes) to yield the product as an oil (0.610 g, 33%). HRMS ESI [M+Na] calc&#39;d=329.2457, observed=329.2474. 
     [2-([{(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}methyl)cyclopropyl]methanol (0.200 g, 0.650 mmol) was dissolved in diethyl ether (3.00 mL), cooled to 0° C. and under an argon atmosphere, charged phosphorus tribromide (0.270 g, 1.00 mmol) and stirred for 15 minutes. The reaction was diluted with hexanes, washed with brine, a saturated sodium bicarbonate solution, and brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to yield crude 1-(bromomethyl)-2-({[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}methyl) cyclopropane (0.110 g, 45%). This material was dissolved in acetonitrile (2.00 mL) and tetrabutylammonium pyrophosphate was added (0.221 g, 0.240 mmol), the reaction under an argon atmosphere for two hours. The mixture was then concentrated in vacuo and purified on a DOWEX50 resin column. The DOWEX50 resin (7.30 g) was stirred in concentrated ammonium hydroxide (30.0 mL) for 20 minutes, then filtered and washed four times with water (100 mL). The resin was suspended in a buffer (20.0 mL of 1:49 2-propano1:25.0 mmolar aqueous ammonium bicarbonate) and poured into a column. The excess 1:49 2-propano1:25.0 mmolar aqueous ammonium bicarbonate buffer was drained from the column and the crude material was applied to the column (dissolved in 3.00 mL of the 1:49 2-propanol:25.0 mmolar aqueous ammonium bicarbonate buffer). The material was eluted with 30.0 mL of the 1:49 2-propanol: 25.0 mmolar aqueous ammonium bicarbonate buffer and lyophilized to a solid (0.113 g, 38%).  31 P NMR (202 MHz, Deuterium Oxide) δ −6.32 (d, J=20.7 Hz), −10.20 (d, J=20.6 Hz). HRMS ESI [M=H] calc&#39;d=465.1813, observed=465.1808. 
     EXAMPLE 32 
     Coupling Class II and Class I Enzymes 
     Enzymes Coleus forskohlii CfTPS2 (SEQ ID NO:69) and  Salvia sclarea  SsSCS (SEQ ID NO:61) were coupled in an in vitro assay to ascertain whether the synthetic unnatural methyl-GGDP (C21) substrate can efficiently yield the corresponding C21 methyl-diterpene. The C21 substrate does not exist in nature and has the following structure. 
     
       
         
         
             
             
         
       
     
     As illustrated in  FIG.  10   , a new methyl-diterpene product, with a structure similar to sclareol, is detected when the  Coleus forskohlii  CfTPS2 and  Salvia sclarea  SsSCS enzymes are used together in an assay with the unnatural methyl-GGDP (C21) substrate. The  Coleus forskohlii  CfTps2 enzyme catalyzed the first step to provide a substrate for the  Salvia sclarea  SsSCS enzyme, which then produced the final product that has a structure similar to sclareol. 
     Many of the foregoing Examples illustrate that single step class I enzymes or single step class II labdane-type enzymes can synthesize irregular type diterpenes and unnatural derivatives thereof. However, this Example demonstrates that modules consisting of coupled class II and class I enzymes can be used in function sequential conversion reaction to prepare new types of diterpenes. 
     All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications. 
     The following statements are intended to describe and summarize various features of the invention according to the foregoing description provided in the specification and figures. 
     Statements: 
     1. A compound of the formula (I) or (II): 
     
       
         
         
             
             
         
       
     
     wherein:
         m is an integer from 0 to 3 (e.g., 1 or 2), with the understanding that if m is 2 or 3, each repeating subunit can be the same or different;   n is an integer from 0 to 1;   the dashed lines ( ) represent a double bond when R 3′  and R 4′  are absent or when R 5′  and R 6′  are absent,   A and A′ are each independently cycloalkyl, aryl or heterocyclyl, each of which can be optionally substituted;   X 1  is a heteroatom, −X 3 -alkyl, -alkyl-X 3 − or alkyl, wherein X 3  is a heteroatom or alkyl or X 1  is:       

     
       
         
         
             
             
         
       
         
         
           
             R 1  and R 2  form a double bond or an epoxide; 
             each R′, R 1′ , R 2 , R 2′ , and R 3 —R 6  is, independently, H, alkyl, halo, aryl, and alkylaryl; 
             R 3′  and R 4′  are absent or R 3′  and R 4′ , together with the carbon atoms to which they are attached, form an epoxide, a cycloalkyl group, an aryl group or a heterocyclyl group; 
             R 5′  and R 6′  are absent or R 5′  and R 6′ , together with the carbon atoms to which they are attached, form an epoxide, a cycloalkyl group, an aryl group or a heterocyclyl group; 
             X 2  is a bond, alkenyl or acyl; and 
             X 4  is a absent, a heteroatom or alkyl;
 
with the proviso that the compound of the formula (I) is not a compound of the formula:
 
           
         
       
    
     
       
         
         
             
             
         
       
     
     2. A compound of Statement 1, wherein the compound of the formula (I) is a compound of the formula: 
     
       
         
         
             
             
         
       
     
     3. A compound of Statement 1, wherein the compound of the formula (II) is a compound of the formula: 
     
       
         
         
             
             
         
       
     
     4. The compound of any preceding Statement, wherein if X 1  is a heteroatom, the heteroatom is oxygen. 
     5. The compound of any preceding Statement, wherein X 3  is oxygen or C 1 -C 5  alkyl, such as —CH 2 — and C 2 -C 3 -alkyl. 
     6. The compound of any preceding Statement, wherein R 3 —R 6  are each H or C 1 -C 5 -alkyl, such as methyl and C 2 -C 3 -alkyl. 
     7. The compound of any preceding Statement, wherein R 3  and R 5  are each H or C 1 -C 5 -alkyl, such as methyl and C 2 -C 3 -alkyl; and R 4  and R 6  are each H. 
     8. The compound of any preceding Statement, wherein m is 1 or 2. 
     9. The compound of any preceding Statement, wherein, m is 0. 
     10. The compound of any preceding Statement, wherein X 2  is an alkenyl group of the formula: 
     
       
         
         
             
             
         
       
     
     or an acyl group of the formula: 
     
       
         
         
             
             
         
       
     
     11. The compound of any preceding Statement, wherein the compound is a compound of the formula: 
     
       
         
         
             
             
         
       
     
     12. The compound of any preceding Statement, wherein the compounds can be enzymatically transformed into a terpenoid. 
     13. The compound of any preceding Statement, wherein the terpenoid comprises a compound core of the formula: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     and derivatives thereof, wherein derivatives can comprise additional double bonds, alkyl groups, hydroxy groups, acyl groups, and the like, dispersed about the cores. 
     14. A method comprising contacting an unnatural substrate with one or more enzymes capable of synthesizing a terpene to generate a primary product. 
     15. The method of Statement 14, wherein the unnatural substrate is a compound of Statements 1-14. 
     16. The method of Statement 14, wherein the one or more enzymes are from species  Tripterygium wilfordii  (Tw),  Euphorbia peplus  (Ep),  Coleus forskohlii  (Cf),  Ajuga reptans  (Ar),  Perovskia atriciplifolia  (Pa),  Nepeta mussini  (Nm),  Origanum majorana  (Om),  Hyptis suaveolens  (Hs),  Grindelia robusta  (Gr),  Leonotis leonurus  (Ll),  Marrubium vulgare  (Mv),  Vitex agnus - castus  (Vac),  Euphorbia peplus  (Ep),  Ricinus communis  (Rc),  Daphne genkwa  (Dg), or  Zea mays  (Zm). 
     17. The method of Statement 14, wherein the enzyme is from species  Salvia sclarea, Coleus forskohlii, Euphorbia peplus, Ajuga reptans, Origanum majoranum, Marrubium vulgare,  or  Kitasatospora griseola.    
     18. The method of Statement 14-17, wherein the primary product is a terpenoid. 
     19. The method of Statement 14-18, wherein one or more of the enzymes has at least 90% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 64, 65, 66, 67, or 69. 
     20. The method of Statement 14-18 or 19, further comprising contacting the primary product with one or more second enzymes. 
     21. The method of Statement 14-20, further comprising generating a second product by one or more second enzymes, where the one or more second enzymes catalyze the formation of the second product by using the primary product as a substrate. 
     22. The method of Statement 20 or 21, wherein the one or more second enzymes at least one of oxidizes, reduces, acylates, and glycosylates the primary product. 
     23. The method of Statement 20, 21 or 22, wherein the one or more second enzymes is an enzyme listed in Table 2. 
     24. The method of Statement 20-22 or 23, wherein one or more of the second enzymes has at least 90% sequence identity to SEQ ID NO: 39, 68, 70, or 71. 
     25. The method of Statement 22-23 or 24, wherein the one or more second enzymes is Cytochrome P450 or a sclareol synthase. 
     26. The method of Statement 14-23 or 24, which is performed in vitro in a cell-free mixture. 
     27. The method of Statement 14-23 or 24, which is performed within a cell that expresses the enzyme. 
     28. A compound of the formula (I)-(IV): 
     
       
         
         
             
             
         
       
     
     wherein:
         each m is independently an integer from 0 to 3, with the understanding that if m is 2 or 3, each repeating subunit can be the same or different;   n is an integer from 0 to 1;   the dashed lines ( ) represent a double bond when R 3′  and R 4′  are absent or when R 5′  and R 6′  are absent,   A and A′ are each independently cycloalkyl, aryl or heterocyclyl, each of which can be optionally substituted;   X 1  is a heteroatom, —X 3 -alkyl, -alkyl-X 3 — or alkyl, wherein X 3  is a heteroatom or alkyl or X 1  is:       

     
       
         
         
             
             
         
       
         
         
           
             R 1  and R 2  form a double bond or an epoxide; 
             each R′, R 1′ , R 2 , R 2′ , and R 3 —R 6  is, independently, H, alkyl, alkoxy, halo, aryl, and alkylaryl; 
             R 3′  and R 4′  are absent or R 3′  and R 4′ , together with the carbon atoms to which they are attached, form an epoxide, a cycloalkyl group, an aryl group or a heterocyclyl group; 
             R 5′  and R 6′  are absent or R 5′  and R 6′ , together with the carbon atoms to which they are attached, form an epoxide, a cycloalkyl group, an aryl group or a heterocyclyl group; 
             X 2  is a bond, alkenyl, alkynyl or acyl; and 
             X 4  is a absent, a heteroatom or alkyl; with the proviso that the compound of the formula (I) is not a compound of the formula: 
           
         
       
    
     
       
         
         
             
             
         
       
     
     29. The compound of Statement 28, wherein X 2  is an alkenyl group of the formula: 
     
       
         
         
             
             
         
       
     
     wherein q is an integer from 1 to 3; or 
     
       
         
         
             
             
         
       
     
     or an acyl group of the formula: 
     
       
         
         
             
             
         
       
     
     30. The compound of Statement 28 or 29, wherein the compound is a compound of the formula: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     The specific methods, devices and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. 
     The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims. 
     Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants. 
     The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention. 
     The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.