Patent Publication Number: US-2022213513-A1

Title: Production of cannabinoids

Description:
TECHNICAL FIELD 
     The present disclosure relates to improved methods of producing cannabinoids. 
     BACKGROUND 
     Cannabinoids are a general class of chemicals that act on cannabinoid receptors and other target molecules to modulate a wide range of physiological behaviour such as neurotransmitter release. Cannabinoids are produced naturally in humans (called endocannabinoids) and by several plant species (called phytocannabinoids) including  Cannabis sativa . Cannabinoids have been shown to have several beneficial medical/therapeutic effects and therefore they are an active area of investigation by the pharmaceutical industry for use as pharmaceutical products for various diseases. 
     Currently the production of cannabinoids for pharmaceutical or other uses is done by chemical synthesis or through the extraction of cannabinoids from plants that are producing these cannabinoids, for example  C. sativa . There are several drawbacks to the current methods of cannabinoid production. The chemical synthesis of various cannabinoids is a costly process when compared to the extraction of cannabinoids from naturally occurring plants. The chemical synthesis of cannabinoids also involves the use of chemicals that are not environmentally friendly, which can be considered as an additional cost to their production. Furthermore, the synthetic chemical production of various cannabinoids has been classified as less pharmacologically active as those extracted from plants such as  C. sativa . Although there are drawbacks to chemically synthesized cannabinoids, the benefit of this production method is that the end product is a highly pure single cannabinoid. This level of purity is preferred for pharmaceutical use. The level of purity required by the pharmaceutical industry is reflected by the fact that no plant extract based cannabinoid production has received FDA approval yet and only synthetic compounds have been approved. 
     In contrast to the synthetic chemical production of cannabinoids, the other method that is currently used to produce cannabinoids is production of cannabinoids in plants that naturally produce these chemicals; the most used plant for this is  C. sativa . In this method, the plant  C. sativa  is cultivated and during the flowering cycle various cannabinoids are produced naturally by the plant. The plant can be harvested and the cannabinoids can be ingested for pharmaceutical purposes in various methods directly from the plant itself or the cannabinoids can be extracted from the plant. There are multiple methods to extract the cannabinoids from the plant  C. sativa . All of these methods typically involve placing the plant,  C. sativa  that contains the cannabinoids, into a chemical solution that selectively solubilizes the cannabinoids into this solution. There are various chemical solutions used to do this such as hexane, cold water extraction methods, C02 extraction methods, and others. This chemical solution, now containing all the different cannabinoids, can then be removed, leaving behind the excess plant material. The cannabinoid containing solution can then be further processed for use. 
     There are several drawbacks of the natural production and extraction of cannabinoids in plants such as  C. sativa . Since there are numerous cannabinoids produced by  C. sativa  it is often difficult to reproduce identical cannabinoid profiles in plants using an extraction process. Furthermore, variations in plant growth will lead to different levels of cannabinoids in the plant itself making reproducible extraction difficult. Different cannabinoid profiles will have different pharmaceutical effects which are not desired for a pharmaceutical product. Furthermore, the extraction of cannabinoids from  C. sativa  extracts produces a mixture of cannabinoids and not a highly pure single pharmaceutical compound. Since many cannabinoids are similar in structure it is difficult to purify these mixtures to a high level resulting in cannabinoid contamination of the end product. 
     There is thus a need to provide improved methods of cannabinoid production. 
     SUMMARY 
     This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims. As described herein, the following claims are made:
     1. A Polyketide Synthase (PKS) enzyme comprising the amino acid sequence selected from:
       a. SEQ ID NO:1 ( C. stellaris -OLAs-dACP1);   b. SEQ ID NO:2 ( C. stellaris -OLAs-dACP2);   c. SEQ ID NO:3 ( C. stellaris -OLAs-wt (wild type  C. stellaris ));   d. SEQ ID NO:6 ( C. grayi -PKS-dACP1);   e. SEQ ID NO:7 ( C. grayi -PKS-dACP2);   f. SEQ ID NO:35 ( P. furfuracea );   g. an PKS enzyme variant of any one of SEQ ID NO:4-5 and 35 ( C. grayi, C. uncialis ), wherein one of the two ACP domains has been inactivated;   h. an PKS enzyme variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 1-7 or 35, wherein said PKS enzyme variant has retained Olivetolic Acid Synthase activity and has inactivated an ACP domain;   i. an PKS enzyme variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity to any one of SEQ ID NOS: 1-7 or 35, wherein said PKS enzyme variant has retained Olivetolic Acid Synthase activity and has inactivated an ACP domain;   j. a PKS enzyme variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the domains selected from: SAT domain, KS domain, AT domain, PT domain, ACP1 domain, ACP2 domain, and TE domain of SEQ ID NOS: 1-7 or 35, wherein said PKS enzyme variant has retained Olivetolic Acid Synthase activity and has inactivated an ACP domain; or   k. any combination of (a)-(j).   
       2. A polynucleotide encoding the PKS enzyme of claim  1 .   3. A composition comprising:
       a. the PKS enzyme of claim  1 ; and   b. a npgA enzyme.   
       4. The composition of claim  3 , wherein said composition is a cell-free composition.   5. The composition of claim  3 , wherein said composition comprises a recombinant microorganism.   6. The composition of claim  5 , wherein said recombinant microorganism:
       a. expresses the PKS enzyme of claim  1 ;   b. expresses the npgA enzyme; and/or   c. comprises the polynucleotide of claim  2 .   
       7. The composition of any one of claims  3 - 6 , wherein said composition further comprises at least one enzyme selected from:
       a. a FAS1 mutant, wherein mutations are selected from I306A, R1834K;   b. a FAS2 mutant, wherein said mutation is selected from G1250S, M1251W;   c. StcJ and StcK;   d. HexA and HexB;   e. ERG10;   f. ERG13;   g. HMGR;   h. tHMGR (truncated HMGR);   i. ERG12;   j. ERG8;   k. ERG19;   l. IDI1;   m. a ERG20 mutant, wherein said mutant is selected from
           i.  S. cerevisiae  ERG20 F96W/N127W  or  Y. lipolytica  ERG20 F88W/N119W  or   ii.  S. cerevisiae  ERG20 K197E  or  Y. lipolytica  ERG20 K189E .   
           n. a mutant NphB (mutNphB) (preferably with mutations at least one of Q161A, G286S, Y288A, A232S);   o. csPT1;   p. csPT4;   q. a tetrahydrocannabinolic acid synthase (THCAS);   r. a cannabidiolic acid synthase (CBDAS);   s. a cannabichromenic acid synthase (CBCAS); or   t. any combination of (a)-(s).   
       8. The composition of any one of claims  5 - 7 , wherein said recombinant microorganism overexpresses a protein selected from:
       a. the PKS enzyme of claim  1 ; and/or   b. the enzyme of claim  7 .   
       9. The composition of claim  8 , wherein said protein is overexpressed by:
       a. operably associating a strong promoter with a polynucleotide encoding the protein; and/or   b. multiple copies of a polynucleotide encoding the protein by the recombinant microorganism.   
       10. The composition of any one of claims  5 - 9 , wherein said recombinant microorganism further comprises inactivation of:
       a. PEX10;   b. CPR1;   c. PEP4 (from  S. cervisae , YALI0F27071p in YL); and/or   d. PRB1 (from  S. cervisae , YALI0B16500p and/or YALI0A06435p in YL).   
       11. The composition of any one of claims  3 - 10 , wherein the composition further comprises any one of:
       a. Compound II, wherein n is 1 (Butyryl-CoA), 2 (Hexanoyl-CoA) or 3 (Octanoyl-CoA);   
       

     
       
         
         
             
             
         
       
     
     and/or
         b. Compound III, wherein n is 1 (Butyric Acid), 2 (Hexanoic Acid) or 3 (Octanoic Acid);       

     
       
         
         
             
             
         
       
         
         12. The composition of any one of claims  3 - 11 , wherein the composition further comprises at least one cannabinoid or cannabinoid precursor. 
         13. The composition of claim  12 , wherein the at least one cannabinoid or cannabinoid precursor comprises CBGA, THCA, CBDA, CBCA, CBD, THC, CBC, CBGVA, THCVA, CBDVA, CBCVA, CBDV, THCV, CBCV, THCA-C7, CBDA-C7, CBGA-C7 CBCA-C7, CBD-C7, THC-C7, CBC-C7, or CBN analog. 
         14. A method of producing Compound I, wherein said method comprises contacting the composition of any one of claims  3 - 13  with a carbohydrate source to enzymatically produce Compound I, wherein Compound I is 
       
    
     
       
         
         
             
             
         
       
         
         
           
             wherein n is selected from 1 (Diviaric Acid), 2 (Olivetolic acid), or 3 (2,4-Dihydroxy-6-geptylbenzoic acid). 
           
         
         15. The method of claim  14 , wherein the carbohydrate source is selected from:
       a. Acetyl-CoA;   b. Malonyl-CoA;   c. Mevalonate;   d. Compound II;   e. Compound III; and/or   f. Compound IV, wherein Compound IV is   
     
       
    
       CH 3 —(CH 2 ) 2n —OH   Compound IV
         wherein n is selected from 1 (propanol), 2 (pentanol), or 3 (heptanol);       16. The method of either claim  14  or  15 , wherein the carbohydrate source is exogenously provided.   17. The method of any one of claims  14 - 16 , wherein said carbohydrate source is provided by enzymatically converting Compound III into Compound II.   18. The method of claim  17 , wherein the enzyme that converts Compound III into Compound II is selected from:
       a. CsAAE1;   b. AAL1ΔSKL; or   c. AAL1.   
       19. The method of claim  14 - 16 , wherein acetyl-CoA and malonyl-CoA is enzymatically converted into Compound II by the combination of enzymes selected from:
       a. StcJ and StcK;   b. HexA and HexB; or   c. MutFas1 and MutFas2.   
       20. The method of any one of claims  14 - 19 , wherein Compound II is enzymatically converted into Compound I.   21. The method of claim  20 , wherein the enzyme that converts Compound II into Compound I is the PKS enzyme of claim  1  and a npgA Enzyme.   22. The method of any one of claims  14 - 21 , wherein said method further comprises enzymatically converting Acetyl-CoA into Mevalonate by:
       a. ERG10;   b. ERG13; or   c. one or both of HMGR or tHMGR.   
       23. The method of claim  22 , wherein Mevalonate is further enzymatically converted into Geranyldiphosphate (GPP) by:
       a. ERG12;   b. ERG8;   c. ERG19;   d. IDI1; and   e. an ERG20 mutant, wherein said mutant is selected from
           i.  S. cerevisiae  ERG20 F96W/N127W  or  Y. lipolytica  ERG20 F88W/N119W  or   ii.  S. cerevisiae  ERG20 K197E  or  Y. lipolytica  ERG20 K189E .   
           
       24. The method of any one of claims  14 - 23 , wherein Geranyldiphosphate is exogenously provided.   25. The method of either claim  24  or  24  wherein said method further comprises enzymatically converting Compound I and Geranyldiphosphate into at least one cannabinoid or cannabinoid precursor.   26. The method of claim  25 , wherein the at least one cannabinoid or cannabinoid precursor comprises CBGA, THCA, CBDA, CBCA, CBD, THC, CBC, CBGVA, THCVA, CBDVA, CBCVA, CBDV, THCV, CBCV, THCA-C7, CBDA-C7, CBGA-C7 CBCA-C7, CBD-C7, THC-C7, CBC-C7, or CBN analog.   27. The method of either claim  25 - 26 , wherein Compound I and Geranyldiphosphate is enzymatically converted into the at least one cannabinoid precursor by mutNphB, csPT1 and/or csPT4.   28. The method of any one of claims  14 - 27 , wherein Compound I, the at least one cannabinoid or cannabinoid precursor, or the CBGA, THCA, CBDA, CBCA, CBD, THC, CBC, CBGVA, THCVA, CBDVA, CBCVA, CBDV, THCV, CBCV, THCA-C7, CBDA-C7, CBGA-C7 CBCA-C7, CBD-C7, THC-C7, CBC-C7, or CBN analog is recovered.   29. The method of any one of claims  14 - 27 , wherein Compound I, the at least one cannabinoid or cannabinoid precursor, or the CBGA, THCA, CBDA, CBCA, CBD, THC, CBC, CBGVA, THCVA, CBDVA, CBCVA, CBDV, THCV, CBCV, THCA-C7, CBDA-C7, CBGA-C7, CBCA-C7, CBD-C7, THC-C7, CBC-C7, or CBN analog is purified.   30. The Compound I, the at least one cannabinoid or cannabinoid precursor, or the CBGA, THCA, CBDA, CBCA, CBD, THC, CBC, CBGVA, THCVA, CBDVA, CBCVA, CBDV, THCV, CBCV, THCA-C7, CBDA-C7, CBGA-C7 CBCA-C7, CBD-C7, THC-C7, CBC-C7, or CBN analog acid produced by the method of any one of claims  14 - 29 .   31. The composition of any one of claims  5 - 13  or the method of any one of claims  14 - 31 , wherein the recombinant microorganism is selected from: bacteria, fungi, yeasts, algae, and archaea.   32. The composition or method of claim  31 , wherein said recombinant microorganism is a yeast.   33. The composition or method of claim  32 , wherein said yeast is oleaginous.   34. The composition or method of claim  33 , wherein the yeast is selected from the genera  Rhodosporidium, Rhodotorula, Yarrowia, Cryptococcus, Candida, Lipomyces  and  Trichosporon.      35. The composition or method of claim  34 , wherein said yeast is a  Yarrowia lipolytica , a  Lipomyces starkey , a  Rhodosporidium toruloides , a  Rhodotorula glutinis , a  Trichosporon fermentans  or a  Cryptococcus curvatus.      36. The composition or method of one of claims  32 - 35 , wherein the yeast comprises at least 5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% dry weight of fatty acids or fats.   37. The composition or method of any one of claims  32 - 36 , wherein the yeast is genetically modified to produce at least 5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% dry weight of fatty acids or fats.   

    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
       Embodiments of the present disclosure will be discussed with reference to the accompanying drawings wherein: 
         FIG. 1A  illustrates a first enzymatic pathway as described herein for producing Compound I from the starting materials of either Compound III and/or Compound II. 
         FIG. 1B  illustrates a second enzymatic pathway as described herein for producing Compound I from the starting materials of either Compound II and/or Acetyl-CoA and Malonyl CoA. 
         FIG. 2  is diagram of the cannabinoid synthesis pathway including nonenzymatic steps starting with a CBGA-Analog; 
         FIG. 3  illustrates the enzymatic pathway as described herein for producing GPP from different carbohydrate sources. 
         FIG. 4  describes the structures for Compound I, II, III and IV. 
         FIGS. 5A-B  describes the structures for Cannabinoid Precursors ( FIG. 5A ) and Cannabinoids ( FIG. 5B ). 
         FIG. 6  is an alignment of SEQ ID NOs: 3-5 showing identical (*) vs conserved amino acid (.) between the three sequences. 
         FIG. 7  provides a list of abbreviations used throughout the specification. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Definitions 
     The following definitions are provided for specific terms which are used in the following written description. 
     As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cannabinoid precursor” includes a plurality of precursors, including mixtures thereof. The term “a polynucleotide” includes a plurality of polynucleotides. 
     As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. “Consisting essentially of” shall mean excluding other elements of any essential significance to the combination. Thus, compositions consisting essentially of produced cannabinoids would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for produced cannabinoids. Embodiments defined by each of these transition terms are within the scope of this invention. 
     The term “about” or “approximately” means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 fold, and more preferably within 2 fold, of a value. Unless otherwise stated, the term ‘about’ means within an acceptable error range for the particular value, such as ±1-20%, preferably ±1-10% and more preferably ±1-5%. 
     Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. 
     As used herein, the terms “polynucleotide” and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term “polynucleotide” includes, for example, single-, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA, recombinant polynucleotides, branched polynucleotides, aptamers, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules (e.g., comprising modified bases, sugars, and/or internucleotide linkers). 
     As used herein, the term “peptide” refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds or by other bonds (e.g., as esters, ethers, and the like). 
     As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, and amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long (e.g., greater than about 10 amino acids), the peptide is commonly called a polypeptide or a protein. While the term “protein” encompasses the term “polypeptide”, a “polypeptide” may be a less than full-length protein. 
     As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA transcribed from the genomic DNA. 
     As used herein, “under transcriptional control” or “operably linked” refers to expression (e.g., transcription or translation) of a polynucleotide sequence which is controlled by an appropriate juxtaposition of an expression control element and a coding sequence. In one aspect, a DNA sequence is “operatively linked” to an expression control sequence when the expression control sequence controls and regulates the transcription of that DNA sequence. 
     As used herein, “coding sequence” is a sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate expression control sequences. The boundaries of a coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, a prokaryotic sequence, cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., yeast, or mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence. 
     As used herein, two coding sequences “correspond” to each other if the sequences or their complementary sequences encode the same amino acid sequences. 
     As used herein, “signal sequence” denotes the endoplasmic reticulum translocation sequence. This sequence encodes a signal peptide that communicates to a cell to direct a polypeptide to which it is linked (e.g., via a chemical bond) to an endoplasmic reticulum vesicular compartment, to enter an exocytic/endocytic organelle, to be delivered either to a cellular vesicular compartment, the cell surface or to secrete the polypeptide. This signal sequence is sometimes clipped off by the cell in the maturation of a protein. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes. 
     As used herein, “hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. 
     As used herein, a polynucleotide or polynucleotide domain (or a polypeptide or polypeptide domain) which has a certain percentage (for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) of “sequence identity” to another sequence means that, when maximally aligned, using software programs routine in the art, that percentage of bases (or amino acids) are the same in comparing the two sequences. 
     Two polypeptide sequences are “substantially homologous” or “substantially similar” when at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% of amino acid residues of the polypeptide match conservative amino acids over a defined length of the polypeptide sequence. 
     Sequences that are similar (e.g., substantially homologous) can be identified by comparing the sequences using standard software available in sequence data banks. 
     Substantially homologous nucleic acid sequences also can be identified in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. For example, stringent conditions can be: hybridization at 5×SSC and 50% formamide at 42° C., and washing at 0.1×SSC and 0.1% sodium dodecyl sulfate at 60° C. Further examples of stringent hybridization conditions include: incubation temperatures of about 25 degrees C. to about 37 degrees C.; hybridization buffer concentrations of about 6×SSC to about 10×SSC; formamide concentrations of about 0% to about 25%; and wash solutions of about 6×SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40 degrees C. to about 50 degrees C.; buffer concentrations of about 9×SSC to about 2×SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples of high stringency conditions include: incubation temperatures of about 55 degrees C. to about 68 degrees C.; buffer concentrations of about 1×SSC to about 0.1×SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1×SSC, 0.1×SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed. Similarity can be verified by sequencing, but preferably, is also or alternatively, verified by function (e.g., ability to traffic to an endosomal compartment, and the like), using assays suitable for the particular domain in question. 
     The terms “percent (%) sequence similarity”, “percent (%) sequence identity”, and the like, generally refer to the degree of identity or similarity between different nucleotide sequences of nucleic acid molecules or amino acid sequences of polypeptides that may or may not share a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.), etc. 
     To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted. 
     The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, J. Mol. Biol. 1990; 215: 403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 1997, 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/on the WorldWideWeb. 
     To determine the percent similarity between two amino acid sequences, the sequences are also aligned for optimal comparison purposes. The percent similarity between the two sequences is a function of the number of conserved amino acids at positions shared by the sequences (i.e., percent similarity=number of conserved amino acids positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are, or are about, of the same length. The percent similarity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence similarity, typically conserved matches are counted. 
     Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1988; 4: 1 1-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. 
     In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 1970, 48:444-453), which has been incorporated into the GAP program in the GCG software package (Accelrys, Burlington, Mass.; available at accelrys.com on the WorldWideWeb), using either a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. 
     Another non-limiting example of how percent identity can be determined is by using software programs such as those described in Current Protocols In Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST. 
     Statistical analysis of the properties described herein may be carried out by standard tests, for example, t-tests, ANOVA, or Chi squared tests. Typically, statistical significance will be measured to a level of p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=0.000001 
     “Conservatively modified variants” of domain sequences also can be provided. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer, et al., 1991, Nucleic Acid Res. 19: 5081; Ohtsuka, et al., 1985, J. Biol. Chem. 260: 2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98). 
     Unless otherwise described, variants of the disclosed gene retain the ability of the wild type protein from which the variant was derived, although the activity may not be at the same level. In preferred embodiments, the variants have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% efficacy compared to the original sequence. In preferred embodiments, the variant has improved activity as compared to the original sequence. For example, variants with improved activity have at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, or at least about 160% efficacy compared to the original sequence. 
     For example, a variant common cannabinoid synthesising protein, such as CBDAS, must retain the ability to cyclize CBGA to produce CBDA with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence. In preferred embodiments, a variant common cannabinoid protein, such as CBDAS, has improved activity over the sequence from which it is derived in that the improved variant common cannabinoid protein has more than 110%, 120%, 130%, 140%, or and 150% improved activity in cyclizing CBGA to produce CBDA, as compared to the sequence from which the improved variant is derived. 
     The term “biologically active fragment”, “biologically active form”, “biologically active equivalent” of and “functional derivative” of a wild-type protein, possesses a biological activity that is at least substantially equal (e.g., not significantly different from) the biological activity of the wild type protein as measured using an assay suitable for detecting the activity. 
     As used herein, the term “isolated” or “purified” means separated (or substantially free) from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. By substantially free or substantially purified, it is meant at least 50% of the population, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%, are free of the components with which they are associated in nature. 
     A cell has been “transformed”, “transduced”, or “transfected” when nucleic acids have been introduced inside the cell. Transforming DNA may or may not be integrated (covalently linked) with chromosomal DNA making up the genome of the cell. For example, the polynucleotide may be maintained on an episomal element, such as a plasmid or a stably transformed cell is one in which the polynucleotide has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the cell to establish cell lines or clones comprised of a population of daughter cells containing the transformed polynucleotide. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations (e.g., at least about 10). 
     A “vector” includes plasmids and viruses and any DNA or RNA molecule, whether self-replicating or not, which can be used to transform or transfect a cell. 
     As used herein, a “genetic modification” refers to any addition, deletion and/or substitution to a cell&#39;s normal nucleotides and/or additional of heterologous sequences. Any method which can achieve the genetic modification are within the spirit and scope of this invention. Art recognized methods include viral mediated gene transfer, liposome mediated transfer, transformation, transfection and transduction. 
     The practice of the present invention employs, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch &amp; Sambrook, In Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames &amp; S. J. Higgins, eds., 1985); Transcription and Translation (B. D. Hames &amp; S. I. Higgins, eds., 1984); Animal Cell Culture (R. I. Freshney, ed., 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984). 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention. 
     Pathway 
     A high-level biosynthetic route to produce cannabinoids and/or cannabinoid precursors is shown in  FIGS. 1-3 . The focus of this pathway is the production of Compound I from Compound II using an PKS Enzyme in combination with a npgA Enzyme. Additional pathways can be added to this core pathway, including the production of (a) Compound II from Compound III; and/or (b) the production of Compound II from Acetyl-CoA and Malonyl CoA; and/or (c) the production of Compound III from Compound IV; and/or (d) the production of Compound III from Compound IV. 
     The biosynthetic routes as shown in  FIGS. 1-3  can be used to produce Compounds described in  FIGS. 4-5 . As shown in the Tables in  FIGS. 4-5 , the compounds comprise identical core structures but comprise different lengths in the C-tails (C-3 Tail, C-5 Tail, or C-7 Tail). Depending on whether the starting materials (e.g., Compound I-IV) comprise a C-3, C-5, or C-7 tail will determine the resulting cannabinoid analogs and/or cannabinoid precursor analogs. Regardless of the length of the C-tail contained in the starting materials, the enzymatic pathways described herein can be used to convert each core structure. 
     Production of Compound I 
     As shown in  FIGS. 1A and 1B , Compound I can be enzymatically produced from Compound II using an PKS Enzyme in combination with a npgA Enzyme. As used herein, an “PKS Enzyme” is defined as any one of the following amino acid sequences:
         a. SEQ ID NO:1 ( C. stellaris -OLAs-dACP1 (sequence on page 4-5));   b. SEQ ID NO:2 ( C. stellaris -OLAs-dACP2 (sequence on page 5));   c. SEQ ID NO:3 ( C. stellaris -OLAs-wt (wild type  C. stellaris ));   d. SEQ ID NO:6 ( C. grayi -PKS-dACP1);   e. SEQ ID NO:7 ( C. grayi -PKS-dACP2);   f. SEQ ID NO:35 ( P. furfuracea );   g. an PKS enzyme variant of any one of SEQ ID NO:4-5 and 35 ( C. stellaris, C. grayi, C. uncialis, P. furfuracea ), wherein one of the two ACP domains have been inactivated;   h. an PKS enzyme variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 1-7 or 35, wherein said PKS enzyme variant has retained Olivetolic Acid Synthase activity and has inactivated an ACP domain;   i. an PKS enzyme variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity to any one of SEQ ID NOS: 1-7 or 35, wherein said PKS enzyme variant has retained Olivetolic Acid Synthase activity and has inactivated an ACP domain;   j. a PKS enzyme variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the domains selected from: SAT domain, KS domain, AT domain, PT domain, ACP1 domain, ACP2 domain, and TE domain of SEQ ID NOS: 1-7 or 35, wherein said PKS enzyme variant has retained Olivetolic Acid Synthase activity and has inactivated an ACP domain; or   k. any combination of (a)-(j).       

     The sequences corresponding to SEQ ID NO:1-7 and 35 are as follows: 
     
       
         
           
               
               
            
               
                   C. Stellaris -OLAs-dACP1 
                   
               
               
                 (SEQ ID NO: 1) 
                   
               
               
                 MTPPNNVVLFGDQTVDPCPVIKQLYRQSRDSLALQAFFRQSYEAVRREIATSEYSDRALFPSFD 
                   
               
               
                   
               
               
                 SIRALAEKQPEKHNEAVSTVLLCIAQLGLLLVHSDQDDSMFDAGPSKTYLVGLCTGMLPAAALA 
               
               
                   
               
               
                 ASSSTSQLLRLAPEIVLVALRLGLEANRRSAQIEASTESWASVVPGMAPQEQQEALAQFNNEFM 
               
               
                   
               
               
                 IPTSKQAYISAESDSTATISGPPSTLVSLFTSSDSFRKARRVKLPITAAFHAPHLRVPDSEKII 
               
               
                   
               
               
                 GSLLNSDEYPLRNDVVIVSTRSGKPIRAQSLGDALQHIILDILREPIRWSRVIEEMIPNLKDQG 
               
               
                   
               
               
                 VILTSAGPVRAADSLRQRMASAGIEVLMSTEMQPLREPRTKPRSSDTATIGYAARLPESETLEE 
               
               
                   
               
               
                 VWKILEDGRDVHKKIPNDRFDVDTHCDPSGKIKNTTYTPYGCFLDRPGFFDARLFNMSPREASQ 
               
               
                   
               
               
                 TDPAQRLLLLTTYEALEMAGYTPDGSPSSAGDRIGTFFGQTLDDYREANASQNIEMYYVSGGIR 
               
               
                   
               
               
                 AFGAGRLNYHFKWEGPSYCVDAACSSSTLSIQMAMSSLRTHECDTAVAGGTNVLTGVDMFSGLS 
               
               
                   
               
               
                 RGSFLSPTGSCKTFDNDADGYCRGDGVGTVILKRLDDAIADGDNIQAVIKSAATNHSAHAVSIT 
               
               
                   
               
               
                 HPHAGAQQNLMRQVLREADVEPSEIDYVEMHGTGTQAGDATEFASVTNVISGRTRDNPLHVGAI 
               
               
                   
               
               
                 KANFGHAEAAAGTNSLVKVLMMMRKNAIPPHVGIKGRINEKFPPLDKINVRINRTMTPFVARAG 
               
               
                   
               
               
                 GDGKRRVLLNNFNATGGNTSLLLEDAPKTDVRGHDLRSAHVIAISAKTSYSFKQNTQRLLEYLQ 
               
               
                   
               
               
                 LNPETQIQDLSYTTTARRMHHVIRKAYAVQSTEQLVQSMKKDISNSSELGATTELSSAIFLFTG 
               
               
                   
               
               
                 QGSQYLGMGRQLFQTNTAFRKSISESDNICVRQGLPSFEWIVTAESSEERVPSPSESQLALVAI 
               
               
                   
               
               
                 ALALASLWQSWGITPKAVIGHSLGEYAALCVAGVLSISDTLYLVGKRAEMMEKKCIANSHSMLA 
               
               
                   
               
               
                 IQSDSESIQQIISGGQMPSCEIACLNGPSNTVVSGSLKDIHSLEEKLNALGTKTTLLKLPFAFH 
               
               
                   
               
               
                 SVQMDPILEDIRALAQNVQFRKPNVPIASTLLGTLVKDHGIITADYLARQARQAVRFQEALQAC 
               
               
                   
               
               
                 KAESIASDDTLWIEVGPHPLCHGMVRSTLGLSPTKALPSLKRDEDCWSTISRSIANAYNSGVKV 
               
               
                   
               
               
                 SWIDYHRDFQGALRLLELPSYAFDLKNYWIQHEGDWSLRKGETTHTNAPPPQASFSTTCLQVIE 
               
               
                   
               
               
                 NETFTQNSASVTFSSQLSEPKLNTAVRGHLVSGIGLCPSSVYADVAFTAAWYIASRMTPSDPVP 
               
               
                   
               
               
                 AMDLSTMEVFRPLIVDSKETPQLLKVSASRNANEQVVNIKISSQDDKGRQEHAHCTVMYGDGHQ 
               
               
                   
               
               
                 WMDEWQRNAYLVESRIDKLTQPSSPGIHRMLKEMIYKQFQTVVTYSPEYHNIDEIFMDCDLNET 
               
               
                   
               
               
                 AANINFQSMAGNGEFIYSPYWIDTVAHLAGFILNANVKTPTDTVFISHGWQSFRIAAPLSDEKT 
               
               
                   
               
               
                 YRGYVRMQPSSGRGVMAGDVYIFDGDEIVVVCKGIKFQQMKRTTLQSLLGVSPAATPISKPIPA 
               
               
                   
               
               
                 KPSGPHPVTARKAAVTQSLSAGFSRVLDTIASEVGVDVSELSDDVKISDVGVD  LLTISILGRL 
               
               
                   
               
               
                 RPETGLDLSSSLFIEHPSIAELRAFFLDKMDVPQATANDDDSDDSSEDDGPGFSRSQSTSTIST 
               
               
                   
               
               
                 PEEPDVVNILMSTIAREVGVEESETQLSTPFAEIGVDSLLTISILDAFKTEIGMNLSANFFHDH 
               
               
                   
               
               
                 PTFADVQKALGAPSTPQKPLDLPLCRLEQSSKPLSQTPRAKSVLLQGRPDKGKPALFLLPDGAG 
               
               
                   
               
               
                 SLFSYISLPSLPSGLPVYGLDSPFHNNPSEYTISFSAVATIYIAAIRAIQPKGPYMLGGWSLGG 
               
               
                   
               
               
                 IHAYETARQLIEQGETISNLIMIDSPCPGTLPPLPAPTLSLLEKAGIFDGLSTSGAPITERTRL 
               
               
                   
               
               
                 HFLGCVRALENYTVTPLPPGKSPGKVTVIWAQEGVLEGREEQGKEYMAATSSGDLNKDMDKAKE 
               
               
                   
               
               
                 WLTGKRTSFGPSGWDKLTGTEVHCHVVSGNHFSIMFPPKVCWQSTSSFSPSMDYDTNAYNLQIT 
               
               
                   
               
               
                 AVAEAVATGLPEK* 
               
               
                   
               
               
                   C. Stellaris -OLAs-dACP2 
               
               
                 (SEQ ID NO: 2) 
                   
               
               
                 MTPPNNVVLFGDQTVDPCPVIKQLYRQSRDSLALQAFFRQSYEAVRREIATSEYSDRALFPSFD 
                   
               
               
                   
               
               
                 SIRALAEKQPEKHNEAVSTVLLCIAQLGLLLVHSDQDDSMFDAGPSKTYLVGLCTGMLPAAALA 
               
               
                   
               
               
                 ASSSTSQLLRLAPEIVLVALRLGLEANRRSAQIEASTESWASVVPGMAPQEQQEALAQFNNEFM 
               
               
                   
               
               
                 IPTSKQAYISAESDSTATISGPPSTLVSLFTSSDSFRKARRVKLPITAAFHAPHLRVPDSEKII 
               
               
                   
               
               
                 GSLLNSDEYPLRNDVVIVSTRSGKPIRAQSLGDALQHIILDILREPIRWSRVIEEMIPNLKDQG 
               
               
                   
               
               
                 VILTSAGPVRAADSLRQRMASAGIEVLMSTEMQPLREPRTKPRSSDTATIGYAARLPESETLEE 
               
               
                   
               
               
                 VWKILEDGRDVHKKIPNDRFDVDTHCDPSGKIKNTTYTPYGCFLDRPGFFDARLFNMSPREASQ 
               
               
                   
               
               
                 TDPAQRLLLLTTYEALEMAGYTPDGSPSSAGDRIGTFFGQTLDDYREANASQNIEMYYVSGGIR 
               
               
                   
               
               
                 AFGAGRLNYHFKWEGPSYCVDAACSSSTLSIQMAMSSLRTHECDTAVAGGTNVLTGVDMFSGLS 
               
               
                   
               
               
                 RGSFLSPTGSCKTFDNDADGYCRGDGVGTVILKRLDDAIADGDNIQAVIKSAATNHSAHAVSIT 
               
               
                   
               
               
                 HPHAGAQQNLMRQVLREADVEPSEIDYVEMHGTGTQAGDATEFASVTNVISGRTRDNPLHVGAI 
               
               
                   
               
               
                 KANFGHAEAAAGTNSLVKVLMMMRKNAIPPHVGIKGRINEKFPPLDKINVRINRTMTPFVARAG 
               
               
                   
               
               
                 GDGKRRVLLNNFNATGGNTSLLLEDAPKTDVRGHDLRSAHVIAISAKTSYSFKQNTQRLLEYLQ 
               
               
                   
               
               
                 LNPETQIQDLSYTTTARRMHHVIRKAYAVQSTEQLVQSMKKDISNSSELGATTELSSAIFLFTG 
               
               
                   
               
               
                 QGSQYLGMGRQLFQTNTAFRKSISESDNICVRQGLPSFEWIVTAESSEERVPSPSESQLALVAI 
               
               
                   
               
               
                 ALALASLWQSWGITPKAVIGHSLGEYAALCVAGVLSISDTLYLVGKRAEMMEKKCIANSHSMLA 
               
               
                   
               
               
                 IQSDSESIQQIISGGQMPSCEIACLNGPSNTVVSGSLKDIHSLEEKLNALGTKTTLLKLPFAFH 
               
               
                   
               
               
                 SVQMDPILEDIRALAQNVQFRKPNVPIASTLLGTLVKDHGIITADYLARQARQAVRFQEALQAC 
               
               
                   
               
               
                 KAESIASDDTLWIEVGPHPLCHGMVRSTLGLSPTKALPSLKRDEDCWSTISRSIANAYNSGVKV 
               
               
                   
               
               
                 SWIDYHRDFQGALRLLELPSYAFDLKNYWIQHEGDWSLRKGETTHTNAPPPQASFSTTCLQVIE 
               
               
                   
               
               
                 NETFTQNSASVTFSSQLSEPKLNTAVRGHLVSGIGLCPSSVYADVAFTAAWYIASRMTPSDPVP 
               
               
                   
               
               
                 AMDLSTMEVFRPLIVDSKETPQLLKVSASRNANEQVVNIKISSQDDKGRQEHAHCTVMYGDGHQ 
               
               
                   
               
               
                 WMDEWQRNAYLVESRIDKLTQPSSPGIHRMLKEMIYKQFQTVVTYSPEYHNIDEIFMDCDLNET 
               
               
                   
               
               
                 AANINFQSMAGNGEFIYSPYWIDTVAHLAGFILNANVKTPTDTVFISHGWQSFRIAAPLSDEKT 
               
               
                   
               
               
                 YRGYVRMQPSSGRGVMAGDVYIFDGDEIVVVCKGIKFQQMKRTTLQSLLGVSPAATPISKPIPA 
               
               
                   
               
               
                 KPSGPHPVTARKAAVTQSLSAGFSRVLDTIASEVGVDVSELSDDVKISDVGVDSLLTISILGRL 
               
               
                   
               
               
                 RPETGLDLSSSLFIEHPSIAELRAFFLDKMDVPQATANDDDSDDSSEDDGPGFSRSQSTSTIST 
               
               
                   
               
               
                 PEEPDVVNILMSIIAREVGVEESEIQLSTPFAEIGVD  LLTISILDAFKTEIGMNLSANFFHDH 
               
               
                   
               
               
                 PTFADVQKALGAPSTPQKPLDLPLCRLEQSSKPLSQTPRAKSVLLQGRPDKGKPALFLLPDGAG 
               
               
                   
               
               
                 SLFSYISLPSLPSGLPVYGLDSPFHNNPSEYTISFSAVATIYIAAIRAIQPKGPYMLGGWSLGG 
               
               
                   
               
               
                 IHAYETARQLIEQGETISNLIMIDSPCPGTLPPLPAPTLSLLEKAGIFDGLSTSGAPITERTRL 
               
               
                   
               
               
                 HFLGCVRALENYTVTPLPPGKSPGKVTVIWAQEGVLEGREEQGKEYMAATSSGDLNKDMDKAKE 
               
               
                   
               
               
                 WLTGKRTSFGPSGWDKLTGTEVHCHVVSGNHFSIMFPPKVCWQSTSSFSPSMDYDTNAYNLQIT 
               
               
                   
               
               
                 AVAEAVATGLPEK 
               
               
                   
               
               
                   C. Stellaris -OLAS 
               
               
                 (SEQ ID NO: 3) 
                   
               
               
                 MTPPNNVVLFGDQTVDPCPVIKQLYRQSRDSLALQAFFRQSYEAVRREIATSEYSDRALFPSFD 
                   
               
               
                   
               
               
                 SIRALAEKQPEKHNEAVSTVLLCIAQLGLLLVHSDQDDSMFDAGPSKTYLVGLCTGMLPAAALA 
               
               
                   
               
               
                 ASSSTSQLLRLAPEIVLVALRLGLEANRRSAQIEASTESWASVVPGMAPQEQQEALAQFNNEFM 
               
               
                   
               
               
                 IPTSKQAYISAESDSTATISGPPSTLVSLFTSSDSFRKARRVKLPITAAFHAPHLRVPDSEKII 
               
               
                   
               
               
                 GSLLNSDEYPLRNDVVIVSTRSGKPIRAQSLGDALQHIILDILREPIRWSRVIEEMIPNLKDQG 
               
               
                   
               
               
                 VILTSAGPVRAADSLRQRMASAGIEVLMSTEMQPLREPRTKPRSSDTATIGYAARLPESETLEE 
               
               
                   
               
               
                 VWKILEDGRDVHKKIPNDRFDVDTHCDPSGKIKNTTYTPYGCFLDRPGFFDARLFNMSPREASQ 
               
               
                   
               
               
                 TDPAQRLLLLTTYEALEMAGYTPDGSPSSAGDRIGTFFGQTLDDYREANASQNIEMYYVSGGIR 
               
               
                   
               
               
                 AFGAGRLNYHFKWEGPSYCVDAACSSSTLSIQMAMSSLRTHECDTAVAGGTNVLTGVDMFSGLS 
               
               
                   
               
               
                 RGSFLSPTGSCKTFDNDADGYCRGDGVGTVILKRLDDAIADGDNIQAVIKSAATNHSAHAVSIT 
               
               
                   
               
               
                 HPHAGAQQNLMRQVLREADVEPSEIDYVEMHGTGTQAGDATEFASVTNVISGRTRDNPLHVGAI 
               
               
                   
               
               
                 KANFGHAEAAAGTNSLVKVLMMMRKNAIPPHVGIKGRINEKFPPLDKINVRINRTMTPFVARAG 
               
               
                   
               
               
                 GDGKRRVLLNNFNATGGNTSLLLEDAPKTDVRGHDLRSAHVIAISAKTSYSFKQNTQRLLEYLQ 
               
               
                   
               
               
                 LNPETQIQDLSYTTTARRMHHVIRKAYAVQSTEQLVQSMKKDISNSSELGATTELSSAIFLFTG 
               
               
                   
               
               
                 QGSQYLGMGRQLFQTNTAFRKSISESDNICVRQGLPSFEWIVTAESSEERVPSPSESQLALVAI 
               
               
                   
               
               
                 ALALASLWQSWGITPKAVIGHSLGEYAALCVAGVLSISDTLYLVGKRAEMMEKKCIANSHSMLA 
               
               
                   
               
               
                 IQSDSESIQQIISGGQMPSCEIACLNGPSNTVVSGSLKDIHSLEEKLNALGTKTTLLKLPFAFH 
               
               
                   
               
               
                 SVQMDPILEDIRALAQNVQFRKPNVPIASTLLGTLVKDHGIITADYLARQARQAVRFQEALQAC 
               
               
                   
               
               
                 KAESIASDDTLWIEVGPHPLCHGMVRSTLGLSPTKALPSLKRDEDCWSTISRSIANAYNSGVKV 
               
               
                   
               
               
                 SWIDYHRDFQGALRLLELPSYAFDLKNYWIQHEGDWSLRKGETTHTNAPPPQASFSTTCLQVIE 
               
               
                   
               
               
                 NETFTQNSASVTFSSQLSEPKLNTAVRGHLVSGIGLCPSSVYADVAFTAAWYIASRMTPSDPVP 
               
               
                   
               
               
                 AMDLSTMEVFRPLIVDSKETPQLLKVSASRNANEQVVNIKISSQDDKGRQEHAHCTVMYGDGHQ 
               
               
                   
               
               
                 WMDEWQRNAYLVESRIDKLTQPSSPGIHRMLKEMIYKQFQTVVTYSPEYHNIDEIFMDCDLNET 
               
               
                   
               
               
                 AANINFQSMAGNGEFIYSPYWIDTVAHLAGFILNANVKTPTDTVFISHGWQSFRIAAPLSDEKT 
               
               
                   
               
               
                 YRGYVRMQPSSGRGVMAGDVYIFDGDEIVVVCKGIKFQQMKRTTLQSLLGVSPAATPISKPIPA 
               
               
                   
               
               
                 KPSGPHPVTARKAAVTQSLSAGFSRVLDTIASEVGVDVSELSDDVKISDVGVDSLLTISILGRL 
               
               
                   
               
               
                 RPETGLDLSSSLFIEHPSIAELRAFFLDKMDVPQATANDDDSDDSSEDDGPGFSRSQSTSTIST 
               
               
                   
               
               
                 PEEPDVVNILMSIIAREVGVEESEIQLSTPFAEIGVDSLLTISILDAFKTEIGMNLSANFFHDH 
               
               
                   
               
               
                 PTFADVQKALGAPSTPQKPLDLPLCRLEQSSKPLSQTPRAKSVLLQGRPDKGKPALFLLPDGAG 
               
               
                   
               
               
                 SLFSYISLPSLPSGLPVYGLDSPFHNNPSEYTISFSAVATIYIAAIRAIQPKGPYMLGGWSLGG 
               
               
                   
               
               
                 IHAYETARQLIEQGETISNLIMIDSPCPGTLPPLPAPTLSLLEKAGIFDGLSTSGAPITERTRL 
               
               
                   
               
               
                 HFLGCVRALENYTVTPLPPGKSPGKVTVIWAQEGVLEGREEQGKEYMAATSSGDLNKDMDKAKE 
               
               
                   
               
               
                 WLTGKRTSFGPSGWDKLTGTEVHCHVVSGNHFSIMFPPKVCWQSTSSFSPSMDYDTNAYNLQIT 
               
               
                   
               
               
                 AVAEAVATGLPEK 
               
               
                   
               
               
                 ( C. Grayi  PKS)(GenBank Accession E9KMQ2.1) 
               
               
                 SEQ ID NO: 4 
                   
               
               
                 MTLPNNVVLFGDQTVDPCPIIKQLYRQSRDSLTLQTLFRQSYDAVRREIATSEASDRALFPSFD 
                   
               
               
                   
               
               
                 SFQDLAEKQNERHNEAVSTVLLCIAQLGLLMIHVDQDDSTFDARPSRTYLVGLCTGMLPAAALA 
               
               
                   
               
               
                 ASSSTSQLLRLAPEIVLVALRLGLEANRRSAQTEASTESWASVVPGMAPQEQQEALAQFNDEFM 
               
               
                   
               
               
                 IPTSKQAYISAESDSSATLSGPPSTLLSLFSSSDIFKKARRIKLPITAAFHAPHLRVPDVEKIL 
               
               
                   
               
               
                 GSLSHSDEYPLRNDVVIVSTRSGKPITAQSLGDALQHIIMDILREPMRWSRVVEEMINGLKDQG 
               
               
                   
               
               
                 AILTSAGPVRAADSLRQRMASAGIEVSRSTEMQPRQEQRTKPRSSDTATIGYAARLPESETLEE 
               
               
                   
               
               
                 VWKILEDGRDVHKKIPSDRFDVDTHCDPSGKIKNTSYTPYGCFLDRPGFFDARLFNMSPREASQ 
               
               
                   
               
               
                 TDPAQRLLLLTTYEALEMAGYTPDGTPSTAGDRIGTFFGQTLDDYREANASQNIEMYYVSGGIR 
               
               
                   
               
               
                 AFGPGRLNYHFKWEGPSYCVDAACSSSTLSIQMAMSSLRAHECDTAVAGGTNVLTGVDMFSGLS 
               
               
                   
               
               
                 RGSFLSPTGSCKTFDNDADGYCRGDGVGSVILKRLDDAIADGDNIQAVIKSAATNHSAHAVSIT 
               
               
                   
               
               
                 HPHAGAQQNLMRQVLREGDVEPADIDYVEMHGTGTQAGDATEFASVTNVITGRTRDNPLHVGAV 
               
               
                   
               
               
                 KANFGHAEAAAGTNSLVKVLMMMRKNAIPPHIGIKGRINEKFPPLDKINVRINRTMTPFVARAG 
               
               
                   
               
               
                 GDGKRRVLLNNFNATGGNTSLLIEDAPKTDIQGHDLRSAHVVAISAKTPYSFRQNTQRLLEYLQ 
               
               
                   
               
               
                 LNPETQLQDLSYTTTARRMHHVIRKAYAVQSIEQLVQSLKKDISSSSEPGATTEHSSAVFLFTG 
               
               
                   
               
               
                 QGSQYLGMGRQLYQTNKAFRKSISESDSICIRQGLPSFEWIVSAEPSEERITSPSESQLALVAI 
               
               
                   
               
               
                 ALALASLWQSWGITPKAVMGHSLGEYAALCVAGVLSISDTLYLVGKRAQMMEKKCIANTHSMLA 
               
               
                   
               
               
                 IQSDSESIQQIISGGQMPSCEIACLNGPSNTVVSGSLTDIHSLEEKLNAMGTKTTLLKLPFAFH 
               
               
                   
               
               
                 SVQMDPILEDIRALAQNVQFRKPIVPIASTLLGTLVKDHGIITADYLTRQARQAVRFQEALQAC 
               
               
                   
               
               
                 RAENIATDDTLWVEVGAHPLCHGMVRSTLGLSPTKALPSLKRDEDCWSTISRSIANAYNSGVKV 
               
               
                   
               
               
                 SWIDYHRDFQGALRLLELPSYAFDLKNYWIQHEGDWSLRKGETTRTTAPPPQASFSTTCLQVIE 
               
               
                   
               
               
                 NETFTQDSASVTFSSQLSEPKLNTAVRGHLVSGTGLCPSSVYADVAFTAAWYIASRMTPSDPVP 
               
               
                   
               
               
                 AMDLSSMEVFRPLIVDSNETSQLLRVSATRNPNEQIVNIKISSQDDKGRQEHAHCTVMYGDGHQ 
               
               
                   
               
               
                 WMEEWQRNAYLIQSRIDKLTQPSSPGIHRMLKEMIYKQFQTVVTYSPEYHNIDEIFMDCDLNET 
               
               
                   
               
               
                 AANIKLQSTAGHGEFIYSPYWIDTVAHLAGFILNANVKTPADTVFISHGWQSFQIAAPLSAEKT 
               
               
                   
               
               
                 YRGYVRMQPSSGRGVMAGDVYIFDGDEIVVVCKGIKFQQMKRTTLQSLLGVSPAATPTSKSIAA 
               
               
                   
               
               
                 KSTRPQLVTVRKAAVTQSPVAGFSKVLDTIASEVGVDVSELSDDVKISDVGVDSLLTISILGRL 
               
               
                   
               
               
                 RPETGLDLSSSLFIEHPTIAELRAFFLDKMDMPQATANDDDSDDSSDDEGPGFSRSQSNSTIST 
               
               
                   
               
               
                 PEEPDVVNVLMSIIAREVGIQESEIQLSTPFAEIGVDSLLTISILDALKTEIGMNLSANFFHDH 
               
               
                   
               
               
                 PTFADVQKALGAAPTPQKPLDLPLARLEQSPRPSSQALRAKSVLLQGRPEKGKPALFLLPDGAG 
               
               
                   
               
               
                 SLFSYISLPSLPSGLPIYGLDSPFHNNPSEFTISFSDVATIYIAAIRAIQPKGPYMLGGWSLGG 
               
               
                   
               
               
                 IHAYETARQLIEQGETISNLIMIDSPCPGTLPPLPAPTLSLLEKAGIFDGLSTSGAPITERTRL 
               
               
                   
               
               
                 HFLGCVRALENYTVTPLPPGKSPGKVTVIWAQDGVLEGREEQGKEYMAATSSGDLNKDMDKAKE 
               
               
                   
               
               
                 WLTGKRTSFGPSGWDKLTGTEVHCHVVGGNHFSIMFPPKVC 
               
               
                   
               
               
                 ( C. Uncialis -PKS)(GenBank Accession AUW31177.1) 
               
               
                 SEQ ID NO: 5 
                   
               
               
                 MTLPNNVVLFGDQTVDPCPIIKQLYRQSRDSLTLQALFRQSYDAVRREIATSEYSDRTLFPSFD 
                   
               
               
                   
               
               
                 SIQGLAEKQTERHNEAVSTVLHCIAQLGLLLIHADQDDFRLDARPSRTYLVGLCTGMLPAAALA 
               
               
                   
               
               
                 ASSSASQLLRLAPEIVLVALRLGLEANRRSAQTEASTESWASVVPGMAPQEQQEALAQFNDEFM 
               
               
                   
               
               
                 IPTSKQAYISAESDSTATLSGPPSTLVSLFSLSDSFRKARRIKLPITAAFHAPHLRLPNVEKII 
               
               
                   
               
               
                 GSLSHSDEYPLRNDVVIISTRSGKPITAQSLGDALQHIILDILREPIRWSTVVEEMINNFEDQG 
               
               
                   
               
               
                 ANLTSVGPVRAADSLRQRMATAGIEILKSTELQPQQEPRTKTRSNDTATIGYAARLPESETLEE 
               
               
                   
               
               
                 AWKILEDGRDVHKKIPSDRFDVDTHCDPSGKIKNTTYTPYGCFLDRPGFFDARLFNMSPREASQ 
               
               
                   
               
               
                 TDPAQRLLLLTTYEALEMAGYTPDGTPSTAGDRIGTFFGQTLDDYREANASQNIEMYYVSGGIR 
               
               
                   
               
               
                 AFGAGRLNYHFKWEGPSYCVDAACSSSTLSIQMAMSSLRAHECDTAVAGGTNVLTGVDMFSGLS 
               
               
                   
               
               
                 RGSFLSPTGSCKTFDNDADGYCRGDGVGSVILKRLDDAVADGDNIQAVIKSAATNHSAHAVSIT 
               
               
                   
               
               
                 HPHAGAQQNLMRQVLREADVEPSEIDYVEMHGTGTQAGDATEFTSVTNVISGRTRDNPLYVGAV 
               
               
                   
               
               
                 KANFGHAEAAAGTNSLVKVLMMMRKNAIPPHIGIKGRINEKFPPLDKINVRINRTMTPFVARAG 
               
               
                   
               
               
                 GDGKRRVLLNNFNATGGNTSLLLEDAPKTDIRGHDPRSAHVIAISAKTPYSFRQNTQRLLEYLQ 
               
               
                   
               
               
                 QNPDTQLQNLSYTTTARRMHHAIRKAYAVQSIEELVQSMKKDVSNSSELGATTEHSTAIFLFTG 
               
               
                   
               
               
                 QGSQYLGMGRQLFQTNTSFRKSISDSDNLCIRQGLPSFEWIVSAEPSEERVPTPSESQLALVAI 
               
               
                   
               
               
                 ALALASLWQSWGITPKAVIGHSLGEYAALCVAGVLSISDTLYLVGKRAEMMEKKCIANTHSMLA 
               
               
                   
               
               
                 VQSASDSIQQIISGGQMPSCEIACLNGPTNTVVSGSLKDIHSLKEKLDTMGTKTTLLKLPFAFH 
               
               
                   
               
               
                 SVQMDPILEDIRALAQNVQFRKPIVPIASTLLGTLVKDHGIITADYLTRQARQAVRFQGALQAC 
               
               
                   
               
               
                 KAESIAGDDTLWIELGPHPLCHGMVRSTLGVSPAKALPSLKRDEDCWSTLSRSIANAYNSGVKM 
               
               
                   
               
               
                 SWIDYHRDFQGALKLLELPSYAFDLKNYWIQHEGDWSLRKGETTRTTAPPPQASFSTTCLQVVE 
               
               
                   
               
               
                 NETFTQDSASVTFSSQLSEPKLNAAIRGHLVSGIGLCPSSVYADVAFTAAWYIASHMTPSDPVP 
               
               
                   
               
               
                 AMDLSTMEVFRPLIVDSNETPQLLKVSASKNSNEQVVNIKISSRDDKGRQEHAHCTVMYGDGHQ 
               
               
                   
               
               
                 WIDEWQRNAYLFESRIAKLTQPSSPGIHRMLKEMIYKQFQTVVTYSREYHNIDEIFMDCDLNET 
               
               
                   
               
               
                 AANIKLQSMAGNGEFIYSPYWIDTIAHLAGFILNANVKTPADTVFISHGWQSFRIAAPLSAEKK 
               
               
                   
               
               
                 YRGYVCMQPSSGRGVMAGDVYLFDGDQIVVVCKGIKFQQMKRTTLQSLLGVSPAATPMSKPITA 
               
               
                   
               
               
                 KSTRPHPVAVRKVVVTQSPGAGFSKVLDTIASEVGVDASELSDDVKISDIGVDSLLTISILGRL 
               
               
                   
               
               
                 RPETGLDLSSSLFIEHPTIAELRAFFLDKMVVPQATVNDDDSDDSSEDGGPGFSRSQSNSTIST 
               
               
                   
               
               
                 PEEPDVVSILMSIIAREVGVEESEIQLSTPFAEIGVDSLLTISILDAFKTEIGMNLSANFFHDH 
               
               
                   
               
               
                 PTVADVQKALGTASTPQKPLDLPLHRVEQNSKPLSQNLRAKSVLLQGRPEKGKPALFLLPDGAG 
               
               
                   
               
               
                 SLFSYISLPSLPSGLPVYGLDSPFHHNPSEYTISFAAVATIYIAAIRAIQPKGPYMLGGWSLGG 
               
               
                   
               
               
                 IHAYETARQLIEQGETISNLIMIDSPCPGTLPPLPAPTLSLLEKAGIFDGLSTSGAPITERTRL 
               
               
                   
               
               
                 HFLGCVRALENYTVTPLPPGKSPGKVTVIWAQEGVLEGREEQGKEYMAATSSGDLNKDMDKAKE 
               
               
                   
               
               
                 WLTGKRTSFGPSGWDKLTGTDVHCHVVGGNHFSIMFPPKVCWRSTFSLSSSIDNDTNAYNLQIA 
               
               
                   
               
               
                 AVAKAVATGLPEK 
               
               
                   
               
               
                 ( C. Grayi -PKS-dACP1) 
               
               
                 SEQ ID NO: 6 
                   
               
               
                 MTLPNNVVLFGDQTVDPCPIIKQLYRQSRDSLTLQTLFRQSYDAVRREIATSEASDRALFPSFD 
                   
               
               
                   
               
               
                 SFQDLAEKQNERHNEAVSTVLLCIAQLGLLMIHVDQDDSTFDARPSRTYLVGLCTGMLPAAALA 
               
               
                   
               
               
                 ASSSTSQLLRLAPEIVLVALRLGLEANRRSAQIEASTESWASVVPGMAPQEQQEALAQFNDEFM 
               
               
                   
               
               
                 IPTSKQAYISAESDSSATLSGPPSTLLSLFSSSDIFKKARRIKLPITAAFHAPHLRVPDVEKIL 
               
               
                   
               
               
                 GSLSHSDEYPLRNDVVIVSTRSGKPITAQSLGDALQHIIMDILREPMRWSRVVEEMINGLKDQG 
               
               
                   
               
               
                 AILTSAGPVRAADSLRQRMASAGIEVSRSTEMQPRQEQRTKPRSSDIAIIGYAARLPESETLEE 
               
               
                   
               
               
                 VWKILEDGRDVHKKIPSDRFDVDTHCDPSGKIKNTSYTPYGCFLDRPGFFDARLFNMSPREASQ 
               
               
                   
               
               
                 TDPAQRLLLLTTYEALEMAGYTPDGTPSTAGDRIGTFFGQTLDDYREANASQNIEMYYVSGGIR 
               
               
                   
               
               
                 AFGPGRLNYHFKWEGPSYCVDAACSSSTLSIQMAMSSLRAHECDTAVAGGTNVLTGVDMFSGLS 
               
               
                   
               
               
                 RGSFLSPTGSCKTFDNDADGYCRGDGVGSVILKRLDDAIADGDNIQAVIKSAATNHSAHAVSIT 
               
               
                   
               
               
                 HPHAGAQQNLMRQVLREGDVEPADIDYVEMHGTGTQAGDATEFASVTNVITGRTRDNPLHVGAV 
               
               
                   
               
               
                 KANFGHAEAAAGTNSLVKVLMMMRKNAIPPHIGIKGRINEKFPPLDKINVRINRTMTPFVARAG 
               
               
                   
               
               
                 GDGKRRVLLNNFNATGGNTSLLIEDAPKTDIQGHDLRSAHVVAISAKTPYSFRQNTQRLLEYLQ 
               
               
                   
               
               
                 LNPETQLQDLSYTTTARRMHHVIRKAYAVQSIEQLVQSLKKDISSSSEPGATTEHSSAVFLFTG 
               
               
                   
               
               
                 QGSQYLGMGRQLYQTNKAFRKSISESDSICIRQGLPSFEWIVSAEPSEERITSPSESQLALVAI 
               
               
                   
               
               
                 ALALASLWQSWGITPKAVMGHSLGEYAALCVAGVLSISDTLYLVGKRAQMMEKKCIANTHSMLA 
               
               
                   
               
               
                 IQSDSESIQQIISGGQMPSCEIACLNGPSNTVVSGSLTDIHSLEEKLNAMGTKTTLLKLPFAFH 
               
               
                   
               
               
                 SVQMDPILEDIRALAQNVQFRKPIVPIASTLLGTLVKDHGIITADYLTRQARQAVRFQEALQAC 
               
               
                   
               
               
                 RAENIATDDTLWVEVGAHPLCHGMVRSTLGLSPTKALPSLKRDEDCWSTISRSIANAYNSGVKV 
               
               
                   
               
               
                 SWIDYHRDFQGALRLLELPSYAFDLKNYWIQHEGDWSLRKGETTRTTAPPPQASFSTTCLQVIE 
               
               
                   
               
               
                 NETFTQDSASVTFSSQLSEPKLNTAVRGHLVSGTGLCPSSVYADVAFTAAWYIASRMTPSDPVP 
               
               
                   
               
               
                 AMDLSSMEVFRPLIVDSNETSQLLRVSATRNPNEQIVNIKISSQDDKGRQEHAHCTVMYGDGHQ 
               
               
                   
               
               
                 WMEEWQRNAYLIQSRIDKLTQPSSPGIHRMLKEMIYKQFQTVVTYSPEYHNIDEIFMDCDLNET 
               
               
                   
               
               
                 AANIKLQSTAGHGEFIYSPYWIDTVAHLAGFILNANVKTPADTVFISHGWQSFQIAAPLSAEKT 
               
               
                   
               
               
                 YRGYVRMQPSSGRGVMAGDVYIFDGDEIVVVCKGIKFQQMKRTTLQSLLGVSPAATPTSKSIAA 
               
               
                   
               
               
                 KSTRPQLVTVRKAAVTQSPVAGFSKVLDTIASEVGVDVSELSDDVKISDVGVD  LLTISILGRL 
               
               
                   
               
               
                 RPETGLDLSSSLFIEHPTIAELRAFFLDKMDMPQATANDDDSDDSSDDEGPGFSRSQSNSTIST 
               
               
                   
               
               
                 PEEPDVVNVLMSIIAREVGIQESEIQLSTPFAEIGVDSLLTISILDALKTEIGMNLSANFFHDH 
               
               
                   
               
               
                 PTFADVQKALGAAPTPQKPLDLPLARLEQSPRPSSQALRAKSVLLQGRPEKGKPALFLLPDGAG 
               
               
                   
               
               
                 SLFSYISLPSLPSGLPIYGLDSPFHNNPSEFTISFSDVATIYIAAIRAIQPKGPYMLGGWSLGG 
               
               
                   
               
               
                 IHAYETARQLIEQGETISNLIMIDSPCPGTLPPLPAPTLSLLEKAGIFDGLSTSGAPITERTRL 
               
               
                   
               
               
                 HFLGCVRALENYTVTPLPPGKSPGKVTVIWAQDGVLEGREEQGKEYMAATSSGDLNKDMDKAKE 
               
               
                   
               
               
                 WLTGKRTSFGPSGWDKLTGTEVHCHVVGGNHFSIMFPPKVC 
               
               
                   
               
               
                 ( C. Grayi -PKS-dACP2) 
               
               
                 SEQ ID NO: 7 
                   
               
               
                 MTLPNNVVLFGDQTVDPCPIIKQLYRQSRDSLTLQTLFRQSYDAVRREIATSEASDRALFPSFD 
                   
               
               
                   
               
               
                 SFQDLAEKQNERHNEAVSTVLLCIAQLGLLMIHVDQDDSTFDARPSRTYLVGLCTGMLPAAALA 
               
               
                   
               
               
                 ASSSTSQLLRLAPEIVLVALRLGLEANRRSAQIEASTESWASVVPGMAPQEQQEALAQFNDEFM 
               
               
                   
               
               
                 IPTSKQAYISAESDSSATLSGPPSTLLSLFSSSDIFKKARRIKLPITAAFHAPHLRVPDVEKIL 
               
               
                   
               
               
                 GSLSHSDEYPLRNDVVIVSTRSGKPITAQSLGDALQHIIMDILREPMRWSRVVEEMINGLKDQG 
               
               
                   
               
               
                 AILTSAGPVRAADSLRQRMASAGIEVSRSTEMQPRQEQRTKPRSSDTATIGYAARLPESETLEE 
               
               
                   
               
               
                 VWKILEDGRDVHKKIPSDRFDVDTHCDPSGKIKNTSYTPYGCFLDRPGFFDARLFNMSPREASQ 
               
               
                   
               
               
                 TDPAQRLLLLTTYEALEMAGYTPDGTPSTAGDRIGTFFGQTLDDYREANASQNIEMYYVSGGIR 
               
               
                   
               
               
                 AFGPGRLNYHFKWEGPSYCVDAACSSSTLSIQMAMSSLRAHECDTAVAGGTNVLTGVDMFSGLS 
               
               
                   
               
               
                 RGSFLSPTGSCKTFDNDADGYCRGDGVGSVILKRLDDAIADGDNIQAVIKSAATNHSAHAVSIT 
               
               
                   
               
               
                 HPHAGAQQNLMRQVLREGDVEPADIDYVEMHGTGTQAGDATEFASVTNVITGRTRDNPLHVGAV 
               
               
                   
               
               
                 KANFGHAEAAAGTNSLVKVLMMMRKNAIPPHIGIKGRINEKFPPLDKINVRINRTMTPFVARAG 
               
               
                   
               
               
                 GDGKRRVLLNNFNATGGNTSLLIEDAPKTDIQGHDLRSAHVVAISAKTPYSFRQNTQRLLEYLQ 
               
               
                   
               
               
                 LNPETQLQDLSYTTTARRMHHVIRKAYAVQSIEQLVQSLKKDISSSSEPGATTEHSSAVFLFTG 
               
               
                   
               
               
                 QGSQYLGMGRQLYQTNKAFRKSISESDSICIRQGLPSFEWIVSAEPSEERITSPSESQLALVAI 
               
               
                   
               
               
                 ALALASLWQSWGITPKAVMGHSLGEYAALCVAGVLSISDTLYLVGKRAQMMEKKCIANTHSMLA 
               
               
                   
               
               
                 IQSDSESIQQIISGGQMPSCEIACLNGPSNTVVSGSLTDIHSLEEKLNAMGTKTTLLKLPFAFH 
               
               
                   
               
               
                 SVQMDPILEDIRALAQNVQFRKPIVPIASTLLGTLVKDHGIITADYLTRQARQAVRFQEALQAC 
               
               
                   
               
               
                 RAENIATDDTLWVEVGAHPLCHGMVRSTLGLSPTKALPSLKRDEDCWSTISRSIANAYNSGVKV 
               
               
                   
               
               
                 SWIDYHRDFQGALRLLELPSYAFDLKNYWIQHEGDWSLRKGETTRTTAPPPQASFSTTCLQVIE 
               
               
                   
               
               
                 NETFTQDSASVTFSSQLSEPKLNTAVRGHLVSGTGLCPSSVYADVAFTAAWYIASRMTPSDPVP 
               
               
                   
               
               
                 AMDLSSMEVFRPLIVDSNETSQLLRVSATRNPNEQIVNIKISSQDDKGRQEHAHCTVMYGDGHQ 
               
               
                   
               
               
                 WMEEWQRNAYLIQSRIDKLTQPSSPGIHRMLKEMIYKQFQTVVTYSPEYHNIDEIFMDCDLNET 
               
               
                   
               
               
                 AANIKLQSTAGHGEFIYSPYWIDTVAHLAGFILNANVKTPADTVFISHGWQSFQIAAPLSAEKT 
               
               
                   
               
               
                 YRGYVRMQPSSGRGVMAGDVYIFDGDEIVVVCKGIKFQQMKRTTLQSLLGVSPAATPTSKSIAA 
               
               
                   
               
               
                 KSTRPQLVTVRKAAVTQSPVAGFSKVLDTIASEVGVDVSELSDDVKISDVGVDSLLTISILGRL 
               
               
                   
               
               
                 RPETGLDLSSSLFIEHPTIAELRAFFLDKMDMPQATANDDDSDDSSDDEGPGFSRSQSNSTIST 
               
               
                   
               
               
                 PEEPDVVNVLMSIIAREVGIQESEIQLSTPFAEIGVD  LLTISILDALKTEIGMNLSANFFHDH 
               
               
                   
               
               
                 PTFADVQKALGAAPTPQKPLDLPLARLEQSPRPSSQALRAKSVLLQGRPEKGKPALFLLPDGAG 
               
               
                   
               
               
                 SLFSYISLPSLPSGLPIYGLDSPFHNNPSEFTISFSDVATIYIAAIRAIQPKGPYMLGGWSLGG 
               
               
                   
               
               
                 IHAYETARQLIEQGETISNLIMIDSPCPGTLPPLPAPTLSLLEKAGIFDGLSTSGAPITERTRL 
               
               
                   
               
               
                 HFLGCVRALENYTVTPLPPGKSPGKVTVIWAQDGVLEGREEQGKEYMAATSSGDLNKDMDKAKE 
               
               
                   
               
               
                 WLTGKRTSFGPSGWDKLTGTEVHCHVVGGNHFSIMFPPKVC 
               
               
                   
               
               
                 ( P. furfuracea -PKS) 
               
               
                 SEQ ID NO: 35 
                   
               
               
                 MTTTSRVVLFGDQTVDPSPLIKQLCRHSTHSLTLQTFLQKTYFAVRQELAICEISDRANFPSFD 
                   
               
               
                   
               
               
                 SILALAETYSQSNESNEAVSTVLLCIAQLGLLLSREYNDNVINDSSCYSTTYLVGLCTGMLPAA 
               
               
                   
               
               
                 ALAFASSTTQLLELAPEVVRISVRLGLEASRRSAQIEKSHESWATLVPGIPLQEQRDILHRFHD 
               
               
                   
               
               
                 VYPIPASKRAYISAESDSTTTISGPPSTLASLFSFSESLRNTRKISLPITAAFHAPHLGSSDTD 
               
               
                   
               
               
                 KIIGSLSKGNEYHLRRDAVIISTSTGDQITGRSLGEALQQVVWDILREPLRWSTVTHAIAAKFR 
               
               
                   
               
               
                 DQDAVLISAGPVRAANSLRREMTNAGVKIVDSYEMQPLQVSQSRNTSGDIAIVGVAGRLPGGET 
               
               
                   
               
               
                 LEEIWENLEKGKDLHKEDRFDVKTHCDPSGKIKNTTLTPYGCFLDRPGFFDARLFNMSPREAAQ 
               
               
                   
               
               
                 TDPAQRLLLLTTYEALEMSGYTPNGSPSSASDRIGTFFGQTLDDYREANASQNIDMYYVTGGIR 
               
               
                   
               
               
                 AFGPGRLNYHFKWEGPSYCVDAACSSSALSVQMAMSSLRARECDTAVAGGTNILTGVDMFSGLS 
               
               
                   
               
               
                 RGSFLSPTGSCKTFDDEADGYCRGEGVGSVVLKRLEDAIAEGDNIQAVIKSAATNHSAHAISIT 
               
               
                   
               
               
                 HPHAGTQQKLIRQVLREADVEADEIDYVEMHGTGTQAGDATEFTSVTKVLSDRTKDNPLHIGAV 
               
               
                   
               
               
                 KANFGHAEAAAGTNSLIKILMMMRKNKIPPHVGIKGRINHKFPPLDKVNVSIDRALVAFKAHAK 
               
               
                   
               
               
                 GDGKRRVLLNNFNATGGNTSLVLEDPPETVTEGEDPRTAWVVAVSAKTSNSFTQNQQRLLNYVE 
               
               
                   
               
               
                 SNPETQLQDLSYTTTARRMHHDTYRKAYAVESMDQLVRSMRKDLSSPSEPTAITGSSPSIFAFT 
               
               
                   
               
               
                 GQGAQYLGMGRQLFETNTSFRQNILDFDRICVRQGLPSFKWLVTSSTSDESVPSPSESQLAMVS 
               
               
                   
               
               
                 IAVALVSLWQSWGIVPSAVIGHSLGEYAALCVAGVLSVSDTLYLVGKRAEMMEKKCIANSHAML 
               
               
                   
               
               
                 AVQSGSELIQQIIHAEKISTCELACSNGPSNTVVSGTGKDINSLAEKLDDMGVKKTLLKLPYAF 
               
               
                   
               
               
                 HSAQMDPILEDIRAIASNVEFLKPTVPIASTLLGSLVRDQGVITAEYLSRQTRQPVKFQEALYS 
               
               
                   
               
               
                 LRSEGIAGDEALWIEVGAHPLCHSMVRSTLGLSPTKALPTLRRDEDCWSTISKSISNAYNSGAK 
               
               
                   
               
               
                 FMWTEYHRDFRGALKLLELPSYAFDLKNYWIQHEGDWSLRKGEKMIASSTPTVPQQTFSTTCLQ 
               
               
                   
               
               
                 KVESETFTQDSASVAFSSRLAEPSLNTAVRGHLVNNVGLCPSSVYADVAFTAAWYIASRMAPSE 
               
               
                   
               
               
                 LVPAMDLSTMEVFRPLIVDKETSQILHVSASRKPGEQVVKVQISSQDMNGSKDHANCTVMYGDG 
               
               
                   
               
               
                 QQWIDEWQLNAYLVQSRVDQLIQPVKPASVHRLLKEMIYRQFQTVVTYSKEYHNIDEIFMDCDL 
               
               
                   
               
               
                 NETAANIRFQPTAGNGNFTYSPYWIDTVAHLAGFVLNASTKTPADTVFISHGWQSFRIAAPLSD 
               
               
                   
               
               
                 EKTYRGYVRMQPIGTRGVMAGDVYIFDGDRIVVLCKGIKFQKMKRNILQSLLSTGHEETPPARP 
               
               
                   
               
               
                 VPSKRTVQGSVTETKAAITPSIKAASGGFSNILETIASEVGIEVSEITDDGKISDLGVDSLLTI 
               
               
                   
               
               
                 SILGRLRSETGLDLPSSLFIAYPTVAQLRNFFLDKVATSQSVFDDEESEMSSSTAGSTPGSSTS 
               
               
                   
               
               
                 HGNQNTTVTTPAEPDVVAILMSIIAREVGIDATEIQPSTPFADLGVDSLLTISILDSFKSEMRM 
               
               
                   
               
               
                 SLAATFFHENPTFTDVQKALGAPSMPQKSLKMPSEFPEMNMGPSNQSVRSKSSILQGRPASNRP 
               
               
                   
               
               
                 ALFLLPDGAGSMFSYISLPALPSGVPVYGLDSPFHNSPKDYTVSFEEVASIFIKEIRAIQPRGP 
               
               
                   
               
               
                 YMLGGWSLGGILAYEASRQLIAQGETITNLIMIDSPCPGTLPPLPSPTLNLLEKAGIFDGLSAS 
               
               
                   
               
               
                 SGPITERTRLHFLGSVRALENYTVKPIPADRSPGKVTVIWAQDGVLEGREDVGGEEWMADSSGG 
               
               
                   
               
               
                 DANADMEKAKQWLTGKRTSFGPSGWDKLTGAEVQCHVVGGNHFSIMFPPKLCGEEKLANASWNN 
               
            
           
         
       
     
     As can be deduced from the alignment shown in  FIG. 6 , variants of SEQ ID NOs:1-7 and 35 are made to retain PKS activity while inactivating one of the two ACP domains which are defined in Table 2: 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                   
                 AA for SEQ 
                   
                 AA for SEQ  
                 AA for SEQ  
               
               
                   
                   
                   
                 ID No: 3  
                 AA for SEQ ID 
                 ID NO: 5  
                 ID NO: 35  
               
               
                 Name 
                 Accession 
                 Description 
                 ( C. Stellaris ) 
                 No: 4 ( C. Grayl ) 
                 ( C. Unicialis ) 
                 ( P. furfuracea ) 
               
               
                   
               
             
            
               
                 PksD 
                 COG3321 
                 Acyl transferase domain in polyketide 
                  367-795 
                  367-795 
                  367-795 
                  370-795 
               
               
                   
                 Cd00833 
                 synthase (PKS) enzymes 
                   
                   
                   
                   
               
               
                 PT_fungal_PKS 
                 TIGR04532 
                 iterative type I PKS product 
                 1273-1587 
                 1273-1587 
                 1273-1587 
                 1276-1590 
               
               
                   
                   
                 template domain 
                   
                   
                   
                   
               
               
                 SAT 
                 pfam16073 
                 Starter unit: ACP transacylase 
                   8-243 
                   8-243 
                   8-243 
                   8-246 
               
               
                   
                   
                 in aflatoxin biosynthesis 
                   
                   
                   
                   
               
               
                 EntF 
                 COG3319 
                 Thioesterase domain of type I 
                 1847-2122 
                 1847-2122 
                 1847-2089 
                 1857-2112 
               
               
                   
                   
                 polyketide synthase or non- 
                   
                   
                   
                   
               
               
                   
                   
                 ribosomal peptide synthetase 
                   
                   
                   
                   
               
               
                 PP-binding  
                 pfam00550 
                 Phosphopantetheine attachment site 
                 1625-1692 
                 1625-1692 
                 1625-1692 
                 1631-1698 
               
               
                 (PKS_PP) 
                 smart00823 
                   
                   
                   
                   
                   
               
               
                 ACP Domain 1 
                   
                   
                   
                   
                   
                   
               
               
                 PP-binding 
                 pfam00550 
                 Phosphopantetheine attachment site 
                 1738-1802 
                 1738-1802 
                 1738-1802 
                 1748-1812 
               
               
                 (PKS_PP) 
                 smart00823 
                   
                   
                   
                   
                   
               
               
                 ACP Domain 2 
                   
                   
                   
                   
                   
                   
               
               
                 PKS_AT 
                 smart00827 
                 Acyl transferase domain in polyketide 
                  893-1195 
                  893-1195 
                  893-1195 
                  894-1196 
               
               
                   
                   
                 synthase (PKS) enzymes 
               
               
                   
               
            
           
         
       
     
     Mutations that inactivate one of two ACP domains can be made by mutating the highly conserved amino acids of the ACP domain, while retaining the PKS activity. Examples of such mutations include:
         a. Substituting the serine at position 1654 or 1766 with any amino acid, such as for example, alanine in SEQ ID NO:3 or the corresponding position in SEQ ID NO:4 and 5 (see for example SEQ ID Nos: 1-2 and 6-7;   b. L1655 to R, H or K; D1653 to R, H or K, L1656 to R, H, K       

     Even though one of the two ACP domains is preferably inactivated in PKS Variant Enzymes, the PKS activity is retained. Examples of amino acids that should be maintained include those that are known to be highly conserved between homologs and/or orthologs. 
     Any of these PKS Enzymes (including the described variants) in combination with a npgA Enzyme can be used to produce Compound I from Compound II in the methods described herein. Variants of the PKS enzymes retain the ability to catalyze the conversion of Compound II into Compound I in combination with a npgA Enzyme, with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence. In preferred embodiments, a variant PKS enzyme, has improved activity over the sequence from which it is derived in that the improved variant has more than 10%, 120%, 130%, 140%, or and 150% improved activity in catalyzing the conversion of Compound II into Compound I as compared to the sequence from which the improved variant is derived. 
     npgA Enzyme 
     The inventors have discovered that the PKS Enzyme require activation of an ACP domain. NpgA can catalyze this reaction. 
     In preferred embodiments, the npgA enzyme comprises the following sequence (SEQ ID NO:8): 
     
       
         
           
               
            
               
                 MVQDTSSASTSPILTRWYIDTRPLTASTAALPLLETLQPADQISVQKYYH 
               
               
                   
               
               
                 LKDKHMSLASNLLKYLFVHRNCRIPWSSIVISRTPDPHRRPCYIPPSGSQ 
               
               
                   
               
               
                 EDSFKDGYTGINVEFNVSHQASMVAIAGTAFTPNSGGDSKLKPEVGIDIT 
               
               
                   
               
               
                 CVNERQGRNGEERSLESLRQYIDIFSEVFSTAEMANIRRLDGVSSSSLSA 
               
               
                   
               
               
                 DRLVDYGYRLFYTYWALKEAYIKMTGEALLAPWLRELEFSNVVAPAAVAE 
               
               
                   
               
               
                 SGDSAGDFGEPYTGVRTTLYKNLVEDVRIEVAALGGDYLFATAARGGGIG 
               
               
                   
               
               
                 ASSRPGGGPDGSGIRSQDPWRPFKKLDIERDIQPCATGVCNCLS 
               
            
           
         
       
     
     Other npgA Enzymes that could be used to enzymatically convert Compound II into Compound I include any one or combination of the following enzymes listed in Table 3 and/or SEQ ID NO:11-12 or 22. 
     Moreover, any of these npgA Enzymes (including variants) can be used in combination with PKS Enzyme described herein to produce Compound I from Compound II in the methods described herein. Variants of the npgA Enzymes retain the ability to catalyze the conversion of Compound II into Compound I in combination with a PKS Enzyme, with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence. In preferred embodiments, a variant npgA enzyme, has improved activity over the sequence from which it is derived in that the improved variant has more than 110%, 120%, 130%, 140%, or and 150% improved activity in catalyzing the conversion of Compound II into Compound I as compared to the sequence from which the improved variant is derived. 
     
       
         
           
               
            
               
                 npgA homolog from  P. furfuracea   
               
               
                 (SEQ ID NO: 11) 
               
               
                 MTYHLCNADDDDGDGQTKAFRWLLDVQALWPAPGGGSQSAQSTAHWATGT 
               
               
                   
               
               
                 AAQHALALLADGERARALRFYRPSDAKLSLGSNLLKHRAIANTCRVPWSE 
               
               
                   
               
               
                 AVISEGANRKPCYKPLGPRSKSLEFNVSHHGSLVALVGCPGEAVKLGVDV 
               
               
                   
               
               
                 VKMNWERDYTTVMKDGFEAWANVYEAVFSEREIKDIAGFVPPIRGTQPDE 
               
               
                   
               
               
                 IRAKLRHFYTHWCLKEAYVKMTGEALLAPWLKDLEFRNVQVPLPASQMHA 
               
               
                   
               
               
                 SGQIGGDWGQTCGGVEIWFYGKRVTDVRLEIQAFREDYMIGTASSSVEMG 
               
               
                   
               
               
                 LSVFKELDVERDVYPTQET 
               
               
                   
               
               
                 npgA homolog from  C. Stellaris   
               
               
                 (SEQ ID NO: 12) 
               
               
                 MNGPKVFRWVLDVQSLWPTPPDGPNGLQPSAREATARWASGKEAQYALSL 
               
               
                   
               
               
                 LASEEQAKVLRFYRPSDAKLSLASCLLKHRAIATTCEIPWSEATIGEDSN 
               
               
                   
               
               
                 RKPCYKPSNPGGNTLEFNVSHHGTLVALVGCPGKAVRLGVDIVRMNWDKD 
               
               
                   
               
               
                 YATVMKEGFQSWAKTYEAVFSDREVQDIAHYVTPKHDDLQDTIRAKLRHF 
               
               
                   
               
               
                 YAHWCLKEAYVKMTGEALLAPWLKDVEFRNVQVPLPTSRAVDGAPEVNLW 
               
               
                   
               
               
                 GQTCTDVEIWAHGNRVTDVQLEIQAFRDDYMIATASSHIGAKFSAFKELD 
               
               
                   
               
               
                 LGKDVYP 
               
               
                   
               
               
                 npgA homolog from  C. Grayi   
               
               
                 (SEQ ID NO: 22) 
               
               
                 MAMTGPKVYRWVLDVQSLWPTPPDGTNHLQPSGREATAQWASGKEARYAL 
               
               
                   
               
               
                 SLLTPEEQAKVLRFYRPSDAKLSLASCLLKRRAIATTCEVPWSEATIGED 
               
               
                   
               
               
                 SNRKPCYKPSNPEGKAVEFNVSHHGSLVALVGCPGKDVSLGVDVVRMNWD 
               
               
                   
               
               
                 KDYAGVMREGFESWARTYEAVFSDREVEDIAHYVAPTHDNVQDTIRAKLR 
               
               
                   
               
               
                 HFYAHWCLKEAYVKMTGEALLAPWLKDVEFRNVQVPLPTGLAADGASENN 
               
               
                   
               
               
                 LWGQTCTDVEIWAHGNRVTDVQLEIQAFRDDYMIATASSHVGAEFSAFRE 
               
               
                   
               
               
                 LDLEKDVYP 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 npgA Enzymes 
               
            
           
           
               
               
               
            
               
                   
                   
                 % identity to SEQ 
               
               
                 Accession No. 
                 Protein Name 
                 ID NO: 8 
               
               
                   
               
               
                 XP_663744.1 
                 hypothetical protein AN6140.2 [ Aspergillus nidulans  FGSC A4] 
                 100.00% 
               
               
                 XP_026607463.1 
                 Uncharacterized protein DSM5745_02284 [ Aspergillus mulundensis ] 
                 75.29% 
               
               
                 OJJ01434.1 
                 hypothetical protein ASPVEDRAFT_82959 [ Aspergillus versicolor  CBS 583.65] 
                 68.35% 
               
               
                 OJJ58831.1 
                 hypothetical protein ASPSYDRAFT_58043 [ Aspergillus sydowii  CBS 593.65] 
                 66.76% 
               
               
                 GAQ06841.1 
                 hypothetical protein ALT_4162 [ Aspergillus lentulus ] 
                 57.79% 
               
               
                 KKK21491.1 
                 hypothetical protein AOCH_005987 [ Aspergillus ochraceoroseus ] 
                 58.13% 
               
               
                 XP_001260366.1 
                 4′-phosphopantetheinyl transferase NpgA [ Aspergillus fischeri  NRRL 181] 
                 57.35% 
               
               
                 CEL00884.1 
                 hypothetical protein ASPCAL00476 [ Aspergillus calidoustus ] 
                 66.28% 
               
               
                 XP_026618747.1 
                 hypothetical protein CDV56_106897 [ Aspergillus thermomutatus ] 
                 55.80% 
               
               
                 KKK11895.1 
                 hypothetical protein ARAM_003790 [ Aspergillus rambellii ] 
                 57.10% 
               
               
                 RHZ72079.1 
                 hypothetical protein CDV55_108504 [ Aspergillus turcosus ] 
                 55.41% 
               
               
                 XP_002378105.1 
                 aflYg/npgA protein, putative [ Aspergillus flavus  NRRL3357] 
                 56.82% 
               
               
                 RAQ52488.1 
                 aflYg/npgA protein [ Aspergillus flavus ] 
                 57.47% 
               
               
                 EDP54396.1 
                 4′-phosphopantetheinyl transferase NpgA [ Aspergillus fumigatus ; A1163] 
                 56.86% 
               
               
                 OXN06337.1 
                 hypothetical protein CDV58_05090 [ Aspergillus fumigatus ] 
                 56.57% 
               
               
                 XP_755193.1 
                 4′-phosphopantetheinyl transferase NpgA/CfwA [ Aspergillus fumigatus  Af293] 
                 56.57% 
               
               
                 XP_022585045.1 
                 hypothetical protein ASPZODRAFT_200027 [ Penicilliopsis zonata  CBS 506.65] 
                 55.16% 
               
               
                 KEY77082.1 
                 4′ phosphopantetheinyl transferase NpgA [ Aspergillus fumigatus  var. RP-2014] 
                 56.16% 
               
               
                 PYI23618.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus violaceofuscus  CBS 115571] 
                 54.78% 
               
               
                 ODM20598.1 
                 hypothetical protein SI65_03651 [ Aspergillus cristatus ] 
                 52.72% 
               
               
                 KJK61502.1 
                 Sfp [ Aspergillus parasiticus  SU-1] 
                 56.82% 
               
               
                 GAO86809.1 
                 L-aminoadipate-semialdehyde dehydrogenase- 
                 56.37% 
               
               
                   
                 phosphopantetheinyl transferase [ Aspergillus udagawae ] 
                   
               
               
                 PIG80832.1 
                 aflYg/npgA protein [ Aspergillus arachidicola ] 
                 56.82% 
               
               
                 XP_025504279.1 
                 hypothetical protein BO66DRAFT_81606 [ Aspergillus aculeatinus  CBS 121060] 
                 52.57% 
               
               
                 RJE25168.1 
                 4′-phosphopantetheinyl transferase NpgA [ Aspergillus sclerotialis ] 
                 55.84% 
               
               
                 XP_001267784.1 
                 4′-phosphopantetheinyl transferase NpgA [ Aspergillus clavatus  NRRL 1] 
                 57.43% 
               
               
                 RWQ96577.1 
                 4′-phosphopantetheinyl transferase NpgA [ Byssochlamys spectabilis ] 
                 52.08% 
               
               
                 RAK81669.1 
                 hypothetical protein BO72DRAFT_444212 [ Aspergillus fijiensis  CBS 313.89] 
                 51.74% 
               
               
                 XP_025431842.1 
                 hypothetical protein BP01DRAFT_356077 [ Aspergillus saccharolyticus  JOP 1030-1] 
                 51.46% 
               
               
                 OJJ31021.1 
                 hypothetical protein ASPWEDRAFT_176122 [ Aspergillus wentii  DTO 134E9] 
                 55.59% 
               
               
                 XP_025576628.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus ibericus  CBS 121593] 
                 54.11% 
               
               
                 XP_020059757.1 
                 hypothetical protein ASPACDRAFT_1852401 [ Aspergillus aculeatus  ATCC 16872] 
                 53.20% 
               
               
                 PYI30524.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus indologenus  CBS 114.80] 
                 54.84% 
               
               
                 XP_015403697.1 
                 putative aflYg/npgA protein [ Aspergillus nomius  NRRL 131371 
                 54.60% 
               
               
                 XP_025470021.1 
                 4′-phosphopantetheinyl transferase NpgA [ Aspergillus sclerotioniger  CBS 115572] 
                 54.46% 
               
               
                 PYI08903.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus sclerotiicarbonarius  CBS 121057] 
                 53.98% 
               
               
                 XP_025446590.1 
                 hypothetical protein BO95DRAFT_478940 [ Aspergillus brunneoviolaceus  CBS 621.78] 
                 52.66% 
               
               
                 XP_023093666.1 
                 unnamed protein product [ Aspergillus oryzae  RIB40] 
                 53.76% 
               
               
                 XP_025495634.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus uvarum  CBS 121591] 
                 55.33% 
               
               
                 EIT78712.1 
                 hypothetical protein A03042_05000 [ Aspergillus oryzae  3.042] 
                 53.48% 
               
               
                 XP_020121487.1 
                 hypothetical protein UA08_03648 [ Talaromyces atroroseus ] 
                 50.42% 
               
               
                 XP_022401752.1 
                 hypothetical protein ASPGLDRAFT_124818 [ Aspergillus glaucus  CBS 516.65] 
                 53.30% 
               
               
                 XP_025530903.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus japonicus  CBS 114.51] 
                 54.21% 
               
               
                 XP_022388698.1 
                 aflYg/npgA protein [ Aspergillus bombycis ] 
                 55.43% 
               
               
                 KUL90071.1 
                 hypothetical protein ZTR_02868 [ Talaromyces verruculosus ] 
                 51.12% 
               
               
                 PCH00357.1 
                 4′-phosphopantetheinyl transferase [ Penicillium  sp. ‘occitanis’] 
                 49.72% 
               
               
                 KFX47391.1 
                 L-aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase 
                 49.73% 
               
               
                   
                 [ Talaromyces marneffei  PM1] 
                   
               
               
                 XP_002146553.1 
                 4′-phosphopantetheinyl transferase NpgA/CfwA 
                 49.73% 
               
               
                   
                 [ Talaromyces marneffei  ATCC 18224] 
                   
               
               
                 CRG90513.1 
                 hypothetical protein PISL3812_07557 [ Talaromyces islandicus ] 
                 52.66% 
               
               
                 PGH13396.1 
                 hypothetical protein AJ79_03675 [ Helicocarpus griseus  UAMH5409] 
                 50.14% 
               
               
                 PLN81137.1 
                 hypothetical protein BDW42DRAFT_102289 [ Aspergillus taichungensis ] 
                 54.24% 
               
               
                 GAD93105.1 
                 4′-phosphopantetheinyl transferase NpgA/CfwA [ Byssochlamys spectabilis  No. 5] 
                 53.95% 
               
               
                 PGH08948.1 
                 4′-phosphopantetheinyl transferase [ Blastomyces parvus ] 
                 48.78% 
               
               
                 XP_024667956.1 
                 hypothetical protein BDW47DRAFT_113120 [ Aspergillus candidus ] 
                 55.90% 
               
               
                 RAO71122.1 
                 hypothetical protein BHQ10_007134 [ Talaromyces amestolkiae]   
                 50.29% 
               
               
                 EEQ83341.1 
                 4′-phosphopantetheinyl transferase NpgA [ Blastomyces dermatitidis  ER-3] 
                 49.59% 
               
               
                 EYE91721.1 
                 hypothetical protein EURHEDRAFT_236841 
                 52.29% 
               
               
                   
                 [ Aspergillus ruber  CBS 135680] 
                   
               
               
                 EQL35867.1 
                 hypothetical protein BDFG_02477 [ Blastomyces dermatitidis  ATCC 26199] 
                 50.14% 
               
               
                 XP_024691353.1 
                 hypothetical protein P168DRAFT_272258 [ Aspergillus campestris  IBT 28561] 
                 56.13% 
               
               
                 GAA86427.1 
                 aflYg/npgA protein [ Aspergillus kawachii  IFO 4308] 
                 51.75% 
               
               
                 EGE81927.1 
                 4′-phosphopantetheinyl transferase NpgA [ Blastomyces dermatitidis  ATCC 18188] 
                 50.14% 
               
               
                 XP_002621466.1 
                 4′-phosphopantetheinyl transferase NpgA [ Blastomyces gilchristii  SLH14081] 
                 50.27% 
               
               
                 OJD18353.1 
                 hypothetical protein AJ78_01597 [ Emergomyces pasteurianus  Ep9510] 
                 49.60% 
               
               
                 XP_024687280.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus novofumigatus  IBT 16806] 
                 56.07% 
               
               
                 GCB28155.1 
                 L-aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase 
                 52.05% 
               
               
                   
                 [ Aspergillus awamori ] 
                   
               
               
                 XP_025454152.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus lacticoffeatus  CBS 101883] 
                 52.05% 
               
               
                 XP_001395469.1 
                 npgA protein [ Aspergillus niger  CBS 513.88] 
                 52.84% 
               
               
                 KLJ10976.1 
                 hypothetical protein EMPG_09807 [ Emmonsia parva  UAMH 139] 
                 50.00% 
               
               
                 XP_026628569.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus welwitschiae ] 
                 51.75% 
               
               
                 OJJ67400.1 
                 hypothetical protein ASPBRDRAFT_200113 [ Aspergillus brasiliensis  CBS 101740] 
                 51.87% 
               
               
                 RDK45378.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus phoenicis  ATCC 13157] 
                 52.63% 
               
               
                 OOF92416.1 
                 hypothetical protein ASPCADRAFT_509391 [ Aspergillus carbonarius  ITEM 5010] 
                 52.57% 
               
               
                 XP_002790645.2 
                 4′-phosphopantetheinyl transferase NpgA [ Paracoccidioides lutzii  Pb01] 
                 49.33% 
               
               
                 PYH95779.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus ellipticus  CBS 707.79] 
                 53.69% 
               
               
                 OJD20335.1 
                 hypothetical protein ACJ73_08332 [ Blastomyces percursus ] 
                 49.59% 
               
               
                 XP_002541282.1 
                 conserved hypothetical protein [ Uncinocarpus reesii  1704] 
                 50.43% 
               
               
                 XP_025565104.1 
                 aflYg/npgA protein [ Aspergillus vadensis  CBS 113365] 
                 53.22% 
               
               
                 ODH48202.1 
                 hypothetical protein GX48_05693 [ Paracoccidioides brasiliensis ] 
                 47.14% 
               
               
                 XP_025535897.1 
                 aflYg/npgA protein [ Aspergillus costaricaensis  CBS 115574] 
                 51.92% 
               
               
                 OAX77444.1 
                 hypothetical protein ACJ72_08257 [ Emmonsia  sp. CAC-2015a] 
                 48.83% 
               
               
                 OXV06433.1 
                 hypothetical protein Egran_05801 [ Elaphomyces granulatus ] 
                 48.78% 
               
               
                 XP_025554268.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus homomorphus  CBS 101889] 
                 50.97% 
               
               
                 GAQ45036.1 
                 aflYg/npgA protein [ Aspergillus niger ] 
                 52.19% 
               
               
                 XP_010760919.1 
                 hypothetical protein PADG_05197 [ Paracoccidioides brasiliensis  P1318] 
                 46.58% 
               
               
                 EEH17147.2 
                 hypothetical protein PABG_07234 [ Paracoccidioides brasiliensis  Pb03] 
                 46.59% 
               
               
                 XP_013324640.1 
                 4′-phosphopantetheinyl transferase NpgA [ Rasamsonia emersonii  CBS 393.64] 
                 52.80% 
               
               
                 OJI80632.1 
                 hypothetical protein ASPTUDRAFT_130475 [ Aspergillus tubingensis  CBS 134.48] 
                 50.73% 
               
               
                 XP_024702426.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus steynii  IBT 23096] 
                 52.68% 
               
               
                 XP_025477897.1 
                 aflYg/npgA protein [ Aspergillus neoniger  CBS 115656] 
                 50.29% 
               
               
                 OXV06984.1 
                 hypothetical protein Egran_05250 [ Elaphomyces granulatus ] 
                 47.34% 
               
               
                 XP_025395965.1 
                 4′-phosphopantetheinyl transferase [ Aspergillus heteromorphus  CBS 117.55] 
                 49.86% 
               
               
                 XP_001218317.1 
                 conserved hypothetical protein [ Aspergillus terreus  NIH2624] 
                 50.14% 
               
               
                 KMP00727.1 
                 phosphopantetheinyl transferase A [ Coccidioides immitis RMSCC  2394] 
                 47.38% 
               
               
                 XP_001247064.2 
                 4′-phosphopantetheinyl transferase NpgA [ Coccidioides immitis  RS] 
                 47.38% 
               
               
                 PGH23632.1 
                 hypothetical protein AJ80_02238 [ Polytolypa hystricis  UAMH7299] 
                 46.83% 
               
               
                 AAU07984.1 
                 putative 4′-phosphopantetheinyl transferase [ Aspergillus fumigatus ] 
                 56.45% 
               
               
                 XP_002478852.1 
                 4′-phosphopantetheinyl transferase NpgA/CfwA [ Talaromyces stipitatus  ATCC 10500] 
                 47.34% 
               
               
                 EEH07682.1 
                 4′-phosphopantetheinyl transferase NpgA [ Histoplasma capsulatum  G186AR] 
                 47.95% 
               
               
                 EFW15615.1 
                 4′-phosphopantetheinyl transferase NpgA [ Coccidioides posadasii  str.  Silveira ] 
                 45.86% 
               
               
                 PGH36127.1 
                 4′-phosphopantetheinyl transferase [ Emmonsia crescens ] 
                 46.90% 
               
               
                   
               
            
           
         
       
     
     Production of Compound II 
     As shown in  FIGS. 1A and 1B , Compound II can be produced by two different mechanisms. 
     First, Compound II can be produced by enzymatically converting Compound III into Compound II by an enzyme selected from AAL1, AAL1ΔSKL, and/or CsAAE1. 
     In preferred embodiments, the AAL1 enzyme comprises the following sequence (SEQ ID NO:9): 
     
       
         
           
               
            
               
                 MPQIIHKSAWGDIPLSTFFYGNVTDYLRSKKSFGSDKIGYIDAETGEGIT 
               
               
                   
               
               
                 YKQLWKLANGISAVLYHHYGIGHARAPVASDHTLGDVVMLHAPNSRFFPS 
               
               
                   
               
               
                 LHYGMLDMGCTITSASVSYDVADLAHQLRVTDASLVLCYQEKENNVRQAI 
               
               
                   
               
               
                 KEAQKDAAFPGITHPVRILLIENLLTMACNISEEKINSAMARKFEYSPQE 
               
               
                   
               
               
                 CTKRIAYLSMSSGTTGGIPKAVRLTHFNMSSCDTLGTLSTPSFSTGDDIR 
               
               
                   
               
               
                 VAAIVPMTHQYGLTKFIFNMCSSHATTVVHRQFDLVKLLESQKKYKLNRL 
               
               
                   
               
               
                 MLVPPVIVKMAKDPAVEPYIPSLYEHVDFITTGAAPLPGSAVTNLLTRIT 
               
               
                   
               
               
                 GNPQGIRHSQSGRPPLTISQGYGLTETSPLCAVFDPLDPDVDFRSAGKAT 
               
               
                   
               
               
                 SHVEIRIVSEDGVDQPQLKLDDLSHLDGMLKRDEPLPVGEVLIRGPMIMD 
               
               
                   
               
               
                 GYHKNRQSSEESFDRSQEDPKTLIHWQDKWLKTGDIGMVDQKGRLMIVDR 
               
               
                   
               
               
                 NKEMIKSMSKQVAPAELESLLLNHDQVIDCAVIGVNSEAKATESARAFLV 
               
               
                   
               
               
                 LKDPSYDAVKIKAWLDGQVPSYKRLYGGVVVLKNEQIPKNPSGKILRRIL 
               
               
                   
               
               
                 RTRKDDFIQGIDVSQL 
               
            
           
         
       
     
     The AAL1ΔSKL sequence is identical to SEQ ID NO:9, except that amino acids 614-616 have been deleted. 
     In preferred embodiments, the CsAAE1 enzyme comprises the following sequence (SEQ ID NO:10): 
     
       
         
           
               
            
               
                 MAYKSLDAISVSDIQALGIASPAAEKLFKEISDIITHYGAATPQTWSRIS 
               
               
                   
               
               
                 KRLLNPDLPFSFHQIMYYGCYKDFGPDPPAWLPDPKTAGFTNVWKLLEKR 
               
               
                   
               
               
                 GYEFLGSNYLDPISSFSAFQEFSVSNPEVYWKTVLDEMSVSFSVPPQCIL 
               
               
                   
               
               
                 REDSPLSNPGGQWLPGAHLNPAKNCLSLNSESSSNDVAITWRDEGSDHLP 
               
               
                   
               
               
                 VSCMTLEELRTEVWSVAYALNALGLDRGAAIAINMPMNVKSVIIYLAIVL 
               
               
                   
               
               
                 AGYVVVSIADSFAPVEISTRLKISQAKAIFTQDLIIRGEKSIPLYSRVVD 
               
               
                   
               
               
                 AQSPMAIVIPTKGSNFSMKLRDGDISWRDFLERVNNLRGNEFAAVEQPVE 
               
               
                   
               
               
                 AYTNILFSSGTTGEPKAIPWINATPLKAAADAWCHMDIRKGDIVAWPTNL 
               
               
                   
               
               
                 GWMMGPWLVYASLLNGACIALYNGSPIGSGFAKFVQDAKVTILGVIPSIV 
               
               
                   
               
               
                 RTWKSTNCTAGYDWSAIRCFGSTGEASNVDEYLWLMGRAHYKPIIEYCGG 
               
               
                   
               
               
                 TEIGGAFITGSLLQPQSLAAFSTPTMGCSLFILGNDGYPIPHNVPGMGEL 
               
               
                   
               
               
                 ALGSLMFGASSSLLNGDHYKVYYKGMPVWNGKILRRHGDVFERTSRGYYH 
               
               
                   
               
               
                 AHGRADDTMNLGGIKVSSVELERLCNAADSSILETAAIGVPPPQGGPERL 
               
               
                   
               
               
                 VIAVVFKHPDNSTPDLEELKKSFNSVVQKKLNPLFRVSRVVPLPSLPRTA 
               
               
                   
               
               
                 TNKVMRRILRQRFVQREQNSKL 
               
            
           
         
       
     
     Moreover, variants of AAL1, AAL1ΔSKL, and/or CsAAE1 can also be used to produce Compound II from Compound III in the methods described herein. Variants of the AAL1, AAL1ΔSKL, and/or CsAAE1 retain the ability to catalyze the conversion of Compound III into Compound II with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence. In preferred embodiments, a variant AAL1, AAL1ΔSKL, and/or CsAAE1 enzyme, has improved activity over the sequence from which it is derived in that the improved variant has more than 110%, 120%, 130%, 140%, or and 150% improved activity in catalyzing the conversion of Compound III into Compound II as compared to the sequence from which the improved variant is derived. 
     The second way in which Compound II can be produce is shown in Table IB. In this situation Acetyl-CoA and Malonyl CoA are enzymatically converted to produce Compound II using a combination of enzymes selected from:
         a. StcJ and StcK;   b. HexA and HexB;   c. MutFas1 and MutFas2;       

     The genes HexA &amp; HexB encode the alpha (hexA) and beta (hexB) subunits of the hexanoate synthase (HexS) from  Aspergillus parasiticus  SU-1 (Hitchman et al. 2001). The genes StcJ and StcK are from  Aspergillus nidulans  and encode yeast-like FAS proteins (Brown et al. 1996). As would be understood by the person skilled in the art, many fungi would have hexanoate synthase or fatty acid synthase genes, which could readily be identified by sequencing of the DNA and sequence alignments with the known genes disclosed herein. Similarly, the skilled person would understand that homologous genes in different organisms may also be suitable. Examples of HexA and HexB homologs as shown in Tables 4 and 5. Examples of FAS1 and FAS2 homologs as shown in Tables 6 and 7. The endogenous yeast genes FAS1 (Fatty acid synthase subunit beta) and FAS2 (Fatty acid synthase subunit alpha) form fatty acid synthase FAS which catalyses the formation of long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH. Mutated FAS produces short-chain fatty acids, such as hexanoic acid. Several different combinations of mutations enable the production of hexanoic acid. The mutations include: FAS1 I306A and FAS2 G1250S; FAS1 I306A and FAS2 G1250S and M1251W; and FAS1 I306A, R1834K and FAS2 G1250S (Gajewski et al. 2017). Mutated FAS2 and FAS1 may be expressed under the control of any suitable promoter, including, but not limited to the alcohol dehydrogenase II promoter of  Y. lipolytica . Alternatively, genomic FAS2 and FAS1 can be directly mutated using, for example, homologous recombination or CRISPR-Cas9 genome editing technology. 
     Accordingly, in certain embodiments, HexA comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:16. In certain embodiments, HexA comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:16. In certain embodiments, HexB comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17. In certain embodiments, HexB comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17. In certain embodiments, StcJ comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:18. In certain embodiments, StcJ comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:18. In certain embodiments, StcK comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:19. In certain embodiments, StcK comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:19. In certain embodiments, FAS2 comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20 and one of the combinations of mutations defined above. In certain embodiments, FAS2 comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20 and one of the combinations of mutations defined above. In certain embodiments, FAS1 comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:21 and one of the combinations of mutations defined above. In certain embodiments, FAS1 comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:21 and one of the combinations of mutations defined above. 
     Variants of the Compound II producing proteins retain the ability to catalyse the formation of long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH. For example, a variant of a Compound II producing protein must retain the ability to catalyse the formation of long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence. In preferred embodiments, a variant of a Compound II producing protein has improved activity over the sequence from which it is derived in that the improved variant common cannabinoid protein has more than 110%, 120%, 130%, 140%, or and 150% improved activity in catalysing the formation of long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH, as compared to the sequence from which the improved variant is derived. 
     The hexanoyl-CoA synthases HexA &amp; HexB, StcJ &amp; StcK, or mutated FAS1&amp;2 may be expressed using, for example, a constitutive TEF intron promoter or native promoter (Wong et al. 2017) and synthesized short terminator (Curran et al. 2015). The production of Compound II may be determined by directly measuring the concentration of Compound II using LC-MS. 
     
       
         
           
               
               
            
               
                 HexA 
                   
               
               
                 SEQ ID NO: 16 
                   
               
               
                 MVIQGKRLAASSIQLLASSLDAKKLCYEYDERQAPGVTQITEEAPTEQPPLSTPPSLPQTPNIS 
                   
               
               
                   
               
               
                 PISASKIVIDDVALSRVQIVQALVARKLKTAIAQLPTSKSIKELSGGRSSLQNELVGDIHNEFS 
               
               
                   
               
               
                 SIPDAPEQILLRDFGDANPTVQLGKTSSAAVAKLISSKMPSDFNANAIRAHLANKWGLGPLRQT 
               
               
                   
               
               
                 AVLLYAIASEPPSRLASSSAAEEYWDNVSSMYAESCGITLRPRQDTMNEDAMASSAIDPAVVAE 
               
               
                   
               
               
                 FSKGHRRLGVQQFQALAEYLQIDLSGSQASQSDALVAELQQKVDLWTAEMTPEFLAGISPMLDV 
               
               
                   
               
               
                 KKSRRYGSWWNMARQDVLAFYRRPSYSEFVDDALAFKVFLNRLCNRADEALLNMVRSLSCDAYF 
               
               
                   
               
               
                 KQGSLPGYHAASRLLEQAITSTVADCPKARLILPAVGPHTTITKDGTIEYAEAPRQGVSGPTAY 
               
               
                   
               
               
                 IQSLRQGASFIGLKSADVDTQSNLTDALLDAMCLALHNGISFVGKTFLVTGAGQGSIGAGVVRL 
               
               
                   
               
               
                 LLEGGARVLVTTSREPATTSRYFQQMYDNHGAKFSELRVVPCNLASAQDCEGLIRHVYDPRGLN 
               
               
                   
               
               
                 WDLDAILPFAAASDYSTEMHDIRGQSELGHRLMLVNVFRVLGHIVHCKRDAGVDCHPTQVLLPL 
               
               
                   
               
               
                 SPNHGIFGGDGMYPESKLALESLFHRIRSESWSDQLSICGVRIGWTRSTGLMTAHDIIAETVEE 
               
               
                   
               
               
                 HGIRTFSVAEMALNIAMLLTPDFVAHCEDGPLDADFTGSLGTLGSIPGFLAQLHQKVQLAAEVI 
               
               
                   
               
               
                 RAVQAEDEHERFLSPGTKPTLQAPVAPMHPRSSLRVGYPRLPDYEQEIRPLSPRLERLQDPANA 
               
               
                   
               
               
                 VVVVGYSELGPWGSARLRWEIESQGQWTSAGYVELAWLMNLIRHVNDESYVGWVDTQTGKPVRD 
               
               
                   
               
               
                 GEIQALYGDHIDNHTGIRPIQSTSYNPERMEVLQEVAVEEDLPEFEVSQLTADAMRLRHGANVS 
               
               
                   
               
               
                 IRPSGNPDACHVKLKRGAVILVPKTVPFVWGSCAGELPKGWTPAKYGIPENLIHQVDPVTLYTI 
               
               
                   
               
               
                 CCVAEAFYSAGITHPLEVFRHIHLSELGNFIGSSMGGPTKTRQLYRDVYFDHEIPSDVLQDTYL 
               
               
                   
               
               
                 NTPAAWVNMLLLGCTGPIKTPVGACATGVESIDSGYESIMAGKTKMCLVGGYDDLQEEASYGFA 
               
               
                   
               
               
                 QLKATVNVEEEIACGRQPSEMSRPMAESRAGFVEAHGCGVQLLCRGDIALQMGLPIYAVIASSA 
               
               
                   
               
               
                 MAADKIGSSVPAPGQGILSFSRERARSSMISVTSRPSSRSSTSSEVSDKSSLTSITSISNPAPR 
               
               
                   
               
               
                 AQRARSTTDMAPLRAALATWGLTIDDLDVASLHGTSTRGNDLNEPEVIETQMRHLGRTPGRPLW 
               
               
                   
               
               
                 AlCQKSVTGHPKAPAAAWMLNGCLQVLDSGLVPGNRNLDTLDEALRSASHLCFPTRTVQLREVK 
               
               
                   
               
               
                 AFLLTSFGFGQKGGQVVGVAPKYFFATLPRPEVEGYYRKVRVRTEAGDRAYAAAVMSQAVVKIQ 
               
               
                   
               
               
                 TQNPYDEPDAPRIFLDPLARISQDPSTGQYRFRSDATPALDDDALPPPGEPTELVKGISSAWIE 
               
               
                   
               
               
                 EKVRPHMSPGGTVGVDLVPLASFDAYKNAIFVERNYTVRERDWAEKSADVRAAYASRWCAKEAV 
               
               
                   
               
               
                 FKCLQTHSQGAGAAMKEIEIEHGGNGAPKVKLRGAAQTAARQRGLEGVQLSISYGDDAVIAVAL 
               
               
                   
               
               
                 GLMSGAS 
               
               
                   
               
               
                 HexB 
               
               
                 SEQ ID NO: 17 
                   
               
               
                 MGSVSREHESIPIQAAQRGAARICAAFGGQGSNNLDVLKGLLELYKRYGPDLDELLDVASNTLS 
                   
               
               
                   
               
               
                 QLASSPAAIDVHEPWGFDLRQWLTTPEVAPSKEILALPPRSFPLNTLLSLALYCATCRELELDP 
               
               
                   
               
               
                 GQFRSLLHSSTGHSQGILAAVAITQAESWPTFYDACRTVLQISFWIGLEAYLFTPSSAASDAMI 
               
               
                   
               
               
                 QDCIEHGEGLLSSMLSVSGLSRSQVERVIEHVNKGLGECNRWVHLALVNSHEKFVLAGPPQSLW 
               
               
                   
               
               
                 AVCLHVRRIRADNDLDQSRILFRNRKPIVDILFLPISAPFHTPYLDGVQDRVIEALSSASLALH 
               
               
                   
               
               
                 SIKIPLYHTGTGSNLQELQPHQLIPTLIRAITVDQLDWPLVCRGLNATHVLDFGPGQTCSLIQE 
               
               
                   
               
               
                 LTQGTGVSVIQLTTQSGPKPVGGHLAAVNWEAEFGLRLHANVHGAAKLHNRMTTLLGKPPVMVA 
               
               
                   
               
               
                 GMTPTTVRWDFVAAVAQAGYHVELAGGGYHAERQFEAEIRRLATAIPADHGITCNLLYAKPTTF 
               
               
                   
               
               
                 SWQISVIKDLVRQGVPVEGITIGAGIPSPEVVQECVQSIGLKHISFKPGSFEAIHQVIQIARTH 
               
               
                   
               
               
                 PNFLIGLQWTAGRGGGHHSWEDFHGPILATYAQIRSCPNILLVVGSGFGGGPDTFPYLTGQWAQ 
               
               
                   
               
               
                 AFGYPCMPFDGVLLGSRMMVAREAHTSAQAKRLIIDAQGVGDADWHKSFDEPTGGVVTVNSEFG 
               
               
                   
               
               
                 QPIHVLATRGVMLWKELDNRVFSIKDTSKRLEYLRNHRQEIVSRLNADFARPWFAVDGHGQNVE 
               
               
                   
               
               
                 LEDMTYLEVLRRLCDLTYVSHQKRWVDPSYRILLLDFVHLLRERFQCAIDNPGEYPLDIIVRVE 
               
               
                   
               
               
                 ESLKDKAYRTLYPEDVSLLMHLFSRRDIKPVPFIPRLDERFETWFKKDSLWQSEDVEAVIGQDV 
               
               
                   
               
               
                 QRIFIIQGPMAVQYSISDDESVKDILHNICNHYVEALQADSRETSIGDVHSITQKPLSAFPGLK 
               
               
                   
               
               
                 VTTNRVQGLYKFEKVGAVPEMDVLFEHIVGLSKSWARTCLMSKSVFRDGSRLHNPIRAALQLQR 
               
               
                   
               
               
                 GDTIEVLLTADSEIRKIRLISPTGDGGSTSKVVLEIVSNDGQRVFATLAPNIPLSPEPSVVFCF 
               
               
                   
               
               
                 KVDQKPNEWTLEEDASGRAERIKALYMSLWNLGFPNKASVLGLNSQFTGEELMITTDKIRDFER 
               
               
                   
               
               
                 VLRQTSPLQLQSWNPQGCVPIDYCVVIAWSALTKPLMVSSLKCDLLDLLHSAISFHYAPSVKPL 
               
               
                   
               
               
                 RVGDIVKTSSRILAVSVRPRGTMLTVSADIQRQGQHVVTVKSDFFLGGPVLACETPFELTEEPE 
               
               
                   
               
               
                 MVVHVDSEVRRAILHSRKWLMREDRALDLLGRQLLFRLKSEKLFRPDGQLALLQVTGSVFSYSP 
               
               
                   
               
               
                 DGSTTAFGRVYFESESCTGNVVMDFLHRYGAPRAQLLELQHPGWTGTSTVAVRGPRRSQSYARV 
               
               
                   
               
               
                 SLDHNPIHVCPAFARYAGLSGPIVHGMETSAMMRRIAEWAIGDADRSRFRSWHITLQAPVHPND 
               
               
                   
               
               
                 PLRVELQHKAMEDGEMVLKVQAFNERTEERVAEADAHVEQETTAYVFCGQGSQRQGMGMDLYVN 
               
               
                   
               
               
                 CPEAKALWARADKHLWEKYGFSILHIVQNNPPALTVHFGSQRGRRIRANYLRMMGQPPIDGRHP 
               
               
                   
               
               
                 PILKGLTRNSTSYTFSYSQGLLMSTQFAQPALALMEMAQFEWLKAQGVVQKGARFAGHSLGEYA 
               
               
                   
               
               
                 ALGACASFLSFEDLISLIFYRGLKMQNALPRDANGHTDYGMLAADPSRIGKGFEEASLKCLVHI 
               
               
                   
               
               
                 IQQETGWFVEVVNYNINSQQYVCAGHFRALWMLGKICDDLSCHPQPETVEGQELRAMVWKHVPT 
               
               
                   
               
               
                 VEQVPREDRMERGRATIPLPGIDIPYHSTMLRGEIEPYREYLSERIKVGDVKPCELVGRWIPNV 
               
               
                   
               
               
                 VGQPFSVDKSYVQLVHGITGSPRLHSLLQQMA 
               
               
                   
               
               
                 StcJ 
               
               
                 SEQ ID NO: 18 
                   
               
               
                 MTQKTIQQVPRQGLELLASTQDLAQLCYIYGEPAEGEDSTADESIINTPQCSTIPEVAVEPEVQ 
                   
               
               
                   
               
               
                 PIPDTPLTAIFIIRALVARKLRRSETEIDPSRSIKELCGGKSTLQNELIGELGNEFQTSLPDRA 
               
               
                   
               
               
                 EDVSLADLDAALGEVSLGPTSVSLLQRVFTAKMPARMTVSNVRERLAEIWGLGFHRQTAVLVAA 
               
               
                   
               
               
                 LAAEPHSRLTSLEAAYQYWDGLNEAYGQSLGLFLRKAISQQAARSDDQGAQAIAPADSLGSKDL 
               
               
                   
               
               
                 ARKQYEALREYLGIRTPTTKQDGLDLADLQQKLDCWTAEFSDDFLSQISRRFDARKTRWYRDWW 
               
               
                   
               
               
                 NSARQELLTICQNSNVQWTDKMREHFVQRAEEGLVEIARAHSLAKPLVPDLIQAISLPPVVRLG 
               
               
                   
               
               
                 RLATMMPRTVVTLKGEIQCEEHEREPSCFVEFFSSWIQANNIRCTIQSNGEDLTSVFINSLVHA 
               
               
                   
               
               
                 SQQGVSFPNHTYLITGAGPGSIGQHIVRRLLTGGARVIVTTSREPLPAAAFFKELYSKCGNRGS 
               
               
                   
               
               
                 QLHLVPFNQASVVDCERLIGYIYDDLGLDLDAILPFAATSQVGAEIDGLDASNEAAFRLMLVNV 
               
               
                   
               
               
                 LRLVGFVVSQKRRRGISCRPTQVVLPLSPNHGILGGDGLYAESKRGLETLIQRFHSESWKEELS 
               
               
                   
               
               
                 ICGVSIGWTRSTGLMAANDLVAETAEKQGRVLTFSVDEMGDLISLLLTPQLATRCEDAPVMADF 
               
               
                   
               
               
                 SGNLSCWRDASAQLAAARASLRERADTARALAQEDEREYRCRRAGSTQEPVDQRVSLHLGFPSL 
               
               
                   
               
               
                 PEYDPLLHPDLVPADAVVVVGFAELGPWGSARIRWEMESRGCLSPAGYVETAWLMNLIRHVDNV 
               
               
                   
               
               
                 NYVGWVDGEDGKPVADADIPKRYGERILSNAGIRSLPSDNREVFQEIVLEQDLPSFETTRENAE 
               
               
                   
               
               
                 ALQQRHGDMVQVSTLKNGLCLVQLQHGATIRVPKSIMSPPGVAGQLPTGWSPERYGIPAEIVQQ 
               
               
                   
               
               
                 VDPVALVLLCCVAEAFYSAGISDPMEIFEHIHLSELGNFVGSSMGGVVNTRALYHDVCLDKDVQ 
               
               
                   
               
               
                 SDALQETYLNTAPAWVNMLYLGAAGPIKTPVGACATALESVDSAVESIKAGQTKICLVGGYDDL 
               
               
                   
               
               
                 QPEESAGFARMKATVSVRDEQARGREPGEMSRPTAASRSGFVESQGCGVQLLCRGDVALAMGLP 
               
               
                   
               
               
                 IYGIIAGTGMASDGIGRSVPAPGQGILTFAQEDAQNPAPSRTALARWGLGIDDITVASLHATST 
               
               
                   
               
               
                 PANDTNEPLVIQREMTHLGRTSGRPLWAICQKFVTGHPKAPAAAWMLNGCLQVLDTGLVPGNRN 
               
               
                   
               
               
                 ADDVDPALRSFSHLCFPIRSIQTDGIKAFLLNSCGFGQKEAQLVGVHPRYFLGLLSEPEFEEYR 
               
               
                   
               
               
                 TRRQLRIAGAERAYISAMMTNSIVCVQSHPPFGPAEMHSILLDPSARICLDSSTNSYRVTKAST 
               
               
                   
               
               
                 PVYTGFQRPHDKREDPRPSTIGVDTVTLSSFNAHENAIFLQRNYTERERQSLQLQSHRSFRSAV 
               
               
                   
               
               
                 ASGWCAKEAVFKCLQTVSKGAGAAMSEIEIVRVQGAPSVLHGDALAAAQKAGLDNIQLSLSYGD 
               
               
                   
               
               
                 DCVVAVALGVRKWCLWPLASIIR 
               
               
                   
               
               
                 StcK 
               
               
                 SEQ ID NO: 19 
                   
               
               
                 MTPSPFLDAVDAGLSRLYACFGGQGPSNWAGLDELVHLSHAYADCAPIQDLLDSSARRLESQQR 
                   
               
               
                   
               
               
                 SHTDRHFLLGAGSNYRPGSTTLLHPHHLPEDLALSPYSFPINTLLSLLHYAITAYSLQLDPGQL 
               
               
                   
               
               
                 RQKLQGAIGHSQGVFVAAAIAISHTDHGWPSFYRAADLALQLSFWVGLESHHASPRSILCANEV 
               
               
                   
               
               
                 IDCLENGEGAPSHLLSVTGLDINHLERLVRKLNDQGGDSLYISLINGHNKFVLAGAPHALRGVC 
               
               
                   
               
               
                 IALRSVKASPELDQSRVPFPLRRSVVDVQFLPVSAPYHSSLLSSVELRVTDAIGGLRLRGNDLA 
               
               
                   
               
               
                 IPVYCQANGSLRNLQDYGTHDILLTLIQSVTVERVNWPALCWAMNDATHVLSFGPGAVGSLVQD 
               
               
                   
               
               
                 VLEGTGMNVVNLSGQSMASNLSLLNLSAFALPLGKDWGRKYRPRLRKAAEGSAHASIETKMTRL 
               
               
                   
               
               
                 LGTPHVMVAGMTPTTCSPELVAAIIQADYHVEFACGGYYNRATLETALRQLSRSIPPHRSITCN 
               
               
                   
               
               
                 VIYASPKALSWQTQVLRRLIMEEGLPIDGITVGAGIPSPEVVKEWIDMLAISHIWFKPGSVDAI 
               
               
                   
               
               
                 DRVLTIARQYPTLPVGIQWTGGRAGGHHSCEDFHLPILDCYARIRNCENVILVAGSGFGGAEDT 
               
               
                   
               
               
                 WPYMNGSWSCKLGYAPMPFDGILLGSRMMVAREAKTSFAVKQLIVEAPGVKDDGNDNGAWAKCE 
               
               
                   
               
               
                 HDAVGGVISVTSEMGQPIHVLATRAMRLWKEFDDRFFSIRDPKRLKAALKQHRVEIINRLNNDF 
               
               
                   
               
               
                 ARPWFAQTDSSKPTEIEELSYRQVLRRLCQLTYVQHQARWIDSSYLSLVHDFLRLAQGRLGSGS 
               
               
                   
               
               
                 EAELRFLSCNTPIELEASFDAAYGVQGDQILYPEDVSLLINLFRRQGQKPVPFIPRLDADFQTW 
               
               
                   
               
               
                 FKKDSLWQSEDVDAVVDQDAQRVCIIQGPVAVRHSRVCDEPVKDILDGITEAHLKMMLKEAASD 
               
               
                   
               
               
                 NGYTWANQRDEKGNRLPGIETSQEGSLCRYYLVGPTLPSTEAIVEHLVGECAWGYAALSQKKVV 
               
               
                   
               
               
                 FGQNRAPNPIRDAFKPDIGDVIEAKYMDGCLREITLYHSLRRQGDPRAIRAALGLIHLDGNKVS 
               
               
                   
               
               
                 VTLLTRSKGKRPALEFKMELLGGTMGPLILKMHRTDYLDSVRRLYTDLWIGRDLPSPTSVGLNS 
               
               
                   
               
               
                 EFTGDRVTITAEDVNTFLAIVGQAGPARCRAWGTRGPVVPIDYAVVIAWTALTKPILLEALDAD 
               
               
                   
               
               
                 PLRLLHQSASTRFVPGIRPLHVGDTVTTSSRITERTITTIGQRVEISAELLREGKPVVRLQTTF 
               
               
                   
               
               
                 IIQRRPEESVSQQQFRCVEEPDMVIRVDSHTKLRVLMSRKWFLLDGPCSDLIGKILIFQLHSQT 
               
               
                   
               
               
                 VFDAAGAPASLQVSGSVSLAPSDTSVVCVSSVGTRIGRVYMEEEGFGANPVMDFLNRHGAPRVQ 
               
               
                   
               
               
                 RQPLPRAGWTGDDAASISFTAPAQSEGYAMVSGDTNPIHVCPLFSRFAGLGQPVVHGLHLSATV 
               
               
                   
               
               
                 RRILEWIIGDNERTRFCSWAPSFDGLVRANDRLRMEIQHFAMADGCMVVHVRVLKESTGEQVMH 
               
               
                   
               
               
                 AEAVLEQAQTTYVFTGQGTQERGMGMALYDTNAAARAVWDRAERHFRSQYGISLLHIVRENPTS 
               
               
                   
               
               
                 LTVNFGSRRGRQIRDIYLSMSDSDPSMLPGLTRDSRSYTFNYPSGLLMSTQFAQPALAVMEIAE 
               
               
                   
               
               
                 YAHLQAQGVVQTQAIFAGHSLGEYSSLGACTTIMPFESLLSLILYRGLKMQNTLPRNANGRTDY 
               
               
                   
               
               
                 GMVAADPSRIRSDFTEDRLIELVRLVSQATGVLLEVVNYNVHSRQYVCAGHVRSLWVLSHACDD 
               
               
                   
               
               
                 LSRSTSPNSPQTMSECIAHHIPSSCSVTNETELSRGRATIPLAGVDIPFHSQMLRGHIDGYRQY 
               
               
                   
               
               
                 LRHHLRVSDIKPEELVGRWIPNVTGKPFALDAPYIRLVQGVTQSRPLLELLRRVEENR 
               
               
                   
               
               
                 FAS alpha|FAS2 
               
               
                 SEQ ID NO: 20 
                   
               
               
                 MRPEIEQELAHTLLVELLAYQFASPVRWIETQDVILAEKRTERIVEIGPADTLGGMARRTLASK 
                   
               
               
                   
               
               
                 YEAYDAATSVQRQILCYNKDAKEIYYDVDPVEEETESAPEAAAAPPTSAAPAAAVVAAPAPAAS 
               
               
                   
               
               
                 APSAGPAAPVEDAPVTALDIVRTLVAQKLKKALSDVPLNKAIKDLVGGKSTLQNEILGDLGKEF 
               
               
                   
               
               
                 GSTPEKPEDTPLDELGASMQATFNGQLGKQSSSLIARLVSSKMPGGFNITAVRKYLETRWGLGP 
               
               
                   
               
               
                 GRQDGVLLLALTMEPASRIGSEPDAKVFLDDVANKYAANSGISLNVPTASGDGGASAGGMLMDP 
               
               
                   
               
               
                 AAIDALTKDQRALFKQQLEIIARYLKMDLRDGQKAFVASQETQKTLQAQLDLWQAEHGDFYASG 
               
               
                   
               
               
                 IEPSFDPLKARVYDSSWNWARQDALSMYYDIIFGRLKVVDREIVSQCIRIMNRSNPLLLEFMQY 
               
               
                   
               
               
                 HIDNCPTERGETYQLAKELGEQLIENCKEVLGVSPVYKDVAVPTGPQTTIDARGNIEYQEVPRA 
               
               
                   
               
               
                 SARKLEHYVKQMAEGGPISEYSNRAKVQNDLRSVYKLIRRQHRLSKSSQLQFNALYKDVVRALS 
               
               
                   
               
               
                 MNENQIMPQENGSTKKPGRNGSVRNGSPRAGKVETIPFLHLKKKNEHGWDYSKKLTGIYLDVLE 
               
               
                   
               
               
                 SAARSGLTFQGKNVLMTGAGAGSIGAEVLQGLISGGAKVIVTTSRYSREVTEYYQAMYARYGAR 
               
               
                   
               
               
                 GSQLVVVPFNQGSKQDVEALVDYIYDTKKGLGWDLDFIVPFAAIPENGREIDSIDSKSELAHRI 
               
               
                   
               
               
                 MLTNLLRLLGSVKAQKQANGFETRPAQVILPLSPNHGTFGNDGLYSESKLALETLFNRWYSENW 
               
               
                   
               
               
                 SNYLTICGAVIGWTRGTGLMSGNNMVAEGVEKLGVRTFSQQEMAFNLLGLMAPAIVNLCQLDPV 
               
               
                   
               
               
                 WADLNGGLQFIPDLKDLMTRLRTEIMETSDVRRAVIKETAIENKVVNGEDSEVLYKKVIAEPRA 
               
               
                   
               
               
                 NIKFQFPNLPTWDEDIKPLNENLKGMVNLDKVVVVTGFSEVGPWGNSRTRWEMEASGKFSLEGC 
               
               
                   
               
               
                 VEMAWIMGLIRHHNGPIKGKTYSGWVDSKTGEPVDDKDVKAKYEKYILEHSGIRLIEPELFKGY 
               
               
                   
               
               
                 DPKKKQLLQEIVIEEDLEPFEASKETAEEFKREHGEKVEIFEVLESGEYTVRLKKGATLLIPKA 
               
               
                   
               
               
                 LQFDRLVAGQVPTGWDARRYGIPEDIIEQVDPVTLFVLVCTAEAMLSAGVTDPYEFYKYVHLSE 
               
               
                   
               
               
                 VGNCIGSGIGGTHALRGMYKDRYLDKPLQKDILQESFINTMSAWVNMLLLSSTGPIKTPVGACA 
               
               
                   
               
               
                 TAVESVDIGYETIVEGKARVCFVGGFDDFQEEGSYEFANMKATSNAEDEFAHGRTPQEMSRPTT 
               
               
                   
               
               
                 TTRAGFMESQGCGMQLIMSAQLALDMGVPIYGIIALTTTATDKIGRSVPAPGQGVLTTARENPG 
               
               
                   
               
               
                 KFPSPLLDIKYRRRQLELRKRQIREWQESELLYLQEEAEAIKAQNPADFVVEEYLQERAQHINR 
               
               
                   
               
               
                 EATRQEKDAQFSLGNNFWKQDSRIAPLRGALATWGLTVDEIGVASFHGTSTVANDKNESDVICQ 
               
               
                   
               
               
                 QMKHLGRKKGNALLGIFQKYLTGHPKGAAGAWMFNGCLQVLDSGLVPGNRNADNVDKVMEKFDY 
               
               
                   
               
               
                 IVYPSRSIQTDGIKAFSVTSFGFGQKGAQVIGIHPKYLYATLDRAQFEAYRAKVETRQKKAYRY 
               
               
                   
               
               
                 FHNGLVNNSIFVAKNKAPYEDELQSKVFLNPDYRVAADKKTSELKYPPKPPVATDAGSESTKAV 
               
               
                   
               
               
                 IESLAKAHATENSKIGVDVESIDSINTSNETFTERILPASEQQYCQNAPSPQSSFAGRWSAKEA 
               
               
                   
               
               
                 VFKSLGVCSKGAGAPLKDIEIENDSNGAPTLHGVAAEAAKEAGVKHISVSISHSDMQAVAVAIS 
               
               
                   
               
               
                 QF 
               
               
                   
               
               
                 FAS beta|FAS1 
               
               
                 SEQ ID NO: 21 
                   
               
               
                 MYGTSTGPQTGINTPRSSQSLRPLILSHGSLEFSFLVPTSLHFHASQLKDTFTASLPEPTDELA 
                   
               
               
                   
               
               
                 QDDEPSSVAELVARYIGHVAHEVEEGEDDAHGTNQDVLKLTLNEFERAFMRGNDVHAVAATLPG 
               
               
                   
               
               
                 ITAKKVLVVEAYYAGRAAAGRPTKPYDSALFRAASDEKARIYSVLGGQGNIEEYFDELREVYNT 
               
               
                   
               
               
                 YTSFVDDLISSSAELLQSLSREPDANKLYPKGLNVMQWLREPDTQPDVDYLVSAPVSLPLIGLV 
               
               
                   
               
               
                 QLAHFAVTCRVLGKEPGEILERFSGTTGHSQGIVTAAAIATATTWESFHKAVANALTMLFWIGL 
               
               
                   
               
               
                 RSQQAYPRTSIAPSVLQDSIENGEGTPTPMLSIRDLPRTAVQEHIDMTNQHLPEDRHISISLVN 
               
               
                   
               
               
                 SARNFVVTGPPLSLYGLNLRLRKVKAPTGLDQNRVPFTQRKVRFVNRFLPITAPFHSQYLYSAF 
               
               
                   
               
               
                 DRIMEDLEDVEISPKSLTIPVYGTKTGDDLRAISDANVVPALVRMITHDPVNWEQTTAFPNATH 
               
               
                   
               
               
                 IVDFGPGGISGLGVLTNRNKDGTGVRVILAGSMDGTNAEVGYKPELFDRDEHSVKYAIDWVKEY 
               
               
                   
               
               
                 GPRLVKNATGQTFVDTKMSRLLGIPPIMVAGMTPTTVPWDFVAATMNAGYHIELAGGGYYNAKT 
               
               
                   
               
               
                 MTEAITKIEKAIPPGRGITVNLIYVNPRAMGWQIPLIGKLRADGVPIEGLTIGAGVPSIEVANE 
               
               
                   
               
               
                 YIETLGIKHIAFKPGSVDAIQQVINIAKANPKFPVILQWTGGRGGGHHSFEDFHQPILQMYSRI 
               
               
                   
               
               
                 RRHENIILVAGSGFGGAEDTYPYLSGNWSSRFGYPPMPFDGCLFGSRMMTAKEAHTSKNAKQAI 
               
               
                   
               
               
                 VDAPGLDDQDWEKTYKGAAGGVVTVLSEMGEPIHKLATRGVLFWHEMDQKIFKLDKAKRVPELK 
               
               
                   
               
               
                 KQRDYIIKKLNDDFQKVWFGRNSAGETVDLEDMTYAEVVHRMVDLMYVKHEGRWIDDSLKKLTG 
               
               
                   
               
               
                 DFIRRVEERFTTAEGQASLLQNYSELNVPYPAVDNILAAYPEAATQLINAQDVQHFLLLCQRRG 
               
               
                   
               
               
                 QKPVPFVPSLDENFEYWFKKDSLWQSEDLEAVVGQDVGRTCILQGPMAAKFSTVIDEPVGDILN 
               
               
                   
               
               
                 SIHQGHIKSLIKDMYNGDETTIPITEYFGGRLSEAQEDIEMDGLTISEDANKISYRLSSSAADL 
               
               
                   
               
               
                 PEVNRWCRLLAGRSYSWRHALFSADVFVQGHRFQTNPLKRVLAPSTGMYVEIANPEDAPKTVIS 
               
               
                   
               
               
                 VREPYQSGKLVKTVDIKLNEKGPIALTLYEGRTAENGVVPLTFLFTYHPDTGYAPIREVMDSRN 
               
               
                   
               
               
                 DRIKEFYYRIWFGNKDVPFYTPTTATFNGGRETITSQAVADFVHAVGNTGEAFVERPGKEVFAP 
               
               
                   
               
               
                 MDFAIVAGWKAITKPIFPRTIDGDLLKLVHLSNGFKMVPGAQPLKVGDVLDTTAQINSIINEES 
               
               
                   
               
               
                 GKIVEVCGTIRRDGKPIMHVTSQFLYRGAYTDFENTFQRKDEVPMQVHLASSRDVAILRSKEWF 
               
               
                   
               
               
                 RLDMDDVELLGQTLTFRLQSLIRFKNKNVFSQVQTMGQVLLELPTKEVIQVASVDYEAGTSHGN 
               
               
                   
               
               
                 PVIDYLQRNGTSIEQPVYFENPIPLSGKTPLVLRAPASNETYARVSGDYNPIHVSRVFSSYANL 
               
               
                   
               
               
                 PGTITHGMYTSAAVRSLVETWAAENNIGRVRGFHVSLVDMVLPNDLITVRLQHVGMIAGRKI1K 
               
               
                   
               
               
                 VEASNKETEDKVLLGEAEVEQPVTAYVFTGQGSQEQGMGMELYATSPVAKEVWDRPSFHWNYGL 
               
               
                   
               
               
                 SIIDIVKNNPKERTVHFGGPRGKAIRQNYMSMTFETVNADGTIKSEKIFKEIDETTTSYTYRSP 
               
               
                   
               
               
                 TGLLSATQFTQPALTLMEKASFEDMRSKGLVQRDSSFAGHSLGEYSALADLADVMLIESLVSVV 
               
               
                   
               
               
                 FYRGLTMQVAVERDEQGRSNYSMCAVNPSRISKTFNEQALQYVVGNISEQTGWLLEIVNYNVAN 
               
               
                   
               
               
                 MQYVAAGDLRALDCLTNLLNYLKAQNIDIPALMQSMSLEDVKAHLVNIIHECVKQTEAKPKPIN 
               
               
                   
               
               
                 LERGFATIPLKGIDVPFHSTFLRSGVKPFRSFLIKKINKTTIDPSKLVGKYIPNVTARPFEITK 
               
               
                   
               
               
                 EYFEDVYRLTNSPRIAHILANWEKYEEGTEGGSRHGGTTAASS 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 1   
               
             
            
               
                   
               
               
                 HEXA HOMOLOGS 
               
            
           
           
               
               
               
            
               
                 Description 
                 Ident 
                 Accession 
               
               
                   
               
               
                 hypothetical protein 
                 99% 
                 KJK60794.1 
               
               
                 [ Aspergillus parasiticus  SU-1] 
                   
                   
               
               
                 sterigmatocystin biosynthesis fatty acid 
                 98% 
                 KOC17633.1 
               
               
                 synthase subunit alpha 
                   
                   
               
               
                 [ Aspergillus flavus  AF70] 
                   
                   
               
               
                 fatty acid synthase alpha subunit 
                 98% 
                 XP_002379948.1 
               
               
                 [ Aspergillus flavus  NRRL3357] 
                   
                   
               
               
                 HexA [ Aspergillus flavus ] 
                 98% 
                 AAS90024.1 
               
               
                 unnamed protein product 
                 98% 
                 XP_001821514.3 
               
               
                 [ Aspergillus oryzae  RIB40] 
                   
                   
               
               
                 sterigmatocystin biosynthesis 
                 97% 
                 PIG79619.1 
               
               
                 fatty acid synthase subunit alpha 
                   
                   
               
               
                 [ Aspergillus arachidicola ] 
                   
                   
               
               
                 sterigmatocystin biosynthesis fatty 
                 92% 
                 XP_022391210.1 
               
               
                 acid synthase subunit alpha 
                   
                   
               
               
                 [ Aspergillus bombycis ] 
                   
                   
               
               
                 sterigmatocystin biosynthesis fatty acid 
                 92% 
                 XP_015404699.1 
               
               
                 synthase subunit alpha  
                   
                   
               
               
                 [ Aspergillus nomius  NRRL 13137] 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2   
               
             
            
               
                   
               
               
                 HEXB HOMOLOGS 
               
            
           
           
               
               
               
            
               
                 Description 
                 Ident 
                 Accession 
               
               
                   
               
               
                 hypothetical protein [ Aspergillus   
                 99% 
                 KJK60796.1 
               
               
                   parasiticus  SU-1] 
                   
                   
               
               
                 fatty acid synthase beta subunit  
                 99% 
                 XP_002379947.1 
               
               
                 [ Aspergillus flavus  NRRL3357] 
                   
                   
               
               
                 HexB [ Aspergillus flavus ] 
                 99% 
                 AAS90085.1 
               
               
                 unnamed protein product [ Aspergillus   
                 98% 
                 XP_001821515.1 
               
               
                   oryzae  RIB40] 
                   
                   
               
               
                 fatty acid synthase beta subunit 
                 98% 
                 KOC17632.1 
               
               
                 [ Aspergillus flavus  AF70] 
                   
                   
               
               
                 fatty acid synthase beta subunit 
                 96% 
                 PIG79622.1 
               
               
                 [ Aspergillus arachidicola ] 
                   
                   
               
               
                 HexB [ Aspergillus flavus ] 
                 96% 
                 AAS90002.1 
               
               
                 enoyl reductase domain of FAS1 
                 98% 
                 EIT81347.1 
               
               
                 [ Aspergillus oryzae  3.042] 
                   
                   
               
               
                 fatty acid synthase beta subunit 
                 89% 
                 XP_022391135.1 
               
               
                 [ Aspergillus bombycis ] 
                   
                   
               
               
                 HexB [ Aspergillus nomius ] 
                 90% 
                 AAS90050.1 
               
               
                 fatty acid synthase beta subunit 
                 90% 
                 XP_015404698.1 
               
               
                 [ Aspergillus nomius  NRRL 13137] 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3   
               
             
            
               
                   
               
               
                 FAS1 HOMOLOGS 
               
            
           
           
               
               
               
            
               
                 Description 
                 Ident 
                 Accession 
               
               
                   
               
               
                 fatty acid synthase, beta subunit [ Aspergillus nidulans ] 
                 100%  
                 AAB41494.1 
               
               
                 hypothetical protein [ Aspergillus nidulans  FGSC A4] 
                 99% 
                 XP_682677.1 
               
               
                 hypothetical protein [ Aspergillus sydowii  CBS 593.65] 
                 94% 
                 OJJ52999.1 
               
               
                 Putative Fatty acid synthase beta subunit dehydratase [ Aspergillus   calidoustus ] 
                 94% 
                 CEN62087.1 
               
               
                 hypothetical protein [ Aspergillus versicolor  CBS 583.65] 
                 93% 
                 OJJ08968.1 
               
               
                 hypothetical protein [ Aspergillus rambellii ] 
                 91% 
                 KKK18959.1 
               
               
                 hypothetical protein [ Aspergillus ochraceoroseus ] 
                 91% 
                 KKK13726.1 
               
               
                 fatty acid synthase beta subunit dehydratase 
                 91% 
                 XP_001213436.1 
               
               
                 [ Aspergillus terreus  NIH2624] 
                   
                   
               
               
                 hypothetical protein [ Aspergillus carbonarius  ITEM 5010] 
                 89% 
                 OOF94457.1 
               
               
                 hypothetical protein [ Aspergillus turcosus ] 
                 90% 
                 OXN14637.1 
               
               
                 fatty acid synthase beta subunit [ Aspergillus sclerotioniger  CBS 115572] 
                 89% 
                 PWY96795.1 
               
               
                 fatty acid synthase beta subunit [ Aspergillus heteromorphus  CBS 117.55] 
                 89% 
                 XP_025394299.1 
               
               
                 fatty acid synthase beta subunit [ Aspergillus   
                 89% 
                 PYI01270.1 
               
               
                   sclerotiicarbonarius  CBS 121057] 
                   
                   
               
               
                 hypothetical protein [ Aspergillus thermomutatus ] 
                 90% 
                 OXS11585.1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4   
               
             
            
               
                   
               
               
                 FAS2 HOMOLOGS 
               
            
           
           
               
               
               
            
               
                 Description 
                 Ident 
                 Accession 
               
               
                   
               
               
                 RecName: Full = Fatty acid synthase subunit alpha; Includes: 
                 100%  
                 P78615.1 
               
               
                 RecName: Full = Acyl carrier; Includes: RecName: Full = 3-oxoacyl- 
                   
                   
               
               
                 [acyl-carrier-protein] reductase; AltName: Full = Beta-ketoacyl 
                   
                   
               
               
                 reductase; Includes: RecName: Full = 3-oxoacyl-[acyl-carrier-protein] 
                   
                   
               
               
                 synthase; AltName: Full = Beta-ketoacyl synthase 
                   
                   
               
               
                 FAS2_PENPA Fatty acid synthase subunit alpha [ Aspergillus   nidulans  FGSC A4] 
                 99% 
                 XP_682676.1 
               
               
                 TPA: Fatty acid synthase, alpha subunit 
                 99% 
                 CBF87553.1 
               
               
                 [Source:UniProtKB/TrEMBL;Acc:P78615] [ Aspergillus nidulans  FGSC A4] 
                   
                   
               
               
                 hypothetical protein ASPVEDRAFT_144895 [ Aspergillus versicolor  CBS 583.65] 
                 93% 
                 OJJ08967.1 
               
               
                 Putative Fatty acid synthase subunit alpha reductase [ Aspergillus   calidoustus ] 
                 93% 
                 CEN62088.1 
               
               
                 hypothetical protein ASPSYDRAFT_564317 [ Aspergillus sydowii  CBS 593.65] 
                 93% 
                 OJJ52998.1 
               
               
                 hypothetical protein BP01DRAFT_383520 [ Aspergillus   
                 91% 
                 XP_025430630.1 
               
               
                   saccharolyticus  JOP 1030-1] 
                   
                   
               
               
                 putative fatty acid synthase alpha subunit FasA [ Aspergillus   
                 91% 
                 PYI32058.1 
               
               
                   indologenus  CBS 114.80] 
                   
                   
               
               
                 hypothetical protein ASPCADRAFT_208136 [ Aspergillus   carbonarius  ITEM 5010] 
                 90% 
                 OOF94458.1 
               
               
                 hypothetical protein ASPACDRAFT_79663 [ Aspergillus aculeatus  ATCC 16872] 
                 90% 
                 XP_020055233.1 
               
               
                 fatty acid synthase alpha subunit FasA [ Aspergillus kawachii  IFO 4308] 
                 91% 
                 GAA92751.1 
               
               
                 putative fatty acid synthase alpha subunit FasA [ Aspergillus fijiensis  CBS 313.89] 
                 90% 
                 RAK72625.1 
               
               
                 putative fatty acid synthase alpha subunit FasA [ Aspergillus   
                 90% 
                 XP_025498650.1 
               
               
                   aculeatinus  CBS 121060] 
                   
                   
               
               
                 putative fatty acid synthase alpha subunit FasA [ Aspergillus   
                 90% 
                 PYI15679.1 
               
               
                   violaceofuscus  CBS 115571] 
                   
                   
               
               
                 fatty acid synthase alpha subunit FasA [ Aspergillus piperis  CBS 112811] 
                 91% 
                 XP_025520376.1 
               
               
                 fatty acid synthase alpha subunit FasA [ Aspergillus vadensis  CBS 113365] 
                 91% 
                 PYH66515.1 
               
               
                 putative fatty acid synthase alpha subunit FasA [ Aspergillus   
                 90% 
                 XP_025442388.1 
               
               
                   brunneoviolaceus  CBS 621.78] 
                   
                   
               
               
                 fatty acid synthase alpha subunit FasA [ Aspergillus neoniger  CBS 115656] 
                 91% 
                 XP_025476115.1 
               
               
                 fatty acid synthase alpha subunit FasA [ Aspergillus costaricaensis  CBS 115574] 
                 91% 
                 RAK83984.1 
               
               
                   
               
            
           
         
       
     
     Production of Compound III 
     The production of Compound III can be enzymatically produced from Compound IV using, for example, ADH alone or with the combination of ADH, FAO and one of 4 FALDH1-4. See, for example Gatter, M., et al., (2014) FEMS Yeast Research 14(6), 858-872 and Salić, A., et al., (2013) Applied Biochemistry and Biotechnology 171(8), 2273-2284. Carbon sources used to produce Compound III from alkans, such as for example hexan, octan. 
     Production of GPP 
       FIG. 3  describes the preferred method of producing GPP. Specifically, GPP may be produced by a mutated farnesyl diphosphate synthase. For example, normally in yeast, the farnesyl diphosphate synthase ERG20 condenses isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to provide geranyl pyrophosphate (GPP) and then condenses two molecules of GPP to provide feranyl pyrophosphate (FPP). However, only a low level of GPP remains as ERG20 converts most of the GPP to FPP. More GPP is required for the commercial scale production of cannabinoids. Accordingly, mutated ERG20 that has a reduced or inability to produce FPP, may be used to increase the production of GPP. Two sets of mutations have been identified in  S. cerevisiae  that increase GPP production. The first mutation is a substitution of K197E and the second is a double substitution of F96W and N127W. As would be readily appreciated by the person skilled in the art, due to the high homology between ERG20 from  S. cerevisiae  and ERG20 from  Y. lipolytica , equivalent mutations may be introduced into ERG20 from  Y. lipolytica . In  Y. lipolytica  the first mutation is a substitution of K189E and the second is a double substitution of F88W and N119W. Introducing  Y. lipolytica  ERG20 (K189E) increases the production of GPP but growth is little bit slower compared to wild type yeast. Introducing  Y. lipolytica  ERG20 (F88W and N119W) produces fast growing clones with a high level of GPP. The sequences for the  Y. lipolytica  and  S. cerevisiae  genes are shown herein, however the skilled person would understand that homologous genes may also be suitable. Examples of ERG20 homologs as shown in Table 8. Accordingly, in certain embodiments, the one or more GPP producing genes comprise: a mutated farnesyl diphosphate synthase; a mutated  S. cerevisiae  ERG20 comprising a K197E substitution; a double mutated  S. cerevisiae  ERG20 comprising F96W and N127W substitutions; a mutated  Y. lipolytica  ERG20 comprising a K189E substitution; or a double mutated  Y. lipolytica  ERG20 comprising F88W and N119W substitutions; or a combination thereof. For the SEQ IDS described herein, mutations are shown with a solid underline. In certain embodiments,  S. cerevisiae  ERG20 (K197E) comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:25. In certain embodiments,  S. cerevisiae  ERG20 (K197E) comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:25. In certain embodiments,  S. cerevisiae  ERG20 (F96W and N127W) comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:26. In certain embodiments,  S. cerevisiae  ERG20 (F96W and N127W) comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:26. The equivalent  Y. lipolytica  amino acid sequences are shown in SEQ ID NOS: 27 and 28. In certain embodiments,  Y. lipolytica  ERG20 (K189E) comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:27. In certain embodiments,  Y. lipolytica  ERG20 (K189E) comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:27. In certain embodiments,  Y. lipolytica  ERG20 (F88W and N119W) comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:28. In certain embodiments,  Y. lipolytica  ERG20 (F88W and N119W) comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:28. 
     Variants of the GPP proteins, such as ERG20, retain the ability to, for example, condense isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to geranyl pyrophosphate (GPP) and yet have reduced GPP to FPP activity. For example, a variant of a GPP protein, such as ERG20, retains the ability to condense isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to geranyl pyrophosphate (GPP) with at least about at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence, while the ability to condense GPP to FPP is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (null mutation) as compared to the sequence from which it is derived. 
     
       
         
           
               
            
               
                 ERG20 (K197E) 
               
               
                 SEQ ID NO: 25 
               
               
                 MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPG 
               
               
                   
               
               
                 GKLNRGLSVVDTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVAD 
               
               
                   
               
               
                 DMMDKSITRRGQPCWYKVPEVGEIAINDAFMLEAAIYKLLKSHFRNEKYY 
               
               
                   
               
               
                 IDITELFHEVTFQTELGQLMDLITAPEDKVDLSKFSLKKHSFIVTFETAY 
               
               
                   
               
               
                 YSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQDDYLDCFGTPE 
               
               
                   
               
               
                 QIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEAKCKK 
               
               
                   
               
               
                 IFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKR 
               
               
                   
               
               
                 SK* 
               
               
                   
               
               
                 ERG20 (F96W and N127W) 
               
               
                 SEQ ID NO: 26 
               
               
                 MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPG 
               
               
                   
               
               
                 GKLNRGLSVVDTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYWLVAD 
               
               
                   
               
               
                 DMMDKSITRRGQPCWYKVPEVGEIAIWDAFMLEAAIYKLLKSHFRNEKYY 
               
               
                   
               
               
                 IDITELFHEVTFQTELGQLMDLITAPEDKVDLSKFSLKKHSFIVTFKTAY 
               
               
                   
               
               
                 YSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQDDYLDCFGTPE 
               
               
                   
               
               
                 QIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEAKCKK 
               
               
                   
               
               
                 IFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKR 
               
               
                   
               
               
                 SK* 
               
               
                   
               
               
                   Y. lipolytica  ERG20 (K189E) 
               
               
                 SEQ ID NO: 27 
               
               
                 MSKAKFESVFPRISEELVQLLRDEGLPQDAVQWFSDSLQYNCVGGKLNRG 
               
               
                   
               
               
                 LSVVDTYQLLTGKKELDDEEYYRLALLGWLIELLQAFFLVSDDIMDESKT 
               
               
                   
               
               
                 RRGQPCWYLKPKVGMIAINDAFMLESGIYILLKKHFRQEKYYIDLVELFH 
               
               
                   
               
               
                 DISFKTELGQLVDLLTAPEDEVDLNRFSLDKHSFIVRYETAYYSFYLPVV 
               
               
                   
               
               
                 LAMYVAGITNPKDLQQAMDVLIPLGEYFQVQDDYLDNFGDPEFIGKIGTD 
               
               
                   
               
               
                 IQDNKCSWLVNKALQKATPEQRQILEDNYGVKDKSKELVIKKLYDDMKIE 
               
               
                   
               
               
                 QDYLDYEEEVVGDIKKKIEQVDESRGFKKEVLNAFLAKIYKRQK 
               
               
                   
               
               
                   Y. lipolytica  ERG20 (F88W and N119W) 
               
               
                 SEQ ID NO: 28 
               
               
                 ASKAKFESVFPRISEELVQLLRDEGLPQDAVQWFSDSLQYNCVGGKLNRG 
               
               
                   
               
               
                 LSVVDTYQLLTGKKELDDEEYYRLALLGWLIELLQAFWLVSDDIMDESKT 
               
               
                   
               
               
                 RRGQPCWYLKPKVGMIAIWDAFMLESGIYILLKKHFRQEKYYIDLVELFH 
               
               
                   
               
               
                 DISFKTELGQLVDLLTAPEDEVDLNRFSLDKHSFIVRYKTAYYSFYLPVV 
               
               
                   
               
               
                 LAMYVAGITNPKDLQQAMDVLIPLGEYFQVQDDYLDNFGDPEFIGKIGTD 
               
               
                   
               
               
                 IQDNKCSWLVNKALQKATPEQRQILEDNYGVKDKSKELVIKKLYDDMKIE 
               
               
                   
               
               
                 QDYLDYEEEVVGDIKKKIEQVDESRGFKKEVLNAFLAKIYKRQK 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 ERG20 HOMOLOGS 
               
            
           
           
               
               
               
            
               
                 Description 
                 Ident 
                 Accession 
               
               
                   
               
               
                 YALI0E05753p [ Yarrowia lipolytica  CLIB122] 
                 99% 
                 XP_503599.1 
               
               
                 hypothetical protein [ Nadsonia fulvescens  var.  elongata  DSM 6958] 
                 71% 
                 ODQ67901.1 
               
               
                 hypothetical protein [ Lipomyces starkeyi  NRRL Y-11557] 
                 70% 
                 ODQ75043.1 
               
               
                 Farnesyl pyrophosphate synthetase [ Galactomyces candidus ] 
                 68% 
                 CDO55796.1 
               
               
                 hypothetical protein [ Kazachstania naganishii  CBS 8797] 
                 68% 
                 XP_022463460.1 
               
               
                 farnesyl pyrophosphate synthase [ Saitoella complicata  NRRL Y-17804] 
                 66% 
                 XP_019025287.1 
               
               
                 hypothetical protein [ Tetrapisispora blattae  CBS 6284] 
                 67% 
                 XP_004179894.1 
               
               
                 hypothetical protein [ Torulaspora delbrueckii]   
                 67% 
                 XP_003680478.1 
               
               
                 unnamed protein product [ Zymoseptoria tritici  ST99CH_1E4] 
                 66% 
                 SMR57088.1 
               
               
                 ERG20 farnesyl diphosphate synthase [ Zymoseptoria tritici  IPO323] 
                 66% 
                 XP_003850094.1 
               
               
                 LAFE_0G04434g1_1 [ Lachancea fermentati ] 
                 68% 
                 SCW03167.1 
               
               
                 ERG20-like protein [ Saccharomyces kudriavzevii  IFO 1802] 
                 66% 
                 EJT43164.1 
               
               
                 hypothetical protein [ Dactylellina haptotyla  CBS 200.50] 
                 66% 
                 EPS37682.1 
               
               
                 CYFA0S07e04962g1_1 [ Cyberlindnera fabianii ] 
                 65% 
                 CDR41679.1 
               
               
                 probable farnesyl pyrophosphate synthetase [ Ramularia collo - cygni ] 
                 65% 
                 XP_023628194.1 
               
               
                 farnesyl pyrophosphate synthetase [ Kluyveromyces marxianus  DMKU3-1042] 
                 65% 
                 XP_022673909.1 
               
               
                 polyprenyl synt-domain-containing protein [ Sphaerulina musiva  SO2202] 
                 67% 
                 XP_016759989.1 
               
               
                   
               
            
           
         
       
     
     High levels of GPP production are dependent on adequate mevalonate production. Hydroxymethylglutaryl-CoA reductase (HMGR) catalyses the production of mevalonate from HMG-CoA and NADPH. HMGR is a rate limiting step in the GPP pathway in yeast. Accordingly, overexpressing HMGR may increase flux through the pathway and increase the production of GPP. HMGR is a GPP pathway gene. Other GPP pathway genes include those genes that are involved in the GPP pathway, the products of which either directly produce GPP or produce intermediates in the GPP pathway, for example, ERG10, ERG13, ERG12, ERG8, ERG19, IDb1 or ERG20, The HMGR1 sequence from  Y. lipolytica  consists of 999 amino acids (aa) (SEQ ID NO: 29), of which the first 500 aa harbor multiple transmembrane domains and a response element for signal regulation. The remaining 499 C-terminal residues contain a catalytic domain and an NADPH-binding region. Truncated HMGR1(tHmgR) has been generated by deleting the N-terminal 500 aa (Gao et al. 2017). tHMGR is able to avoid self-degradation mediated by its N-terminal domain and is thus stabilized in the cytoplasm, which increases flux through the GPP pathway. The N-terminal 500 aa are shown with a dashed underline in SEQ ID NO:29. The N-terminal 500 aa are deleted in SEQ ID NO:30. In certain embodiments, the one or more GPP pathway genes comprise a hydroxymethylglutaryl-CoA reductase (HMGR); a truncated hydroxymethylglutaryl-CoA reductase (tHMGR); or a combination thereof. The sequence for the  Y. lipolytica  gene are shown herein, however the skilled person would understand that homologous genes may also be suitable. Examples of HMGR homologs as shown in Table 9. In certain embodiments, HMGR comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:29. In certain embodiments, HMGR comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:29. In certain embodiments, tHmgR comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:30. In certain embodiments, tHmgR comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:30. 
     The GPP producing and GPP pathway genes may be expressed using, for example, a constitutive TEF intron promoter or native promoter (Wong et al. 2017) and synthesized short terminator (Curran et al. 2015). Increased production of GPP can be determined by overexpressing a single heterologous gene encoding linalool synthase and then determining the production of linalool using, for example, a colorimentric assay (Ghorai 2012). Increased production of GPP may be indicated by a linalool concentration of at least 0.5 mg/L, 0.7 mg/L, 0.9 mg/L or preferably at least about 1 mg/L. 
     
       
         
           
               
            
               
                 HMGR1 (underlined sequence is removed in tHMGR1) 
               
               
                 SEQ ID NO: 29 
               
               
                 
                   MLQAAIGKIVGFAVNRPIHTVVLTSIVASTAYLAILDIAIPGFEGTQPIS 
                 
               
               
                   
               
               
                 
                   YYHPAAKSYDNPADWTHIAEADIPSDAYRLAFAQIRVSDVQGGEAPTIPG 
                 
               
               
                   
               
               
                 
                   AVAVSDLDHRIVMDYKQWAPWTASNEQIASENHIWKHSFKDHVAFSWIKW 
                 
               
               
                   
               
               
                 
                   FRWAYLRLSTLIQGADNFDIAVVALGYLAMHYTFFSLFRSMRKVGSHFWL 
                 
               
               
                   
               
               
                 
                   ASMALVSSTFAFLLAVVASSSLGYRPSMITMSEGLPFLVVAIGFDRKVNL 
                 
               
               
                   
               
               
                 
                   ASEVLTSKSSQLAPMVQVITKIASKALFEYSLEVAALFAGAYTGVPRLSQ 
                 
               
               
                   
               
               
                 
                   FCFLSAWILIFDYMFLLTFYSAVLAIKFEINHIKRNRMIQDALKEDGVSA 
                 
               
               
                   
               
               
                 
                   AVAEKVADSSPDAKLDRKSDVSLFGASGAIAVFKIFMVLGFLGLNLINLT 
                 
               
               
                   
               
               
                 
                   AIPHLGKAAAAAQSVTPITLSPELLHAIPASVPVVVTFVPSVVYEHSQLI 
                 
               
               
                   
               
               
                 
                   LQLEDALTTFLAACSKTIGDPVISKYIFLCLMVSTALNVYLFGATREVVR 
                 
               
               
                   
               
               
                 TQSVKVVEKHVPIVIEKPSEKEEDTSSEDSIELTVGKQPKPVTETRSLDD 
               
               
                   
               
               
                 LEAIMKAGKTKLLEDHEVVKLSLEGKLPLYALEKQLGDNTRAVGIRRSII 
               
               
                   
               
               
                 SQQSNTKTLETSKLPYLHYDYDRVFGACCENVIGYMPLPVGVAGPMNIDG 
               
               
                   
               
               
                 KNYHIPMATTEGCLVASTMRGCKAINAGGGVTTVLTQDGMTRGPCVSFPS 
               
               
                   
               
               
                 LKRAGAAKIWLDSEEGLKSMRKAFNSTSRFARLQSLHSTLAGNLLFIRFR 
               
               
                   
               
               
                 TTTGDAMGMNMISKGVEHSLAVMVKEYGFPDMDIVSVSGNYCTDKKPAAI 
               
               
                   
               
               
                 NWIEGRGKSVVAEATIPAHIVKSVLKSEVDALVELNISKNLIGSAMAGSV 
               
               
                   
               
               
                 GGFNAHAANLVTAIYLATGQDPAQNVESSNCITLMSNVDGNLLISVSMPS 
               
               
                   
               
               
                 IEVGTIGGGTILEPQGAMLEMLGVRGPHIETPGANAQQLARIIASGVLAA 
               
               
                   
               
               
                 ELSLCSALAAGHLVQSHMTHNRSQAPTPAKQSQADLQRLQNGSNICTRS 
               
               
                   
               
               
                 tHmgR 
               
               
                 SEQ ID NO: 30 
               
               
                 TQSVKVVEKHVPIVIEKPSEKEEDTSSEDSIELTVGKQPKPVTETRSLDD 
               
               
                   
               
               
                 LEAIMKAGKTKLLEDHEVVKLSLEGKLPLYALEKQLGDNTRAVGIRRSII 
               
               
                   
               
               
                 SQQSNTKTLETSKLPYLHYDYDRVFGACCENVIGYMPLPVGVAGPMNIDG 
               
               
                   
               
               
                 KNYHIPMATTEGCLVASTMRGCKAINAGGGVTTVLTQDGMTRGPCVSFPS 
               
               
                   
               
               
                 LKRAGAAKIWLDSEEGLKSMRKAFNSTSRFARLQSLHSTLAGNLLFIRFR 
               
               
                   
               
               
                 TTTGDAMGMNMISKGVEHSLAVMVKEYGFPDMDIVSVSGNYCTDKKPAAI 
               
               
                   
               
               
                 NWIEGRGKSVVAEATIPAHIVKSVLKSEVDALVELNISKNLIGSAMAGSV 
               
               
                   
               
               
                 GGFNAHAANLVTAIYLATGQDPAQNVESSNCITLMSNVDGNLLISVSMPS 
               
               
                   
               
               
                 IEVGTIGGGTILEPQGAMLEMLGVRGPHIETPGANAQQLARIIASGVLAA 
               
               
                   
               
               
                 ELSLCSALAAGHLVQSHMTHNRSQAPTPAKQSQADLQRLQNGSNICIRS 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 HMGR HOMOLOGS 
               
            
           
           
               
               
               
            
               
                 Description 
                 Ident 
                 Accession 
               
               
                   
               
               
                 YALI0E04807p [ Yarrowia lipolytica  CLIB122] 
                 100%  
                 XP_503558.1 
               
               
                 hypothetical protein [ Nadsonia fulvescens  var. elongata DSM 6958] 
                 75% 
                 ODQ65159.1 
               
               
                 hypothtical protein [ Galactomyces candidum ] 
                 74% 
                 CDO55526.1 
               
               
                 hypothetical protein 
                 74% 
                 ODQ70929.1 
               
               
                 [ Lipomyces starkeyi  NRRL Y-11557] 
                   
                   
               
               
                 hypothetical protein [ Meyerozyma guilliermondii  ATCC 6260] 
                 76% 
                 EDK40614.2 
               
               
                 HMG1 [ Sugiyamaella lignohabitans ] 
                 73% 
                 XP_018736018.1 
               
               
                 hypothetical protein [ Meyerozyma guilliermondii  ATCC 6260] 
                 76% 
                 XP_001482757.1 
               
               
                 hypothetical protein [ Babjeviella inositovora  NRRL Y-12698] 
                 76% 
                 XP_018984841.1 
               
               
                 DEHA2D09372p [ Debaryomyces hansenii  CBS767] 
                 75% 
                 XP_458872.2 
               
               
                 3-hydroxy-3-methylglutaryl-coenzyme 
                 75% 
                 KTB22480.1 
               
               
                 A reductase 1 [[ Candida ]  glabrata ] 
                   
                   
               
               
                 hypothetical protein 
                 72% 
                 XP_001643950.1 
               
               
                 [ Vanderwaltozyma polyspora  DSM 70294] 
                   
                   
               
               
                 LAFE_0A01552g1_1 [ Lachancea fermentati ] 
                 76% 
                 SCV99364.1 
               
               
                 hypothetical protein [ Debaryomyces fabryi ] 
                 75% 
                 XP_015466829.1 
               
               
                 uncharacterized protein [ Kuraishia capsulata  CBS 1993] 
                 76% 
                 XP_022457391.1 
               
               
                 uncharacterized protein [ Candida ]  glabrata ] 
                 75% 
                 XP_449268.1 
               
               
                   
               
            
           
         
       
     
     Cannabinoid Precursor or Cannabinoid Producing Genes 
     The production of the cannabinoids tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) involves the prenylation of OA with GPP to CBGA (as shown in  FIGS. 1A and 1B ) by an aromatic prenyltransferase, and then CBDA, THCA or CBCA by CBDAS, THCAS or CBCAS, respectively. 
     As described herein CBGA-analogs may be produced by a membrane-bound CBGA synthase (CBGAS) from  C. sativa . CBGAS is also known as geranylpyrophosphate olivetolate geranyltransferase, of which there are several forms, CsPT1, CsPT3 and CsPT4. In certain embodiments, the one or more cannabinoid precursor or cannabinoid producing genes comprise: a soluble aromatic prenyltransferase; a cannabigerolic acid synthase (CBGAS); or a combination thereof; either alone or in combination with the cannabinoid producing genes: tetrahydrocannabinolic acid synthase (THCAS); cannabidiolic acid synthase (CBDAS); cannabichromenic acid synthase (CBCAS); or any combination thereof. The sequences for the  Cannabis sativa  genes CBGAS, THCAS, CBDAS and CBCAS are shown herein, however the skilled person would understand that homologous genes may also be suitable. 
     In certain embodiments, CBGA synthase comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:31. In certain embodiments, CBGA synthase comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:32. In certain embodiments, CBGA synthase comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:33. In certain embodiments, CBGA synthase comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOS: 31, 32 or 33. CBGA may also be formed by heterologous expression of a soluble aromatic prenyltransferase. In certain embodiments, the soluble aromatic prenyltransferase is NphB from  Streptomyces  sp. strain CL190 (ie wild type NphB) (Bonitz et al., 2011; Kuzuyama et al., 2005; Zirpel et al., 2017). In certain embodiments, the soluble aromatic prenyltransferase is NphB, comprising at least one mutation selected from (a) Q161A; (b) G286S; (c) Y288A; (d) A232S; (e) Y288A+G286S; (f) Y288A+G286S+Q161A; (g) Q161A+G286S; (h) Q161A+Y288A; or (i) Y288A+A232S. It is expected that the mutants of NphB (e.g., Q161A) produces more CBGA that wild type NphB (Muntendam 2015). 
     Wild type NphB produces 15% CBGA and 85% of another by-product. The sequence for the  Streptomyces  sp. strain CL190 gene NphB is shown herein, however the skilled person would understand that homologous genes may also be suitable. In certain embodiments, NphB comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:34. In certain embodiments, NphB comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:34. 
     Variants of the cannabinoid precursor or cannabinoid producing protein, such as NphB variant (e.g., at least one of Q161A, G286S, Y288A, A232S), retains the ability to attach geranyl groups to aromatic substrates—such as converting Compound I and GPP to CBGA-analog. For example, a variant Cannabinoid precursor or cannabinoid producing protein, such as NphB variant (e.g., at least one of Q161A, G286S, Y288A, A232S), must retain the ability to attach geranyl groups to aromatic substrates, such as converting Compound I and GPP to CBGA-analog, with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence. In preferred embodiments, a variant of a Cannabinoid precursor or cannabinoid producing protein, such as NphB variant (e.g., at least one of Q161A, G286S, Y288A, A232S), has improved activity over the sequence from which it is derived in that the improved variant common cannabinoid protein has more than 110%, 120%, 130%, 140%, or and 150% improved activity in attach geranyl groups to aromatic substrates, such as converting Compound I and GPP to CBGA-analog, as compared to the sequence from which the improved variant is derived. 
     The cannabinoid precursor or cannabinoid producing genes CBGAS, soluble aromatic prenyltransferase, CBGAS, THCAS, CBDAS and CBCAS may be expressed using, for example, a constitutive TEF intron promoter or native promoter (Wong et al. 2017) and synthesized short terminator (Curran et al. 2015). The production of one or more cannabinoid precursors or cannabinoids may be determined using a variety of methods. For example, if all of the precursors are available in the yeast cell, then the presence of the product, such as THCA, may be determined using HPLC or gas chromatography (GC). Alternatively, if only a portion of the cannabinoid synthesis pathway present, then cannabinoids will not be present and the activity of one or more genes can be checked by adding a gene and precursor. For example, to check CBGAS activity, Compound I and GPP are added to a crude cellular lysate. For checking CBCAS, THCAS or CBDAS activity, a CBGA-analog is added to a crude cellular lysate. A crude lysate or purified proteins may be used. Further, it may be necessary to use an aqueous/organic two-liquid phase setup in order to solubilize the hydrophobic substrate (eg CBGA) and to allow in situ product removal. 
     
       
         
           
               
            
               
                 CsPT1 
               
               
                 SEQ ID NO: 31 
               
               
                 MGLSSVCTFSFQTNYHTLLNPHNNNPKTSLLCYRHPKTPIKYSYNNFPSK 
               
               
                   
               
               
                 HCSTKSFHLQNKCSESLSIAKNSIRAATTNQTEPPESDNHSVATKILNFG 
               
               
                   
               
               
                 KACWKLQRPYTIIAFTSCACGLFGKELLHNTNLISWSLMFKAFFFLVAIL 
               
               
                   
               
               
                 CIASFTTTINQIYDLHIDRINKPDLPLASGEISVNTAWIMSIIVALFGLI 
               
               
                   
               
               
                 ITIKMKGGPLYIFGYCFGIFGGIVYSVPPFRWKQNPSTAFLLNFLAHIIT 
               
               
                   
               
               
                 NFTFYYASRAALGLPFELRPSFTFLLAFMKSMGSALALIKDASDVEGDTK 
               
               
                   
               
               
                 FGISTLASKYGSRNLTLFCSGIVLLSYVAAILAGIIWPQAFNSNVMLLSH 
               
               
                   
               
               
                 AILAFWLILQTRDFALTNYDPEAGRRFYEFMWKLYYAEYLVYVFI 
               
               
                   
               
               
                 CsPT3 
               
               
                 SEQ ID NO: 32 
               
               
                 MGLSLVCTFSFQTNYHTLLNPHNKNPKNSLLSYQHPKTPIIKSSYDNFPS 
               
               
                   
               
               
                 KYCLTKNFHLLGLNSHNRISSQSRSIRAGSDQIEGSPHHESDNSIATKIL 
               
               
                   
               
               
                 NFGHTCWKLQRPYVVKGMISIACGLFGRELFNNRHLFSWGLMWKAFFALV 
               
               
                   
               
               
                 PILSFNFFAAIMNQIYDVDIDRINKPDLPLVSGEMSIETAWILSIIVALT 
               
               
                   
               
               
                 GLIVTIKLKSAPLFVFIYIFGIFAGFAYSVPPIRWKQYPFTNFLITISSH 
               
               
                   
               
               
                 VGLAFTSYSATTSALGLPFVWRPAFSFIIAFMTVMGMTIAFAKDISDIEG 
               
               
                   
               
               
                 DAKYGVSTVATKLGARNMTFVVSGVLLLNYLVSISIGIIWPQVFKSNIMI 
               
               
                   
               
               
                 LSHAILAFCLIFQTRELALANYASAPSRQFFEFIWLLYYAEYFVYVFI 
               
               
                   
               
               
                 CsPT4 
               
               
                 SEQ ID NO: 33 
               
               
                 MVFSSVCSFPSSLGTNFKLVPRSNFKASSSHYHEINNFINNKPIKFSYFS 
               
               
                   
               
               
                 SRLYCSAKPIVHRENKFTKSFSLSHLQRKSSIKAHGEIEADGSNGTSEFN 
               
               
                   
               
               
                 VMKSGNAIWRFVRPYAAKGVLFNSAAMFAKELVGNLNLFSWPLMFKILSF 
               
               
                   
               
               
                 TLVILCIFVSTSGINQIYDLDIDRLNKPNLPVASGEISVELAWLLTIVCT 
               
               
                   
               
               
                 ISGLTLTIITNSGPFFPFLYSASIFFGFLYSAPPFRWKKNPFTACFCNVM 
               
               
                   
               
               
                 LYVGTSVGVYYACKASLGLPANWSPAFCLLFWFISLLSIPISIAKDLSDI 
               
               
                   
               
               
                 EGDRKFGIITFSTKFGAKPIAYICHGLMLLNYVSVMAAAIIWPQFFNSSV 
               
               
                   
               
               
                 ILLSHAFMAIWVLYQAWILEKSNYATETCQKYYIFLWIIFSLEHAFYLFM 
               
               
                   
               
               
                 NphB 
               
               
                 SEQ ID NO: 34 
               
               
                 MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQDTLVEGGSVVVF 
               
               
                   
               
               
                 SMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQK 
               
               
                   
               
               
                 HLPVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAE 
               
               
                   
               
               
                 LFARYGLDKV A MTSMDYKKRQVNLYFSELSAQTLEAESVLALVRELGLHV 
               
               
                   
               
               
                 PNELGLKFCKRSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSDEGDIE 
               
               
                   
               
               
                 KFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQRGLLK 
               
               
                   
               
               
                 AFDSLED 
               
            
           
         
       
     
     Producing a CBGA-analog is an initial step in producing many cannabinoids. Once a CBGA-analog is produced, a single additional enzymatic step is required to turn the CBGA-analog into many other cannabinoids (ie, CBDA-analog, THCA-analog, CBCA-analog, etc.). The acidic forms of the cannabinoids can be used as a pharmaceutical product or the acidic cannabinoids can be turned into their neutral form for use, for example Cannabidiol (CBD) is produced from CBDA through decarboxylation. The resulting cannabinoid products will be used in the pharmaceutical/nutraceutical industry to treat a wide range of health issues. 
     The genes for tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS) and cannabichromenic acid synthase (CBCAS) may be derived from  C. sativa , however, the skilled person would understand that homologous genes may also be suitable. In certain embodiments, THCAS comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13. In certain embodiments, THCAS comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:13. In certain embodiments, CBDAS comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:14. In certain embodiments, CBDAS comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:14. In certain embodiments, CBCAS comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15. In certain embodiments, CBCAS comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:15. Accordingly, in certain embodiments, the one or more cannabinoid precursor or cannabinoid producing genes comprise soluble aromatic prenyltransferase, cannabigerolic acid synthase (CBGAS), tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS) and cannabichromenic acid synthase (CBCAS). 
     
       
         
           
               
            
               
                 THCAS 
               
               
                 SEQ ID NO: 13 
               
               
                 NPRENFLKCFSKHIPNNVANPKLVYTQHDQLYMSILNSTIQNLRFISDTT 
               
               
                   
               
               
                 PKPLVIVTPSNNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYISQVPFV 
               
               
                   
               
               
                 VVDLRNMHSIKIDVHSQTAWVEAGATLGEVYYWINEKNENLSFPGGYCPT 
               
               
                   
               
               
                 VGVGGHFSGGGYGALMRNYGLAADNIIDAHLVNVDGKVLDRKSMGEDLFW 
               
               
                   
               
               
                 AIRGGGGENFGIIAAWKIKLVAVPSKSTIFSVKKNMEIHGLVKLFNKWQN 
               
               
                   
               
               
                 IAYKYDKDLVLMTHFITKNITDNHGKNKTTVHGYFSSIFHGGVDSLVDLM 
               
               
                   
               
               
                 NKSFPELGIKKTDCKEFSWIDTTIFYSGVVNFNTANFKKEILLDRSAGKK 
               
               
                   
               
               
                 TAFSIKLDYVKKPIPETAMVKILEKLYEEDVGAGMYVLYPYGGIMEEISE 
               
               
                   
               
               
                 SAIPFPHRAGIMYELWYTASWEKQEDNEKHINWVRSVYNFTTPYVSQNPR 
               
               
                   
               
               
                 LAYLNYRDLDLGKTNHASPNNYTQARIWGEKYFGKNFNRLVKVKTKVDPN 
               
               
                   
               
               
                 NFFRNEQSIPPLPPHHH 
               
               
                   
               
               
                 CBDAS 
               
               
                 SEQ ID NO: 14 
               
               
                 NPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHNLRFTSDTT 
               
               
                   
               
               
                 PKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMSYISQVPFV 
               
               
                   
               
               
                 IVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNENLSLAAGYCPT 
               
               
                   
               
               
                 VCAGGHFGGGGYGPLMRNYGLAADNIIDAHLVNVHGKVLDRKSMGEDLFW 
               
               
                   
               
               
                 ALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNI 
               
               
                   
               
               
                 AYKYDKDLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMN 
               
               
                   
               
               
                 KSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAGQNG 
               
               
                   
               
               
                 AFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISES 
               
               
                   
               
               
                 AIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSKNPRL 
               
               
                   
               
               
                 AYLNYRDLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNN 
               
               
                   
               
               
                 FFRNEQSIPPLPRHRH 
               
               
                   
               
               
                 CBCAS 
               
               
                 SEQ ID NO: 15 
               
               
                 NPQENFLKCFSEYIPNNPANPKFIYTQHDQLYMSVLNSTIQNLRFTSDTT 
               
               
                   
               
               
                 PKPLVIVTPSNVSHIQASILCSKKVGLQIRTRSGGHDAEGLSYISQVPFA 
               
               
                   
               
               
                 IVDLRNMHTVKVDIHSQTAWVEAGATLGEVYYWINEMNENFSFPGGYCPT 
               
               
                   
               
               
                 VGVGGHFSGGGYGALMRNYGLAADNIIDAHLVNVDGKVLDRKSMGEDLFW 
               
               
                   
               
               
                 AIRGGGGENFGIIAAWKIKLVVVPSKATIFSVKKNMEIHGLVKLFNKWQN 
               
               
                   
               
               
                 IAYKYDKDLMLTTHFRTRNITDNHGKNKTTVHGYFSSIFLGGVDSLVDLM 
               
               
                   
               
               
                 NKSFPELGIKKTDCKELSWIDTTIFYSGVVNYNTANFKKEILLDRSAGKK 
               
               
                   
               
               
                 TAFSIKLDYVKKLIPETAMVKILEKLYEEEVGVGMYVLYPYGGIMDEISE 
               
               
                   
               
               
                 SAIPFPHRAGIMYELWYTATWEKQEDNEKHINWVRSVYNFTTPYVSQNPR 
               
               
                   
               
               
                 LAYLNYRDLDLGKTNPESPNNYTQARIWGEKYFGKNFNRLVKVKTKADPN 
               
               
                   
               
               
                 NFFRNEQSIPPLPPRHH 
               
            
           
         
       
     
     Fatty Acid and Fat Producing Genes: 
     For successful process development and application of THCAS, the properties of the reactants (cannabinoids and enzyme) have to be taken into account, since they determine preferences for process variables and reaction conditions. In  C. sativa  L., the THCAS is active in specialized structures called trichomes (Sirikantaramas et al., 2005). These glandular trichomes harbor a storage cavity (Mahlberg and Kim, 1992), containing the hydrophobic and for plant cells toxic cannabinoids in oil droplets (Morimoto et al., 2007). In this manner, the plant solves solubility and toxicity issues of the cannabinoids (Kim and Mahlberg, 2003). A similar strategy have used for biotechnological cannabinoid production, since multi-phase production systems are one of the applied concepts in reaction engineering to avoid limitations caused by toxicity, volatility, or low solubility of substrates and/or products (Willrodt et al., 2015). It was shown that THCAS is active in a two—liquid phase setup using hexane as organic phase for continuous substrate supply and in situ product removal (1.5 U g—1 total protein)(Lange e t al., 2015b). In another study, whole cells of  P. pastoris  were able to produce THCA with a maximal space—time—yield of 0.059 g L −1  h −1  (Zirpel et al., 2015). 
     The similar environment can be reproduced inside of  Y. lipolitica  which has incorporated lipid bodies. In this case lipid bodies will perform the role of lipid droplets in plants. Cannabinoids are almost not soluble in the aquatic phase. At the same time, they have a great solubility in oils (lipids). By using strains with a large content of lipids and lipid bodies we are providing a safe (not toxic) storage for produced cannabinoids. 
     Thus, the production of fatty acids and fats in yeast may be increased by expressing rate limiting genes in the lipid biosynthesis pathway.  Y. lipolytica  naturally produces Acetyl-CoA. The overexpression of ACC increases the amount of Malonyl-CoA, which is the first step in fatty acid production. In certain embodiments, the one or more genetic modifications that result in increased production of fatty acids or fats comprise Acetyl-CoA carboxylase (ACC1) and Diacylglyceride acyl-transferase (DGA1). The sequences for the native  Y. lipolytica  genes are shown herein, however the skilled person would understand that homologous genes may also be suitable. Examples of DGA1 homologs as shown in Table 8. In certain embodiments, ACC comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:23. In certain embodiments, ACC1 comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:23. In certain embodiments, DGA1 comprises a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:24. In certain embodiments, DGA1 comprises a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:24. 
     ACC1 and DGA1 may be overexpressed in yeast by adding extra copies of the genes driven by native or stronger promoters. Alternatively, native promoters may be substituted by stronger promoters such as TEFin, hp4d, hp8d and others, as would be appreciated by the person skilled in the art. The overexpression of ACC and DGA1 may be determined by quantitative PCR, Microarrays, or next generation sequencing technologies, such as RNA-seq. Alternatively, the product of increased enzyme levels will be increased production of fatty acids. Fatty acid production may be determined using chemical titration, thermometric titration, measurement of metal-fatty acid complexes using spectrophotometry, enzymatic methods or using a fatty acid binding protein. 
     Variants of the fatty acid and fat producing proteins, such as ACC1 retain the ability to produce malonyl-CoA from acetyl-CoA plus bicarbonate. For example, a variant of a fatty acid and fat producing protein, such as ACC1, must retain the ability to produce malonyl-CoA from acetyl-CoA plus bicarbonate with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% efficacy compared to the original sequence. In preferred embodiments, a variant of a fatty acid and fat producing protein, such as ACC1, has improved activity over the sequence from which it is derived in that the improved variant common cannabinoid protein has more than 110%, 120%, 130%, 140%, or and 150% improved activity in producing malonyl-CoA from acetyl-CoA plus bicarbonate, as compared to the sequence from which the improved variant is derived. 
     
       
         
           
               
            
               
                 ACC1 
               
               
                 SEQ ID NO: 23 
               
               
                 MRLQLRTLTRRFFSMASGSSTPDVAPLVDPNIHKGLASHFFGLNSVHTAK 
               
               
                   
               
               
                 PSKVKEFVASHGGHTVINKVLIANNGIAAVKEIRSVRKWAYETFGDERAI 
               
               
                   
               
               
                 SFTVMATPEDLAANADYIRMADQYVEVPGGTNNNNYANVELIVDVAERFG 
               
               
                   
               
               
                 VDAVWAGWGHASENPLLPESLAASPRKIVFIGPPGAAMRSLGDKISSTIV 
               
               
                   
               
               
                 AQHAKVPCIPWSGTGVDEVVVDKSTNLVSVSEEVYTKGCTTGPKQGLEKA 
               
               
                   
               
               
                 KQIGFPVMIKASEGGGGKGIRKVEREEDFEAAYHQVEGEIPGSPIFIMQL 
               
               
                   
               
               
                 AGNARHLEVQLLADQYGNNISLFGRDCSVQRRHQKIIEEAPVTVAGQQTF 
               
               
                   
               
               
                 TAMEKAAVRLGKLVGYVSAGTVEYLYSHEDDKFYFLELNPRLQVEHPTTE 
               
               
                   
               
               
                 MVTGVNLPAAQLQIAMGIPLDRIKDIRLFYGVNPHTTTPIDFDFSGEDAD 
               
               
                   
               
               
                 KTQRRPVPRGHTTACRITSEDPGEGFKPSGGTMHELNFRSSSNVWGYFSV 
               
               
                   
               
               
                 GNQGGIHSFSDSQFGHIFAFGENRSASRKHMVVALKELSIRGDFRTTVEY 
               
               
                   
               
               
                 LIKLLETPDFEDNTITTGWLDELISNKLTAERPDSFLAVVCGAATKAHRA 
               
               
                   
               
               
                 SEDSIATYMASLEKGQVPARDILKTLFPVDFIYEGQRYKFTATRSSEDSY 
               
               
                   
               
               
                 TLFINGSRCDIGVRPLSDGGILCLVGGRSHNVYWKEEVGATRLSVDSKTC 
               
               
                   
               
               
                 LLEVENDPTQLRSPSPGKLVKFLVENGDHVRANQPYAEIEVMKMYMTLTA 
               
               
                   
               
               
                 QEDGIVQLMKQPGSTIEAGDILGILALDDPSKVKHAKPFEGQLPELGPPT 
               
               
                   
               
               
                 LSGNKPHQRYEHCQNVLHNILLGFDNQVVMKSTLQEMVGLLRNPELPYLQ 
               
               
                   
               
               
                 WAHQVSSLHTRMSAKLDATLAGLIDKAKQRGGEFPAKQLLRALEKEASSG 
               
               
                   
               
               
                 EVDALFQQTLAPLFDLAREYQDGLAIHELQVAAGLLQAYYDSEARFCGPN 
               
               
                   
               
               
                 VRDEDVILKLREENRDSLRKVVMAQLSHSRVGAKNNLVLALLDEYKVADQ 
               
               
                   
               
               
                 AGTDSPASNVHVAKYLRPVLRKIVELESRASAKVSLKAREILIQCALPSL 
               
               
                   
               
               
                 KERTDQLEHILRSSVVESRYGEVGLEHRTPRADILKEVVDSKYIVFDVLA 
               
               
                   
               
               
                 QFFAHDDPWIVLAALELYIRRACKAYSILDINYHQDSDLPPVISWRFRLP 
               
               
                   
               
               
                 TMSSALYNSVVSSGSKTPTSPSVSRADSVSDFSYTVERDSAPARTGAIVA 
               
               
                   
               
               
                 VPHLDDLEDALTRVLENLPKRGAGLAISVGASNKSAAASARDAAAAAASS 
               
               
                   
               
               
                 VDTGLSNICNVMIGRVDESDDDDTLIARISQVIEDFKEDFEACSLRRITF 
               
               
                   
               
               
                 SFGNSRGTYPKYFTFRGPAYEEDPTIRHIEPALAFQLELARLSNFDIKPV 
               
               
                   
               
               
                 HTDNRNIHVYEATGKNAASDKRFFTRGIVRPGRLRENIPTSEYLISEADR 
               
               
                   
               
               
                 LMSDILDALEVIGTTNSDLNHIFINFSAVFALKPEEVEAAFGGFLERFGR 
               
               
                   
               
               
                 RLWRLRVTGAEIRMMVSDPETGSAFPLRAMINNVSGYVVQSELYAEAKND 
               
               
                   
               
               
                 KGQWIFKSLGKPGSMHMRSINTPYPTKEWLQPKRYKAHLMGTTYCYDFPE 
               
               
                   
               
               
                 LFRQSIESDWKKYDGKAPDDLMTCNELILDEDSGELQEVNREPGANNVGM 
               
               
                   
               
               
                 VAWKFEAKTPEYPRGRSFIVVANDITFQIGSFGPAEDQFFFKVTELARKL 
               
               
                   
               
               
                 GIPRIYLSANSGARIGIADELVGKYKVAWNDETDPSKGFKYLYFTPESLA 
               
               
                   
               
               
                 TLKPDTVVTTEIEEEGPNGVEKRHVIDYIVGEKDGLGVECLRGSGLIAGA 
               
               
                   
               
               
                 TSRAYKDIFTLTLVTCRSVGIGAYLVRLGQRAIQIEGQPIILTGAPAINK 
               
               
                   
               
               
                 LLGREVYSSNLQLGGTQIMYNNGVSHLTARDDLNGVHKIMQWLSYIPASR 
               
               
                   
               
               
                 GLPVPVLPHKTDVWDRDVTFQPVRGEQYDVRWLISGRTLEDGAFESGLFD 
               
               
                   
               
               
                 KDSFQETLSGWAKGVVVGRARLGGIPFGVIGVETATVDNTTPADPANPDS 
               
               
                   
               
               
                 IEMSTSEAGQVWYPNSAFKTSQAINDFNHGEALPLMILANWRGFSGGQRD 
               
               
                   
               
               
                 MYNEVLKYGSFIVDALVDYKQPIMVYIPPTGELRGGSWVVVDPTINSDMM 
               
               
                   
               
               
                 EMYADVESRGGVLEPEGMVGIKYRRDKLLDTMARLDPEYSSLKKQLEESP 
               
               
                   
               
               
                 DSEELKVKLSVREKSLMPIYQQISVQFADLHDRAGRMEAKGVIREALVWK 
               
               
                   
               
               
                 DARRFFFWRIRRRLVEEYLITKINSILPSCTRLECLARIKSWKPATLDQG 
               
               
                   
               
               
                 SDRGVAEWFDENSDAVSARLSELKKDASAQSFASQLRKDRQGTLQGMKQA 
               
               
                   
               
               
                 LASLSEAERAELLKGL 
               
               
                   
               
               
                 DGA1 
               
               
                 SEQ ID NO: 24 
               
               
                 MTIDSQYYKSRDKNDTAPKIAGIRYAPLSTPLLNRCETFSLVWHIFSIPT 
               
               
                   
               
               
                 FLTIFMLCCAIPLLWPFVIAYVVYAVKDDSPSNGGVVKRYSPISRNFFIW 
               
               
                   
               
               
                 KLFGRYFPITLHKTVDLEPTHTYYPLDVQEYHLIAERYWPQNKYLRAIIS 
               
               
                   
               
               
                 TIEYFLPAFMKRSLSINEQEQPAERDPLLSPVSPSSPGSQPDKWINHDSR 
               
               
                   
               
               
                 YSRGESSGSNGHASGSELNGNGNNGTTNRRPLSSASAGSTASDSTLLNGS 
               
               
                   
               
               
                 LNSYANQIIGENDPQLSPTKLKPTGRKYIFGYHPHGIIGMGAFGGIATEG 
               
               
                   
               
               
                 AGWSKLFPGIPVSLMTLTNNFRVPLYREYLMSLGVASVSKKSCKALLKRN 
               
               
                   
               
               
                 QSICIVVGGAQESLLARPGVMDLVLLKRKGFVRLGMEVGNVALVPIMAFG 
               
               
                   
               
               
                 ENDLYDQVSNDKSSKLYRFQQFVKNFLGFTLPLMHARGVFNYDVGLVPYR 
               
               
                   
               
               
                 RPVNIVVGSPIDLPYLPHPTDEEVSEYHDRYIAELQRIYNEHKDEYFIDW 
               
               
                   
               
               
                 TEEGKGAPEFRMIE 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5   
               
             
            
               
                   
               
               
                 DGA1 HOMOLOGS 
               
            
           
           
               
               
               
            
               
                 Description 
                 Ident 
                 Accession 
               
               
                   
               
               
                 YALIOE32769p [ Yarrowia lipolytica   
                 l00%  
                 XP_504700.1 
               
               
                 CLIB122] 
                   
                   
               
               
                 Diacylglycerol acyltransferase 
                 44% 
                 CDO57007.1 
               
               
                 [ Galactomyces candidus]   
                   
                   
               
               
                 hypothetical protein 
                 60% 
                 ODQ70106.1 
               
               
                 [ Lipomyces starkeyi  NRRL Y-11557] 
                   
                   
               
               
                 DAGAT-domain-containing protein 
                 60% 
                 ODQ67305.1 
               
               
                 [ Nadsonia fulvescens  var.  elongata   
                   
                   
               
               
                 DSM 6958] 
                   
                   
               
               
                 hypothetical protein 
                 65% 
                 ODV90514.1 
               
               
                 [Tortispora caseinolytica NRRL Y-17796] 
                   
                   
               
               
                 diacylglycerol acyltransferase 
                 60% 
                 XP_019022950.1 
               
               
                 [Saitoella complicata NRRL Y-17804] 
                   
                   
               
               
                 uncharacterized protein 
                 51% 
                 XP_022458761.1 
               
               
                 KUCA_T00002736001 
                   
                   
               
               
                 [ Kuraishia capsulata  CBS 1993] 
                   
                   
               
               
                 diacylglycerol O-acyltransferas-like 
                 55% 
                 XP_024728739.1 
               
               
                 protein 2B [ Meliniomyces bicolor  E] 
                   
                   
               
               
                 Diacylglycerol O-acyltransferase 
                 57% 
                 OEJ83128.1 
               
               
                 1 [ Hanseniaspora osmophila ] 
                   
                   
               
               
                 DAGAT-domain-containing protein 
                 49% 
                 XP_020048004.1 
               
               
                 [ Ascoidea rubescens  DSM 1968] 
               
               
                   
               
            
           
         
       
     
     NADPH Balance 
     NADPH is extremely critical for a production of fatty acids. It is required 16 molecules of NADPH to produce one stearic acid. By using NADPH, cells create an excess of NADH. NADPH is also important for production of fatty acids and cannabinoids. Four molecules of NADPH is required to produce 1 molecule of GPP. 
     Thus, to produce one Hexanoyl-CoA, 4 molecules of NADPH is required. Production of OLA from Hexanoyl-CoA does not require any additional NADPH. Therefore, we will need 8 molecules of NADPH to directly produce 1 molecule of a cannabinoid precursor. Preferred methods of overexpressing NADP+ include, but are not limited to use of glucose-6-phosphate dehydrogenase, which is encoded by, for example ZWF1 (see, for example, Yuzbasheva, E. Y., et al., New Biotechnology 39 (Pt A), 18-21, or use of GAPC and/or MCE2 (see, for example, Qiao, K., et al., (2017) Nature Biotechnology 35(2), 173-177. 
     Recombinant Microorganisms 
     As described above, the microorganism employed in a method of the invention or contained in the composition of the invention may be a microorganism which has been genetically modified by the introduction of a nucleic acid molecule encoding a corresponding enzyme. Thus, in a preferred embodiment, the microorganism is a recombinant microorganism which has been genetically modified to have an increased activity of at least one enzyme described above for the conversions of the method according to the present invention. This can be achieved e.g. by transforming the microorganism with a nucleic acid encoding a corresponding enzyme. Preferably, the nucleic acid molecule introduced into the microorganism is a nucleic acid molecule which is heterologous with respect to the microorganism, i.e. it does not naturally occur in said microorganism. 
     The term “microorganism” in the context of the present invention refers to bacteria, as well as to fungi, such as yeasts, and also to algae and archaea. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the process according to the invention are bacteria of the genus  Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas  or  Escherichia . In a particularly preferred embodiment, the bacterium belongs to the genus  Escherichia  and even more preferred to the species  Escherichia coli . In another preferred embodiment the bacterium belongs to the species  Pseudomonas putida  or to the species  Zymomonas mobilis  or to the species  Corynebacterium glutamicum  or to the species  Bacillus subtilis . It is also possible to employ an extremophilic bacterium such as  Thermus thermophilus , or anaerobic bacteria from the family Clostridiae. 
     It is also conceivable to use in the method according to the invention a combination of microorganisms wherein different microorganisms express different enzymes as described above. 
     In the context of the present invention, an “increased activity” means that the expression and/or the activity of an enzyme in the genetically modified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-modified microorganism. In even more preferred embodiments, the increase in expression and/or activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-modified microorganism. 
     The term “increased” expression/activity also covers the situation in which the corresponding non-modified microorganism does not express a corresponding enzyme so that the corresponding expression/activity in the non-modified microorganism is zero. Preferably, the concentration of the overexpressed enzyme is at least 5%, 10%, 20%, 30%, or 40% of the total host cell protein. Additionally, as would be appreciated by the person skilled in the art, increased expression of a gene may provide increased the activity of the gene product. In certain embodiments, overexpression of a gene can increase the activity of the gene product by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 95%, or about 200%. 
     Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. In one embodiment, the measurement of the level of expression is done by measuring the amount of the corresponding protein. Corresponding methods are well known to the person skilled in the art and include Western Blot, ELISA etc. In another embodiment the measurement of the level of expression is done by measuring the amount of the corresponding RNA. Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot. 
     In addition, it is possible to insert different mutations into the polynucleotides by methods usual in molecular biology (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA), leading to the synthesis of polypeptides possibly having modified biological properties. The introduction of point mutations is conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity or the regulation of the polypeptide. Similarly, CRISPR-Cas9 genome editing technology can be used to modify the disclosed sequences to produce enzyme variants. 
     The transformation of the host cell with a polynucleotide or vector as described above can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc. 
     The disclosed genes may be under the control of any suitable promoter. Many native promoters are available, for example, for  Y. lipolytica , native promoters are available from the genes for translational elongation factor EF-1 alpha, acyl-CoA: diacylglycerol acyltransferase, acetyl-CoA-carboxylase 1, ATP citrate lyase 2, fatty acid synthase subunit beta, fatty acid synthase subunit alpha, isocitrate lyase 1, POX4 fatty-acyl coenzyme A oxidase, ZWF1 glucose-6-phosphate dehydrogenase, gytosolic NADP-specific isocitrate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, the TEF intron promoter or native promoter (Wong et al. 2017), a synthesized short terminator (Curran et al. 2015), or the alcohol dehydrogenase II promoter of  Y. lipolytica . Any suitable terminator may be used. Short synthetic terminators are particularly suitable and are readily available, see for example, MacPherson et al. 2016. 
     Methods of detecting increase production of Compound I may be determined using high-performance liquid chromatography (HPLC) or Liquid chromatography-mass spectrometry (LC/MS). For example, as yeast do not produce OA endogenously, the presence of OA indicates that the PKS Enzyme is functioning. 
     Genetically Modified Yeast Strains 
     In another preferred embodiment the microorganism is a fungus, more preferably a fungus of the genus  Saccharomyces, Schizosaccharomyces, Aspergillus, Trichoderma, Kluyveromyces  or  Pichia  and even more preferably of the species  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger, Trichoderma reesei, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia pastoris, Pichia torula  or  Pichia utilis.    
     In further preferred embodiments, genetically modified yeasts comprising one or more genetic modifications that result in the production of at least one cannabinoid or cannabinoid precursor and methods for their creation. The disclosed yeast may produce various cannabinoids from a simple sugar source, for example, where the main carbon source available to the yeast is a sugar (glucose, galactose, fructose, sucrose, honey, molasses, raw sugar, etc.). Genetic engineering of the yeast involves inserting various genes that produce the appropriate enzymes and/or altering the natural metabolic pathway in the yeast to achieve the production of a desired compound. Through genetic engineering of yeast, these metabolic pathways can be introduced into these yeast and the same metabolic products that are produced in the plant  C. sativa  can be produced by the yeast. The benefit of this method is that once the yeast is engineered, the production of the cannabinoid is low cost and reliable, only a specific cannabinoid is produced or a subset is produced, depending on the organism and the genetic manipulation. The purification of the cannabinoid is straightforward since there is only a single cannabinoid or a selected few cannabinoids present in the yeast. The process is a sustainable process which is more environmentally friendly than synthetic production. 
     In the past, there have been multiple attempts to produce cannabinoids in yeasts. At present, no one has been able reach a reasonable price for production due to extremely low yield. We have identified how the yield can be increased. 
     In preferred embodiments, the biosynthetic pathways shown in  FIGS. 1-3  are produced in yeast having at least 5% dry weight of fatty acids or fats, such as oily yeasts, for example,  Y. Lipolytica.    
     Additionally and as described below, we also propose (1) making additional genetic modifications that will increase oil production level in the engineered yeast; (2) add additional genes from the cannabinoid production pathway in combination with genes from alternative pathways that produce cannabinoid intermediates, such as for example NphB; (3) increase production of GPP by, for example, genetically mutating ERG20 and/or by using equivalent genes from alternative pathways; (4) increase production of compounds from fatty acid pathway for use in the cannabinoid production pathway, for example, increase the production of malonyl-CoA by overexpressing ACC1. 
     Cannabinoids have a limited solubility in water solutions. Yet, they have a high solubility in hydrophobic liquids like lipids, oils or fats. If hydrophobic media is limited or completely removed than a CBGA-analog will not be solubilized and will have limited availability to following cannabinoid synthetases. As an example, in the paper (Zirpel et al. 2015) it was shown that purified THCA synthase is almost unable to convert CBGA into THCA. In the same paper the authors demonstrated that unpurified yeast lysate converts CBGA much more efficiently. The authors also demonstrated that CBGA was dissolved in the lipid fraction. In another paper (Lange et al. 2016) the authors made the next step in improving a cell free process. They used a two-phase reaction with an organic, hydrophobic phase and aquatic phase. The authors demonstrated a high yield of THCA from CBGA. They found that CBGA was dissolved in organic phase. They also demonstrated that THCA was moved back to the organic phase. We can therefore conclude that a hydrophobic phase is required for successful synthesis and that cannabinoids are mostly present in the organic phase. 
     Production of cannabinoid in traditional yeast, like  S. cerevisiae, K. phaffii, K. marxianus , results in the cannabinoids, like the main mass of lipids to be deposited in the lipid membrane. These types of yeast almost have no oily bodies. In such a case, any cannabinoids that are produced will be dissolved in this membrane. Too many cannabinoids will destabilize a membrane which will cause cell death. It was reported that in the best conditions, with high sugar content and without nitrogen supply, these yeasts can have a maximum of 2-3% dry weight of oils (ie fats and fatty acids). 
     However, there are several non-traditional yeasts, like  Y. lipolytica . The natural form of  Y. lipolytica  can have up to 17% dry weight of oils. The main mass of oil is located in oily bodies. Cannabinoids dissolved in such bodies will not cause membrane instability. As a result,  Y. lipolytica  can have a much higher cannabinoid production level. Several works have demonstrated modifications for  Y. lipolytica  which can bring the lipid content above 80% of dry mass (Qiao et al. 2015). 
     Therefore, we propose that cannabinoids can be produced to some percentage of the oil content in yeast. This gives a correlation—more oil means more cannabinoid production. 
     A review paper (Angela et al. 2017) analysed different types of yeast as a potential producers for cannabinoids. TABLE 1 is adapted from the summary table in Angela et al. 2017, in which the authors compared 4 yeasts types by different parameters. Yet, they completely ignored oil content, theoretical maximal limit of production and minimal cost of goods for production. The far right two columns show maximum oil amount as a percentage of dry weight, and the production cost if there is only 1% of cannabinoid in the oil. The bottom row shows an embodiment of a modified  Yarrowia lipolytica  of the present disclosure. Finally, the authors in Angela et al. 2017 considered that acetyl-CoA pool engineering had optimization potential; +. However, we have found that YL has large concentration of acetyl-CoA without modifications. 
     Therefore, in preferred embodiments, we are proposing to use oily yeasts as a backbone for cannabinoid and/or cannabinoid precursor production. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                     COMPARISON OF DIFFERENT MICROBIAL EXPRESSION HOSTS REGARDING THEIR CAPACITY OF HETEROOGOUS 
               
               
                 CANNABINOID BIOSYNTHESIS 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Maximal 
                 Production 
               
               
                   
                   
                   
                 plant 
                   
                   
                   
                   
                 oil 
                 cost with  
               
               
                   
                 Genetic 
                 Strains, 
                 protein 
                 Post- 
                   
                 Hexanoic 
                 acetyl-CoA 
                 amount  
                 only 1% of 
               
               
                   
                 tools 
                 promoters, 
                 expression 
                 translational 
                 GPP 
                 acid 
                 pool  
                 % of dry 
                 cannabinoids  
               
               
                   
                 available 
                 vectors 
                 capacity 
                 modifications 
                 engineering 
                 engineering 
                 engineering 
                 weight 
                 from oils 
               
               
                   
               
               
                 
                   E. 
                   coli 
                 
                 +++ 
                 +++ 
                 + 
                 − 
                 ++ 
                 + 
                 + 
                  2% 
                 $12.50 
               
               
                 
                   S. 
                   cerevisiae 
                 
                 +++ 
                 +++ 
                 ++ 
                 ++ 
                 +++ 
                 ++ 
                 +++ 
                  2% 
                 $12.50 
               
               
                 
                   P. 
                   Pastoris 
                 
                 + 
                 ++ 
                 +++ 
                 ++ 
                 + 
                 ++ 
                   
                  3% 
                  $8.33 
               
               
                 
                   K. 
                   marxianus 
                 
                 ++ 
                 + 
                 ++ 
                 ++ 
                   
                   
                   
                  3% 
                  $8.33 
               
               
                 
                   Y. 
                   Lipolica 
                 
                 + 
                 + 
                 ++ 
                 ++ 
                 + 
                 ++ 
                 +, YL has large 
                 17% 
                  $1.47 
               
               
                   
                   
                   
                   
                   
                   
                   
                 concentration of 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 ac-CoA without 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 modifications 
                   
                   
               
               
                 
                   Y. 
                   L.  
                 
                 + 
                 + 
                 ++ 
                 ++ 
                 + 
                 ++ 
                 +, YL has large 
                 80% 
                  $0.31 
               
               
                 modified 
                   
                   
                   
                   
                   
                   
                 concentration of  
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 ac-CoA without 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 modifications 
               
               
                   
               
               
                 *maximal oil % means how much oils can be produced in the best cultivation conditions. % calculated from dried mass. 
               
               
                 Table 1 adapted from Carvalho, Ângela, et al. “Designing microorganisms for heterologous biosynthesis of cannbinoids.”  FEMS yeast research  17.4 (2017). 
               
               
                 1. +++, many publications available, well estabilshed; ++, publications available, optimization potential; +, first publications available, not yet established/not working; −, not possible; ‘empty’, not yet described. 
               
            
           
         
       
     
     As described above, in certain embodiments, the yeast comprises at least 5% dry weight of fatty acids or fats. Accordingly, the yeast may be oleaginous. Any oleaginous yeast may be suitable, however, particularly suitable yeast may be selected from the genera  Rhodosporidium, Rhodotorula, Yarrowia, Cryptococcus, Candida, Lipomyces  and  Trichosporon . In certain embodiments, the yeast is a  Yarrowia lipolytica , a  Lipomyces starkey , a  Rhodosporidium toruloides , a  Rhodotorula glutinis , a  Trichosporon fermentans  or a  Cryptococcus curvatus . The yeast may be naturally oleaginous. Accordingly, in certain embodiments, the yeast comprises at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% dry weight of fatty acids or fats. The yeast may also be genetically modified to accumulate or produce more fatty acids or fats. Accordingly, in certain embodiments, the yeast is genetically modified to produce at least 5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% dry weight of fatty acids or fats. 
     Cell-Free Production 
     The method according to the present invention can also be carried out in a cell-free system (e.g., in vitro). An in vitro reaction is understood to be a reaction in which no cells are employed, i.e. an acellular reaction. Thus, in vitro preferably means in a cell-free system. The term “in vitro” in one embodiment means in the presence of isolated enzymes (or enzyme systems optionally comprising possibly required cofactors). In one embodiment, the enzymes employed in the method are used in purified form. 
     For carrying out the method in vitro the substrates for the reaction and the enzymes are incubated under conditions (buffer, temperature, cosubstrates, cofactors etc.) allowing the enzymes to be active and the enzymatic conversion to occur. The reaction is allowed to proceed for a time sufficient to produce the respective product. The production of the respective products can be measured by methods known in the art, such as gas chromatography possibly linked to mass spectrometry detection. 
     The enzymes described herein may be in any suitable form allowing the enzymatic reaction to take place. They may be purified or partially purified or in the form of crude cellular extracts or partially purified extracts. It is also possible that the enzymes are immobilized on a suitable carrier. 
     Carbohydrate Sources 
     In another aspect of the present disclosure, there is provided method of producing at least one cannabinoid or cannabinoid precursor comprising contacting the compositions as described herein with a carbohydrate source under conditions and for a time sufficient to produce the at least one cannabinoid or cannabinoid precursor. 
     Specifically, examples of the culture conditions for producing at least one cannabinoid or cannabinoid precursor include a batch process and a fed batch or repeated fed batch process in a continuous manner, but are not limited thereto. Carbon sources that may be used for producing at least one cannabinoid or cannabinoid precursor may include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, xylose and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, chicken fat and beef tallow; fatty acids such as palmitic acid, stearic acid, oleic acid and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as gluconic acid, acetic acid, malic acid and pyruvic acid, but these are not limited thereto. These substances may be used alone or in a mixture. Nitrogen sources that may be used in the present disclosure may include peptone, yeast extract, meat extract, malt extract, corn steep liquor, defatted soybean cake, and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, but these are not limited thereto. These nitrogen sources may also be used alone or in a mixture. Phosphorus sources that may be used in the present disclosure may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or corresponding sodium-containing salts, but these are not limited thereto. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate, which is may be required for the growth. Lastly, in addition to the above-described substances, essential growth factors such as amino acids and vitamins may be used. Such a variety of culture methods is disclosed, for example, in the literature (“Biochemical Engineering” by James M. Lee, Prentice-Hall International Editions, pp 138-176). 
     Basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acidic compounds such as phosphoric acid or sulfuric acid may be added to the culture medium in a suitable manner to adjust the pH of the culture medium. In addition, an anti-foaming agent such as fatty acid polyglycol ester may be used to suppress the formation of bubbles. In certain embodiments, the culture medium is maintained in an aerobic state, accordingly, oxygen or oxygen-containing gas (e.g., air) may be injected into the culture medium. The temperature of the culture medium may be usually 20° C. to 35° C., preferably 25° C. to 32° C., but may be changed depending on conditions. The culture may be continued until the maximum amount of a desired cannabinoid precursor or cannabinoid is produced, and it may generally be achieved within 5 hours to 160 hours. The cannabinoid precursor or cannabinoid may be released into the culture medium or contained in the recombinant microorganisms. 
     The method of the present disclosure for producing at least one cannabinoid or cannabinoid precursor may include a step of recovering the at least one cannabinoid or cannabinoid precursor from the microorganism or the medium. Methods known in the art, such as centrifugation, filtration, anion-exchange chromatography, crystallization, HPLC, etc., may be used for the method for recovering at least one cannabinoid or cannabinoid precursor from the microorganism or the culture, but the method is not limited thereto. The step of recovering may include a purification process. Specifically, following an overnight culture, 1 L cultures are pelleted by centrifugation, resuspended, washed in PBS and pelleted. The cells are lysed by either chemical or mechanical methods or a combination of methods. Mechanical methods can include a French Press or glass bead milling or other standard methods. Chemical methods can include enzymatic cell lysis, solvent cell lysis, or detergent based cell lysis. A liquid-liquid extraction of the cannabinoids is performed using the appropriate chemical solvent in which the cannabinoids are highly soluble and the solvent is not miscible in water. Examples include hexane, ethyl acetate, and cyclohexane, preferably solvents with straight or branched alkane chains (C5-C8) or mixtures thereof. 
     In certain embodiments, the at least one cannabinoid or cannabinoid precursor comprises a CBGA-analog, a THCA-analog, a CBDA-analog or a CBCA-analog. The production of one or more cannabinoid precursors or cannabinoids may be determined using a variety of methods as described herein. An example protocol for analysing a CBDA-analog is as follows:
         1. Remove solvent from samples under vacuum.   2. Re-suspend dry samples in either 100 uL of dry hexane or dry ethyl acetate   3. Add 20 uL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)   4. Briefly mix   5. Heat solution to 60° C. for 10-15 minutes   6. GC-MS Method
           a. Instrument Agilent 6890-5975 GC-MS (Model Number: Agilent 19091S-433)   b. Column HP-5MS 5% Phenyl Methyl Siloxane   c. OVEN:
               i. Initial temp: 100° C. (On) Maximum temp: 300° C.   ii. Initial time: 3.00 min Equilibration time: 0.50 min   iii. Ramps:
                   # Rate Final temp Final time   1-30.00 280 1.00   2-70.00 300 5.00   3-0.0 (Off)   
                   iv. Post temp: 0° C.   v. Post time: 0.00 min   vi. Run time: 15.29 min   
               
               

     In a third aspect of the present disclosure, there is provided a cannabinoid precursor, cannabinoid or a combination thereof produced using the methods described herein. In certain embodiments, the at least one cannabinoid or cannabinoid precursor comprises a CBGA-analog, a THCA-analog, a CBDA-analog or a CBCA-analog. 
     EXAMPLES 
     Example 1: Vector Construction and Transformation 
       Y. lipolytica  episomal plasmids comprise a centromere, origin and bacteria replicative backbone. Fragments for these regions were synthesized by Twist Bioscience and cloned to make an episomal parent vector pBM-pa. Plasmids were constructed by Gibson Assembly, Golden gate assembly, ligation or sequence- and ligation-independent cloning (SLIC). Genomic DNA isolation from bacteria ( E. coli ) and yeast ( Yarrowia lipolytica ) were performed using Wizard Genomic DNA purification kit according to manufacturer&#39;s protocol (Promega, USA). Synthetic genes were codon-optimized using GeneGenie or Genscript (USA) and assembled from gene fragments purchased from TwistBioscience. All the engineered  Y. lipolytica  strains were constructed by transforming the corresponding plasmids. All gene expression cassettes were constructed using a TEF intron promoter and synthesized short terminator. Up to six expression cassettes were cloned into episomal expression vectors through SLIC. 
       E. coli  minipreps were performed using the Zyppy Plasmid Miniprep Kit (Zymo Research Corporation). Transformation of  E. coli  strains was performed using Mix &amp; Go Competent Cells (Zymo research, USA). Transformation of  Y. lipolytica  with episomal expression plasmids was performed using the Zymogen Frozen EZ Yeast Transformation Kit II (Zymo Research Corporation), and spread on selective plates. Transformation of  Y. lipolytica  with linearized cassettes was performed using LiOAc method. Briefly,  Y. lipolytica  strains were inoculated from glycerol stocks directly into 10 ml YPD media, grown overnight and harvested at an OD600 between 9 and 15 by centrifugation at 1,000 g for 3 min. Cells were washed twice in sterile water. Cells were dispensed into separate microcentrifuge tubes for each transformation, spun down and resuspended in 1.0 ml 100 mM LiOAc. Cells were incubated with shaking at 30° C. for 60 min, spun down, resuspended in 90 ul 100 mM LiOAc and placed on ice. Linearized DNA (1-5 mg) was added to each transformation mixture in a total volume of 10 ul, followed by 25 ul of 50 mg/ml boiled salmon sperm DNA. Cells were incubated at 30° C. for 15 min with shaking, before adding 720 μl PEG buffer (50% PEG8000, 100 mM LiOAc, pH=6.0) and 45 μl 2 M Dithiothreitol. Cells were incubated at 30° C. with shaking for 60 min, heat-shocked for 10 min in a 39° C. water bath, spun down and resuspended in 1 ml sterile water. Cells (200 μl) were plated on appropriate selection plates. 
     Example 2: Yeast Culture Conditions 
       E. coli  strain DH10B was used for cloning and plasmid propagation. DH10B was grown at 37° C. with constant shaking in Luria-Bertani Broth supplemented with 100 mg/L of ampicillin for plasmid propagation.  Y. lipolytica  strains W29 was used as the base strain for all experiments.  Y. lipolytica  was cultivated at 30° C. with constant agitation. Cultures (2 ml) of  Y. lipolytica  used in large-scale screens were grown in a shaking incubator at speed 250 rpm for 1 to 3 days, and larger culture volumes were shaken in 50 ml flasks or fermented in a bioreactor. 
     For colony screening and cell propagation,  Y. lipolytica  grew on YPD liquid media contained 10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose, or YPD agar plate with addition of 20 g/L of agar. Medium was often supplemented with 150 to 300 mg/L Hygromycin B or 250 to 500 mg/L nourseothricin for selection, as appropriate. For cannabinoid producing strains, modified YPD media with 0.1 to 1 g/L yeast extract was used for promoting lipid accumulation and often supplemented with 0.2 g/L and 5 g/L ammonium sulphate as alternative nitrogen source. 
     Example 3: Cannabinoid Isolation 
       Y. lipolytica  culture from the shaking flask experiment or bioreactor are pelleted and homogenized in acetonitrile followed by incubation on ice for 15 min. Supernatants are filtered (0.45 μm, Nylon) after centrifugation (13,100 g, 4° C., 20 min) and analyzed by HPLC-DAD. Quantification of products are based on integrated peak areas of the UV-chromatograms at 225 nm. Standard curves are generated for CBGA and THCA. The identity of all compounds can be confirmed by comparing mass and tandem mass spectra of each sample with coeluting standards analysed by Bruker Compact™ ESI-Q-TOF using positive ionization mode. 
     Example 4: Gene Combinations 
     Embodiment 1 
       Y. lipolytica  ERG20 comprising F88W and N119W substitutions; tHMGR; OLS: OAC; CBGAS; THCAS; HexA and HexB. 
     Embodiment 2 
       Y. lipolytica  ERG20 comprising F88W and N119W substitutions; HMGR; OLS: OAC; NphB Q161A; THCAS; FAS1 I306A, M1251W and FAS2 G1250S. 
     Embodiment 3 
       S. cerevisiae  ERG20 comprising a K197E substitution; OLS: OAC; NphB Q161A; CBDAS; StcJ and StcK. 
     Embodiment 4 
       Y. lipolytica  ERG20 comprising a K189E substitution; HMGR; OLS: OAC; CBGAS; CBCAS; HexA and HexB. 
     Embodiment 5 
       Y. lipolytica  ERG20 comprising a K189E substitution; tHMGR; OLS: OAC; CBGAS; CBDAS; StcJ and StcK. 
     The genetically modified yeast of the present disclosure enable the production of cannabinoid precursors and cannabinoids. The accumulation of fatty acids or fats in the yeast of at least 5% dry weight provides a storage location for the cannabinoid precursors and cannabinoids removed from the plasma membrane. This reduces the accumulation of cannabinoid precursors and cannabinoids in the plasma membrane, reducing membrane destabilisation and reducing the chances of cell death. Oily yeast such as  Y. lipolytica  can be engineered to have a fatty acid or fat (eg lipid) content above 80% dry weight, compared to 2-3% for yeast such as  S. cerevisiae . Accordingly, cannabinoid precursor and cannabinoid production can be much higher in oily yeast, particularly oily yeast engineered to have a high fatty acid or fat (eg lipid) content. 
     The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge. 
     It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the disclosure as set forth and defined by the following claims. 
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