# Patent

## Title
Production of carotenoids in oleaginous yeast and fungi

## Abstract

The present invention provides systems for producing engineered oleaginous yeast or fungi that express carotenoids.

## Background
RELATED APPLICATIONS
This application is a divisional application which claims the benefit of U.S. Ser. No. 11/385,580, filed Mar. 20, 2006, which claims the benefit of U.S. Provisional Application No. 60/663,621, filed Mar. 18, 2005, the contents of which are hereby incorporated by reference in their entirety for all purposes.


BACKGROUND OF THE INVENTION
Carotenoids are organic pigments ranging in color from yellow to red that are naturally produced by certain organisms, including photosynthetic organisms (e.g., plants, algae, cyanobacteria), and some fungi. Carotenoids are responsible for the orange color of carrots, as well as the pink in flamingos and salmon, and the red in lobsters and shrimp. Animals, however, cannot produce carotenoids and must receive them through their diet.
Carotenoid pigments (e.g., β-carotene and astaxanthin) are used industrially as ingredients for food and feed stocks, both serving a nutritional function and enhancing consumer acceptability. For example, astaxanthin is widely used in salmon aquaculture to provide the orange coloration characteristic of their wild counterparts. Some carotenoids are also precursors of vitamin A. Also, carotenoids have antioxidant properties, and may have various health benefits (see, for example, Jyonouchi et al., Nutr. Cancer 16:93, 1991; Giovannucci et al., J. Natl. Cancer Inst. 87:1767, 1995; Miki, Pure Appl. Chem. 63:141, 1991; Chew et al., Anticancer Res. 19:1849, 1999; Wang et al., Antimicrob. Agents Chemother. 44:2452, 2000). Some carotenoids such as β-carotene, lycopene, and lutein are currently sold as nutritional supplements.
In general, the biological systems that produce carotenoids are industrially intractable and/or produce the compounds at such low levels that commercial scale isolation is not practicable. Thus, most carotenoids used in industry are produced by chemical synthesis. There is a need for improved biological systems that produce carotenoids. Some efforts have previously been made to genetically engineer certain bacteria or fungi to produce higher levels of carotenoids (see, for example, Misawa et al., J. Biotechnol. 59:169, 1998; Visser et al., FEMS Yeast Research 4:221, 2003). However, improved systems, allowing higher levels of production and greater ease of isolation, are needed.
SUMMARY OF THE INVENTION
The present invention provides improved systems for the biological production of carotenoids. In one aspect, the invention encompasses the discovery that it is desirable to produce carotenoids in oleaginous organisms. Without wishing to be bound by any particular theory, the present inventors propose that biological systems may be able to accumulate higher levels of carotenoids if the compounds are sequestered in lipid bodies. Regardless of whether absolute levels are higher, however, carotenoids that are accumulated within lipid bodies in oleaginous organisms are readily isolatable through isolation of the lipid bodies.
The present invention therefore provides oleaginous fungi (including, for example, yeast or other unicellular fungi) that produce one or more carotenoids. The present invention also provides methods of constructing such yeast and fungi, methods of using such yeast and fungi to produce carotenoids, and methods of preparing carotenoid-containing compositions, such as food or feed additives, or nutritional supplements, using carotenoids produced in such oleaginous yeast or fungi. In particular, the present invention provides systems and methods for generating yeast and fungi containing one or more oleaginic and/or carotenogenic modifications that increase the oleaginicity and/or alter their carotenoid-producing capabilities as compared with otherwise identical organisms that lack the modification(s).
The present invention further encompasses the general recognition that lipid-accumulating systems are useful for the production and/or isolation of lipophilic agents (such as, but not limited to isoprenoids, or isoprenoid-derived compounds). Thus, according to the present invention, it is desirable to engineer organisms to produce such lipophilic agents and/or to accumulate lipid.
Various other aspects of the present invention will be apparent to those of ordinary skill in the art from the present description, including the appended claims.



BRIEF DESCRIPTION OF THE DRAWING
FIG. 1A-1D depicts certain common carotenoids.
FIG. 2 depicts how sufficient levels of acetyl-CoA and NADPH may be accumulated in the cytosol of oleaginous organisms to allow for production of significant levels of cytosolic lipids. Enzymes: 1, pyruvate decarboxylase; 2, malate dehydrogenase; 3, malic enzyme; 4, pyruvate dehydrogenase; 5, citrate synthase; 6, ATP-citrate lyase; 7, citrate/malate translocase.
FIGS. 3A and 3B depict the mevalonate isoprenoid biosynthesis pathway, which typically operates in eukaryotes, including fungi.
FIG. 4 depicts the mevalonate-independent isoprenoid biosynthesis pathway, also known as the DXP pathway, which typically operates in bacteria and in the plastids of plants.
FIG. 5 depicts intermediates in the isoprenoid biosynthesis pathway and how they feed into biosynthetic pathways of other biomolecules, including carotenoids as well as non-carotenoid compounds such as sterols, steroids, and vitamins, such as vitamin E or vitamin K.
FIGS. 6A-6D illustrate various carotenoid biosynthetic pathways. FIG. 6A highlights branches leading to various cyclic and acyclic xanthophylls; FIG. 6B shows certain X. dendrorhous pathways that generate dicyclic and monocyclic carotenoids, including astaxanthin; FIG. 6C shows interconnecting pathways for converting β-carotene into any of a variety of other carotenoids, including astaxanthin; FIG. 6D depicts possible routes of synthesis of cyclic carotenoids and common plant and algal xanthophylls from neurosporene.
FIG. 7 and subparts FIGS. 7A-7I show an alignment of certain representative fungal HMG-CoA reductase polypeptides. As can be seen, these polypeptides show very high identity across the catalytic region, and also have complex membrane spanning domains. In some embodiments of the invention, these membrane-spanning domains are disrupted or are removed, so that, for example, a hyperactive version of the polypeptide may be produced.
FIGS. 8A-8D depict schematic representations of plasmids generated and described in detail in the exemplification.



DEFINITIONS
Carotenogenic modification: The term “carotenogenic modification”, as used herein, refers to a modification of a host organism that adjusts production of one or more carotenoids, as described herein. For example, a carotenogenic modification may increase the production level of one or more carotenoids, and/or may alter relative production levels of different carotenoids. In principle, an inventive carotenogenic modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of one or more carotenoids in a host organism produced by that organism as compared with the level produced in an otherwise identical organism not subject to the same modification. In most embodiments, however, the carotenogenic modification will comprise a genetic modification, typically resulting in increased production of one or more selected carotenoids. In some embodiments, the selected carotenoid is one or more of astaxanthin, β-carotene, canthaxanthin, lutein, lycopene, phytoene, zeaxanthin, and/or modifications of zeaxanthin or astaxanthin (e.g., glucoside, esterified zeaxanthin or astaxanthin). In some embodiments, the selected carotenoid is one or more xanthophylls, and/or a modification thereof (e.g., glucoside, esterified xanthophylls). In certain embodiments, the selected xanthophyll is selected from the group consisting of astaxanthin, lutein, zeaxanthin, lycopene, and modifications thereof. In some embodiments, the selected carotenoid is one or more of astaxanthin, β-carotene, canthaxanthin, lutein, lycopene, and zeaxanthin and/or modifications of zeaxanthin or astaxanthin. In some embodiments, the carotenoid is β-carotene. In some embodiments, the selected carotenoid is astaxanthin. In some embodiments, the selected carotenoid is other than β-carotene.
Carotenogenic polypeptide: The term “carotenogenic polypeptide”, as used herein, refers to any polypeptide that is involved in the process of producing carotenoids in a cell, and may include polypeptides that are involved in processes other than carotenoid production but whose activities affect the extent or level of production of one or more carotenoids, for example by scavenging a substrate or reactant utilized by a carotenoid polypeptide that is directly involved in carotenoid production. Carotenogenic polypeptides include isoprenoid biosynthesis polypeptides, carotenoid biosynthesis polypeptides, and isoprenoid biosynthesis competitor polypeptides, as those terms are defined herein. The term also encompasses polypeptides that may affect the extent to which carotenoids are accumulated in lipid bodies.
Carotenoid: The term “carotenoid” is understood in the art to refer to a structurally diverse class of pigments derived from isoprenoid pathway intermediates. The commitment step in carotenoid biosynthesis is the formation of phytoene from geranylgeranyl pyrophosphate. Carotenoids can be acyclic or cyclic, and may or may not contain oxygen, so that the term carotenoids include both carotenes and xanthophylls. In general, carotenoids are hydrocarbon compounds having a conjugated polyene carbon skeleton formally derived from the five-carbon compound IPP, including triterpenes (C30 diapocarotenoids) and tetraterpenes (C40 carotenoids) as well as their oxygenated derivatives and other compounds that are, for example, C35, C50, C60, C70, C80 in length or other lengths. Many carotenoids have strong light absorbing properties and may range in length in excess of C200. C30 diapocarotenoids typically consist of six isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule so that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1,5-positional relationship. Such C30 carotenoids may be formally derived from the acyclic C30H42 structure, having a long central chain of conjugated double bonds, by: (i) hydrogenation (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification/glycosylation, or any combination of these processes. C40 carotenoids typically consist of eight isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule so that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1,5-positional relationship. Such C40 carotenoids may be formally derived from the acyclic C40H56 structure, having a long central chain of conjugated double bonds, by (i) hydrogenation, (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification/glycosylation, or any combination of these processes. The class of C40 carotenoids also includes certain compounds that arise from rearrangements of the carbon skeleton, or by the (formal) removal of part of this structure. More than 600 different carotenoids have been identified in nature; certain common carotenoids are depicted in FIG. 1. Carotenoids include but are not limited to: antheraxanthin, adonirubin, adonixanthin, astaxanthin, canthaxanthin, capsorubrin, β-cryptoxanthin, α-carotene, β-carotene, β,ψ-carotene, δ-carotene, ε-carotene, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, γ-carotene, ψ-carotene, 4-keto-γ-carotene, ζ-carotene, α-cryptoxanthin, deoxyflexixanthin, diatoxanthin, 7,8-didehydroastaxanthin, didehydrolycopene, fucoxanthin, fucoxanthinol, isorenieratene, β-isorenieratene, lactucaxanthin, lutein, lycopene, myxobactone, neoxanthin, neurosporene, hydroxyneurosporene, peridinin, phytoene, rhodopin, rhodopin glucoside, 4-keto-rubixanthin, siphonaxanthin, spheroidene, spheroidenone, spirilloxanthin, torulene, 4-keto-torulene, 3-hydroxy-4-keto-torulene, uriolide, uriolide acetate, violaxanthin, zeaxanthin-β-diglucoside, zeaxanthin, and C30 carotenoids. Additionally, carotenoid compounds include derivatives of these molecules, which may include hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups. Further, included carotenoid compounds include ester (e.g., glycoside ester, fatty acid ester) and sulfate derivatives (e.g., esterified xanthophylls).
Carotenoid biosynthesis polypeptide: The term “carotenoid biosynthesis polypeptide” refers to any polypeptide that is involved in the synthesis of one or more carotenoids. To mention but a few, these carotenoid biosynthesis polypeptides include, for example, polypeptides of phytoene synthase, phytoene dehydrogenase (or desaturase), lycopene cyclase, carotenoid ketolase, carotenoid hydroxylase, astaxanthin synthase, carotenoid epsilon hydroxylase, lycopene cyclase (beta and epsilon subunits), carotenoid glucosyltransferase, and acyl CoA:diacyglycerol acyltransferase. Representative examples of carotenoid biosynthesis polypeptide sequences are presented in Tables 17-25.
Gene: The term “gene”, as used herein, generally refers to a nucleic acid encoding a polypeptide, optionally including certain regulatory elements that may affect expression of one or more gene products (i.e., RNA or protein).
Heterologous: The term “heterologous”, as used herein to refer to genes or polypeptides, refers to a gene or polypeptide that does not naturally occur in the organism in which it is being expressed. It will be understood that, in general, when a heterologous gene or polypeptide is selected for introduction into and/or expression by a host cell, the particular source organism from which the heterologous gene or polypeptide may be selected is not essential to the practice of the present invention. Relevant considerations may include, for example, how closely related the potential source and host organisms are in evolution, or how related the source organism is with other source organisms from which sequences of other relevant polypeptides have been selected.
Host cell: As used herein, the “host cell” is a yeast or fungal cell that is manipulated according to the present invention to accumulate lipid and/or to express one or more carotenoids as described herein. A “modified host cell”, as that term is used herein, is a host cell that contains at least one oleaginic modification and/or at least one carotenogenic modification according to the present invention.
Isolated: The term “isolated”, as used herein, means that the isolated entity has been separated from at least one component with which it was previously associated. When most other components have been removed, the isolated entity is “purified”. Isolation and/or purification may be performed using any techniques known in the art including, for example, fractionation, extraction, precipitation, or other separation.
Isoprenoid biosynthesis competitor polypeptide: The term “isoprenoid biosynthesis competitor polypeptide”, as used herein, refers to a polypeptide whose expression in a cell reduces the level of geranylgeranyl diphosphate (GGPP) available to enter the carotenoid biosynthesis pathway. For example, isoprenoid biosynthesis competitor polypeptides include enzymes that act on isoprenoid intermediates prior to GGPP, such that less GGPP is generated (see, for example, FIG. 5). Squalene synthase is but one isoprenoid biosynthesis competitor polypeptide according to the present invention; representative squalene synthase sequences are presented in Table 16. Prenyldiphosphate synthase enzymes and para-hydroxybenzoate (PHB) polyprenyltransferase are yet additional isoprenoid biosynthesis competitor polypeptides according to the present invention; representative prenyldiphosphate synthase enzymes and PHB polyprenyltransferase polypeptides are presented in Table 29 and 30 respectively.
Isoprenoid biosynthesis polypeptide: The term “isoprenoid biosynthesis polypeptide” refers to any polypeptide that is involved in the synthesis of isoprenoids. For example, as discussed herein, acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, IPP isomerase, FPP synthase, and GGPP synthase, are all involved in the mevalonate pathway for isoprenoid biosynthesis. Each of these proteins is also an isoprenoid biosynthesis polypeptide for purposes of the present invention, and sequences of representative examples of these enzymes are provided in Tables 7-15.
Isoprenoid pathway: The “isoprenoid pathway” is understood in the art to refer to a metabolic pathway that either produces or utilizes the five-carbon metabolite isopentyl pyrophosphate (IPP). As discussed herein, two different pathways can produce the common isoprenoid precursor IPP—the “mevalonate pathway” and the “non-mevalonate pathway”. The term “isoprenoid pathway” is sufficiently general to encompass both of these types of pathway. Biosynthesis of isoprenoids from IPP occurs by polymerization of several five-carbon isoprene subunits. Isoprenoid metabolites derived from IPP are of varying size and chemical structure, including both cyclic and acyclic molecules. Isoprenoid metabolites include, but are not limited to, monoterpenes, sesquiterpenes, diterpenes, sterols, and polyprenols such as carotenoids.
Oleaginic modification: The term “oleaginic modification”, as used herein, refers to a modification of a host organism that adjusts the desirable oleaginy of that host organism, as described herein. In some cases, the host organism will already be oleaginous in that it will have the ability to accumulate lipid to at least about 20% of its dry cell weight. It may nonetheless be desirable to apply an oleaginic modification to such an organism, in accordance with the present invention, for example to increase (or, in some cases, possibly to decrease) its total lipid accumulation, or to adjust the types or amounts of one or more particular lipids it accumulates (e.g., to increase relative accumulation of triacylglycerol). In other cases, the host organism may be non-oleaginous (though may contain some enzymatic and regulatory components used in other organisms to accumulate lipid), and may require oleaginic modification in order to become oleaginous in accordance with the present invention. The present invention also contemplates application of oleaginic modification to non-oleaginous host strains such that their oleaginicity is increased even though, even after being modified, they may not be oleaginous as defined herein. In principle, the oleaginic modification may be any chemical, physiological, genetic, or other modification that appropriately alters oleaginy of a host organism as compared with an otherwise identical organism not subjected to the oleaginic modification. In most embodiments, however, the oleaginic modification will comprise a genetic modification, typically resulting in increased production and/or activity of one or more oleaginic polypeptides. In some embodiments, the oleaginic modification comprises at least one chemical, physiological, genetic, or other modification; in other embodiments, the oleaginic modification comprises more than one chemical, physiological, genetic, or other modification. In certain aspects where more than one modification is utilized, such modifications can comprise any combination of chemical, physiological, genetic, or other modification (e.g., one or more genetic modification and chemical or physiological modification).
Oleaginic polypeptide: The term “oleaginic polypeptide”, as used herein, refers to any polypeptide that is involved in the process of lipid accumulation in a cell and may include polypeptides that are involved in processes other than lipid biosynthesis but whose activities affect the extent or level of accumulation of one or more lipids, for example by scavenging a substrate or reactant utilized by an oleaginic polypeptide that is directly involved in lipid accumulation. For example, as discussed herein, acetyl-CoA carboxylase, pyruvate decarboxylase, isocitrate dehydrogenase, ATP-citrate lyase, malic enzyme, and AMP deaminase, among other proteins, are all involved in lipid accumulation in cells. In general, reducing the activity of pyruvate decarboxylase or isocitrate dehydrogenase, and/or increasing the activity of acetyl CoA carboxylase, ATP-citrate lyase, malic enzyme and/or AMP deaminase is expected to promote oleaginy. Each of these proteins is an oleaginic polypeptide for purposes of the present invention, and sequences of representative examples of these enzymes are provided in Tables 1-6.
Oleaginous: The term “oleaginous”, refers to the ability of an organism to accumulate lipid to at least about 20% of its dry cell weight. In certain embodiments of the invention, oleaginous yeast or fungi accumulate lipid to at least about 25% of their dry cell weight. In other embodiments, inventive oleaginous yeast or fungi accumulate lipid within the range of about 20-45% of their dry cell weight. In some embodiments, oleaginous organisms may accumulate lipid to as much as about 70% of their dry cell weight. In some embodiments of the invention, oleaginous organisms may accumulate a large fraction of total lipid accumulation in the form of triacylglycerol. In certain embodiments, the majority of the accumulated lipid is in the form of triacylglycerol. Alternatively or additionally, the lipid may accumulate in the form of intracellular lipid bodies, or oil bodies. In certain embodiments, the present invention utilizes yeast or fungi that are naturally oleaginous. In some aspects, naturally oleaginous organisms are manipulated (e.g., genetically, chemically, or otherwise) so as to further increase the level of accumulated lipid in the organism. In other embodiments, yeast or fungi that are not naturally oleaginous are manipulated (e.g., genetically, chemically, or otherwise) to accumulate lipid as described herein. For the purposes of the present invention, Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and Candida utilis are not naturally oleaginous fungi.
Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. However, the term is also used to refer to specific functional classes of polypeptides, such as, for example, oleaginic polypeptides, carotenogenic polypeptides, isoprenoid biosynthesis polypeptides, carotenoid biosynthesis polypeptides, and isoprenoid biosynthesis competitor polypeptides. For each such class, the present specification provides several examples of known sequences of such polypeptides. Those of ordinary skill in the art will appreciate, however, that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions (e.g., isocitrate dehydrogenase polypeptides often share a conserved AMP-binding motif; HMG-CoA reductase polypeptides typically include a highly conserved catalytic domain (see, for example, FIG. 7); acetyl coA carboxylase typically has a carboxyl transferase domain; see, for example, Downing et al., Chem. Abs. 93:484, 1980; Gil et al., Cell 41:249, 1985; Jitrapakdee et al. Curr Protein Pept Sci. 4:217, 2003; U.S. Pat. No. 5,349,126, each of which is incorporated herein by reference in its entirety), usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein.
Source organism: The term “source organism”, as used herein, refers to the organism in which a particular polypeptide sequence can be found in nature. Thus, for example, if one or more heterologous polypeptides is/are being expressed in a host organism, the organism in which the polypeptides are expressed in nature (and/or from which their genes were originally cloned) is referred to as the “source organism”. Where more than one heterologous polypeptides are being expressed in a host organism, one or more source organism(s) may be utilized for independent selection of each of the heterologous polypeptide(s). It will be appreciated that any and all organisms that naturally contain relevant polypeptide sequences may be used as source organisms in accordance with the present invention. Representative source organisms include, for example, animal, mammalian, insect, plant, fungal, yeast, algal, bacterial, cyanobacterial, archaebacterial and protozoal source organisms.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION
As noted above, the present invention encompasses the discovery that carotenoids can desirably be produced in oleaginous yeast and fungi. According to the present invention, strains that both (i) accumulate lipid, often in the form of cytoplasmic oil bodies and typically to at least about 20% of their dry cell weight; and (ii) produce carotenoid(s) at a level at least about 1%, and in some embodiments at least about 3-20%, of their dry cell weight, are generated through manipulation of host cells (i.e., strains, including, e.g., naturally-occurring strains, strains which have been previously modified, etc.). These manipulated host cells are then used to produce carotenoids, so that carotenoids that partition into the lipid bodies can readily be isolated.
In general, it will be desirable to balance oleaginy and carotenoid production in inventive cells such that, as soon as a minimum desirable level of oleaginy is achieved, substantially all further carbon which is capable of being utilized and diverted into biosynthesis of products is diverted into a carotenoid production pathway. In some embodiments of the invention, this strategy involves engineering cells to be oleaginous; in other embodiments, it involves engineering cells to accumulate a higher level of lipid, particularly cytoplasmic lipid, than they would accumulate in the absence of such engineering even though the engineered cells may not become “oleaginous” as defined herein. In other embodiments, the extent to which an oleaginous host cell accumulates lipid is actually reduced so that remaining carbon can be utilized in carotenoid production.
Host Cells
Those of ordinary skill in the art will readily appreciate that a variety of yeast and fungal strains exist that are naturally oleaginous or that naturally produce carotenoids. Any of such strains may be utilized as host strains according to the present invention, and may be engineered or otherwise manipulated to generate inventive oleaginous, carotenoid-producing strains. Alternatively, strains that naturally are neither oleaginous nor carotenoid-producing may be employed. Furthermore, even when a particular strain has a natural capacity for oleaginy or for carotenoid production, its natural capabilities may be adjusted as described herein, so as to change the production level of lipid and/or carotenoid. In certain embodiments engineering or manipulation of a strain results in modification of a type of lipid and/or carotenoid which is produced. For example, a strain may be naturally oleaginous and/or carotenogenic, however engineering or modification of the strain may be employed so as to change the type of lipid which is accumulated and or to change the type of carotenoid which is produced.
When selecting a particular yeast or fungal strain for use in accordance with the present invention, it will generally be desirable to select one whose cultivation characteristics are amenable to commercial scale production. For example, it will generally (though not necessarily always) be desirable to avoid filamentous organisms, or organisms with particularly unusual or stringent requirements for growth conditions. However, where conditions for commercial scale production can be applied which allow for utilization of filamentous organisms, these may be selected as host cells. In some embodiments of the invention, it will be desirable to utilize edible organisms as host cells, as they may optionally be formulated directly into food or feed additives, or into nutritional supplements, as desired. For ease of production, some embodiments of the invention utilize host cells that are genetically tractable, amenable to molecular genetics (e.g., can be efficiently transformed, especially with established or available vectors; optionally can incorporate and/or integrate multiple genes, for example sequentially; and/or have known genetic sequence; etc), devoid of complex growth requirements (e.g., a necessity for light), mesophilic (e.g., prefer growth temperatures with in the range of about 25-32° C.), able to assimilate a variety of carbon and nitrogen sources and/or capable of growing to high cell density. Alternatively or additionally, various embodiments of the invention utilize host cells that grow as single cells rather than multicellular organisms (e.g., as mycelia).
In general, when it is desirable to utilize a naturally oleaginous organism in accordance with the present invention, any modifiable and cultivatable oleaginous organism may be employed. In certain embodiments of the invention, yeast or fungi of genera including, but not limited to, Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon, and Yarrowia are employed. In certain particular embodiments, organisms of species that include, but are not limited to, Blakeslea trispora, Candida pulcherrima, C. revkaufi, C. tropicalis, Cryptococcus curvatus, Cunninghamella echinulata, C. elegans, C. japonica, Lipomyces starkeyi, L. lipoferus, Mortierella alpina, M. isabellina, M ramanniana, M vinacea, Mucor circinelloides, Phycomyces blakesleanus, Pythium irregulare, Rhodosporidium torulo ides, Rhodotorula glutinis, R. gracilis, R. graminis, R. mucilaginosa, R. pinicola, Trichosporon pullans, T. cutaneum, and Yarrowia lipolytica are used.
Of these naturally oleaginous strains, some also naturally produce carotenoids and some do not. In most cases, only low levels (less than about 0.05% dry cell weight) of carotenoids are produced by naturally-occurring carotenogenic, oleaginous yeast or fungi. Higher levels of β-carotene are sometimes produced, but high levels of other carotenoids are generally not observed.
In general, any organism that is naturally oleaginous and non-carotenoid-producing (e.g., produce less than about 0.05% dry cell weight, do not produce the carotenoid of interest) may be utilized as a host cell in accordance with the present invention. In some embodiments, the organism is a yeast or fungus from a genus such as, but not limited to, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Pythium, Trichosporon, and Yarrowia; in some embodiments, the organism is of a species including, but not limited to, Mortierella alpina and Yarrowia lipolytica. 
Comparably, the present invention may utilize any naturally oleaginous, carotenoid-producing organism as a host cell. In general, the present invention may be utilized to increase carbon flow into the isoprenoid pathway in naturally carotenoid-producing organisms (particularly for organisms other than Blakeslea and Phycomyces), and/or to shift production from one carotenoid (e.g., β-carotene) to another (e.g., astaxanthin). Introduction of one or more carotenogenic modifications (e.g., increased expression of one or more endogenous or heterologous carotenogenic polypeptides), in accordance with the present invention, can achieve these goals.
In certain embodiments of the invention, the utilized oleaginous, carotenoid-producing organism is a yeast or fungus, for example of a genus such as, but not limited to, Blakeslea, Mucor, Phycomyces, Rhodosporidium, and Rhodotorula; in some embodiments, the organism is of a species such as, Mucor circinelloides and Rhodotorula glutinis. 
When it is desirable to utilize strains that are naturally non-oleaginous as host cells in accordance with the present invention, genera of non-oleaginous yeast or fungi include, but are not limited to, Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces, Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia); in some embodiments, the organism is of a species including, but not limited to, Aspergillus nidulans, A. niger, A. terreus, Botrytis cinerea, Cercospora nicotianae, Fusarium fujikuroi (Gibberella zeae), Kluyveromyces lactis, K. lactis, Neurospora crassa, Pichia pastoris, Puccinia distincta, Saccharomyces cerevisiae, Sclerotium rolfsii, Trichoderma reesei, and Xanthophyllomyces dendrorhous (Phaffia rhodozyma).
It will be appreciated that the term “non-oleaginous”, as used herein, encompasses both strains that naturally have some ability to accumulate lipid, especially cytoplasmically, but do not do so to a level sufficient to qualify as “oleaginous” as defined herein, as well as strains that do not naturally have any ability to accumulate extra lipid, e.g., extra-membranous lipid. It will further be appreciated that, in some embodiments of the invention, it will be sufficient to increase the natural level of oleaginy of a particular host cell, even if the modified cell does not qualify as oleaginous as defined herein.
As with the naturally oleaginous organisms, some of the naturally non-oleaginous fungi naturally produce carotenoids, whereas others do not. Genera of naturally non-oleaginous fungi that do not naturally produce carotenoids (e.g., produce less than about 0.05% dry cell weight, do not produce carotenoid of interest) may desirably be used as host cells in accordance with the present invention include, but are not limited to, Aspergillus, Kluyveromyces, Penicillium, Saccharomyces, and Pichia; species include, but are not limited to, Aspergillus niger and Saccharomyces cerevisiae. Genera of naturally non-oleaginous fungi that do naturally produce carotenoids and that may desirably be used as host cells in accordance with the present invention include, but are not limited to, Botrytis, Cercospora, Fusarium (Gibberella), Neurospora, Puccinia, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia); species include, but are not limited to, Xanthophyllomyces dendrorhous (Phaffia rhodozyma).
As discussed above, any of a variety of organisms may be employed as host cells in accordance with the present invention. In certain embodiments of the invention, host cells will be Yarrowia lipolytica cells. Advantages of Y. lipolytica include, for example, tractable genetics and molecular biology, availability of genomic sequence (see, for example. Sherman et al. Nucleic Acids Res. 32 (Database issue):D315-8, 2004), suitability to various cost-effective growth conditions, and ability to grow to high cell density. In addition, Y. lipolytica is naturally oleaginous, such that fewer manipulations may be required to generate an oleaginous, carotenoid-producing Y. lipolytica strain than might be required for other organisms. Furthermore, there is already extensive commercial experience with Y. lipolytica. 
Saccharomyces cerevisiae is also a useful host cell in accordance with the present invention, particularly due to its experimental tractability and the extensive experience that researchers have accumulated with the organism. Although cultivation of Saccharomyces under high carbon conditions may result in increased ethanol production, this can generally be managed by process and/or genetic alterations.
Additional useful hosts include Xanthophyllomyces dendrorhous (Phaffia rhodozyma), which is experimentally tractable and naturally carotenogenic. Xanthophyllomyces dendrorhous (Phaffia rhodozyma) strains can produce several carotenoids, including astaxanthin.
Aspergillus niger and Mortierella alpina accumulate large amounts of citric acid and fatty acid, respectively; Mortierella alpina is also oleaginous.
Neurospora or Gibberella are also useful. They are not naturally oleaginous and tend to produce very low levels of carotenoids, thus extensive modification may be required in accordance with the present invention. Neurospora and Gibberella are considered relatively tractable from an experimental standpoint. Both are filamentous fungi, such that production at commercial scales can be a challenge necessary to overcome in utilization of such strains.
Mucor circinelloides is another available useful species. While its molecular genetics are generally less accessible than are those of some other organisms, it naturally produces β-carotene, thus may require less modification than other species available.
Molecular genetics can be performed in Blakeslea, though significant effort may be required. Furthermore, cost-effective fermentation conditions can be challenging, as, for example, it may be required that the two mating types are mixed. Fungi of the genus Phycomyces are also possible sources which have the potential to pose fermentation process challenges, and these fungi are also may be less amenable to manipulate than several other potential host organisms.
Those of ordinary skill in the art will appreciate that the selection of a particular host cell for use in accordance with the present invention will also affect, for example, the selection of expression sequences utilized with any heterologous polypeptide to be introduced into the cell, and will also influence various aspects of culture conditions, etc. Much is known about the different gene regulatory requirements, protein targeting sequence requirements, and cultivation requirements, of different host cells to be utilized in accordance with the present invention (see, for example, with respect to Yarrowia, Barth et al. FEMS Microbiol Rev. 19:219, 1997; Madzak et al. J. Biotechnol. 109:63, 2004; see, for example, with respect to Xanthophyllomyces, Verdoes et al. Appl Environ Microbiol 69: 3728-38, 2003; Visser et al. FEMS Yeast Res 4: 221-31, 2003; Martinez et al. Antonie Van Leeuwenhoek. 73(2):147-53, 1998; Kim et al. Appl Environ Microbiol. 64(5):1947-9, 1998; Wery et al. Gene. 184(1):89-97, 1997; see, for example, with respect to Saccharomyces, Guthrie and Fink Methods in Enzymology 194:1-933, 1991). In certain aspects, for example, targeting sequences of the host cell (or closely related analogs) may be useful to include for directing heterologous proteins to subcellular localization. Thus, such useful targeting sequences can be added to heterologous sequence for proper intracellular localization of activity. In other aspects (e.g., addition of mitochondrial targeting sequences), heterologous targeting sequences may be eliminated or altered in the selected heterologous sequence (e.g., alteration or removal of source organism plant chloroplast targeting sequences).
Engineering Oleaginy
All living organisms synthesize lipids for use in their membranes and various other structures. However, most organisms do not accumulate in excess of about 10% of their dry cell weight as total lipid, and most of this lipid generally resides within cellular membranes.
Significant biochemical work has been done to define the metabolic enzymes necessary to confer oleaginy on microorganisms (primarily for the purpose of engineering single cell oils as commercial sources of arachidonic acid and docosahexaenoic acid; see for example Ratledge Biochimie 86:807, 2004, the entire contents of which are incorporated herein by reference). Although this biochemical work is compelling, prior to the present invention, there have been no reports of de novo oleaginy being established through genetic engineering with the genes encoding the key metabolic enzymes.
It should be noted that oleaginous organisms typically only accumulate lipid when grown under conditions of carbon excess and nitrogen or other nutrient limitation. Under these conditions, the organism readily depletes the limiting nutrient but continues to assimilate the carbon source. The “excess” carbon is channeled into lipid biosynthesis so that lipids (usually triacylglycerols) accumulate in the cytosol, typically in the form of bodies.
In general, it is thought that, in order to be oleaginous, an organism must produce both acetyl-CoA and NADPH in the cytosol, which can then be utilized by the fatty acid synthase machinery to generate lipids. In at least some oleaginous organisms, acetyl-CoA is generated in the cytosol through the action of ATP-citrate lyase, which catalyzes the reaction:

citrate+CoA+ATP→acetyl-CoA+oxaloacetate+ADP+Pi.  (1)

Of course, in order for ATP-citrate lyase to generate appropriate levels of acetyl-CoA in the cytosol, it must first have an available pool of its substrate citric acid. Citric acid is generated in the mitochondria of all eukaryotic cells through the tricarboxylic acid (TCA) cycle, and can be moved into the cytosol (in exchange for malate) by citrate/malate translocase.
In most oleaginous organisms, and in some non-oleaginous organisms, the enzyme isocitrate dehydrogenase, which operates as part of the TCA cycle in the mitochondria, is strongly AMP-dependent. Thus, when AMP is depleted from the mitochondria, this enzyme is inactivated. When isocitrate dehydrogenase is inactive, isocitrate accumulates in the mitochondria. This accumulated isocitrate is then equilibrated with citric acid, presumably through the action of aconitase. Therefore, under conditions of low AMP, citrate accumulates in the mitochondria. As noted above, mitochondrial citrate is readily transported into the cytosol.
AMP depletion, which in oleaginous organisms is believed to initiate the cascade leading to accumulation of citrate (and therefore acetyl-CoA) in the cytoplasm, occurs as a result of the nutrient depletion mentioned above. When oleaginous cells are grown in the presence of excess carbon source but under conditions limiting for nitrogen or some other nutrient(s), the activity of AMP deaminase, which catalyzes the reaction:

AMP→inosine 5′-monophosphate+NH3  (2)

is strongly induced. The increased activity of this enzyme depletes cellular AMP in both the cytosol and the mitochondria. Depletion of AMP from the mitochondria is thought to inactivate the AMP-dependent isocitrate dehydrogenase, resulting in accumulation of citrate in the mitochondria and, therefore, the cytosol. This series of events is depicted diagrammatically in FIG. 2.

As noted above, oleaginy requires both cytosolic acetyl-CoA and cytosolic NADPH. It is believed that, in many oleaginous organisms, appropriate levels of cytosolic NADPH are provided through the action of malic enzyme (Enzyme 3 in FIG. 2). Some oleaginous organisms (e.g., Lipomyces and some Candida) do not appear to have malic enzymes, however, so apparently other enzymes can provide comparable activity, although it is expected that a dedicated source of NADPH is probably required for fatty acid synthesis (see, for example, Wynn et al., Microbiol 145:1911, 1999; Ratledge Adv. Appl. Microbiol. 51:1, 2002, each of which is incorporated herein by reference in its entirety).
Thus, according to the present invention, the oleaginy of a host organism may be enhanced by modifying the expression or activity of one or more polypeptides involved in generating cytosolic acetyl-CoA and/or NADPH. For example, modification of the expression or activity of one or more of acetyl-CoA carboxylase, pyruvate decarboxylase, isocitrate dehydrogenase, ATP-citrate lyase, malic enzyme, and AMP-deaminase can enhance oleaginy in accordance with the present invention. Exemplary polypeptides which can be utilized or derived so as to enhance oleaginy in accordance with the present invention include, but are not limited to those acetyl-CoA carboxylase, pyruvate decarboxylase, isocitrate dehydrogenase, ATP-citrate lyase, malic enzyme, and AMP-deaminase polypeptides provided in Table 1, Table 2, Table 3, Table 4, Table 5, and Table 6, respectively.
In some embodiments of the invention, where an oleaginous host cell is employed, enzymes and regulatory components relevant to oleaginy are already in place but could be modified, if desired, by for example altering expression or activity of one or more oleaginic polypeptides and/or by introducing one or more heterologous oleaginic polypeptides. In those embodiments of the invention where a non-oleaginous host cell is employed, it is generally expected that at least one or more heterologous oleaginic polypeptides will be introduced.
The present invention contemplates not only introduction of heterologous oleaginous polypeptides, but also adjustment of expression or activity levels of heterologous or endogenous oleaginic polypeptides, including, for example, alteration of constitutive or inducible expression patterns. In some embodiments of the invention, expression patterns are adjusted such that growth in nutrient-limiting conditions is not required to induce oleaginy. For example, genetic modifications comprising alteration and/or addition of regulatory sequences (e.g., promoter elements, terminator elements) may be utilized to confer particular regulation of expression patterns. Such genetic modifications may be utilized in conjunction with endogenous genes (e.g., for regulation of endogenous oleagenic polypeptide(s)); alternatively, such genetic modifications may be included so as to confer regulation of expression of at least one heterologous polypeptide (e.g., oleagenic polypeptide(s)). For example, promoters including, but not limited to Tef1, Gpd1 promoters can be used in conjunction with endogenous genes and/or heterolous genes for modification of expression patterns of endogenous oleaginic polypeptides and/or heterolous oleagenic polypeptides. Similarly, exemplary terminator sequences include, but are not limited to, use of Y. lipolytica XPR2 terminator sequences.
In some embodiments, at least one oleaginic polypeptide is introduced into a host cell. In some embodiments of the invention, a plurality (e.g., two or more) of different oleaginic polypeptides is introduced into the same host cell. In some embodiments, the plurality of oleaginic polypeptides contains polypeptides from the same source organism; in other embodiments, the plurality includes polypeptides independently selected from different source organisms.
Representative examples of a variety of oleaginic polypeptides that may be introduced into or modified within host cells according to the present invention, include, but are not limited to, those provided in Tables 1-6. As noted above, it is expected that at least some of these polypeptides (e.g., malic enzyme and ATP-citrate lyase) should desirably act in concert, and possibly together with one or more components of fatty acid synthase, such that, in some embodiments of the invention, it will be desirable to utilize two or more oleaginic polypeptides from the same source organism.
In general, source organisms for oleaginic polypeptides to be used in accordance with the present invention include, but are not limited to, Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia, Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces, Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia). In some embodiments, the source species for acetyl CoA carboxylase, ATP-citrate lyase, malice enzyme and/or AMP deaminase polypeptides include, but are not limited to, Aspergillus nidulans, Cryptococcus neoformans, Fusarium fujikuroi, Kluyveromyces lactis, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Ustilago maydis, and Yarrowia lipolytica; in some embodiments, source species for pyruvate decarboxylase or isocitrate dehydrogenase polypeptides include, but are not limited to Neurospora crassa, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), Aspergillus niger, Saccharomyces cerevisiae, Mucor circinelloides, Rhodotorula glutinis, Candida utilis, Mortierella alpina and Yarrowia 
Engineering Carotenoid Production
Carotenoids are synthesized from isoprenoid precursors, some of which are also involved in the production of steroids and sterols. The most common isoprenoid biosynthesis pathway, sometimes referred to as the “mevalonate pathway”, is generally depicted in FIG. 3. As shown, acetyl-CoA is converted, via hydroxymethylglutaryl-CoA (HMG-CoA), into mevalonate. Mevalonate is then phosphorylated and converted into the five-carbon compound isopentenyl pyrophosphate (IPP). Following isomerization of IPP into dimethylallyl pyrophosphate (DMAPP), three sequential condensation reactions with additional molecules of IPP generate the ten-carbon molecule geranyl pyrophosphate (GPP), followed by the fifteen-carbon molecule farnesyl pyrophosphate (FPP), and finally the twenty-carbon compound geranylgeranyl pyrophosphate (GGPP).
An alternative isoprenoid biosynthesis pathway, that is utilized by some organisms (particularly bacteria) and is sometimes called the “mevalonate-independent pathway”, is depicted in FIG. 4. This pathway is initiated by the synthesis of 1-deoxy-D-xyloglucose-5-phosphate (DOXP) from pyruvate and glyceraldehyde-3-phosphate. DOXP is then converted, via a series of reactions shown in FIG. 4, into IPP, which isomerizes into DMAPP and is then converted, via GPP and FPP, into GGPP as shown in FIG. 3 and discussed above.
Various proteins involved in isoprenoid biosynthesis have been identified and characterized in a number of organisms. Moreover, various aspects of the isoprenoid biosynthesis pathway are conserved throughout the fungal, bacterial, plant and animal kingdoms. For example, polypeptides corresponding to the acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, IPP isomerase, FPP synthase, and GGPP synthase shown in FIG. 3 have been identified in and isolated from a wide variety of organisms and cells. Representative examples of a wide variety of such polypeptides are provided in Tables 7-15. One or more of the polypeptides selected from those provided in any one of Tables 7-15 may be utilized or derived for use in the methods and compositions in accordance with the present invention.
According to the present invention, carotenoid production in a host organism may be adjusted by modifying the expression or activity of one or more proteins involved in isoprenoid biosynthesis. In some embodiments, such modification involves introduction of one or more heterologous isoprenoid biosynthesis polypeptides into the host cell; alternatively or additionally, modifications may be made to the expression or activity of one or more endogenous or heterologous isoprenoid biosynthesis polypeptides. Given the considerable conservation of components of the isoprenoid biosynthesis polypeptides, it is expected that heterologous isoprenoid biosynthesis polypeptides will often function even in significantly divergent organisms. Furthermore, should it be desirable to introduce more than one heterologous isoprenoid biosynthesis polypeptide, in many cases polypeptides from different source organisms will function together. In some embodiments of the invention, a plurality of different heterologous isoprenoid biosynthesis polypeptides is introduced into the same host cell. In some embodiments, this plurality contains only polypeptides from the same source organism (e.g., two or more sequences of, or sequences derived from, the same source organism); in other embodiments the plurality includes polypeptides independently selected from from different source organisms (e.g., two or more sequences of, or sequences derived from, at least two independent source organisms).
In some embodiments of the present invention that utilize heterologous isoprenoid biosynthesis polypeptides, the source organisms include, but are not limited to, fungi of the genera Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia, Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces, Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces, Schizosaccharomyces, Sclerotium, Trichoderms, Ustilago, and Xanthophyllomyces (Phaffia). In certain embodiments, the source organisms are of a species including, but not limited to, Cryptococcus neoformans, Fusarium fujikuroi, Kluyverimyces lactis, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Ustilago maydis, and Yarrowia lipolytica.
As noted above, the isoprenoid biosynthesis pathway is also involved in the production of non-carotenoid compounds, such as sterols, steroids, and vitamins, such as vitamin E or vitamin K. Proteins that act on isoprenoid biosynthesis pathway intermediates, and divert them into biosynthesis of non-carotenoid compounds are therefore indirect inhibitors of carotenoid biosynthesis (see, for example, FIG. 5, which illustrates points at which isoprenoid intermediates are channeled into other biosynthesis pathways). Such proteins are therefore considered isoprenoid biosynthesis competitor polypeptides. Reductions of the level or activity of such isoprenoid biosynthesis competitor polypeptides are expected to increase carotenoid production in host cells according to the present invention.
In some embodiments of the present invention, production or activity of endogenous isoprenoid biosynthesis competitor polypeptides may be reduced or eliminated in host cells. In some embodiments, this reduction or elimination of the activity of an isoprenoid biosynthesis competitor polypeptide can be achieved by treatment of the host organism with small molecule inhibitors of enzymes of the ergosterol biosynthetic pathway. Enzymes of the ergosterol biosynthetic pathway include, for example, squalene synthase, squalene epoxidase, 2,3-oxidosqualene-lanosterol cyclase, cytochrome P450 lanosterol 14α-demethylase, C-14 sterol reductase, C-4 sterol methyl oxidase, SAM:C-24 sterol methyltransferase, C-8 sterol isomerase, C-5 sterol desaturase, C-22 sterol desaturase, and C-24 sterol reductase. Each of these enzymes is considered an isoprenoid biosynthesis competitor polypeptide. Regulators of these enzymes may also be considered isoprenoid biosynthesis competitor polypeptides (e.g., the yeast proteins Sut1 (Genbank Accession JC4374 GI:2133159) and Mot3 (Genbank Accession NP—013786 GI:6323715), which may or may not have homologs in other organisms.
In other embodiments, reduction or elimination of the activity of an isoprenoid biosynthesis competitor polypeptide can be achieved by decreasing activity of the ubiquinone biosynthetic pathway. The commitment step in ubiquinone biosynthesis is the formation of para-hydroxybenzoate (PHB) from tyrosine or phenylalanine in mammals or chorismate in bacteria, followed by condensation of PHB and isoprene precursor, resulting in addition of the prenyl group. This reaction is catalyzed by PHB-polyprenyltransferase. The isoprenoid side chain of ubiquinone is determined by the prenyldiphosphate synthase enzyme. The 3-decaprenyl-4-hydroxybenzoic acid resulting from the condensation of PHB and decaprenyldiphosphate reaction undergoes further modifications, which include hydroxylation, methylation and decarboxylation, in order to form ubiquinone (CoQ10). Thus, inhibition of prenyldiphosphate synthase leading from farnesyldiphosphate to extended isoprenoids, or inhibition of PHB polyprenyltransferase may be useful in increasing the amount of isoprenoid available for carotenoid biosynthesis. (Examples of prenyldiphosphate synthase and PHB-polyprenyltransferase enzymes are depicted in Tables 29 and 30, respectively).
Known small molecule inhibitors of isoprenoid biosynthesis competitor enzymes include, but are not limited to, zaragosic acid (including analogs thereof such as TAN1607A (Biochem Biophys Res Commun 1996 Feb. 15; 219(2):515-520)), RPR 107393 (3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1-azabicyclo[2-2-2]octane dihydrochloride; J Pharmacol Exp Ther. 1997 May; 281(2):746-52), ER-28448 (5-{N-[2-butenyl-3-(2-methoxyphenyl)]-N-methylamino}-1,1-penthylidenebis(phosphonic acid) trisodium salt; Journal of Lipid Research, Vol. 41, 1136-1144, July 2000), BMS-188494 (The Journal of Clinical Pharmacology, 1998; 38:1116-1121), TAK-475 (1-[2-[(3R,5 S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-1,2,3,5-tetrahydro-2-oxo-5-(2,3-dimethoxyphenyl)-4,1-benzoxazepine-3-yl]acetyl]piperidin-4-acetic acid; Eur J. Pharmacol. 2003 Apr. 11; 466(1-2):155-61), YM-53601 ((E)-2-[2-fluoro-2-(quinuclidin-3-ylidene) ethoxy]-9H-carbazole monohydrochloride; Br J. Pharmacol. 2000 September; 131(1):63-70), or squalestatin I that inhibit squalene synthase; terbinafine that inhibits squalene epoxidase; various azoles that inhibit cytochrome P450 lanosterol 14α-demethylase; and fenpropimorph that inhibits the C-14 sterol reductase and the C-8 sterol isomerase. In other embodiments, heterologous isoprenoid biosynthesis competitor polypeptides may be utilized (whether functional or non-functional; in some embodiments, dominant negative mutants are employed).
One particular isoprenoid biosynthesis competitor polypeptide useful according to the present invention is squalene synthase which has been identified and characterized from a variety of organisms; representative examples of squalene synthase polypeptide sequences are included in Table 16. In some embodiments of the invention that utilize squalene synthase (or modifications of squalene synthase) source organisms include, but are not limited to, Neurospora crassa, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), Aspergillus niger, Saccharomyces cerevisiae, Mucor circinelloides, Rhotorula glutinis, Candida utilis, Mortierella alpina, and Yarrowia lipolytica. 
The carotenoid biosynthesis pathway branches off from the isoprenoid biosynthesis pathway at the point where GGPP is formed. The commitment step in carotenoid biosynthesis is the formation of phytoene by the head-to-head condensation of two molecules of GGPP, catalyzed by phytoene synthase (often called crtB; see FIG. 6). A series of dehydrogenation reactions, each of which increases the number of conjugated double bonds by two, converts phytoene into lycopene via neurosporene. The pathway branches at various points, both before and after lycopene production, so that a wide range of carotenoids can be generated. For example, action of a cyclase enzyme on lycopene generates γ-carotene; action of a desaturase instead produces 3,4-didehydrolycopene. γ-carotene is converted to β-carotene through the action of a cyclase. β-carotene can be processed into any of a number of products (see, for example, FIG. 6C), including astaxanthin (via echinone, hydroxyechinone, and phoenicoxanthin).
According to the present invention, carotenoid production in a host organism may be adjusted by modifying the expression or activity of one or more proteins involved in carotenoid biosynthesis. As indicated, in some embodiments, it will be desirable to utilize as host cells organisms that naturally produce one or more carotenoids. In some such cases, the focus will be on increasing production of a naturally-produced carotenoid, for example by increasing the level and/or activity of one or more proteins involved in the synthesis of that carotenoid and/or by decreasing the level or activity of one or more proteins involved in a competing biosynthetic pathway. Alternatively or additionally, in some embodiments it will be desirable to generate production of one or more carotenoids not naturally produced by the host cell.
According to some embodiments of the invention, it will be desirable to introduce one or more heterologous carotenogenic polypeptides into a host cell. As will be apparent to those of ordinary skill in the art, any of a variety of heterologous polypeptides may be employed; selection will consider, for instance, the particular carotenoid whose production is to be enhanced. The present invention contemplates not only introduction of heterologous carotenogenic polypeptides, but also adjustment of expression or activity levels of heterologous or endogenous carotenogenic polypeptides, including, for example, alteration of constitutive or inducible expression patterns. In some embodiments of the invention, expression patterns are adjusted such that growth in nutrient-limiting conditions is not required to induce oleaginy. For example, genetic modifications comprising alteration and/or addition of regulatory sequences (e.g., promoter elements, terminator elements) may be utilized to confer particular regulation of expression patterns. Such genetic modifications may be utilized in conjunction with endogenous genes (e.g., for regulation of endogenous carotenogenic); alternatively, such genetic modifications may be included so as to confer regulation of expression of at least one heterologous polypeptide (e.g., carotenogenic polypeptide(s)). For example, promoters including, but not limited to Tef1, Gpd1 promoters can be used in conjunction with endogenous genes and/or heterolous genes for modification of expression patterns of endogenous carotenogenic polypeptide(s) and/or heterolous carotenogenic polypeptide(s). Similarly, exemplary terminator sequences include, but are not limited to, use of Y. lipolytica XPR2 terminator sequences.
As indicated in FIG. 6 and in the literature, proteins involved in carotenoid biosynthesis include, but are not limited to, phytoene synthase, phytoene dehydrogenase, lycopene cyclase, carotenoid ketolase, carotenoid hydroxylase, astaxanthin synthase (a single multifunctional enzyme found in some source organisms that typically has both ketolase and hydroxylase activities), carotenoid epsilon hydroxylase, lycopene cyclase (beta and epsilon subunits), carotenoid glucosyltransferase, and acyl CoA:diacyglycerol acyltransferase. Representative example sequences for these carotenoid biosynthesis polypeptides are provided in Tables 17-25.
Xanthophylls can be distinguished from other carotenoids by the presence of oxygen containing functional groups on their cyclic end groups. For instance, lutein and zeaxanthin contain a single hydroxyl group on each of their terminal ring structures, while astaxanthin contains both a keto group and a hydroxyl on each terminal ring. This property makes xanthophylls more polar than carotenes such as beta-carotene and lycopene, and thus dramatically reduces their solubility in fats and lipids. Naturally occurring xanthophylls are often found as esters of the terminal hydroxyl groups, both mono- and diesters of fatty acids. They also occur as glucosides in certain species of bacteria. The solubility and dispersibility of xanthophylls can be greatly modified by the addition of ester moieties, and it is known that esterification can also affect the absorbability and/or bioavailability of a given carotenoid. It is an objective of this invention to maximize the amount of a particular xanthophyll accumulating within the intracellular triacylglyceride fraction of oleaginous yeasts, and one mechanism for achieving this goal is to increase the hydrophobic nature of the xanthophyll product that accumulates. One way of achieving this is to engineer the production of fatty-acyl mono- and/or diesters of the target xanthophyll compound.
A variety of enzymes can function to esterify carotenoids. For example, carotenoid glucosyltransferases have been identified in several bacterial species (see, e.g., Table 24). In addition, acyl CoA:diacyglycerol acyltransferase (DGAT) and acyl CoA:monoacylglycerol acyltransferases (MGAT), which function in the final steps of triacylglycerol biosynthesis, are likely to serve an additional role in the esterification of xanthophylls. Representative DGAT polypeptides are shown in Table 25. Furthermore, other enzymes may specifically modify carotenoids and molecules of similar structure (e.g. sterols) and be available for modification and ester production.
In some embodiments of the invention, potential source organisms for carotenoid biosynthesis polypeptides include, but are not limited to, genera of naturally oleaginous or non-oleaginous fungi that naturally produce carotenoids. These include, but are not limited to, Botrytis, Cercospora, Fusarium (Gibberella), Mucor, Neurospora, Phycomyces, Puccina, Rhodotorula, Sclerotium, Trichoderma, and Xanthophyllomyces. Exemplary species include, but are not limited to, Neurospora crassa, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), Mucor circinelloides, and Rhodotorula glutinis. Of course, carotenoids are produced by a wide range of diverse organisms such as plants, algae, yeast, fungi, bacteria, cyanobacteria, etc. Any such organisms may be source organisms for carotenoid biosynthesis polypeptides according to the present invention.
It will be appreciated that the particular carotenogenic modification to be applied to a host cell in accordance with the present invention will be influenced by which carotenoid(s) is desired to be produced. For example, isoprenoid biosynthesis polypeptides are relevant to the production of most carotenoids. Carotenoid biosynthesis polypeptides are also broadly relevant. Ketolase is particularly relevant for production of canthaxanthin, as hydroxylase is for production of lutein and zeaxanthin, among others. Both hydroxylase and ketolase (or astaxanthin synthase) are particularly useful for production of astaxanthin.
Production and Isolation of Carotenoids
As discussed above, accumulation of lipid bodies in oleaginous organisms is generally induced by growing the relevant organism in the presence of excess carbon source and limiting nitrogen. Specific conditions for inducing such accumulation have previously been established for a number of different oleaginous organisms (see, for example, Wolf (ed.) Nonconventional yeasts in biotechnology Vol. 1, Springer-Verlag, Berlin, Germany, pp. 313-338; Lipids 18(9):623, 1983; Indian J. Exp. Biol. 35(3):313, 1997; J. Ind. Microbial. Biotechnol. 30(1):75, 2003; Bioresour Technol. 95(3):287, 2004, each of which is incorporated herein by reference in its entirety).
In general, it will be desirable to cultivate inventive modified host cells under conditions that allow accumulation of at least about 20% of their dry cell weight as lipid. In other embodiments, the inventive modified host cells are grown under conditions that permit accumulation of at least about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or even 80% or more of their dry cell weight as lipid. In certain embodiments, the host cells utilized are cells which are naturally oleaginous, and induced to produce lipid to the desired levels. In other embodiments, the host cells are cells which naturally produce lipid, but have been engineered to increase production of lipid such that desired levels of lipid production and accumulation are achieved.
In certain embodiments, the host cells of the invention are not naturally oleaginous, but have been engineered to produce lipid such that desired levels of lipid production are obtained. Those of ordinary skill in the art will appreciate that, in general, growth conditions that are effective for inducing lipid accumulation in a source organism, may well also be useful for inducing lipid accumulation in a host cell into which the source organism's oleaginic polypeptides have been introduced. Of course, modifications may be required in light of characteristics of the host cell, which modifications are within the skill of those of ordinary skill in the art.
It will also be appreciated by those of ordinary skill in the art that it will generally be desirable to ensure that production of the desired carotenoid by the inventive modified host cell occurs at an appropriate time in relation to the induction of oleaginy such that the carotenoid(s) accumulate(s) in the lipid bodies. In some embodiments, it will be desirable to induce production of the carotenoid(s) in a host cell which does not naturally produce the carotenoid(s), such that detectable levels of the carotenoid(s) is/are produced. In certain aspects the host cells which do not naturally produce a certain carotenoid(s) are capable of production of other carotenoid(s) (e.g. certain host cells may, for example, naturally produce β-carotene but may not naturally produce astaxanthin); in other aspects the host cells do not naturally produce any carotenoid(s). In other embodiments, it will be desirable to increase production levels of carotenoid(s) in a host cell which does naturally produce low levels of the carotenoid(s), such that increased detectable levels of the carotenoid(s) are produced. In certain aspects, the host cells which do naturally produce the carotenoid(s) (e.g., β-carotene) also produce additional carotenoid(s) (e.g., astaxanthin, etc.); in still other aspects, the cells which naturally produce the carotenoid(s) (e.g., β-carotene) do not produce additional carotenoid(s).
In certain embodiments of the invention, it will be desirable to accumulate carotenoids to levels (i.e., considering the total amount of all produced carotenoids together) that are greater than at least about 1% of the dry weight of the cells. In some embodiments, the total carotenoid accumulation in the lipid bodies will be to a level at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20% or more of the total dry weight of the cells. In certain embodiments of the invention, it will be desirable to achieve total levels of carotenoid accumulation in the lipid bodies (i.e., considering the total amount of all produced carotenoids together) that are greater than at least about 1% of the dry weight of the cells. In some embodiments, the total carotenoid accumulation in the lipid bodies will be to a level at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20% or more of the total dry weight of the cells.
Bacterial carotenogenic genes have already been demonstrated to be transferrable to other organisms, and are therefore particularly useful in accordance with the present invention (see, for example, Miura et al., Appl. Environ. Microbiol. 64:1226, 1998). In other embodiments, it may be desirable to utilize genes from other source organisms such as plant, alga, or microalgae; these organisms provide a variety of potential sources for ketolase and hydroxylase polypeptides. Still additional useful source organisms include fungal, yeast, insect, protozoal, and mammalian sources of polypeptides.
In certain embodiments, the Mucor circinelloides multi-functional phytoene synthase/lycopene cyclase and the Neurospora crassa phytoene dehydrogenase genes can be expressed in Yarrowia lipolytica. Subsequent overexpression of the catalytic domain from N. crassa hydroxymethylglutaryl-CoA reductase and/or treatment of the modified Y. lipolytica strains with the squalene synthase inhibitor zaragozic acid further increases carotenoid production. Finally, Paracoccus marcusii genes encoding carotenoid hydroxylase and carotenoid ketolase enzymes are expressed in Y. lipolytica β-carotene-producing strains, and this modification results in the accumulation of astaxanthin. Similar approaches to enhance carotenoid production could be employed in other oleaginous or non-oleaginous host organisms can be undertaken, using the same, homologous, or functionally similar carotogenic polypeptides.
It should be noted that, for inventive organisms that produce more than one carotenoid, it will sometimes be possible to adjust the relative amounts of individual carotenoids produced by adjusting growth conditions. For example, it has been reported that controlling the concentration of dissolved oxygen in a culture during cultivation can regulate relative production levels of certain carotenoids such as β-carotene, echinenone, β-cryptoxanthin, 3-hydroxyechinenone, asteroidenone, canthaxanthin, zeaxanthin, adonirubin, adonixanthin and astaxanthin (see, for example, U.S. Pat. No. 6,825,002 to Tsubokura et al., the entire contents of which are incorporated herein by reference).
Particularly for embodiments of the present invention directed toward production of astaxanthin, it will often be desirable to utilize one or more genes from a natural astaxanthin-producing organism. Where multiple heterologous polypeptides are to be expressed, it may be desirable to utilize the same source organism for all, or to utilize closely related source organisms.
One advantage provided by the present invention is that, in addition to allowing the production of high levels of carotenoids, the present invention allows those produced compounds to be readily isolated because they accumulate in the lipid bodies within oleaginous organisms. Methods and systems for isolating lipid bodies have been established for a wide variety of oleaginous organisms (see, for example, U.S. Pat. Nos. 5,164,308; 5,374,657; 5,422,247; 5,550,156; 5,583,019; 6,166,231; 6,541,049; 6,727,373; 6,750,048; and 6,812,001, each of which is incorporated herein by reference in its entirety). In brief, cells are typically recovered from culture, often by spray drying, filtering or centrifugation. In some instances, cells are homogenized and then subjected to supercritical liquid extraction or solvent extraction (e.g., with solvents such as chloroform, hexane, methylene chloride, methanol, isopropanol, ethyl acetate, etc.), yielding a crude oil suspension. This oil suspension may optionally be refined as known in the art. Refined oils may be used directly as feed or food additives. Alternatively or additionally, carotenoids can be isolated from the oil using conventional techniques.
Given the sensitivity of carotenoids generally to oxidation, many embodiments of the invention employ oxidative stabilizers (e.g., tocopherols, vitamin C; ethoxyquin; vitamin E, BHT, BHA, TBHQ, etc, or combinations thereof) during and/or after carotenoid isolation. Alternatively or additionally, microencapsulation, for example with proteins, may be employed to add a physical barrier to oxidation and/or to improve handling (see, for example, U.S. Patent Application 2004/0191365).
Uses
Carotenoids produced according to the present invention can be utilized in any of a variety of applications, for example exploiting their biological or nutritional properties (e.g., anti-oxidant, anti-proliferative, etc.) and/or their pigment properties. For example, according to the present invention, carotenoids may be used in pharmaceuticals (see, for example, Bertram, Nutr. Rev. 57:182, 1999; Singh et al., Oncology 12:1643, 1998; Rock, Pharmacol. Ther. 75:185, 1997; Edge et al, J. Photochem Photobiol 41:189, 1997; U.S. Patent Application 2004/0116514; U.S. Patent Application 2004/0259959), food supplements (see, for example, Koyama et al, J. Photochem Photobiol 9:265, 1991; Bauernfeind, Carotenoids as colorants and vitamin A precursors, Academic Press, NY, 1981; U.S. Patent Application 2004/0115309; U.S. Patent Application 2004/0234579), electro-optic applications, animal feed additives (see, for example, Krinski, Pure Appl. Chem. 66:1003, 1994; Polazza et al., Meth. Enzymol. 213:403, 1992), cosmetics (as anti-oxidants and/or as cosmetics, including fragrances; see for example U.S. Patent Application 2004/0127554), etc. Carotenoids produced in accordance with the present invention may also be used as intermediates in the production of other compounds (e.g., steroids, etc.).
For example, astaxanthin and/or esters thereof may be useful in a variety of pharmaceutical applications and health foods including treatment of inflammatory diseases, asthma, atopic dermatitis, allergies, multiple myeloma, arteriosclerosis, cardiovascular disease, liver disease, cerebrovascular disease, thrombosis, neoangiogenesis-related diseases, including cancer, rheumatism, diabetic retinopathy; macular degeneration and brain disorder, hyperlipidemia, kidney ischemia, diabetes, hypertension, tumor proliferation and metastasis; and metabolic disorders. Additionally, carotenoids and astaxanthin may be useful in the prevention and treatment of fatigue, for improving kidney function in nephropathy from inflammatory diseases, as well as prevention and treatment of other life habit-related diseases. Still further, astaxanthin has been found to play a role as inhibitors of various biological processes, including interleukin inhibitors, phosphodiesterase inhibitors inhibitors, phospholipase A2 inhibitors, cyclooxygenase-2 inhibitors, matrix metalloproteinase inhibitors, capillary endothelium cell proliferation inhibitors, lipoxygenase inhibitors. See, e.g., Japanese Publication No. 2006022121, published 20060126 (JP Appl No. 2005-301156 filed 20051017); Japanese Publication No. 2006016408, published 20060119 (JP Appl No. 2005-301155 filed 20051017); Japanese Publication No. 2006016409, published 20060119 (JP Appl No. 2005-301157 filed 20051017); Japanese Publication No. 2006016407, published 20060119 (JP Appl No. 2005-301153 filed 20051017); Japanese Publication No. 2006008717, published 20060112 (JP Appl No. 2005-301151 filed 20051017); Japanese Publication No. 2006008716, published 20060112 (JP Appl No. 2005-301150 filed 20051017); Japanese Publication No. 2006008720, published 20060112 (JP Appl No. 2005-301158 filed 20051017); Japanese Publication No. 2006008719, published 20060112 (JP Appl No. 2005-301154 filed 20051017); Japanese Publication No. 2006008718, published 20060112 (JP Appl No. 2005-301152 filed 20051017); Japanese Publication No. 2006008713, published 20060112 (JP Appl No. 2005-301147 filed 20051017); Japanese Publication No. 2006008715, published 20060112 (JP Appl No. 2005-301149 filed 20051017); Japanese Publication No. 2006008714, published 20060112 (JP Appl No. 2005-301148 filed 20051017); and Japanese Publication No. 2006008712, published 20060112 (JP Appl No. 2005-301146 filed 20051017).
It will be appreciated that, in some embodiments of the invention, carotenoids produced by manipulated host cells as described herein are incorporated into a final product (e.g., food or feed supplement, pharmaceutical, cosmetic, dye-containing item, etc.) in the context of the host cell. For example, host cells may be lyophilized, freeze dried, frozen or otherwise inactivated, and then whole cells may be incorporated into or used as the final product. The host cell may also be processed prior to incorporation in the product to increase bioavailability (e.g., via lysis). Alternatively or additionally, a final product may incorporate only a portion of the host cell (e.g., fractionated by size, solubility), separated from the whole. For example, in some embodiments of the invention, lipid droplets are isolated from the host cells and are incorporated into or used as the final product. In other embodiments, the carotenoids themselves, or individual carotenoid compounds are isolated and reformulated into the final product.
As stated above, fatty acid and glucoside esters are the predominant carotenoid esters found in nature, whereas additional esters (e.g. with organic acids or inorganic phosphate) can be synthesized to generate useful product forms. For delivery, carotenoid esters can also be formulated as salts of the ester form. See, e.g., US Publication No. 20050096477.
The amount of carotenoid incorporated into a given product may vary dramatically depending on the product, and the particular carotenoid(s) involved. Amounts may range, for example, from less than 0.01% by weight of the product, to more than 1%, 10%, 20%, 30% or more; in some cases the carotenoid may comprise 100% of the product.
In some embodiments of the invention, one or more produced carotenoids is incorporated into a component of food or feed (e.g., a food supplement). Types of food products into which carotenoids can be incorporated according to the present invention are not particularly limited, and include beverages such as teas, juices, and liquors; confections such as jellies and biscuits; fat-containing foods and beverages such as dairy products; processed food products such as rice and soft rice (or porridge); infant formulas; or the like. In some embodiments of this aspect of the invention, it may be useful to incorporate the carotenoids within bodies of edible lipids as it may facilitate incorporation into certain fat-containing food products.
Examples of feedstuffs into which carotenoids produced in accordance with the present invention may be incorporated include, for instance, pet foods such as cat foods, dog foods and the like, feeds for aquarium fish, cultured fish or crustaceans, etc., feed for farm-raised animals (including livestock and further including fish or crustaceans raised in aquaculture). Food or feed material into which the carotenoid(s) produced in accordance with the present invention is incorporated is preferably palatable to the organism which is the intended recipient. This food or feed material may have any physical properties currently known for a food material (e.g., solid, liquid, soft).
In some embodiments of the invention, one or more produced carotenoids is incorporated into a cosmetic product. Examples of such cosmetics include, for instance, skin cosmetics (e.g., lotions, emulsions, creams and the like), lipsticks, anti-sunburn cosmetics, makeup cosmetics, fragrances, products for daily use (e.g., toothpastes, mouthwashes, bad breath preventive agents, solid soaps, liquid soaps, shampoos, conditioners), etc.
In some embodiments, one or more produced carotenoids is incorporated into a pharmaceutical. Examples of such pharmaceuticals include, for instance, various types of tablets, capsules, drinkable agents, troches, gargles, etc. In some embodiments, the pharmaceutical is suitable for topical application. Dosage forms are not particularly limited, and include capsules, oils, granula, granula subtilae, pulveres, tabellae, pilulae, trochisci, or the like. Oils and oil-filled capsules may provide additional advantages both because of their lack of ingredient decomposition during manufacturing, and because inventive carotenoid-containing lipid droplets may be readily incorporated into oil-based formulations.
Pharmaceuticals according to the present invention may be prepared according to techniques established in the art including, for example, the common procedure as described in the United States Pharmacopoeia, for example.
Carotenoids produced according to the present invention may be incorporated into any pigment-containing product including, for example, fabric, paint, etc. They may also be incorporated into a product which is an environmental indicator, or an instrument such as a biosensor for use as a detection agent.
EXEMPLIFICATION
Table 26 below describes certain Yarrowia lipolytica strains used in the following exemplification:







TABLE 26





Yarrowia lipolytica strains.

















NRRL Y-1095
Wild type diploid



ATCC76861
MATB ura2-21 lyc1-5 LYS1-5B



ATCC76982
MATB ade 1 leu2-35 lyc1-5 xpr2



ATCC201249
MATA ura3-302 leu2-270 lys8-11 PEX17-HA



MF346
MATA ura2-21
ATCC76861 × ATCC201249


MF350
MATB ura2-21 leu2-35 ade l
ATCC76982 × MF346









(The genotypes at LYC1, LYS1, XPR2, and PEX17 were not determined in crosses nor verified for ATCC strains.)
All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. or Ausubel et al. (Sambrook J, Fritsch E F, Maniatis T (eds). 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York; Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K (eds). 1998. Current Protocols in Molecular Biology. Wiley: New York).
Example 1
Production of Plasmids for Carotenoid Strain Construction
Plasmids were generated for construction of carotenoid producing strains. The following subparts describe production of plasmids encoding carotenogenic polypeptides. Plasmids used in these studies and details of their construction are described in Table 27. Additional plasmid construction details and descriptions of their use are found in the text of the relevant subsection. All PCR amplifications used NRRL Y-1095 genomic DNA as template unless otherwise specified. The URA5 gene described below is allelic with the ura2-21 auxotrophy above. The GPD1 and TEF1 promoters are from Y. lipolytica as is the XPR2 terminator.
GGS1 is the gene encoding the Y. lipolytica gene encoding geranylgeranylpyrophosphate synthase. The nucleic acid coding sequence, and encoded Ggs1 protein of pMB4591 and pMB4683 are as follows:







               atggattataacagcgcggatttcaaggagatatg


 


gggcaaggccgccgacaccgcgctgctgggaccgtacaactacctcgcca


 


acaaccggggccacaacatcagagaacacttgatcgcagcgttcggagcg


 


gttatcaaggtggacaagagcgatctcgagaccatttcgcacatcaccaa


 


gattttgcataactcgtcgctgcttgttgatgacgtggaagacaactcga


 


tgctccgacgaggcctgccggcagcccattgtctgtttggagtcccccaa


 


accatcaactccgccaactacatgtactttgtggctctgcaggaggtgct


 


caagctcaagtcttatgatgccgtctccattttcaccgaggaaatgatca


 


acttgcatagaggtcagggtatggatctctactggagagaaacactcact


 


tgcccctcggaagacgagtatctggagatggtggtgcacaagaccggtgg


 


actgtttcggctggctctgagacttatgctgtcggtggcatcgaaacagg


 


aggaccatgaaaagatcaactttgatctcacacaccttaccgacacactg


 


ggagtcatttaccagattctggatgattacctcaacctgcagtccacgga


 


attgaccgagaacaagggattctgcgaagatatcagcgaaggaaagtttt


 


cgtttccgctgattcacagcatacgcaccaacccggataaccacgagatt


 


ctcaacattctcaaacagcgaacaagcgacgcttcactcaaaaagtacgc


 


cgtggactacatgagaacagaaaccaagagtttcgactactgcctcaaga


 


ggatacaggccatgtcactcaaggcaagttcgtacattgatgatctagca


 


gcagctggccacgatgtctccaagctacgagccattttgcattattttgt


 


gtccacctctgactgtgaggagagaaagtactttgaggatgcgcagtga


 


               mdynsadfkeiwgkaadtallgpynylannrghni


 


rehliaafgavikvdksdletishitkilhnssllvddvednsmlrrglp


 


aahclfgvpqtinsanymyfvalqevlklksydavsifteeminlhrgqg


 


mdlywretltcpsedeylemvvhktgglfrlalrlmlsvaskqedhekin


 


fdlthltdtlgviyqilddylnlqsteltenkgfcedisegkfsfplihs


 


irtnpdnheilnilkqrtsdaslkkyavdymrtetksfdyclkriaamsl


 


kassyiddlaaaghdvsklrailhyfvstsdceerkyfedaq













TABLE 27







Plasmids










Plasmid
Backbone
Insert
Oligos or source





pMB4529
PCR2.1
3.4 kb ADE1 PCR product
MO4475 & MO4476


pMB4534
PCR2.1
2.1 kb LEU2 PCR product
MO4477 & MO4478


pMB4535
PCR2.1
1.2 kb URA5 PCR product
MO4471 & MO4472


pMB4589
pMB4535 (KpnI + SpeI)
1.2 kb GPD1 promoter (KpnI +
MO4568 &




NotI); 0.14 kb XPR2
MO4591; MO4566




terminator (NotI + SpeI)
& MO4593


pMB4590
pMB4535 (KpnI + SpeI)
0.4 kb TEF1 promoter (KpnI +
MO4571 &




NotI); 0.14 kb XPR2
MO4592; MO4566




terminator (NotI + SpeI)
& MO4593


pMB4591
pMB4590 (NheI + MluI)
1.0 kb GGS1 ORF (XbaI +
MO4534 & MO4544




MluI)


pMB4597
pMB4534 (Acc65I + SpeI)
GPD1 promoter & XPR2
From pMB4589




terminator (Acc65I + SpeI)


pMB4603
pMB4597 (RsrII + MluI)
Residual backbone
From pMB4590




& TEF1 promoter (RsrII +




MluI)


pMB4616
pMB4529 (RsrII + SpeI)
Residual backbone
From pMB4589




& GPD1 promoter & XPR2




terminator (RsrII + SpeI)


pMB4629
pMB4616 (RsrII + MluI)
Residual backbone
From pMB4590




& TEF1 promoter (RsrII +




MluI)


pMB4631
pMB4603 (KpnI + NheI)
1.2 kb GPD1 promoter (KpnI +
MO4568 & MO4659




NheI);


pMB4628
pMB4603
Carp
See 1A


pMB4637
pMB4629 (NheI + MluI)
1.5 kb hmg1trunc ORF (XbaI +
See 1D




MluI)


pMB4638
pMB4629
carB(i−)
See 1B


pMB4660
pMB4638 (+URA3)
carB(i−)
See 1C


pMB4662
pMB4631 (SpeI + XhoI)
1.8 kb URA3 fragment (SpeI +
MO4684 & MO4685




BsaI
See 1C


pMB4683
pMB4662 (Acc65I + MluI)
1.4 kb tef1p-GGS1 fragment
From pMB4591




(Acc65I + MluI)


pMB4692
pMB4662 (Acc65I + MluI)
0.4 kb TEF1 promoter
See 1E




(Acc65I + NheI); 0.55 kb crtZ




ORF (XbaI + MluI)


pMB4698
pMB4629 (NheI + MluI)
0.9 kb crtW ORF (XbaI +
See 1F




MluI)


pMB4599
pBluescriptSKII-
1.9 kb carRP gene
MO4525 & MO4541



(EcoRV)


pMB4606
pBluescriptSKII-
1.9 kb carB gene
MO4530 &



(EcoRV)

MO4542


pMB4613
pMB4599 (Acc65I +
carRP(i−)
See text



PpuMI)


pMB4619
pBluescriptSKII-
carB(i−)
See text



(BamHI + Acc65I))









Certain oligonucleotides referred to in Table 27 above are as follows:








MO4471
5′-CTGGGTGACCTGGAAGCCTT


 


MO4472
5′-AAGATCAATCCGTAGAAGTTCAG


 


MO4475
5′-AAGCGATTACAATCTTCCTTTGG


 


MO4476
5′-CCAGTCCATCAACTCAGTCTCA


 


MO4477
5′-GCATTGCTTATTACGAAGACTAC


 


MO4478
5′-CCACTGTCCTCCACTACAAACAC


 


MO4534
5′-CACAAACGCGTTCACTGCGCATCCTCAAAGT


 


MO4544
5′-CACAATCTAGACACAAATGGATTATAACAGCGCGGAT


 


MO4566
5′-CACAAACTAGTTTGCCACCTACAAGCCAGAT


 


MO4568
5′-CACAAGGTACCAATGTGAAAGTGCGCGTGAT


 


MO4571
5′-CACAAGGTACCAGAGACCGGGTTGGCGG


 


MO4591
5′-CACAAGCGGCCGCGCTAGCATGGGGATCGATCTCTTATAT


 


MO4592
5′-CACAAGCGGCCGCGCTAGCGAATGATTCTTATACTCAGAAG


 


MO4593
5′-CACAAGCGGCCGCACGCGTGCAATTAACAGATAGTTTGCC


 


MO4659
5′-CACAAGCTAGCTGGGGATGCGATCTCTTATATC






1A: Production of pMB4628 (tef1p-carRP LEU2) encoding phytoene synthase/lycopene cyclase: Intron-containing carRP was amplified from M. circinelloides (ATCC 90680) genomic DNA using M04525 and M04541:










MO4525
5′-CACAAACGCGTTTAAATGGTATTTAGATTTCTCATT



 



MO4541
5′-CACAATCTAGACACAAATGCTGCTCACCTACATGGA






and the resulting 1.9 kb fragment was phosphorylated with T4 polynucleotide kinase. The resulting fragment was blunt-end ligated into pBluescriptSKII— cleaved with EcoRV, yielding pMB4599. The 1.9 kb XbaI-MluI fragment from pMB4599 was inserted into NheI- and MluI-cleaved pMB4603, yielding pMB4628. The intron containing nucleic acid coding sequence, and encoded CarRP protein of pMB4628 are as follows:








               atgctgctcacctacatggaagtccacctctacta


 


cacgctgcctgtgctgggcgtcctgtcctggctgtcgcggccgtactaca


 


cagccaccgatgcgctcaaattcaaatttctgacactggttgccttcacg


 


accgcctccgcctgggacaactacattgtctaccacaaggcgtggtccta


 


ctgccccacctgcgtcaccgctgtcattggctacgtgcccttggaggagt


 


acatgttcttcatcatcatgactctgttgaccgtggcattcaccaatctg


 


gtgatgcgctggcacctgcacagcttctttatcaggcctgaaacgcccgt


 


catgcagtccgtcctggtccgtcttgtccccataacagccttattaatca


 


ctgcatacaaggcttgggtaagcaaacaaacaaatgatgtgccgcatcgc


 


attttaatattaaccattgcatacacagcatttggcggtccctggaaagc


 


cactgttctacggatcatgcattttgtggtacgcctgtccggttttggcc


 


ttattgtggtttggtgctggcgagtacatgatgcgtcgtccgctggcggt


 


gctcgtctccattgcgctgcccacgctgtttctctgctgggtcgatgtcg


 


tcgctattggcgccggcacatgggacatttcgctggccacaagcaccggc


 


aagttcgtcgtgccccacctgcccgtggaggaattcatgttctttgcgct


 


aattaataccgttttggtatttggtacgtgtgcgatcgatcgcacgatgg


 


cgatcctccacctgttcaaaaacaagagtccttatcagcgcccataccag


 


cacagcaagtcgttcctccaccagatcctcgagatgacctgggccttctg


 


tttacccgaccaagtgctgcattcagacacattccacgacctgtccgtca


 


gctgggacatcctgcgcaaggcctccaagtccttttacacggcctctgct


 


gtctttcccggcgacgtgcgccaagagctcggtgtgctatacgccttttg


 


cagagccacggacgatctctgcgacaacgagcaggtccctgtgcagacgc


 


gaaaggagcagctgatactgacacatcagttcgtcagcgatctgtttggc


 


caaaagacaagcgcgccgactgccattgactgggacttttacaacgacca


 


actgcctgcctcgtgcatctctgccttcaagtcgttcacccgtttgcgcc


 


atgtgctggaagctggagccatcaaggaactgctcgacgggtacaagtgg


 


gatttggagcgtcgctccatcagggatcaggaggatctcagatattactc


 


agcttgtgtcgccagcagtgttggtgaaatgtgcactcgcatcatactgg


 


cccacgccgacaagcccgcctcccgccagcaaacacagtggatcattcag


 


cgtgcgcgtgaaatgggtctggtactccaatatacaaacattgcaagaga


 


cattgtcaccgacagcgaggaactgggcagatgctacctgcctcaggatt


 


ggcttaccgagaaggaggtggcgctgattcaaggcggccttgcccgagaa


 


attggcgaggagcgattgctctcactgtcgcatcgcctcatctaccaggc


 


agacgagctcatggtggttgccaacaagggcatcgacaagctgcccagcc


 


attgtcaaggcggcgtgcgtgcggcctgcaacgtctatgcttccattggc


 


accaagctcaagtcttacaagcaccactatcccagcagagcacatgtcgg


 


caattcgaaacgagtggaaattgctcttcttagcgtatacaacctttaca


 


ccgcgccaattgcgactagtagtaccacacattgcagacagggaaaaatg


 


agaaatctaaataccatttaa


 


               mlltymevhlyytlpvlgvlswlsrpyytatdalk


 


fkfltlvafttasawdnyivyhkawsycptcytavigyvpleeymffiim


 


tlltvaftnlvmrwhlhsffirpetpvmqsvlvrlvpitallitaykawh


 


lavpgkplfygscilwyacpvlallwfgageymmrrplavlvsialptlf


 


lcwvdvvaigagtwdislatstgkfvvphlpveefmffalintvlvfgtc


 


aidrtmailhlfknkspyqrpyqhsksflhqilemtwafclpdqvlhsdt


 


fhdlsvswdilrkasksfytasavfpgdvrqelgvlyafcratddlcdne


 


qvpvqtrkeqlilthqfvsdlfgqktsaptaidwdfyndqlpascisafk


 


sftrlrhvleagaikelldgykwdlerrsirdqedlryysacvassvgem


 


ctriilahadkpasrqqtqwiiqraremglvlqytniardivtdseelgr


 


cylpqdwltekevaliqgglareigeerllslshrliyqadelmvvankg


 


idklpshcqggvraacnvyasigtklksykhhypsrahvgnskrveiall


 


svynlytapiatsstthcrqgkmrnlnti






Alternatively, pMB4599 was also used as a template for PCR amplification using MO4318, MO4643, MO4644, and MO4639 and








MO4318
5′-GTAAAACGACGGCCAGT


 


MO4643
5′-CACACGGTCTCATGCCAAGCCTTGTATGCAGTGATTAA


 


MO4639
5′-CCACTGTGTTTGCTGGCGG


 


MO4644
5′-CACACGGTCTCTGGCATTTGGCGGTCCCTGGAAA






producing fragments of 0.5 and 0.95 kb, that were subsequently cleaved with Acc65I and BsaI, and BsaI and PpuMI, respectively. These fragments were ligated to pMB4599 that had been digested with Acc65I and PpuMI, yielding pMB4613, harboring intronless carRP. The 1.85 kb XbaI-MluI fragment from pMB4613 can be inserted into NheI- and MluI-cleaved pMB4603 to yield pCarRPdelI.

1B: Production of pMB4638 (tef1 p-carB ADE1), encoding phytoene dehydrogenase: Intron-containing carB was amplified from M. circinelloides (ATCC 90680) genomic DNA using MO4530 and MO4542:








MO4530
5′-CACAAACGCGTTTAAATGACATTAGAGTTATGAAC


 


MO4542
5′-CACAATCTAGACACAAATGTCCAAGAAACACATTGTC






and the resulting 1.9 kb fragment was phosphorylated with T4 polynucleotide kinase and blunt-end ligated into pBS-SKII- cleaved with EcoRV, yielding pMB4606. pMB4606 was then used as a template for PCR amplification using MO4318 and MO4648, and MO4646 and MO4647, and MO4343 and MO4645:











MO4318
5′-GTAAAACGACGGCCAGT



 



MO4648
5′-CACAAGGTCTCAAGCACGCATCCCGGAACTG



 



MO4646
5′-CACACGGTCTCAGGCATGTCGCCCTACGATGC



 



MO4647
5′-CACACGGTCTCATGCTTGCACCCACAAAGAATAGG



 



MO4343
5′-CAGGAAACAGCTATGAC



 



MO4645
5′-CACACGGTCTCTTGCCCATATACATGGTCTGAAACG






producing fragments of 0.4 and 0.85 and 0.7 kb, that were subsequently cleaved with Acc65I and BsaI, and BsaI, and BsaI and BamHI, respectively. These fragments were ligated to pBS-SKII- that had been cut with Acc65I and BamHI, yielding pMB4619, harboring intronless carB. The 1.75 kb XbaI-MluI fragment from pMB4619 was inserted into NheI- and MluI-cleaved pMB4629, yielding pMB4638. The resulting nucleic acid coding sequence and encoded CarB protein of pMB4638 are as follows:








               atgtccaagaaacacattgtcattatcggtgctgg


 


cgtgggtggcacggctacagctgctcgtttggcccgcgaaggcttcaagg


 


tcactgtggtggagaaaaacgactttggtggcggccgctgctccttgatc


 


catcaccagggccatcgctttgatcagggcccgtcgctctacctgatgcc


 


caagtactttgaggacgcctttgccgatctggacgagcgcattcaagacc


 


acctggagctgctgcgatgcgacaacaactacaaggtgcactttgacgac


 


ggtgagtcgatccagctgtcgtctgacttgacacgcatgaaggctgaatt


 


ggaccgcgtggagggcccccttggttttggccgattcctggatttcatga


 


aagagacacacatccactacgaaagcggcaccctgattgcgctcaagaag


 


aatttcgaatccatctgggacctgattcgcatcaagtacgctccagagat


 


ctttcgcttgcacctgtttggcaagatctacgaccgcgcttccaagtact


 


tcaagaccaagaagatgcgcatggcattcacgtttcagaccatgtatatg


 


ggcatgtcgccctacgatgcgcctgctgtctacagcctgttgcagtacac


 


cgagttcgctgaaggcatctggtatccccgtggcggcttcaacatggtgg


 


ttcagaagctagaggcgattgcaaagcaaaagtacgatgccgagtttatc


 


tacaatgcgcctgttgccaagattaacaccgatgatgccaccaaacaagt


 


gacaggtgtaaccttggaaaatggccacatcatcgatgccgatgcggttg


 


tgtgtaacgcagatctggtctatgcttatcacaatctgttgcctccctgc


 


cgatggacgcaaaacacactggcttccaagaaattgacgtcttcttccat


 


ttcctctactggtccatgtccaccaaggtgcctcaattggacgtgcacaa


 


catctttttggccgaggcttatcaggagagctttgacgaaatcttcaagg


 


actttggcctgccttctgaagcctccttctacgtcaatgtgccctctcgc


 


atcgatccttctgctgctcccgacggcaaggactctgtcattgtcttggt


 


gcctattggtcatatgaagagcaagacgggcgatgcttccaccgagaact


 


acccggccatggtggacaaggcacgcaagatggtgctggctgtgattgag


 


cgtcgtctgggcatgtcgaatttcgccgacttgattgagcatgagcaagt


 


caatgatcccgctgtatggcagagcaagttcaatctgtggagaggctcaa


 


ttctgggtttgtctcatgatgtgcttcaggtgctgtggttccgtcccagc


 


acaaaggattctaccggtcgttatgataacctattctttgtgggtgcaag


 


cacgcatcccggaactggtgttcccattgtccttgcaggaagcaagctca


 


cctctgaccaagttgtcaagagctttggaaagacgcccaagccaagaaag


 


atcgagatggagaacacgcaagcacctttggaggagcctgatgctgaatc


 


gacattccctgtgtggttctggttgcgcgctgccttttgggtcatgttta


 


tgttcttttacttcttccctcaatccaatggccaaacgcccgcatctttt


 


atcaataatttgttacctgaagtattccgcgttcataactctaatgtcat


 


ttaa


 


               mskkhiviigagvggtataarlaregfkvtvvekn


 


dfgggrcslihhqghrfdqgpslylmpkyfedafadlderiqdhlellrc


 


dnnykvhfddgesiqlssdltrmkaeldrvegplgfgrfldfmkethihy


 


esgtfialkknfesiwdlirikyapeifrlhlfgkiydraskyfktkkmr


 


maftfqtmymgmspydapavysllqytefaegiwyprggfnmvvqkleai


 


akqkydaefiynapvakintddatkqvtgvtlenghiidadavvcnadlv


 


yayhnllppcrwtqntlaskkltsssisfywsmstkvpqldvhniflaea


 


yqesfdeifkdfglpseasfyvnvpsridpsaapdgkdsvivlvpighmk


 


sktgdastenypamvdkarkmvlavierrlgmsnfadlieheqvndpavw


 


qskfnlwrgsilglshdvlqvlwfrpstkdstgrydnlffvgasthpgtg


 


vpivlagskltsdqvvksfgktpkprkiementqapleepdaestfpvwf


 


wlraafwvmfmffyffpqsngqtpasfinnllpevfrvhnsnvi






1C. Production of pMB4660 (tef1 p-carB URA3) encoding phytoene dehydrogenase: The 4.3 kb XhoI-NotI fragment and the 1.8 kb NotI-SpeI fragment from pMB4638 were ligated to the 1.9 kb BsaI- and SpeI-cleaved URA3 gene generated by PCR amplification of Y lipolytica genomic DNA using MO4684 and MO4685 to create pMB4660:










MO4684
5′-CATTCACTAGTGGTGTGTTCTGTGGAGCATTC



 



MO4685
5′-CACACGGTCTCATCGAGGTGTAGTGGTAGTGCAGTG






The resulting nucleic acid coding sequence and encoded CarB(i) protein of pMB4660 are as follows:








               atgtccaagaaacacattgtcattatcggtgctgg


 


cgtgggtggcacggctacagctgctcgtttggcccgcgaaggcttcaagg


 


tcactgtggtggagaaaaacgactttggtggcggccgctgctccttgatc


 


catcaccagggccatcgctttgatcagggcccgtcgctctacctgatgcc


 


caagtactttgaggacgcctttgccgatctggacgagcgcattcaagacc


 


acctggagctgctgcgatgcgacaacaactacaaggtgcactttgacgac


 


ggtgagtcgatccagctgtcgtctgacttgacacgcatgaaggctgaatt


 


ggaccgcgtggagggcccccttggttttggccgattcctggatttcatga


 


aagagacacacatccactacgaaagcggcaccctgattgcgctcaagaag


 


aatttcgaatccatctgggacctgattcgcatcaagtacgctccagagat


 


ctttcgcttgcacctgtttggcaagatctacgaccgcgcttccaagtact


 


tcaagaccaagaagatgcgcatggcattcacgtttcagaccatgtatatg


 


ggcatgtcgccctacgatgcgcctgctgtctacagcctgttgcagtacac


 


cgagttcgctgaaggcatctggtatccccgtggcggcttcaacatggtgg


 


ttcagaagctagaggcgattgcaaagcaaaagtacgatgccgagtttatc


 


tacaatgcgcctgttgccaagattaacaccgatgatgccaccaaacaagt


 


gacaggtgtaaccttggaaaatggccacatcatcgatgccgatgcggttg


 


tgtgtaacgcagatctggtctatgcttatcacaatctgttgcctccctgc


 


cgatggacgcaaaacacactggcttccaagaaattgacgtcttcttccat


 


ttccttctactggtccatgtccaccaaggtgcctcaattggacgtgcaca


 


acatctttttggccgaggcttatcaggagagctttgacgaaatcttcaag


 


gactttggcctgccttctgaagcctccttctacgtcaatgtgccctctcg


 


catcgatccttctgctgctcccgacggcaaggactctgtcattgtcttgg


 


tgcctattggtcatatgaagagcaagacgggcgatgcttccaccgagaac


 


tacccggccatggtggacaaggcacgcaagatggtgctggctgtgattga


 


gcgtcgtctgggcatgtcgaatttcgccgacttgattgagcatgagcaag


 


tcaatgatcccgctgtatggcagagcaagttcaatctgtggagaggctca


 


attctgggtttgtctcatgatgtgcttcaggtgctgtggttccgtcccag


 


cacaaaggattctaccggtcgttatgataacctattctttgtgggtgcaa


 


gcacgcatcccggaactggtgttcccattgtccttgcaggaagcaagctc


 


acctctgaccaagttgtcaagagctttggaaagacgcccaagccaagaaa


 


gatcgagatggagaacacgcaagcacctttggaggagcctgatgctgaat


 


cgacattccctgtgtggttctggttgcgcgctgccttttgggtcatgttt


 


atgttcttttacttcttccctcaatccaatggccaaacgcccgcatcttt


 


tatcaataatttgttacctgaagtattccgcgttcataactctaatgtca


 


tttaa


 


               mskkhiviigagvggtataarlaregfkvtvvekn


 


dfgggrcslihhqghrfdqgpslylmpkyfedafadlderiqdhlellrc


 


dnnykvhfddgesiqlssdltrmkaeldrvegplgfgrfldfmkethihy


 


esgtlialkknfesiwdlirikyapeifrlhlfgkiydraskyfktkkmr


 


maftfqtmymgmspydapavysllqytefaegiwyprggfnmvvqkleai


 


akqkydaefiynapvakintddatkqvtgvtlenghiidadavvcnadlv


 


yayhnllppcrwtqntlaskkltsssisfywsmstkvpqldvhniflaea


 


yqesfdeifkdfglpseasfyvnvpsridpsaapdgkdsvivlvpighmk


 


sktgdastenypamvdkarkmvlavierrlgmsnfadlieheqvndpavw


 


qskfnlwrgsilglshdvlqvlwfrpstkdstgrydnlffvgasthpgtg


 


vpivlagskltsdqvvksfgktpkprkiementqapleepdaestfpvwf


 


wlraafwvmfmffyffpqsngqtpasfinnllpevfrvhnsnvi






1D. Production of pMB4637 and pTef-HMG encoding a truncated HMG1. For production of a truncated variant of the HMG-CoA reductase gene, which also encodes a 77 amino acid leader sequence derived from S. cerevisiae, the following oligonucleotides are synthesized:








PRIMER O
5′-TTCTAGACACAAAAATGGCTGCAGACCAATTGGTGA


 


PRIMER P
5′-CATTAATTCTTCTAAAGGACGTATTTTCTTATC


 


PRIMER Q
5′-GTTCTCTGGACGACCTAGAGG


 


MO4658
5′-CACACACGCGTACACCTATGACCGTATGCAAAT






Primers O and P are used to amplify a 0.23 kb fragment encoding Met-Ala followed by residues 530 to 604 of the Hmg1 protein of S. cerevisiae, using genomic DNA as template. Primers Q and MO4658 are used to amplify a 1.4 kb fragment encoding the C-terminal 448 residues of the Hmg1 protein of Y. lipolytica, using genomic DNA as template. These fragments are ligated to the appropriate cloning vector, and the resultant plasmids, designated pOP and pQMO4658, are verified by sequencing. The OP fragment is liberated with XbaI and AseI, and the QMO4658 fragment is liberated with MaeI and MluI. These fragments are then ligated to the ADE1 TEF1p expression vector pMB4629 cut with XbaI and MluI to produce pTefHMG.

Alternatively, the native HMG1 gene from Y. lipolytica may be modified without S. cerevisiae sequences as described in the table above using primers MO4658 (described above) and MO4657, to create pMB4637:








MO4657
5′-CACACTCTAGACACAAAAATGACCCAGTCTGTGAAGGTGG






The resulting nucleic acid coding sequence and encoded Hmg1trune protein of pMB4637 are as follows:








               atgacccagtctgtgaaggtggttgagaagcacgt


 


tcctatcgtcattgagaagcccagcgagaaggaggaggacacctcttctg


 


aagactccattgagctgactgtcggaaagcagcccaagcccgtgaccgag


 


acccgttctctggacgacctagaggctatcatgaaggcaggtaagaccaa


 


gcttctggaggaccacgaggttgtcaagctctctctcgagggcaagcttc


 


ctttgtatgctcttgagaagcagcttggtgacaacacccgagctgttggc


 


atccgacgatctatcatctcccagcagtctaataccaagactttagagac


 


ctcaaagcttccttacctgcactacgactacgaccgtgtttttggagcct


 


gttgcgagaacgttattggttacatgcctctccccgttggtgttgctggc


 


cccatgaacattgatggcaagaactaccacattcctatggccaccactga


 


gggttgtcttgttgcctcaaccatgcgaggttgcaaggccatcaacgccg


 


gtggcggtgttaccactgtgcttactcaggacggtatgacacgaggtcct


 


tgtgtttccttcccctctctcaagcgggctggagccgctaagatctggct


 


tgattccgaggagggtctcaagtccatgcgaaaggccttcaactccacct


 


ctcgatttgctcgtctccagtctcttcactctacccttgctggtaacctg


 


ctgtttattcgattccgaaccaccactggtgatgccatgggcatgaacat


 


gatctccaagggcgtcgaacactctctggccgtcatggtcaaggagtacg


 


gcttccctgatatggacattgtgtctgtctcgggtaactactgcactgac


 


aagaagcccgcagcgatcaactggatcgaaggccgaggcaagagtgttgt


 


tgccgaagccaccatccctgctcacattgtcaagtctgttctcaaaagtg


 


aggttgacgctcttgttgagctcaacatcagcaagaatctgatcggtagt


 


gccatggctggctctgtgggaggtttcaatgcacacgccgcaaacctggt


 


gaccgccatctaccttgccactggccaggatcctgctcagaatgtcgagt


 


cttccaactgcatcacgctgatgagcaacgtcgacggtaacctgctcatc


 


tccgtttccatgccttctatcgaggtcggtaccattggtggaggtactat


 


tttggagccccagggggctatgctggagatgcttggcgtgcgaggtcctc


 


acatcgagacccccggtgccaacgcccaacagcttgctcgcatcattgct


 


tctggagttcttgcagcggagctttcgctgtgttctgctcttgctgccgg


 


ccatcttgtgcaaagtcatatgacccacaaccggtcccaggctcctactc


 


cggccaagcagtctcaggccgatctgcagcgtctacaaaacggttcgaat


 


atttgcatacggtcatag


 


               mtqsvkvvekhvpiviekpsekeedtssedsielt


 


vgkqpkpvtetrslddleaimkagktklledhevvklslegklplyalek


 


qlgdntravgirrsiisqqsntktletsklpylhydydrvfgaccenvig


 


ymplpvgvagpmnidgknyhipmattegclvastmrgckainagggvttv


 


ltqdgmtrgpcvsfpslkragaakiwldseeglksmrkafnstsrfarlq


 


s1hstlagnllfirfrtttgdamgmnmiskgvehslavmvkeygfpdmdi


 


vsysgnyctdkkpaainwiegrgksvvaeatipahivksvlksevdalve


 


lnisknligsamagsvggfnahaanlvtaiylatgqdpaqnvessncitl


 


msnvdgnllisvsmpsievgtigggtilepqgamlemlgvrgphietpga


 


naqq1ariiasgvlaaelslcsalaaghlvqshmthnrsqaptpakqsqa


 


dlqrlqngsnicirs






1E. Production of pMB4692 (URA3 tef1p-crtZ) encoding carotene hydroxylase. The following carotene hydroxylase (CrtZ) ORF sequence was synthesized; based on protein sequence of Novosphingobium aromaticivorans, using Y. lipolytica codon bias:







5′-


 


ttctagacacaaaaatgggtggagccatgcagaccctcgctgctatcctg


 


atcgtcctcggtacagtgctcgctatggagtttgtcgcttggtcttctca


 


taagtatatcatgcatggcttcggatggggatggcatagagaccatcacg


 


agccccatgagggatttcttgagaagaatgacttatacgccatcgttggc


 


gctgccctctcgatactcatgtttgccctcggctctcccatgatcatggg


 


cgctgacgcctggtggcccggaacctggatcggactcggtgtcctcttct


 


atggtgtcatctataccctcgtgcaccacggtctggtgcaccaacgatgg


 


tttagatgggtgcctaaacgaggttacgccaaacgactcgtgcaggccca


 


taagctgcaccacgccaccattggcaaggaaggaggcgtctcattcggtt


 


tcgtgttcgcccgagatcccgccgttctgaagcaggagcttcgagctcaa


 


cgagaagcaggtatcgccgtgctgcgagaggctgtggacggctagacgcg


 


t






This sequence was cleaved using XbaI and MluI and ligated, along with an Acc651-NheI TEF1 promoter fragment from pMB4629, to pMB4662 cut with Acc65I and MluI to produce pMB4692. The nucleic acid coding sequence is depicted in bold underline above. The resulting encoded crtZ protein of pMB4692 is as follows:







               mggamqtlaailivlgtvlamefvawsshkyimhg


 


fgwgwhrdhhephegflekndlyaivgaalsilmfalgspmimgadawwp


 


gtwiglgvlfygviytlvhdglvhqrwfrwvpkrgyakrlvqahklhhat


 


igkeggvsfgfvfardpavlkqelraqreagiavlreavdg






1F. Production of pMB4698 (ADE1 tef1p-crtW), encoding carotene ketolase. The following carotene ketolase (CrtW) ORF sequence was synthesized, based on protein sequence of an environmental sequence isolated from the Sargasso Sea (Genbank accession AACY01034193.1):







5′-


 


ttctagacacaaaaatgactcgatctatttcctggccttccacctactgg


 


cacctccagccctcctgttcttcttgggtcgcaaacgaattctctcctca


 


agcccgaaaaggtctcgtcctcgctggtctcattggttccgcttggctgc


 


ttactctcggacttggcttttcccttcccctccatcaaacgagctggctt


 


ctcatcggttgtctcgttctccttagatctttcctgcacaccggactttt


 


tatcgttgcccatgacgctatgcacgcttctcttgttcctgaccaccctg


 


gccttaaccgttggattggacgtgtctgtcttctcatgtatgctggactc


 


tcctacaaaagatgctgccgaaatcaccgtcgacaccaccaagcccctga


 


aacagttgaagaccctgactaccaacgatgcactaacaacaatatcctcg


 


actggtacgttcactttatgggaaattacctcggatggcaacaattgctt


 


aatctctcttgcgtttggctcgctctcaccttccgtgtttctgactactc


 


tgctcaattcttccacctgctccttttctctgtccttcctctcatcgtct


 


cctcctgtcaactcttcctcgtgggaacctggctgccacaccgacgaggc


 


gctactactcgacccggcgttaccactcgatccctgaacttccaccctgc


 


tctttccttcgctgcttgctaccacttcggttaccaccgtgaacaccatg


 


aatctccctctactccttggttccaacttcctaaactccgagaaggttct


 


ctcatctaaacgcgt






This sequence was cleaved using XbaI and MluI and ligated to pMB4629 cut with NheI and MluI to produce pMB4698. The nucleic acid coding sequence is depicted in bold underline above. The resulting encoded crtW protein of pMB4698 is as follows:

mtrsiswpstywhlqpscsswvanefspqarkglvlagligsawlltlglgfslplhqtswlligclvllrsflhtglfiva hdamhaslvpdhpglnrwigrvcllmyaglsykrccmhnhhqapetvedpdyqrannnildwyvhfmgnylgwqqllnlscvwlal tfrvsdysaqffhlllfsvlplivsscqlflvgtwlphrrgattrpgvttrslnfhpalsfaacyhfgyhrehhespstpwfqlpklregsli
Example 2
Engineering Yarrowia lipolytica for Increased Carotenoid Production
2A. Production of Y. lipolytica expressing geranylgeranylpyrophosphate synthase and phytoene dehydrogenase: MF350 (MATB ura2-21 leu2-35 ade1) was transformed with pMB4591 (tef1p-GGSI) that had been cleaved upstream of URA5 with SspI; a Ura+ transformant carrying the plasmid at the ura2 locus was identified and named MF364. It was subsequently transformed with pMB4638 (tef1p-carB) that had been cleaved at ADE1 with SspI and a prototrophic transformant was chosen that harbored the plasmid at the ade1 locus. This strain was named MF502.
2B. Production of Y. lipolytica expressing geranylgeranylpyrophosphate synthase, phytoene dehydrogenase and phytoene synthase/lycopene cyclase MF502 was transformed with pMB4628 (tef1p-carRP) that had been treated with SspI. Nine prototrophic colonies were chosen that were uncolored, orange, or very orange on the transformation plate (YNB agar with 1% glucose and 0.1% glutamate [YNBglut]) after two to three days of growth. Two, MF597 and MF600 (the very orange ones), produced greater than 4 mg carotene per g dry cell weight (DCW) after four days of growth in YPD at 30° C. Southern analysis reveals a different single KpnI-HindIII band in genomic DNA from MF597 and MF600, neither of which suggested that homologous integration occurred at leu2-270.
2C. Production of Y lipolytica expressing phytoene synthase/lycopene cyclase and phytoene dehydrogenase: ATCC201249 (MATA ura3-302 leu2-270 lys8-11) was transformed with SspI-cleaved pMB4628. Hundreds of Leu+ colonies were pooled, re-grown, and transformed with pMB4660 (tef1p-carB) that had been cleaved upstream of URA3 with SalI. One colony that was noticeably yellow after 5 days at 30° C. on YNBglut plus 0.6 mM lysine was selected, named MF447, and found to produce 0.2 mg carotene per gram dry cell weight after 4 days of growth in YPD.
MF447 was challenged with 1 g/L 5-fluoroorotic acid and Ura− segregants selected. Surprisingly, they were all found to retain the identical yellow appearance of their parent, implying that the loss of a functional URA3 gene did not coincide with the loss of a functional CarB enzyme. Southern analysis demonstrates that two fragments from a KpnI-HindIII digest of MF447 DNA contain URA3p-hybridizing sequences, only one of which also hybridizes to carB. The other is absent in MF578, the Ura3− segregant chosen for further manipulation. Plasmid rescue and analysis of the DNA sequence encompassing the carRP intron in strains MF447, MF597 (example 2c), and MF600 (example 2c) revealed that exons 1 and 2 were contiguous and were each separated by an intron sequence that lacked the original internal SspI site (present in pMB4628).
2D. Production of Y. lipolytica expressing phytoene synthase/lycopene cyclase, phytoene dehydrogenase and geranylgeranylpyrophosphate synthase: MF578 was transformed with pMB4683 (tef1p-GGS1) that had been cleaved with SalI (upstream of URA3) or with StuI (within the GGS1 ORF). Ura+Leu+ colonies in both cases appeared bright orange on YNBglut+Lys and on YPD, and several produced greater than 4 mg carotene per gram of dry cell weight when grown as above. One, MF633, contained a single copy of the plasmid at the GGS1 locus, as inferred from Southern analysis. The others arose by non-homologous or more complex integrations.
2E. Production of Y. lipolytica expressing phytoene synthase/lycopene cyclase, phytoene dehydrogenase and geranylgeranylpyrophosphate synthase: MF364 is crossed with MF578, and spores from the resulting diploid are plated on YPD for two to three days at 30° C. Orange Leu+ Ade− Ura− colonies are screened for the presence of tefp-carB, tefp-carRP, and tefp-GGS1 by PCR, and for high carotenoid (>4 mg/g dry cell weight) production after growth in YPD liquid medium. Colonies meeting these criteria, as well as displaying resistance to 5-fluorootic acid, an indication that they harbor the ura3-302 allele, are chosen for further studies and hereafter referred to as GBRPua strains. Such a strain is selected for further analysis and modification.
Example 3
Extraction of Carotenoids from Yarrowia lipolytica Cells
Shake-flask testing of generated strains was conducted using YPD medium (1% yeast extract, 2% peptone, 2% glucose). 20 ml cultures in 125 ml flasks were grown at 30° C. Y. lipolytica cells were harvested from 72-96 hour cultures, and extractions were performed to determine carotenoid form and quantity. 1.8 ml of culture was placed into an Eppendorf tube. Cells were pelleted and washed twice with 1 ml H2O. After the second wash, the resuspended cells were transferred to a pre-weighed snap-cap tube with a hole poked in the top, and the cells were lyophilized overnight. After drying to completion, the tube was weighed in order to calculate dry cell weight. 0.25 ml from the same shake flask culture was placed into a 2 ml screw-cap tube for carotenoid extraction. Cells were pelleted and the supernatant was aspirated. Pelleted cells may be frozen at −80° C. and stored. An equal volume of cubic zirconia beads was added to cell pellets, along with 1 ml ice-cold extraction solvent (a 50/50 v/v mix of hexane and ethyl acetate containing 0.01% butylhydroxytoluene (BHT)). The mixture was then agitated (Mini-BeadBeater-8, BioSpec Products, Inc.) at maximum speed for 5 minutes at 4° C. The mixture was then spun at maximum speed for 1 minute, and the supernatant was collected and deposited in a cold 16 ml glass vial. The remaining cell debris was re-extracted at least three times, without the addition of zirconia beads; all supernatants were pooled in the 16 ml glass vial. Following extraction, the glass vial was spun for 5 minutes at 2000 rpm at 4° C. in a Sorvall tabletop centrifuge, and the supernatant was transferred to a new cold 16 ml glass vial. A Speed Vac was used to concentrate the supernatant (room temperature in dark), and the samples were stored at −20° C. or −80° C. until immediately before HPLC analysis. Prior to HPLC analysis, the samples were resuspended in 1 ml ice-cold solvent and then transferred to a cold amber vial. Throughout the protocol, care was taken to avoid contact with oxygen, light, heat, and acids.
Example 4
Quantification of Carotenoid Production by HPLC
For carotenoid analysis, samples were resuspended in ice-cold extraction solvent (a 50/50 v/v mix of hexane and ethyl acetate containing 0.01% butylhydroxytoluene (BHT)). An Alliance 2795 HPLC (Waters) equipped with a Waters XBridge C18 column (3.5 μm, 2.1×50 mm) and Thermo Basic 8 guard column (2.1×10 mm) was used to resolve carotenoid at 25° C.; authentic carotenoid samples were used as standards. The mobile phases and flow rates are shown below (Solvent A=Ethyl Acetate; Solvent B=Water; Solvent C=Methanol; Solvent D=Acetonitrile). The injection volume was 10 μL. The detector is a Waters 996 photodiode array detector. The retention times for lipophilic molecules include astaxanthin (1.159 min), zeaxanthin (1.335), β-apo-8′-carotenal (2.86 min), ergosterol (3.11 min), lycopene (3.69 min), β-Carotene (4.02 min), and phytoene (4.13 min). Astaxanthin, zeaxanthin, β-apo-8′-carotenal, lycopene and β-Carotene are detected at 475 nm, whereas ergosterol and phytoene were detected at 286 nm.







TABLE 28







Retention Times for Lipophilic Molecules













Time (min)
Flow (mL/min)
% A
% B
% C
% D
Curve

















0.50
0.0
20.0
0.0
80.0



3.00
1.00
20.0
0.0
0.0
80.0
6


4.50
1.00
80.0
0.0
20.0
0.0
6


5.50
1.00
0.0
0.0
60.0
40.0
6


6.50
1.00
0.0
0.0
80.0
20.0
6


7.50
1.00
0.0
0.0
100.0
0.0
6


8.50
1.00
0.0
0.0
100.0
0.0
6


9.50
1.00
0.0
20.0
0.0
80.0
6


10.50
0.50
0.0
20.0
0.0
80.0
6









Example 5
Expression of a Truncated Form of HMG-CoA Reductase Results in Increased Carotenoid Production
In order to increase carotenoid production, carbon flow through the isoprenoid pathway is enhanced by introducing a truncated variant of the HMG-CoA reductase gene.
In one approach, a truncated variant of the HMG-CoA reductase gene which also encodes a 77 amino acid leader sequence derived from S. cerevisiae Hmg1 is introduced into a GRPBua strain (described in Example 2E above). Plasmid pTefHMG can be cleaved with SnaBI, BbvCI, or Bsu361 to direct integration at the ade1 locus, or with BamHI to direct integration at the HMG1 locus, or with EcoRV to promote random integration, in the GRPBua strains, restoring them to adenine prototrophy. Resulting Ade+ transformants are screened for increased carotenoid production.
Alternatively, the native HMG1 gene from Y. lipolytica may be modified without S. cerevisiae sequences as described in Example I D above, to create pMB4637. This plasmid can be digested as described for pTefHMG and transformed into GRPBua strains, and resulting transformants screened as described for increased carotenoid production.
In still another approach, a truncated variant of the N. crassa HMG-CoA reductase gene may be utilized and introduced into Y. lipolytica strains. In order to generate a plasmid suitable for expression of the heterologous HMG-CoA reductase, p641 P (Yeast 2001; 18 (2001): 97-113) is modified by replacing the ICL1 promoter with the GPD promoter, and by the addition of sequences conferring resistance to phleomycin. Y. lipolytica genomic DNA is amplified with two primers.







GPDdist:


5′ CACACGGTacctgtaggttgggttgggtg


 


GPDprox:


5′ CACACGGATCCtgtttaattcaagaatgaatatagagaagagaag,






and the resulting fragment (0.7 kb) is cleaved with BamHI and KpnI, and ligated to BamHI- and KpnI-cleaved p641P, creating the plasmid “p641 Pgpd”. The ble gene under the control of the A. nidulans GPD promoter is then excised from pBCphleo (Silar, Fungal Genetics Newsletter 42:73) as a 3.2 kb BclI-BamHI fragment and inserted into the unique BamHI site of “p641 Pgpd”, in the orientation that preserves the BamHI site proximal to the GPD promoter, to create “p641 Pgpdble”,

N. crassa genomic DNA is amplified with two primers:









Neuhmg fwd:



5′ CACACGGATCCACATCAACAatggcatctgccacccttcccc



 



Neuhmg rev:



5′ CACACGGATCcaagtgctgacgcggaacttg,






and the resulting fragment is cleaved with BamHI and inserted into BamHI-digested “p641 Pgpdble” in the correct orientation. The resulting plasmid, “pZg”, contains sequences encoding a truncated cytosolic catalytic domain of hydroxymethylglutaryl-CoA reductase from N. crassa (Genbank accession: XP—324892) under the control of the constitutive GPD promoter. This plasmid can be introduced into the Y. lipolytica strain created in Example 2E above, and transformants are selected by their resistance to phleomycin (100 μg/ml). Resulting transformants are tested for n-carotene production, as described above.

Example 6
Introduction of Heterologous carotene hydroxylase and carotene ketolase Genes into Y.  lipolytica strains producing carotenoid for production of astaxanthin
For introduction of carotene hydroxylase and carotene ketolase into carotenoid producting Y. lipolytica, pMB4692 and pMB4698, described as in Example 1E and 1F above, can be sequentially introduced into the GRPBua strain (described in Example 2E). For the introduction of pMB4692, the plasmid may be cleaved with SalI or BsrGI to direct integration at the ura3 locus, or with XbaI to promote random integration, selecting for uracil prototrophy. GRPBua Ura+ transformants harboring pMB4692 are screened for zeaxanthin production in YPD. Zeaxanthin-producing cells are transformed with pMB4698 (which can be cleaved with PpuMI, SspI or BbvC1 to direct integration at the ade1 locus, or with EcoRV to promote random integration) and prototrophic colonies are screened for astaxanthin production.
Alternatively, the order of plasmid transformation may be reversed wherein pMB4698 is transformed first and transformants are selected for adenine prototrophy. GRPBua Ade+ transformants harboring pMB4698 are screened for canthaxanthin production. Canthaxanthin-producing GRPBua[pMB4698] cells are transformed with pMB4692 and prototrophic colonies are screened for astaxanthin production.
In another approach, the carotenoid ketolase and carotenoid hydroxylase genes from P. marcusii can be introduced into the strains described in Example 2 above, in order to convert β-carotene into astaxanthin. P. marcusii genomic DNA is amplified with two primers.









CrtZfwd:



5′ CACACCGTCTCAAatgaccaatttcctgatcgtcgtc



 



CrtZrev:



5′ CACACAGATCtcacgtgcgctcctgcgcc,






and the resulting fragment is cleaved with BsmBI, modified with the Klenow fragment of DNA polymerase, and cleaved with BglII. This fragment is inserted into PmlI- and BamHI-cleaved pINA1269 (J. Mol. Microbiol. Biotechnol. 2 (2000): 207-216), containing the hp4d promoter, the XPR2 terminator, the selectable LEU2 gene, and sequences necessary for selection and propagation in E. coli. The resulting plasmid “pA” contains sequences encoding carotene hydroxylase from P. marcusii (crtZ gene) (Genbank accession: CAB56060.1) under the control of the hp4d promoter.

“pYEG1TEF” is modified by substituting the LIP2 terminator for the XPR2 terminator as follows. pINAl291 is digested with AvrII, modified with the Klenow fragment of DNA polymerase, and cleaved with EcoRI, and the small LIP2t containing fragment is ligated to “pYEG1TEF” that has been digested with SacII, modified with T4 DNA polymerase in the presence of dNTP, and cleaved with EcoRI. The resulting plasmid is named “pYEGITEF-LIP2t”.
In order to amplify the carotenoid ketolase gene, P. marcusii genomic DNA is amplified with two primers.









CrtWfwd:



5′ CACACCCTAGGCCatgagcgcacatgccctgc



 



CrtWrev:



5′ CACACAAGCTTtcatgcggtgtcccccttg,






and the resulting fragment is cleaved with AvrII and HindIII, and inserted into AvrII- and HindIII II-cleaved “pYEG1TEF-LIP2t”. The resulting plasmid, “pBt”, contains sequences encoding the carotene ketolase (crtW gene) (Genbank accession: CAB56059.1) under the control of the constitutive TEF1 promoter.

In order to combine the two expression cassettes into a single plasmid, “pBt” is cleaved with ClaI, modified with the Klenow fragment of DNA polymerase, and cleaved with EcoRI, and the crtW-containing fragment is isolated, mixed with the phosphorylated oligonucleotide adaptor pair:









5′ AATTCGCGGCCGCT



and



 



5′ AGCGGCCGCG,






cleaved with NotI, and ligated to NotI-digested “pA”. The resulting plasmid, “pABt”, contains both the TEF1p/crtW/LIP2t cassette and the hp4d/crtZ/XPR2t cassette as well as the selectable LEU2 gene.

“pABt” can be introduced into the Y. lipolytica strain described above in Example 4 (TEF1p/a1-1/XPR2t; hp4d/carRP/LIP2t; GPDp/HMGRtrunc), and transformants selected for leucine prototrophy.
Example 7
Partial Inactivation of Y. Lipolytica Erg9 Gene Encoding Squalene Synthase Results in Increased Carotenoid Production
7A. In order to partially inactivate the ERG9 gene encoding squalene synthase, the neighboring FOL3 gene is disrupted, resulting in a folinic acid requirement. This strain is then transformed with a mutagenized fragment of DNA partially spanning the two genes, and For transformants are screened for decreased squalene synthase activity.
The following oligonucleotides are synthesized:










PRIMER K
5′-CCTTCTAGTCGTACGTAGTCAGC;



 



PRIMER L
5′-CCACTGATCTAGAATCTCTTTCTGG






and used to amplify a 2.3 kb fragment from Y. lipolytica genomic DNA spanning most of the FOL3 gene, using Pfu polymerase. The resulting fragment is cleaved with XbaI and phosphorylated, then ligated into pBluescriptSK− that has been cleaved with KpnI, treated with T4 DNA polymerase (T4pol) in the presence of dNTPs, and subsequently cleaved with XbaI. The resultant plasmid, designated pBS-fol3, is then cleaved with Acc651 and EcoRI, treated with T4pol as above, and ligated to the 3.4 kb EcoRV-SpeI ADE1 fragment (treated with T4pol) from pMB4529.

The resulting plasmid, pBSfol3Δade, can be cleaved with BsiWI and XbaI to liberate a 5.5 kb fragment that is used to transform the GRBPua strains described above to adenine prototrophy. Resulting Ade+ transformants are screened for a folinic acid requirement, and for homologous integration by PCR analysis.
Strains that harbor the resultant fol3ΔADE1 allele can be transformed with a 3.5 kb DNA fragment generated by mutagenic PCR amplification using the primers:










PRIMER M
5′-GGCTCATTGCGCATGCTAACATCG;



 



PRIMER N
5′-CGACGATGCTATGAGCTTCTAGACG,






and Y. lipolytica genomic DNA as template. The resulting fragment containing the N-terminal three-quarters of the FOL3 ORF and the C-terminal nine-tenths of the ERG9 ORF is used to transform strains. The resulting Fol+Ade− transformants are screened for decreased squalene synthase activity by sensitivity to agents such as zaragozic acid, itraconazole, or fluconazole. Additionally, the resulting transformants are screened for increased carotenoid production.

7B. Alternatively, the PCR fragment produced in 7A could be cloned and altered in such a way as to remove the 3′-untranslated region of ERG9 gene. Replacement of the fol3ΔADE1 disruption by this fragment results in decreased expression of squalene synthase [Schuldiner et al. (2005), Cell 123:507-519][Muhlrad and Parker (1999), RNA 5:1299-1307], which can be confirmed as in 7A. This approach may also be used in a Fol+Ade− strain, using the ADE1 marker to disrupt the ERG9 3′-UTR.
7C. In still another approach, partially defective ERG9 alleles can be identified in S. cerevisiae using plasmid shuffling techniques [Boeke et al. (1987), Methods Enzymol. 154:164-175], and using drug sensitivities as a phenotype. Defective genes can be transferred to Y. lipolytica using standard molecular genetic techniques.
Example 8
Treatment of Y. Lipolytica Strains Producing Carotenoid with Inhibitor of an Isoprenoid Biosynthesis Competitor Polypeptide Results in Increased Carotenoid Production
Cultures produced in Example 2 are treated with the squalene synthase inhibitor, zaragozic acid (zaragozic acid at 0.5 μM) and monitored for β-carotene production, as described above.
Example 9
Constructing an Oleaginous strain of Saccharomyces cerevisiae 
The genes encoding the two subunits of ATP-citrate lyase from N. crassa, the AMP deaminase from Saccharomyces cerevisiae, and the cytosolic malic enzyme from M. circinelloides are overexpressed in S. cereviseae strains in order to increase the total lipid content. Similar approaches to enhance lipid production could be employed in other host organisms such as Xanthophyllomyces dendrorhous (Phaffia rhodozyma), using the same, homologous, or functionally similar oleaginic polypeptides.
Qiagen RNAEasy kits (Qiagen, Valencia, Calif.) are used to prepare messenger RNA from lyophilized biomass prepared from cultures of N. crassa. Subsequently, RT-PCR is performed in two reactions containing the mRNA template and either of the following primer pairs.








acl1:



1fwd:
5′ CACACGGATCCTATAatgccttccgcaacgaccg


 


1rev:
5′ CACACACTAGttaaatttggacctcaacacgaccc


 


acl2:



2fwd:
5′ CACACGGATCCAATATAAatgtctgcgaagagcatcctcg


 


2rev:
5′ CACACGCATGCttaagcttggaactccaccgcac






The resulting fragment from the acl1 reaction is cleaved with SpeI and BamHI, and that from the acl2 reaction is cleaved with BamHI and SphI, and both are ligated together into YEp24 that has been digested with NheI and SphI, creating the plasmid “p12”. The bi-directional GAL1-10 promoter is amplified from S. cerevisiae genomic DNA using the primers.







gal10:


5′ CACACGGATCCaattttcaaaaattcttactttttttttggatggac


 


gal1:


5′ CACACGGATCCttttttctccttgacgttaaagtatagagg,






and the resulting 0.67 kb fragment is cleaved with BamHI and ligated in either orientation to BamHI-digested “p12” to create “p1ga12” and “p2ga11”, containing GAL1-acl1/GAL10-acl2 and GAL10-acl1/GAL1-acl2, respectively (Genbank accession: acl1: CAB91740.2; acl2: CAB91741.2).

In order to amplify the S. cereviseae gene encoding AMP deaminase and a promoter suitable for expressing this gene, S. cerevisiae genomic DNA is amplified using two primer pairs in separate reactions:







AMD1 ORF:


AMD1FWD:


5′ CACACGAGCTCAAAAatggacaatcaggctacacagag


 


AMD1rev:


5′ CACACCCTAGGtcacttttcttcaatggttctcttgaaattg


 


GAL7p:


gal7prox:


5′ CACACGAGCTCggaatattcaactgtttttttttatcatgttgatg


 


gal7dist:


5′ CACACGGAtccttcttgaaaatatgcactctatatcttttag,






and the resulting fragment from the AMD I reaction (2.4 kb) is cleaved with SacI and AvrII, and that from the GAL7 reaction (0.7 kb) is cleaved with BamHI and SphI, and both are ligated together into YEp13 that has been digested with NheI and BamHI, creating the plasmid “pAMPD”. This plasmid carries the S. cerevisiae gene, AMDI, encoding AMP deaminase, under the control of the galactose-inducible GAL7 promoter.

Messenger RNA is prepared from lyophilized biomass of M. circinelloides, as described above, and the mRNA template is used in a RT-PCR reaction with two primers:









MAEfwd:



5′ CACACGCTAGCTACAAAatgttgtcactcaaacgcatagcaac



 



MAErev:



5′ CACACGTCGACttaatgatctcggtatacgagaggaac,






and the resulting fragment is cleaved with NheI and SalI, and ligated to XbaI- and XhoI-digested pRS413TEF (Mumberg, D. et al. (1995) Gene, 156:119-122), creating the plasmid “pTEFMAE”, which contains sequences encoding the cytosolic NADP+-dependant malic enzyme from M. circinelloides (E.C. 1.1.1.40; mce gene; Genbank accession: AY209191) under the control of the constitutive TEF1 promoter.

The plasmids “p1ga12”, “pAMPD”, and “pTEFMAE” are sequentially transformed into a strain of S. cereviseae to restore prototrophy for uracil (“p1ga12”), leucine (“pAMPD”), and histidine (“pTEFMAE”) (Guthrie and Fink Methods in Enzymology 194:1-933, 1991). The resulting transformants are tested for total lipid content following shake flask testing in either synthetic complete (SC) medium lacking uracil, leucine and histidine, as described in Example 3, or in a 2-step fermentation process. In the 2-step process, 1.5 ml of cells from an overnight 2 ml roll tube culture containing SC medium lacking uracil, leucine and histidine are centrifuged, washed in distilled water, and resuspended in 20 ml of a nitrogen-limiting medium suitable for lipid accumulation (30 g/L glucose, 1.5 g/L yeast extract, 0.5 g/L NH4Cl, 7 g/L KH2PO4, 5 g/L Na2HPO4-12H2O, 1.5 g/L MgSO4-7H2O, 0.08 g/L FeCl3-6H2O, 0.01 g/L ZnSO4-7H2O, 0.1 g/L CaCl2-2H2O, 0.1 mg/L MnSO4-5H2O, 0.1 mg/L CuSO4-5H2O, 0.1 mg/L Co(NO3)2-6H2O; pH 5.5 (J Am Oil Chem Soc 70:891-894 (1993)).
Intracellular lipid content of the modified and control S. cerevisiae strains is analyzed using the fluorescent probe, Nile Red (J Microbiol Meth (2004) 56:331-338). In brief, cells diluted in buffer are stained with Nile Red, excited at 488 nm, and the fluorescent emission spectra in the wavelength region of 400-700 nm are acquired and compared to the corresponding spectra from cells not stained with Nile Red. To confirm results from the rapid estimation method, the total lipid content is determined by gas chromatographic analysis of the total fatty acids directly transmethylesterified from dried cells, as described (Appl Microbiol Biotechnol. 2002 November; 60(3):275-80). Non-transformed S. cerevisiae strains produce 6% and 10% total lipid (dry cell weight basis) after growth in YPD and lipid accumulation medium, respectively. Yeast strains expressing the multiple oleaginic polypeptides produce 17% and 25% total lipid following growth in YPD and lipid accumulation medium, respectively.
Example 10
Introduction of Heterologous Carotene Hydroxylase into Y. Lipolytica Strains Producing Carotenoid for Production of Zeaxanthin
MF578 (tef-carRP tef-carB) was transformed with pMB4692 that had been cleaved with SalI. Several Ura+ colonies inferred to contain tef-crtZ by PCR analysis were able to produce zeaxanthin in YPD shake flasks, and in one case, all of the carotene was depleted.
The following tables are referenced throughout the description:







TABLE 1







Examples of acetyl-CoA carboxylase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
XP_410263
49097606


2
XP_329580
32418204


3
XP_386756
46124405


4
XP_367702
39972623


5
XP_501721
50548503


6
EAK99708
46440402


7
XP_457211
50413128


8
NP_982612
45184894


9
XP_449236
50293649


10
NP_593271
19114183


11
NP_014413
6324343


12
XP_455355
50310667


13
T42531
11272737


14
AAA20073
171504


15
EAL20176
50257469


16
XP_571316
58268320


17
XP_402244
49076566


18
S60200
2133343


19
BAA24410
2804173


20
P32874
1708192


21
S55089
7438088


22
NP_990836
45382859


23
CAE01471
32526576


24
AAR37018
40019048


25
NP_001...
57164283


26
NP_776649
27806341


27
CAI25271
56205878


28
XP_109883
51828611


29
NP_942134
38679971


30
NP_942131
38679960


31
NP_942135
38679974


32
NP_942136
38679977


33
AAP94122
33112885


34
NP_071529
11559962


35
2006242A
740964


36
AAS13685
42405896


37
NP_598665
48976025


38
Q13085
2493311


39
XP_548250
57091783


40
XP_314071
58385597


41
CAG08536
47226520


42
NP_724636
24586460


43
NP_610342
24586458


44
NP_001084
4501855


45
NP_446374
16758804


46
EAL63219
60465120


47
NP_921034
37533464


48
T07084
7438099


49
AAP78896
32264940


50
AAO62903
29123370


51
BAA07012
1100253


52
AAL02056
15558947


53
AAG40563
11869927


54
D86483
25293894


55
T07920
7438090


56
A57710
2130099


57
AAO62902
29123376


58
2208491A
1588584


59
T09538
7438102


60
CAC19875
12057067


61
AAP78897
32264942


62
T02235
7438095


63
AAG40564
11869928


64
E86483
25293893


65
CAC84161
20975574


66
T07081
7438097


67
CAC19876
12057069
















TABLE 2







Examples of pyruvate decarboxylase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
1QPBB
7245977


2
CAA54522
871533


3
1PYDB
515237


4
CAA28380
4109


5
1PVDB
1127233


6
CAA33709
4114


7
AAN77243
25992752


8
NP_013235
6323163


9
Q6FJA3
57012668


10
S36363
486942


11
Q12629
52788279


12
AAP75898
37359468


13
S70684
2131152


14
NP_011601
6321524


15
AAQ73618
34500072


16
NP_983270
45185554


17
AAF78895
8745337


18
CAB65554
6689662


19
AAP75899
37359470


20
NP_982469
45184751


21
CAA97091
1945321


22
S50700
1086157


23
XP_446491
50288125


24
XP_462338
50427451


25
AAC03164
17066784


26
EAK96569
46437219


27
XP_457131
50412425


28
AAC03165
2734883


29
XP_459224
50421349


30
CAH56494
52673248


31
XP_502647
50550349


32
NP_010203
6320123


33
BAA04886
1786148


34
XP_449074
50293325


35
EAL04098
46444826


36
CAD60727
27803024


37
T38759
25777585


38
XP_331173
32421459


39
NP_594083
19114995


40
XP_401609
49075036


41
XP_390010
46136637


42
XP_409025
49095128


43
NP_984350
45188127


44
AAD16178
4323053


45
P87208
2501326


46
EAL18331
50255598


47
XP_567475
58260130


48
AAM73540
21666011


49
AAM73539
21666009


50
XP_502508
50550071


51
CAA93158
1177659


52
XP_412533
49123327


53
P51844
1706333


54
XP_455842
50311631


55
CAA61155
3688422


56
XP_444902
50284947


57
CAA47319
4118
















TABLE 3







Examples of isocitrate dehydrogenase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
O13285
3023996


2
EAK91676
46432179


3
O13285
3023996


4
EAK94305
46434909


5
XP_451683
50303483


6
XP_459772
50422415


7
O13294
27805482


8
XP_460289
50423413


9
XP_390523
46137663


10
XP_367343
39971905


11
XP_323176
32405126


12
XP_445447
50286037


13
AAK76730
15027826


14
NP_010217
6320137


15
NP_984921
45190667


16
AAK76731
15027827


17
P79089
3023999


18
NP_013275
6323203


19
XP_407136
49091350


20
NP_982520
45184802


21
XP_446953
50289047


22
XP_445184
50285511


23
XP_455638
50311227


24
AAA64516
736722


25
NP_970434
42525054


26
AAT93173
51013759


27
XP_569233
58264154


28
XP_569234
58264156


29
XP_403726
49080406


30
XP_503571
50552322


31
XP_458151
50428131


32
O13302
13124301


33
XP_409927
49096934


34
XP_385909
46122711


35
XP_365293
39967489


36
NP_983873
45187650


37
XP_455266
50310493


38
NP_594397
19115309


39
XP_324955
32408949


40
CAE81942
38636405


41
NP_014361
6324291


42
XP_446479
50288101


43
XP_567378
58259936


44
XP_398944
49069310


45
XP_502479
50550013


46
EAK96238
46436883


47
EAK96305
46436951


48
XP_461797
50426401


49
XP_328403
32415850


50
CAF31997
42820684


51
XP_389756
46136129


52
XP_363786
39952139


53
AAL73035
18463935


54
XP_405140
49086142


55
NP_595203
19111995


56
NP_014779
6324709


57
XP_447564
50290265


58
NP_985684
45198655


59
XP_566837
58258849


60
XP_454086
50308171


61
XP_398943
49069308
















TABLE 4







Examples of ATP-citrate lyase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
XP_327071
32413182


2
O93988
30912679


3
XP_370222
39977669


4
XP_406573
49090008


5
XP_504787
50554757


6
Q9P7W3
30912748


7
XP_398620
49068662


8
NP_596202
19112994


9
XP_567460
58260100


10
NP_001008
56118260


11
XP_418154
50760837


12
AAH84253
54038148


13
NP_942127
38569423


14
NP_001087
38569421


15
P53396
20141248


16
AAL34316
17028103


17
NP_001002
50540366


18
AAH84776
54311201


19
S21173
105392


20
AAT94429
51092031


21
AAD34754
28372804


22
AAH21502
18204829


23
XP_319323
58392375


24
NP_725514
24653990


25
EAL26601
54637198


26
CAE56725
39579419


27
CAE64663
39593194


28
XP_511495
55645405


29
CAF95829
47210997


30
AAO22565
27754223


31
AAL33788
17065616


32
CAB46077
5304837


33
CAF96044
47204726


34
AAK13318
13160653


35
AAQ75159
34558815


36
AAQ75128
34558783


37
XP_537640
57091075


38
XP_327069
32413178


39
CAB76164
7160184


40
XP_370223
39977671


41
XP_386215
46123323


42
CAA10666
7159697


43
XP_406572
49090004


44
XP_503231
50551515


45
NP_593246
19114158


46
XP_398620
49068662


47
XP_567460
58260100


48
AAT94429
51092031


49
NP_725514
24653990


50
AAD34754
28372804


51
EAL26601
54637198


52
XP_319323
58392375


53
AAH84776
54311201


54
BAB00624
9229902


55
NP_001008
56118260


56
AAH84253
54038148


57
AAH56378
38614162


58
NP_001087
38569421


59
NP_942127
38569423


60
P53396
20141248


61
XP_511495
55645405


62
NP_058683
8392839


63
NP_001002
50540366


64
S21173
105392


65
NP_508280
17551266


66
CAE64663
39593194


67
CAE56725
39579419


68
NP_506267
17557344


69
XP_537640
57091075


70
CAF96059
47204551


71
F96633
25404292


72
AAM91141
22136126


73
NP_849634
30681854


74
AAO23582
27764922


75
AAM65078
21593129


76
CAC86996
15919089


77
AAQ75158
34558814


78
AAQ75127
34558782
















TABLE 5







Examples of malic enzyme polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
NP_012896
6322823


2
XP_448858
50292851


3
XP_454793
50309563


4
NP_986598
45201028


5
XP_460887
50424595


6
EAK97738
46438407


7
XP_504112
50553402


8
XP_330094
32419237


9
XP_380981
46107844


10
XP_411070
49102552


11
XP_362875
39946676


12
NP_587760
19075260


13
NP_978189
42780942


14
YP_035982
49481098


15
YP_027934
49184682


16
YP_018438
47527089


17
ZP_002365
47565532


18
YP_083209
52143619


19
XP_571672
58269032


20
NP_391586
16080758


21
YP_092693
52786864


22
NP_831516
30019885


23
YP_093460
52787631


24
YP_081030
52082239


25
NP_822689
29828055


26
O34389
33517449


27
EAL19111
50256386


28
NP_825047
29830413


29
ZP_002340
47096498


30
NP_928837
37525493


31
NP_230833
15641201


32
NP_934257
37679648


33
NP_761613
27366085


34
AC1314
25283688


35
YP_055602
50842375


36
YP_095310
52841511


37
ZP_002315
47093832


38
AC1686
25283689


39
YP_126594
54294179


40
YP_123567
54297198


41
EAJ76260
44510091


42
YP_114273
53803890


43
NP_797637
28898032


44
YP_040250
49483026


45
ZP_001276
53693400


46
YP_044961
50083451


47
YP_128226
54295811


48
NP_719387
24375344


49
XP_572853
58271394


50
NP_252161
15598667


51
ZP_001368
46164263


52
YP_125345
54298976


53
NP_793695
28871076


54
YP_096964
52843165


55
EAH92280
44245125


56
YP_154988
56459707


57
EAI68195
44354928


58
YP_070054
51595863


59
YP_133025
54303032


60
NP_969623
42524243


61
NP_856009
31793516


62
DECARBOXY
ATING))


63
NP_935035
37680426


64
YP_050922
50121755


65
E70705
7431223


66
NP_216848
57116971


67
DECARBOXY
ATING))


68
YP_143786
55980489


69
YP_130202
54309182


70
NP_415996
16129438


71
NP_819843
29654151


72
NP_753809
26247769


73
NP_707611
56479957


74
F85728
25283682


75
YP_163690
56552851


76
YP_150562
56413487


77
NP_720610
24378655


78
NP_460525
16764910


79
ZP_003193
48865537


80
NP_784797
28377905


81
T13496
7431227


82
AAV65766
55793550


83
A97096
25283683


84
YP_193951
58337366


85
H97096
25283684


86
ZP_003237
48870993


87
ZP_001460
41689468


88
D86737
25283676


89
ZP_002870
48825851


90
ZP_001439
34762975


91
1922245A
737262


92
YP_169914
56708018


93
YP_055027
50841800


94
ZP_000625
23023297


95
NP_296302
15807565


96
NP_285599
15807938


97
YP_132069
54302076


98
CAA50716
467569


99
ZP_002906
48833596


100
ZP_003155
48861632


101
NP_773109
27381580


102
AAQ95658
37622953


103
CAC19505
56204311


104
AAH80660
51873855


105
P40927
729986


106
AAT02533
46850200


107
BAC37086
26346875


108
T02763
7431235


109
XP_387367
46125627


110
AAC50613
1465733


111
CAA39421
669118


112
CAA39420
669117


113
NP_032641
6678912


114
CAA39419
581228


115
AAB01380
1335389


116
JC4160
1085347


117
E96828
25283677


118
BAD87910
57899974


119
EAJ77083
44511304


120
P13697
266504


121
NP_036732
7106353


122
YP_065939
51246055


123
CAC18164
16944467


124
XP_322953
32404680


125
AAK91502
18460985


126
AAQ88396
37147841


127
NP_001003
57525624


128
1GQ2P
21465488


129
AAO26053
28195290


130
AAH84250
54038006


131
XP_362590
39946106


132
AAH03287
13096987


133
Q29558
2497785


134
XP_532217
57094622


135
P28227
126734


136
NP_496968
17537199


137
NP_914533
34906372


138
AAD10504
4096786


139
AAO67523
50897495


140
P43279
1170871


141
AAK83074
15077109


142
AAP33011
30575690


143
AAN86690
27357017


144
P78715
41017288


145
AAP32204
30526303


146
AAV31249
54287505


147
T06402
7431232


148
Q99KE1
55583978


149
XP_399922
49071266


150
P36444
547886


151
AAO30034
28059162


152
AAK83073
15077107


153
NP_002387
4505145


154
AAA33487
168528


155
BAA74735
4239891


156
NP_989634
45383538


157
1GZ3D
31615316


158
AAW56450
57791240


159
AAT02534
46850202


160
S29742
422339


161
1O0SB
34811253


162
P27443
126732


163
T06401
7431231


164
AAL16175
16226466


165
AAF73006
8118507


166
AAK97530
15420975


167
EAI90348
44385841


168
P51615
1708924


169
AAA19575
169327


170
S43718
1084300


171
P34105
1346485


172
AAS38597
42733630


173
BAC54101
27530932


174
AAT02535
46850204


175
CAB66003
6706333


176
AAH84860
54311418


177
CAA39422
669119


178
NP_916713
34910732


179
CAA56354
510876


180
DEFBC
7427668


181
JC5967
7431234


182
NP_197960
15239517


183
NP_651959
21356279


184
CAB64263
6634090


185
BAB20887
54606800


186
EAL27424
54638022


187
NP_006671
5729920


188
AAB08874
1561774


189
1PJLH
33358128


190
1GZ4D
22218682


191
1QR6B
5822327


192
1PJ3D
39654475


193
P22178
126736


194
XP_410305
49097690


195
AAH22472
18490280
















TABLE 6







Examples of AMP deaminase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
AAA34420
171053


2
XP_446684
50288509


3
NP_983153
45185436


4
XP_453337
50306727


5
EAL02322
46443037


6
XP_460211
50423261


7
XP_503822
50552824


8
XP_413009
49131023


9
XP_360256
39941438


10
XP_381547
46108978


11
XP_330167
32419447


12
CAB97316
16945394


13
T50996
11359582


14
NP_595153
19111945


15
EAL22226
50259553


16
XP_402237
49076548


17
CAA62797
995562


18
AAF65407
7638159


19
XP_537039
57088163


20
AAH49119
29145073


21
XP_569691
58265070


22
AAD56303
5922018


23
NP_004028
21264318


24
A44313
345738


25
CAI19307
56206061


26
AAA62126
644509


27
CAI19305
56206059


28
XP_310497
58424203


29
CAI19306
56206060


30
AAC50308
608499


31
CAG06825
47229629


32
NP_727741
45551453


33
NP_727739
45551452


34
NP_727740
24641890


35
AAN09337
22832227


36
T01259
7484807


37
XP_506591
51963676


38
NP_850294
30687456


39
CAG07509
47228777


40
NP_494974
32564190


41
T15771
7497030


42
CAE59064
39596837


43
NP_494973
32564194


44
BAA06505
1321635


45
NP_000471
4502079


46
S68147
2134756


47
AAH56380
38614134


48
O08739
2494043


49
NP_113732
13928736


50
O09178
2494044


51
XP_420973
50747746


52
NP_956142
41054127


53
CAG01709
47222742


54
NP_957187
41053780


55
XP_392957
48104570


56
AAH07183
13938134


57
CAG05605
47220579


58
NP_620231
20302047


59
XP_540247
57098851


60
CAF99638
47230445


61
XP_513671
55587796


62
CAI18828
56203368


63
CAI18829
56203369


64
CAI18830
56203370


65
EAA19931
23484684


66
CAH99706
56500932


67
XP_131103
38076931


68
CAH77387
56523366
















TABLE 7







Examples of acetoacetyl-CoA thiolase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
P10551
135758


2
Q04677
418002


3
Q12598
34925109


4
T10247
7433657


5
T42741
11257345


6
AAL18924
16417944


7
AAM67058
21618008


8
AAO51605
28829030


9
AAU95618
53854350


10
AAU95619
53854352


11
BAA97003
8777413


12
CAE76429
38567134


13
EAK90852
46431255


14
EAL32264
54643520


15
NP_015297
6325229


16
NP_568694
30695411


17
NP_572414
24640423


18
NP_596686
19113478


19
NP_851154
30695409


20
NP_908411
34894172


21
NP_974900
42573608


22
NP_974901
42573610


23
NP_984262
45188039


24
XP_389497
46134945


25
XP_401186
49074048


26
XP_405546
49087148


27
XP_449306
50293789


28
XP_449306
50293789


29
XP_450298
50899020


30
XP_453599
50307241


31
XP_460741
50424309


32
XP_500646
50546253
















TABLE 8







Examples of HMG-CoA synthase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
B55729
1083370


2
P54869
1708235


3
S13887
86312


4
S27197
284048


5
AAA37076
387072


6
AAF89580
9621905


7
AAH00297
33991031


8
AAH31363
21618633


9
AAH42929
27552834


10
AAH79694
50925193


11
AAH83543
54035469


12
AAO52569
28830079


13
AAP35966
30583443


14
BAB23657
12836439


15
BAC04559
21754758


16
BAC05233
21758044


17
CAA52032
1772495


18
CAC18553
11602786


19
CAG33131
48145817


20
CAH92111
55730782


21
CAI22408
56205097


22
EAK97451
46438115


23
EAL25034
54635631


24
NP_002121
54020720


25
NP_013580
6323509


26
NP_032282
31560689


27
NP_058964
8393538


28
NP_593859
19114771


29
NP_666054
31981842


30
NP_725570
24654139


31
NP_775117
27465521


32
NP_957379
41055180


33
NP_983739
45187516


34
NP_990742
45382279


35
NP_999545
47523816


36
XP_315872
58387870


37
XP_323241
32405256


38
XP_368218
39973655


39
XP_389442
46134253


40
XP_397202
48141273


41
XP_402977
49078452


42
XP_409060
49095198


43
XP_446972
50289085


44
XP_453529
50307101


45
XP_456470
50405663


46
XP_506052
50557288


47
XP_513693
55587844


48
XP_536483
57085299


49
XP_569805
58265298


50
XP_571930
58269548
















TABLE 9







Examples of HMG-CoA reductase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
A23586
90238


2
O74164
11132850


3
P51639
1708252


4
P54960
1708251


5
Q12649
18276268


6
Q29512
2495262


7
Q9Y7D2
11133211


8
S30338
422383


9
S72194
7450066


10
AAA36989
387052


11
AAA37077
305355


12
AAA49740
214237


13
AAD20975
9817458


14
AAH74197
49257596


15
AAL09351
15824453


16
AAO85434
29468180


17
AAP72015
32165622


18
AAR02862
45272118


19
AAT92819
51013051


20
BAC20567
23574646


21
CAA63970
4376229


22
CAE47850
41581201


23
CAF92135
47213283


24
CAH92577
55731745


25
EAK94577
46435190


26
EAL20195
50257490


27
AAF80374
8886086


28
NP_013555
6323483


29
NP_013636
6323565


30
NP_032281
56119096


31
NP_037266
40538852


32
NP_588235
19075735


33
NP_985010
45190756


34
NP_989816
45383193


35
NP_999724
47551099


36
XP_324892
32408825


37
XP_364130
39955070


38
XP_389373
46134115


39
XP_400629
49072680


40
XP_405730
49087632


41
XP_407954
49092986


42
XP_449268
50293713


43
XP_451740
50303597


44
XP_458872
50420671


45
XP_503558
50552167


46
XP_536323
57084803


47
XP_571450
58268588
















TABLE 10







Examples of mevalonate kinase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
XP_386088
46123069


2
XP_408006
49093090


3
XP_370449
39978123


4
EAL04797
46445529


5
XP_322935
32404644


6
NP_001007
55925207


7
XP_460851
50424525


8
XP_567851
58260882


9
XP_567850
58260880


10
AAQ02416
33303805


11
CAA53059
450346


12
AAH16140
16359371


13
AAH05606
13542811


14
XP_403111
49078786


15
XP_452532
50305147


16
CAG08527
47226511


17
XP_446138
50287417


18
AAO51522
28828936


19
NP_985191
45190937


20
XP_500956
50546973


21
NP_013935
6323864


22
AAD45421
5578718


23
NP_920723
37532842


24
NP_851084
30690651


25
AAL18925
16417946


26
NP_788338
28573850


27
AAU20834
51988124


28
AAU87813
52839819


29
AAU20835
51988125


30
YP_183887
57641409


31
NP_143478
14591399


32
BAA24409
2804172


33
NP_126232
14520757


34
XP_522574
55639331


35
NP_071114
11499870


36
XP_423949
50797461


37
NP_633786
21227864


38
ZP_002971
48840229


39
EAH50787
44170778


40
NP_615566
20089491


41
1VISA
40890012


42
EAK03559
44549994


43
NP_248080
15669275


44
1KKHA
20150886


45
Q50559
2497518


46
CAF88123
47200914


47
NP_275189
15678075


48
EAI88745
44383877


49
ZP_002040
46141948


50
XP_543435
57105916


51
EAI38920
44313360


52
NP_148611
14602065


53
EAD08953
43286228


54
EAD45697
43361720


55
YP_134862
55377012


56
NP_720650
24378695


57
NP_614276
20094429


58
E84270
25409931


59
NP_691146
23097680


60
ZP_003233
48870579


61
AAG02440
9937386


62
EAD12278
43292898


63
NP_498328
17555862


64
EAB31483
42928976


65
ZP_003319
50590618


66
NP_814642
29375488


67
AC1434
25514495


68
ZP_003577
53796847


69
EAD82048
43454743


70
CAE73618
39586491


71
YP_012624
46906235


72
NP_988455
45358898


73
ZP_002348
47097293


74
ZP_002862
48824993


75
ZP_002307
47093020


76
NP_597102
19173299


77
CAD24422
20429111


78
NP_785308
28378416


79
EAA39098
29247539


80
NP_819638
29653946


81
EAH49746
44168765


82
EAH49745
44168764


83
NP_378182
15922513


84
ZP_000459
23002259


85
H90181
25393827


86
YP_054120
50405028


87
BAB07790
9695270


88
AAG02435
9937379


89
NP_560495
18313828


90
YP_187834
57866187


91
EAK40782
44602942


92
CAC51370
15212070


93
AAG02424
9937364


94
YP_185521
57651465


95
YP_040044
49482820


96
YP_194037
58337452


97
D86675
25400965


98
NP_763916
27467279


99
CAF89434
47197810


100
EAF38333
43767792


101
EAK46841
44611394


102
H89827
25507776


103
ZP_003149
48861061


104
EAK17824
44570143


105
EAH86276
44235719


106
YP_118418
54024176


107
ZP_003196
48865749


108
AAG02430
9937372


109
NP_269075
15674901


110
NP_802520
28896170


111
AAL97579
19748102


112
ZP_003666
56808907


113
NP_965060
42519130


114
NP_819639
29653947


115
EAD97024
43484567


116
BAD86800
57753870
















TABLE 11







Examples of phosphomevalonate kinase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
AAA34596
171479


2
XP_452514
50305111


3
NP_985210
45190956


4
XP_446144
50287429


5
XP_462340
50427455


6
EAL04096
46444824


7
EAL03941
46444668


8
XP_503619
50552418


9
XP_389940
46136497


10
XP_329795
32418634


11
XP_369652
39976529


12
XP_406448
49089559


13
NP_593421
19114333


14
XP_568385
58261950


15
EAL17628
50254887


16
AAL18926
16417948


17
BAD43274
51969164


18
BAD44652
51971975


19
XP_398375
49068172


20
BAD44486
51971643


21
F90479
25393214


22
YP_194039
58337454
















TABLE 12







Examples of mevalonate pyrophosphate decarboxylase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
AAT93171
51013755


2
1FI4A
13786942


3
XP_455548
50311049


4
XP_445335
50285813


5
XP_456912
50409853


6
NP_986435
45200865


7
AAF19399
6625790


8
XP_328845
32416734


9
XP_505041
50555265


10
NP_594027
19114939


11
XP_364905
39963452


12
XP_390600
46137817


13
XP_408551
49094180


14
AAA34506
7544604


15
EAL18927
50256200


16
XP_568247
58261674


17
XP_402794
49077992


18
AAH81784
51980639


19
EAL00166
46440864


20
NP_619597
20149736


21
NP_112324
13592005


22
BAC40852
26354448


23
XP_546783
57087071


24
Q99JF5
23814095


25
AAH63907
39645379


26
CAF99534
47230341


27
AAP35576
30582699


28
AAP36301
30584105


29
AAL18927
16417950


30
AAV32433
54292590


31
AAP68208
31711704


32
AAM64988
21593039


33
NP_566995
18410026


34
XP_423130
50771155


35
AAM65192
21593243


36
NP_001007
55925435


37
NP_573068
28571205


38
BAD27942
50252009


39
T47584
11281655


40
XP_307373
31196851


41
CAE73245
39591192


42
NP_496966
17537201


43
XP_393230
48121058


44
G90479
25393662


45
NP_496967
17537203


46
NP_691147
23097681


47
EAL29282
54640164


48
AD1434
25515042


49
ZP_002308
47093021


50
YP_012625
46906236


51
ZP_002348
47097294


52
NP_819637
29653945


53
NP_376888
15921219


54
ZP_003319
50590617


55
NP_585805
19074299


56
YP_187835
57866188


57
CAD24423
20429112


58
AAG02431
9937373


59
NP_763917
27467280


60
AAG02446
9937394


61
ZP_002863
48824994


62
AAG02441
9937387


63
YP_185522
57651466


64
A89828
25505863


65
NP_814641
29375487


66
YP_040045
49482821


67
NP_785307
28378415


68
ZP_003196
48865750


69
ZP_003233
48870580


70
E86675
25400967


71
EAE31110
43552684


72
BAB07791
9695271


73
CAC51371
15212071


74
ZP_000459
23002258


75
NP_965061
42519131


76
BAD86801
57753871


77
YP_194038
58337453


78
YP_118419
54024177


79
EAK18820
44571499


80
EAI85935
44379784


81
NP_721336
24379381


82
D95044
25388338


83
AAG02456
9937408


84
C97914
25511486


85
EAK47683
44612560


86
EAB86425
43039778


87
YP_140971
55822530


88
YP_139081
55820639


89
BAD07376
40882372


90
NP_968512
42523132


91
EAI06705
44265427


92
YP_060018
50914046


93
AAG02451
9937401


94
NP_269076
15674902


95
ZP_003666
56808906


96
NP_688323
22537472


97
NP_735832
25011437


98
EAC40267
43149093


99
AAL97580
19748103


100
EAI76915
44367119


101
EAD35042
43339207


102
YP_073129
51598941


103
EAI90092
44385501


104
BAB07818
9711347


105
EAD72850
43433025


106
NP_212820
15595031


107
YP_124337
54297968


108
YP_096056
52842257


109
EAA39903
29248368


110
EAH06252
44088237


111
YP_127354
54294939


112
EAD45753
43361830


113
NP_802519
28896169
















TABLE 13







Examples of IPP isomerase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
NP_015208
6325140


2
XP_448008
50291151


3
NP_983828
45187605


4
XP_455121
50310203


5
XP_462358
50427491


6
EAL01685
46442395


7
XP_504974
50555131


8
XP_328425
32415894


9
XP_367200
39971619


10
XP_389898
46136413


11
XP_404716
49085144


12
CAD37150
21627818


13
NP_595164
19111956


14
XP_566641
58258457


15
XP_402453
49077100


16
O35586
6225528


17
AAP36609
30584713


18
AAF37873
7188790


19
NP_445991
16758306


20
O42641
6225529


21
BAA33979
3790386


22
Q13907
6225527


23
AAH22418
48257241


24
AAH19227
48257312


25
AAH57827
35505325


26
NP_004499
40018633


27
AAH89786
58477715


28
CAH91844
55730243


29
XP_418561
50732281


30
AAH06999
48257093


31
CAF98782
47225155


32
NP_808875
29366820


33
XP_507622
55633353


34
AAH82648
52139082


35
NP_001011
58332496


36
AAF29976
6856556


37
AAG10423
9971806


38
O48964
6225525


39
AAF29973
6856550


40
AAF29977
6856558


41
AAQ84167
35186998


42
AAF29974
6856552


43
Q39472
6225526


44
S49588
1085973


45
AAL91980
19568939


46
BAB40973
13603406


47
AAF29975
6856554


48
T52027
25493162


49
AAL91979
19568937


50
T46812
11362218


51
T51248
11362217


52
BAB40974
13603408


53
O48965
6225532


54
XP_225509
34877710


55
XP_506401
51963472


56
AAF29978
6856560


57
AAH76541
50369278


58
AAT94033
51038230


59
XP_225502
34876517


60
Q39471
6225533


61
AAB67743
1213450


62
NP_197148
22326844


63
BAB09611
9759126


64
AAD41766
5305669


65
AAB67741
1213442


66
XP_395125
48101420


67
AAN28784
23505849


68
AAF36996
7110585


69
BAB16690
15289752


70
AAQ14869
33340598


71
BAC65421
28971819


72
S71369
2129625


73
AAF29979
6856562


74
AAF29980
6856564


75
AAP21674
30267831


76
Q39664
6225534


77
NP_650962
24648688


78
AAM50284
21429130


79
XP_321388
58395620


80
Q9BXS1
20978506


81
T07979
7484383


82
XP_225508
34876527


83
AAT92102
51011386


84
XP_225507
34876555


85
XP_344623
34876537


86
S44843
630677


87
XP_225498
27687955


88
AAT08468
47013849


89
EAI79636
44370808


90
CAE75055
39587401


91
EAL04047
46444775


92
XP_225528
34876543


93
XP_544282
57040602


94
XP_225511
27688013


95
P26173
114853


96
EAJ04069
44405322


97
EAH27496
44127513


98
AAF91499
9653280


99
AAM48661
21328655


100
EAK17826
44570145


101
EAD59515
43391069


102
YP_128702
54307682


103
EAK66656
44639203


104
YP_118189
54023947


105
T50740
11282665


106
ZP_002077
46193541


107
EAK16470
44568229


108
YP_165403
56695056


109
EAD08775
43285885


110
YP_195623
58616494


111
EAI38918
44313358


112
NP_930583
37527239


113
YP_160254
56478665


114
EAH69842
44206571


115
EAK26254
44582307


116
AAR24381
38569721


117
AAM48607
21328600


118
EAD82049
43454744


119
ZP_001924
45914126


120
YP_056780
50843553


121
YP_050880
50121713


122
EAF29235
43749645


123
NP_630823
21225044


124
Q82MJ7
34582349


125
ZP_003374
52010110


126
AAS75819
45737905


127
Q8KP37
30913023


128
XP_507621
55633351


129
XP_344621
34876521


130
XP_346322
34880719


131
YP_152060
56414985


132
AAT42442
48429280


133
Q9KK75
13878536


134
NP_806649
29143307


135
YP_063124
50955836


136
Q8FND7
46395593


137
CAF20647
41326485


138
Q8NN99
23821718


139
Q7X5H2
46395586


140
NP_336246
15841209


141
Q83MJ9
46395588


142
P60923
46395576


143
Q8FE75
31563050


144
1R67A
38493022


145
Q9KWD1
13878537


146
Q7VEU0
46395585


147
B84333
25410326


148
NP_417365
16130791


149
E85944
25355426


150
1HZTA
15826050


151
1PVFB
50513321


152
EAD63579
43403471


153
1I9AB
13786886


154
YP_012992
46906603


155
ZP_002293
47091503


156
EAI37194
44310821


157
YP_137864
55380014


158
CAD92056
42516867


159
1OW2B
42543244
















TABLE 14







Examples of FPP synthase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
Q92250
2497455


2
XP_363065
39948036


3
XP_386960
46124813


4
Q92235
3122099


5
XP_412149
49116518


6
XP_503599
50552378


7
NP_593299
19114211


8
CAD42869
21955860


9
XP_448787
50292709


10
NP_012368
6322294


11
T42081
7433997


12
EAK93751
46434339


13
XP_451300
50302727


14
XP_571137
58267962


15
XP_460720
50424267


16
NP_984739
45190485


17
BAD15361
46367743


18
S71433
7433991


19
CAA65643
1523990


20
XP_399061
49069544


21
S71432
7433990


22
AAH68912
46249832


23
1FPS
1065289


24
P08836
3915686


25
AAH83515
53733369


26
1UBX
1942050


27
1UBY
1942051


28
AAF37872
7188788


29
NP_803463
29135293


30
AAK63847
14488053


31
AAV58896
55710092


32
T06272
7433988


33
JC4846
2117737


34
P05369
120478


35
O24241
25452945


36
O24242
25452946


37
AAH59125
37590777


38
AAH48497
28913418


39
AAP74720
32329199


40
CAG11850
47225367


41
AAM51429
21436457


42
AAP74719
32329197


43
AAM08927
20135548


44
XP_537252
57089113


45
AAQ56011
34013692


46
AAQ14872
33340604


47
AAQ14871
33340602


48
AAD17204
4324960


49
AAH87886
56789674


50
AAK68152
14573639


51
AAA52423
182399


52
S66470
2129849


53
CAA29064
4725


54
CAI12715
55957735


55
BAA03523
40788949


56
P14324
1346031


57
S66471
2129850


58
AAA35820
182405


59
CAA59170
1491641


60
BAB16687
15289750


61
CAA72793
1922251


62
CAH91070
55728661


63
AAK58594
14279425


64
AAB07264
1146159


65
Q09152
21431776


66
O64905
6016044


67
BAB60822
14422406


68
S52009
1076319


69
NP_917118
34911542


70
AAD32648
4894899


71
AAA40960
203582


72
AAR27053
38684029


73
AAU43998
52353430


74
AAL82595
18958450


75
NP_917069
34911444


76
XP_228802
34879769


77
BAD81810
56785155


78
AAN62522
24796660


79
NP_595334
19112126


80
T52066
25458583


81
AAL49067
17946048


82
CAA08919
3395483


83
XP_547662
57089869


84
EAL26135
54636732


85
BAB60821
14422404


86
AAP74721
32329201


87
XP_496902
51466663


88
XP_474182
50929309


89
CAA87327
1160178


90
BAD20729
47776234


91
BAC53873
30984142


92
BAB69490
15991313


93
NP_974565
42572937


94
CAA08918
5678609


95
AAP86267
32527731


96
AAO17735
30522953


97
AAK71861
14647139


98
AAL73357
18478919


99
AAO63552
29124957


100
CAI00471
56498227


101
NP_701155
23508486


102
XP_474180
50929305


103
AAL73358
18478922


104
EAH48995
44167328


105
NP_493027
17508563


106
CAE71711
39580204


107
XP_487220
51766977
















TABLE 15







Examples of GGPP synthase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
AAT92871
51013155


2
XP_447025
50289191


3
NP_984623
45190369


4
XP_390273
46137163


5
XP_404791
49085320


6
XP_368486
39974191


7
Q92236
6831550


8
AAO85432
29468176


9
XP_572774
58271236


10
XP_502923
50550901


11
AAK11525
13021716


12
XP_326920
32412880


13
CAF32032
42820719


14
BAD29965
50355599


15
XP_384767
46117498


16
BAD29970
50355631


17
CAB89115
7649674


18
CAG09545
47229030


19
CAI13753
55960163


20
AAH69913
47124116


21
AAH67768
45709211


22
XP_455003
50309979


23
P56966
9296978


24
NP_001007
56090562


25
AAT65717
49409613


26
NP_956329
41053321


27
BAA90525
6899844


28
XP_405729
49087630


29
AAK11531
13021724


30
XP_412280
49119197


31
AAC05273
2944400


32
NP_523958
24660002


33
XP_402074
49076128


34
EAL30191
54641441


35
XP_536340
57084951


36
XP_424685
50811194


37
AAH06798
13905030


38
AAP06018
29841005


39
XP_460338
50423511


40
AAC05595
2957271


41
EAK92197
46432727


42
XP_535573
57108760


43
AAH83212
53734594


44
XP_486466
51827552


45
CAH18006
51469024


46
CAA75568
3549881


47
XP_397455
48143654


48
XP_410947
49101294


49
XP_381914
46109712


50
XP_364478
39959279


51
XP_360889
39942704


52
XP_369218
39975655


53
XP_406544
49089926


54
XP_367595
39972409


55
XP_363775
39952117


56
XP_368486
39974191


57
XP_390273
46137163


58
Q92236
6831550


59
AAK11525
13021716


60
CAF32032
42820719


61
XP_404791
49085320


62
AAO85432
29468176


63
BAD29965
50355599


64
BAD29970
50355631


65
BAA90525
6899844


66
AAT65717
49409613


67
XP_384767
46117498


68
CAB89115
7649674


69
XP_572774
58271236


70
AAK11531
13021724


71
XP_502923
50550901


72
CAI13753
55960163


73
CAG09545
47229030


74
XP_412280
49119197


75
P56966
9296978


76
NP_001007
56090562


77
AAH69913
47124116


78
AAH67768
45709211


79
NP_956329
41053321


80
EAL30191
54641441


81
XP_424685
50811194


82
XP_536340
57084951


83
NP_523958
24660002


84
AAC05273
2944400


85
XP_405729
49087630


86
AAC05595
2957271


87
XP_402074
49076128


88
AAP06018
29841005


89
AAH06798
13905030


90
XP_535573
57108760


91
AAH83212
53734594


92
AAP21085
30097620


93
NP_984623
45190369


94
XP_447025
50289191


95
AAT92871
51013155


96
XP_486466
51827552


97
XP_410947
49101294


98
XP_397455
48143654


99
XP_455003
50309979


100
EAK92197
46432727


101
XP_381914
46109712


102
XP_460338
50423511


103
CAH18006
51469024


104
XP_360889
39942704


105
XP_406544
49089926


106
XP_364478
39959279


107
XP_363775
39952117


108
XP_367595
39972409


109
XP_369218
39975655


110
C39273
483124


111
BAB79600
18143445


112
BAA14124
216682


113
AAN85596
27228290


114
AAA32797
413730


115
Q08291
585326


116
S52584
1073293


117
S53722
1076576


118
AAC44848
1842242


119
BAA19583
1944371


120
S71230
2129674


121
BAA23157
2578822


122
AAC77874
3885426


123
CAB38744
4490594


124
BAA78047
4958920


125
BAA82613
5631295


126
CAB56064
5912297


127
BAA86284
6277254


128
T11021
7447356


129
AAF78199
8650415


130
AAG10424
9971808


131
CAC10561
10637876


132
T50879
11279298


133
BAB01343
11994221


134
Q42698
13431546


135
Q43133
13431547


136
P54976
13878921


137
BAB50600
14023995


138
BAB60678
14325238


139
BAB60820
14422402


140
NP_189589
15228704


141
NP_188651
15231055


142
NP_188069
15231869


143
NP_188073
15231881


144
AAL01997
15553715


145
AAL01998
15553717


146
NP_252732
15599238


147
NP_245470
15602398


148
NP_390308
16079484


149
NP_440010
16329282


150
NP_440010
16329282


151
AAL17614
17352451


152
NP_520343
17546941


153
AAL76349
18645048


154
AAM21638
20386366


155
AAM21639
20386368


156
NP_622916
20807745


157
AAM48650
21328644


158
NP_659794
21492720


159
AAM64496
21592547


160
AAM65107
21593158


161
NP_680811
22297564


162
ZP_000474
23003800


163
ZP_001252
23469933


164
NP_698760
23502633


165
E84566
25313373


166
F85434
25313385


167
AC1245
25313389


168
E83997
25313393


169
G84566
25313395


170
AH2910
25315863


171
D87505
25398795


172
A89932
25505949


173
F97685
25520741


174
AI3285
25527013


175
BAC42571
26450928


176
NP_785195
28378303


177
NP_790546
28867927


178
AAO63392
28950937


179
AAO93113
29893480


180
NP_833891
30022260


181
AAP59037
31621279


182
ZP_001374
32039216


183
NP_864766
32471772


184
NP_875521
33240579


185
NP_881399
33593755


186
NP_884694
33597051


187
NP_888456
33600896


188
NP_893187
33861626


189
NP_894940
33863380


190
NP_896835
33865276


191
NP_896835
33865276


192
AAQ65086
34365549


193
NP_945877
39933601


194
NP_946867
39934591


195
NP_952815
39996864


196
AAR37805
40062934


197
AAR37858
40062988


198
AAR98495
41018904


199
AAR99082
41059107


200
NP_965349
42519419


201
NP_980544
42783297


202
EAA96348
42858148


203
EAB36506
42939031


204
EAB36642
42939300


205
EAC39208
43146996


206
EAD26007
43320598


207
EAE43084
43576643


208
EAE70061
43630884


209
EAF70308
43832107


210
EAG88494
44055952


211
EAH52060
44173220


212
EAH78354
44221788


213
EAH84117
44231960


214
EAI11762
44272832


215
EAI49391
44328289


216
EAI54846
44336042


217
EAI68356
44355138


218
EAI68713
44355672


219
EAI69401
44356609


220
EAI73873
44362658


221
EAJ73634
44506168


222
EAJ77351
44511694


223
EAK70639
44644254


224
ZP_001751
45523854


225
AAS76253
45752710


226
ZP_001957
45916757


227
1RTRB
46015556


228
ZP_001863
46105954


229
ZP_002002
46107045


230
ZP_001711
46132567


231
ZP_002073
46192680


232
ZP_002074
46192861


233
AAS82860
46241274


234
ZP_002108
46308696


235
YP_010568
46579760


236
BAD18313
47076770


237
ZP_002315
47093750


238
ZP_002335
47095946


239
AAT35222
47531118


240
ZP_002401
47569437


241
ZP_002435
47573473


242
ZP_002626
48728941


243
ZP_002702
48765678


244
ZP_002705
48766028


245
ZP_002732
48768894


246
ZP_002914
48834438


247
ZP_003024
48848203


248
ZP_003129
48858958


249
ZP_003177
48863841


250
ZP_003225
48869790


251
AAT51323
49086036


252
ZP_003301
49236117


253
YP_034222
49476181


254
YP_040995
49483771


255
YP_043579
49486358


256
AAT71982
50253560


257
AAT90315
50952782


258
YP_066435
51246551


259
YP_075673
51892982


260
YP_085511
52141318


261
YP_092166
52786337


262
ZP_001298
53691368


263
YP_105136
53716444


264
YP_111769
53722784


265
ZP_003630
54030933


266
YP_129021
54308001


267
AAV74395
56122554


268
AAV74396
56122556


269
YP_148246
56420928


270
YP_156518
56461237


271
YP_162590
56551751


272
YP_171470
56750769


273
YP_175959
56964228


274
YP_186407
57650478


275
YP_190690
58038726


276
AAW66658
58201026


277
YP_194187
58337602


278
YP_197469
58579257


279
YP_201938
58582922


280
YP_196510
58617311
















TABLE 16







Examples of squalene synthase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
AAA34597
171481


2
CAA42583
3686


3
Q9HGZ6
51704336


4
BAB12207
9955387


5
XP_453457
50306959


6
Q752X9
51701405


7
O74165
51701378


8
XP_458579
50420093


9
EAK95451
46436082


10
P78589
2499979


11
Q9Y753
51701459


12
XP_407513
49092104


13
XP_364394
39958237


14
Q7S4Z6
51701416


15
CAD60581
27764301


16
XP_389557
46135731


17
NP_595363
19112155


18
B48057
477750


19
NP_034321
34328173


20
CAH92517
55731622


21
AAF00038
6002565


22
P53798
1706773


23
NP_004453
31542632


24
AAP36671
30584837


25
1EZFC
11514497


26
AAH09251
14328083


27
AAH84016
54035372


28
I52090
2136196


29
XP_420043
50745256


30
AAH81810
51858605


31
CAE48363
50841455


32
XP_569783
58265254


33
XP_569782
58265252


34
XP_534557
57105080


35
XP_401989
49075920
















TABLE 17







Examples of phytoene dehydrogenase polypeptides.









Genbank


Row
ACCESSION











1
1613414B


2
1613414F


3
1904206A


4
2121278A


5
A86203


6
A96612


7
A99470


8
AAA24820


9
AAA34001


10
AAA50313


11
AAA64981


12
AAA91161


13
AAA99519


14
AAC44798


15
AAC44850


16
AAC48983


17
AAF78201


18
AAG10426


19
AAG14399


20
AAG28700


21
AAG50743


22
AAH85048


23
AAK51545


24
AAK51557


25
AAK64299


26
AAL02000


27
AAL15300


28
AAL38046


29
AAL73986


30
AAL80005


31
AAL91366


32
AAM45380


33
AAM48646


34
AAM63349


35
AAM94364


36
AAN75037


37
AAN85599


38
AAO24235


39
AAO46892


40
AAO46894


41
AAO53257


42
AAO53258


43
AAO64750


44
AAO93135


45
AAP59036


46
AAP79175


47
AAQ04224


48
AAQ04225


49
AAQ65246


50
AAQ65246


51
AAQ88931


52
AAR37797


53
AAR37802


54
AAR37850


55
AAR37855


56
AAR86105


57
AAR98491


58
AAR98494


59
AAR98733


60
AAS17750


61
AAT01639


62
AAT35222


63
AAT74579


64
AAT74580


65
AAT76050


66
AAT76434


67
AAT90316


68
AAU34019


69
AAW23161


70
AB2035


71
AB2064


72
AC2446


73
AF1557


74
AF2029


75
AG2103


76
AG2509


77
AH1199


78
AI2185


79
AI2273


80
B55548


81
B84327


82
B90061


83
BAA14127


84
BAA20276


85
BAA76534


86
BAB10768


87
BAB50520


88
BAB51896


89
BAB68552


90
BAB79603


91
BAB82461


92
BAB82462


93
BAB98016


94
BAC75676


95
BAC77668


96
BAC77671


97
BAD07279


98
BAD07280


99
BAD07287


100
BAD07288


101
CAA52098


102
CAA60479


103
CAA66626


104
CAB38739


105
CAB38743


106
CAB40843


107
CAB56041


108
CAB56062


109
CAB59726


110
CAB65434


111
CAB94794


112
CAC85667


113
CAD19989


114
CAD27442


115
CAD55814


116
CAE00192


117
CAE83576


118
CAF19330


119
CAF21094


120
CAF21337


121
CAH91165


122
E90061


123
EAA90383


124
EAA98598


125
EAB09790


126
EAB14136


127
EAB18725


128
EAB29729


129
EAB30992


130
EAB41377


131
EAB54727


132
EAB76679


133
EAB87028


134
EAB92587


135
EAB94459


136
EAB96864


137
EAC01884


138
EAC38895


139
EAC60360


140
EAD05874


141
EAD05999


142
EAD20520


143
EAE06978


144
EAE70773


145
EAF04985


146
EAF51354


147
EAF62819


148
EAF75453


149
EAG09111


150
EAG19412


151
EAG23070


152
EAG25053


153
EAG25054


154
EAG29279


155
EAG39845


156
EAG56100


157
EAG63013


158
EAG68633


159
EAG71574


160
EAG89835


161
EAH04928


162
EAH04936


163
EAH08586


164
EAH22597


165
EAH22853


166
EAH31648


167
EAH55579


168
EAH68071


169
EAH73302


170
EAH79041


171
EAH86965


172
EAH97108


173
EAH99977


174
EAI01660


175
EAI03576


176
EAI06784


177
EAI11087


178
EAI15261


179
EAI15547


180
EAI17521


181
EAI21398


182
EAI29728


183
EAI38468


184
EAI43591


185
EAI51589


186
EAI58453


187
EAI72974


188
EAI77885


189
EAI78272


190
EAI80262


191
EAI83937


192
EAI86664


193
EAJ00517


194
EAJ05570


195
EAJ08238


196
EAJ15524


197
EAJ18144


198
EAJ20649


199
EAJ21683


200
EAJ24413


201
EAJ28774


202
EAJ30522


203
EAJ35157


204
EAJ37407


205
EAJ39929


206
EAJ54356


207
EAJ54959


208
EAJ56207


209
EAJ58447


210
EAJ59958


211
EAJ63347


212
EAJ66054


213
EAJ67637


214
EAJ69812


215
EAJ74441


216
EAJ76472


217
EAJ76473


218
EAJ80355


219
EAJ80839


220
EAJ81408


221
EAJ86174


222
EAJ87600


223
EAJ88203


224
EAJ88682


225
EAJ92341


226
EAJ94774


227
EAJ97555


228
EAJ97958


229
EAK07654


230
EAK08513


231
EAK08529


232
EAK10609


233
EAK10614


234
EAK12902


235
EAK13034


236
EAK15092


237
EAK22483


238
EAK23222


239
EAK24187


240
EAK24674


241
EAK28785


242
EAK34731


243
EAK34742


244
EAK36883


245
EAK37522


246
EAK42705


247
EAK43213


248
EAK52580


249
EAK53452


250
EAK58759


251
EAK62665


252
EAK63558


253
F84187


254
F90272


255
G87635


256
G90413


257
H83880


258
H84320


259
JC7723


260
NP_060220


261
NP_080435


262
NP_193157


263
NP_214383


264
NP_276913


265
NP_293819


266
NP_294534


267
NP_294585


268
NP_295972


269
NP_338490


270
NP_376437


271
NP_377056


272
NP_388895


273
NP_441167


274
NP_441254


275
NP_442491


276
NP_442727


277
NP_562475


278
NP_568712


279
NP_601630


280
NP_601630


281
NP_616426


282
NP_624522


283
NP_626360


284
NP_630834


285
NP_643053


286
NP_647302


287
NP_659552


288
NP_661086


289
NP_661546


290
NP_661701


291
NP_662300


292
NP_681023


293
NP_681127


294
NP_682351


295
NP_693380


296
NP_693382


297
NP_737250


298
NP_763380


299
NP_786524


300
NP_822198


301
NP_822828


302
NP_827278


303
NP_851528


304
NP_857496


305
NP_868798


306
NP_869339


307
NP_870237


308
NP_874530


309
NP_874561


310
NP_874977


311
NP_892236


312
NP_892265


313
NP_892458


314
NP_893232


315
NP_894882


316
NP_895385


317
NP_895793


318
NP_895829


319
NP_896854


320
NP_896994


321
NP_898304


322
NP_898346


323
NP_902647


324
NP_923340


325
NP_923639


326
NP_923813


327
NP_925079


328
NP_931515


329
NP_936379


330
NP_940208


331
NP_945754


332
NP_946860


333
NP_946866


334
NP_948851


335
NP_962004


336
NP_968600


337
NP_974222


338
NP_974545


339
O49901


340
P17059


341
P54971


342
P54978


343
P54979


344
P54981


345
P54982


346
P74306


347
Q01671


348
Q02861


349
Q38893


350
Q40406


351
Q9FV46


352
Q9SE20


353
Q9SMJ3


354
Q9ZTN9


355
Q9ZTP4


356
S29314


357
S32171


358
S49624


359
S52586


360
S65060


361
T10701


362
T31463


363
T46822


364
T48646


365
T50745


366
T50749


367
T50893


368
T50910


369
T51119


370
T51123


371
XP_324732


372
XP_383241


373
XP_401825


374
XP_470568


375
XP_473486


376
XP_477063


377
XP_525801


378
XP_540198


379
YP_006049


380
YP_013621


381
YP_024310


382
YP_041986


383
YP_041988


384
YP_044561


385
YP_044564


386
YP_062471


387
YP_117947


388
YP_120612


389
YP_135077


390
YP_136483


391
YP_145331


392
YP_145348


393
YP_171014


394
YP_172823


395
YP_173078


396
YP_173207


397
YP_184572


398
YP_187368


399
YP_187371


400
YP_187371


401
YP_187371


402
ZP_000490


403
ZP_000509


404
ZP_000518


405
ZP_000566


406
ZP_000627


407
ZP_000627


408
ZP_001073


409
ZP_001081


410
ZP_001091


411
ZP_001116


412
ZP_001117


413
ZP_001119


414
ZP_001124


415
ZP_001510


416
ZP_001591


417
ZP_001593


418
ZP_001602


419
ZP_001614


420
ZP_001645


421
ZP_001650


422
ZP_001722


423
ZP_001746


424
ZP_001752


425
ZP_001770


426
ZP_001777


427
ZP_001787


428
ZP_001837


429
ZP_001867


430
ZP_002073


431
ZP_002077


432
ZP_002339


433
ZP_002680


434
ZP_002705


435
ZP_002771


436
ZP_002892


437
ZP_002916


438
ZP_002963


439
ZP_003022


440
ZP_003036


441
ZP_003107


442
ZP_003202


443
ZP_003258


444
ZP_003268


445
ZP_003269


446
ZP_003276


447
ZP_003283


448
ZP_003557


449
ZP_003559


450
ZP_003565


451
ZP_003577


452
ZP_003593


453
ZP_003595


441
ZP_003685
















TABLE 18







Examples of phytoene synthase and lycopene cyclase polypeptides.










Genbank



Row
Accession
Genbank GI












1
1613414C
227040


2
A49558
1076590


3
AAA19428
506623


4
AAA32836
413732


5
AAA64982
148413


6
AAB87738
29893495


7
AAC44849
1842243


8
AAD38051
13542332


9
AAF78202
8650418


10
AAF82616
9081847


11
AAG10427
9971814


12
AAG28701
11066678


13
AAK07734
18476085


14
AAK07735
18476089


15
AAK15621
13195243


16
AAL02001
15553721


17
AAL76346
18645045


18
AAL82578
21326700


19
AAM45379
21360353


20
AAM48647
21328641


21
AAM62787
21553694


22
AAM94363
22296799


23
AAN85600
27228294


24
AAO24767
27903500


25
AAO39835
28403302


26
AAO46895
37729028


27
AAO47570
33465823


28
AAO73816
33465821


29
AAP22038
30349414


30
AAP55451
32350232


31
AAP55453
32350236


32
AAP55461
32350252


33
AAP55471
32350272


34
AAP55484
32350298


35
AAP55486
32350302


36
AAP56083
32349564


37
AAP56124
32349646


38
AAP56127
32349652


39
AAP56136
32349670


40
AAP56148
32349694


41
AAP56155
32349708


42
AAP56156
32349710


43
AAP56157
32349712


44
AAP56158
32349714


45
AAP79176
32307542


46
AAQ91837
37499616


47
AAR08445
38037628


48
AAR31885
39842609


49
AAR37803
40062932


50
AAR37856
40062986


51
AAR86104
40456029


52
AAR87868
40557193


53
AAR98492
41018901


54
AAS02284
41394357


55
AAS17009
42491736


56
AAS18307
42521626


57
AAT28184
47498515


58
AAT35222
47531118


59
AAT38473
47779181


60
AAT46069
48686711


61
AAT74581
50313418


62
AAT90319
50952786


63
AAV74394
56122551


64
AAW23162
56698928


65
AC2035
25366683


66
AC2035
25366683


67
BAB18514
11344507


68
BAB79604
18143449


69
BAD07278
40809739


70
BAD07286
40809755


71
BAD62106
54291340


72
BAD62107
54291341


73
C90061
25506636


74
CAA47625
19347


75
CAA68575
19341


76
CAB07958
1934837


77
CAB38740
4490590


78
CAB51949
5690074


79
CAB56063
5912296


80
CAB86388
7453011


81
CAB93661
8250520


82
CAB94795
8574392


83
CAC19567
11990226


84
CAC27383
12584564


85
CAD19988
18307500


86
CAD29284
57282088


87
CAE76609
38567321


88
E37802
95606


89
E84320
25410251


90
EAA98758
42863045


91
EAB01965
42869439


92
EAB04170
42873822


93
EAB07138
42879858


94
EAB09791
42885235


95
EAB19826
42905452


96
EAB35029
42936011


97
EAB41375
42948740


98
EAB78706
43024004


99
EAB92586
43052355


100
EAC06949
43081493


101
EAC18360
43104624


102
EAC25793
43119723


103
EAC29883
43128092


104
EAC32813
43133973


105
EAC33105
43134560


106
EAC38486
43145552


107
EAC52233
43173313


108
EAC60029
43189028


109
EAC68026
43204953


110
EAC96197
43261031


111
EAD08701
43285745


112
EAD20866
43310220


113
EAD32755
43334458


114
EAD38008
43345761


115
EAD50152
43370658


116
EAD50402
43371147


117
EAD81123
43452903


118
EAD93882
43478303


119
EAE12860
43516265


120
EAE16121
43522884


121
EAE31084
43552634


122
EAE35665
43561764


123
EAE44717
43579862


124
EAE46627
43583580


125
EAE47846
43586023


126
EAE72264
43635190


127
EAE76009
43642573


128
EAE86335
43662748


129
EAE89581
43669163


130
EAF18881
43728007


131
EAF64277
43819669


132
EAF67931
43827263


133
EAF84745
43861327


134
EAF94004
43880040


135
EAG06083
43903395


136
EAG21950
43933540


137
EAG43625
43973477


138
EAG50171
43985555


139
EAG57517
43999205


140
EAG62787
44009110


141
EAG65580
44014171


142
EAG68110
44018715


143
EAG72283
44026322


144
EAG78750
44037938


145
EAG80445
44041116


146
EAG93220
44064453


147
EAH04927
44085694


148
EAH08972
44093217


149
EAH10377
44095788


150
EAH22151
44117864


151
EAH31461
44134654


152
EAH50033
44169323


153
EAH64480
44196848


154
EAH79040
44223009


155
EAH99976
44255671


156
EAI02786
44259828


157
EAI02787
44259829


158
EAI03575
44260943


159
EAI05900
44264266


160
EAI61004
44344824


161
EAI70669
44358327


162
EAI83938
44377067


163
EAJ05110
44406802


164
EAJ05569
44407471


165
EAJ08876
44412338


166
EAJ35156
44449986


167
EAJ38900
44455130


168
EAJ49645
44470504


169
EAJ54357
44477026


170
EAJ60475
44485647


171
EAJ64125
44492007


172
EAJ67499
44497025


173
EAJ76471
44510405


174
EAJ76950
44511114


175
EAJ78637
44513596


176
EAJ78787
44513824


177
EAJ79616
44515082


178
EAJ80356
44516200


179
EAJ81914
44518489


180
EAJ87417
44526623


181
EAK08514
44557109


182
EAK08523
44557119


183
EAK12901
44563097


184
EAK22180
44576315


185
EAK24859
44580262


186
EAK28345
44585276


187
EAK34732
44594324


188
EAK34736
44594329


189
EAK37296
44597942


190
EAK37521
44598256


191
EAK56335
44624430


192
G84363
25410528


193
NP_274195
15677043


194
NP_284085
15794263


195
NP_294586
15805888


196
NP_388961
16078144


197
NP_441168
16330440


198
NP_443763
16519643


199
NP_624523
21218744


200
NP_630832
21225053


201
NP_662273
21674208


202
NP_682350
22299103


203
NP_693381
23099915


204
NP_786525
28379633


205
NP_822199
29827565


206
NP_822829
29828195


207
NP_851527
30795077


208
NP_868799
32475805


209
NP_874560
33239618


210
NP_879992
33592348


211
NP_884101
33596458


212
NP_889809
33602249


213
NP_892264
33860703


214
NP_895828
33864268


215
NP_898345
33866786


216
NP_902648
34498433


217
NP_902649
34498434


218
NP_924690
37521313


219
NP_931516
37528171


220
NP_946861
39934585


221
NP_949079
39936803


222
NP_962005
41409169


223
NP_968601
42523221


224
O07333
3913360


225
P08196
585746


226
P21683
30923192


227
P37269
585009


228
P37271
27735222


229
P37272
585749


230
P53797
1709885


231
P54975
1706137


232
P54977
1706139


233
P65860
54041032


234
Q9SSU8
8928282


235
Q9UUQ6
34922667


236
S22474
7489041


237
S32170
321671


238
S52587
1073300


239
S56668
2129505


240
S68307
2130144


241
T10702
7484346


242
T46594
11291807


243
T50746
11356347


244
T50895
11291816


245
XP_324765
32408567


246
XP_383242
46114448


247
XP_403902
49080862


248
YP_006040
46255128


249
YP_103126
53723680


250
YP_112342
53723357


251
YP_117945
54023703


252
YP_120611
54026369


253
YP_136628
55378778


254
YP_136629
55378779


255
YP_145340
55978284


256
YP_145343
55978287


257
YP_160917
56479328


258
YP_160918
56479329


259
YP_162605
56551766


260
YP_172822
56752121


261
YP_187369
57652299


262
YP_192648
58040684


263
ZP_000044
22956752


264
ZP_001091
53688068


265
ZP_001591
53763709


266
ZP_001657
45514234


267
ZP_001690
46132223


268
ZP_001746
45523280


269
ZP_001837
53771530


270
ZP_001867
45546711


271
ZP_002096
46204978


272
ZP_002248
46324513


273
ZP_002450
47575031


274
ZP_002680
48763469


275
ZP_002710
48766450


276
ZP_002791
48782680


277
ZP_002892
48832182


278
ZP_002916
48834623


279
ZP_003036
48849426


280
ZP_003269
48893702


281
ZP_003351
52007802


282
ZP_003487
53730362


283
ZP_003501
53759405


284
ZP_003591
53798896


285
ZP_003628
54030691
















TABLE 19







Examples of carotenoid ketolase polypeptides.










Accession



Row
Number
GI Number












1
AAA99932
609575


2
AAB48668
1870215


3
AAC25611
2541936


4
AAF78203
8650419


5
AAH16427
16741158


6
AAN03484
22597194


7
AAN85497
26541510


8
AAN86030
33439708


9
AAO64399
28976134


10
AAQ23139
33621091


11
AAT35222
47531118


12
AAT35555
47558911


13
AAV41372
55139370


14
AB2307
25530134


15
AF2204
25533132


16
BAB54999
14028447


17
BAB58879
14270087


18
BAC98366
37360914


19
CAA60478
2654318


20
CAB56059
5912292


21
D87673
25398945


22
EAA79304
42823978


23
EAA80363
42826055


24
EAA81403
42828115


25
EAA84711
42834481


26
EAB82380
43031476


27
EAB86624
43040184


28
EAC05755
43079085


29
EAD12219
43292778


30
EAD71182
43427899


31
EAD94927
43480380


32
EAF11582
43712986


33
EAF98072
43888329


34
EAG19345
43928738


35
EAG38273
43963688


36
EAG79800
44039853


37
EAG96474
44070318


38
EAH00349
44077315


39
EAH36448
44143633


40
EAH40683
44151265


41
EAH53180
44175316


42
EAH96648
44250729


43
EAI05260
44263397


44
EAI17468
44281329


45
EAI53009
44333409


46
EAI54054
44334878


47
EAI67818
44354362


48
EAI68153
44354875


49
EAI89684
44384943


50
EAJ27674
44439188


51
EAJ45589
44464684


52
EAJ45589
44464684


53
EAJ67118
44496466


54
EAJ74221
44507022


55
EAJ74653
44507662


56
EAJ88396
44528064


57
EAJ88887
44528792


58
EAK06069
44553531


59
EAK11467
44561166


60
EAK16824
44568733


61
EAK28828
44585942


62
EAK28828
44585942


63
EAK31112
44589271


64
EAK42591
44605441


65
NP_045063
11465545


66
NP_081575
27754029


67
NP_338204
15843167


68
NP_440788
16330060


69
NP_441220
16330492


70
NP_682690
22299443


71
NP_770721
27379192


72
NP_848964
30468077


73
NP_857223
31794730


74
NP_881760
33594116


75
NP_882469
33594826


76
NP_886657
33599097


77
NP_895643
33864083


78
NP_896386
33864827


79
NP_897461
33865902


80
NP_924674
37521297


81
NP_927525
37524181


82
NP_947075
39934799


83
P54972
1706150


84
Q39982
2498257


85
Q44261
2498256


86
T31123
11361063


87
XP_330780
32420673


88
XP_368852
39974923


89
XP_380194
46102628


90
XP_383758
46115480


91
XP_405100
49086048


92
XP_409222
49095522


93
YP_102417
53725671


94
YP_108945
53719959


95
YP_132414
54302421


96
YP_154670
56459389


97
YP_166682
56696325


98
YP_168846
56698471


99
YP_172377
56751676


100
ZP_001068
23124870


101
ZP_001112
53688676


102
ZP_001607
53764743


103
ZP_001757
46118877


104
ZP_001787
53736018


105
ZP_002218
46321435


106
ZP_002456
47575608


107
ZP_003028
48848557


108
ZP_003107
48856640


109
ZP_003264
48893204


110
ZP_003458
53688805


111
ZP_003513
53763576
















TABLE 20







Examples of carotenoid hydroxylase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
AAC44852
1842246


2
AAC49443
1575296


3
AAD54243
5852870


4
AAG10430
9971820


5
AAG10793
9988836


6
AAG33636
11245486


7
AAL80006
19071768


8
AAM44971
21280903


9
AAM51300
21436107


10
AAM77007
21734857


11
AAN85601
27228295


12
AAO53295
28911949


13
AAS48097
44887642


14
AAS55552
45184599


15
AAS88426
46326968


16
AAT48741
49036137


17
AAT84408
50844570


18
AAV85452
56267980


19
AAV85453
56267982


20
BAA14129
216687


21
BAB79605
18143450


22
BAC77670
31790567


23
BAD07283
40809749


24
BAD07291
40809765


25
CAA70427
2956671


26
CAA70888
2956717


27
CAB55625
5870598


28
CAB55626
5870600


29
CAB56060
5912293


30
CAC06712
9968545


31
CAC95130
33145986


32
EAB30128
42926157


33
EAC49462
43167766


34
EAC86129
43241003


35
EAD61089
43395962


36
EAD76156
43443111


37
EAD88640
43467793


38
EAE27903
43546376


39
EAE28203
43546980


40
EAE78743
43647896


41
EAF12173
43714211


42
EAH29370
44130906


43
EAH44202
44158360


44
EAI00766
44256844


45
EAI29017
44298625


46
EAJ30844
44443849


47
EAJ72524
44504516


48
EAK10611
44559981


49
EAK53455
44620561


50
EAK63955
44635271


51
H90469
25394049


52
NP_745389
26989964


53
NP_922503
37536402


54
P54973
1706152


55
Q44262
2498258


56
S52982
1073291


57
XP_473611
50928167


58
YP_024309
48478603


59
ZP_003055
48851297


60
ZP_003107
48856620
















TABLE 21







Examples of astaxanthin synthase polypeptides and putative


astaxanthin synthase polypeptides.










Genbank



Row
ACCESSION
Genbank GI












1
AAM56288
21501451


2
XP_571276
58268240


3
EAL20013
50257304


4
XP_401804
49075484


5
XP_397817
49067054


6
XP_399595
49070612


7
XP_403279
49079218


8
XP_382294
46110473


9
XP_406021
49088382


10
XP_381224
46108332


11
XP_391479
46139577


12
XP_569261
58264210


13
EAL22841
50260180


14
XP_359866
39940658
















TABLE 22







Examples of carotenoid epsilon hydroxylase polypeptides.









ACCESSION
GI
PROTEIN DESCRIPTION





ABB52076
79155148
putative epsilon-ring carotene hydroxylase




[Daucus carota subsp. sativus]


BAD94136
62319017
Cytochrom P450-like protein




[Arabidopsis thaliana]


ABD28565
87162770
E-class P450, group I [Medicago truncatula]


AAT28222
47498772
putative 97B2-like cytochrome P450




[Ginkgo biloba]


ABC68396
85001685
cytochrome P450 monooxygenase




CYP97A [Glycine max]


ABC59110
84514203
cytochrome P450 monooxygenase




CYP97B [Medicago truncatula]


NP_190881
42565881
LUT1 (LUTEIN DEFICIENT 1); oxygen




binding [Arabidopsis thaliana]


ABB47954
78708979
cytochrome P450 monooxygenase, putative




[Oryza sativa (japonica cultivar-group)]


NP_922604
37536604
putative cytochrome P450 monooxygenase




[Oryza sativa (japonica cultivar-group)]
















TABLE 23







Examples of lycopene cyclase polypeptides, beta and epsilon subunits.









ACCESSION
GI
PROTEIN DESCRIPTION












AAK07431
12746307
lycopene epsilon-cyclase [Adonis palaestina]


ABB52073
79154988
putative lycopene epsilon cyclase [Daucus carota subsp.




sativus]


Q38932
27735211
Lycopene epsilon cyclase, chloroplast precursor


AAB53336
1399181
lycopene epsilon cyclase


AAG10428
9971816
epsilon cyclase [Tagetes erecta]


AAK07434
12746313
lycopene epsilon-cyclase [Lactuca sativa]


AAM45382
21360359
epsilon cyclase [Tagetes erecta]


O65837
11132841
Lycopene epsilon cyclase, chloroplast precursor


AAL69394
18419661
lycopene epsilon-cyclase [Spinacia oleracea]


BAE79549
87299433
lycopene epsilon-cyclase [Chrysanthemum x morifolium]


XP_463351
50901836
putative lycopene epsilon-cyclase [Oryza sativa (japonica




cultivar-group)]


AAS48096
44887640
epsilon lycopene cyclase [Citrus sinensis]


AAX92679
62638188
lycopene epsilon cyclase [Citrus maxima]


AAL92114
19569601
lycopene epsilon-cyclase [Citrus x paradisi]


AAK07433
12746311
lycopene epsilon-cyclase [Solanum tuberosum]


AAL47019
17864021
lycopene epsilon-cyclase [Citrus sinensis]


AAT46065
48686703
chloroplast lycopene epsilon-cyclase precursor




[Chlamydomonas reinhardtii]


BAD07293
40809769
lycopene epsilon-cyclase [Citrus limon]


BAD07285
40809753
lycopene epsilon-cyclase [Citrus sinensis]


BAD07277
40809737
lycopene epsilon-cyclase [Citrus unshiu]


EAJ62839
44489138
unknown [environmental sequence]


BAE43547
73993068
putative lycopene beta cyclase [Taxodium distichum var.




distichum]


BAE43550
73993074
putative lycopene beta cyclase [Taxodium distichum var.




distichum]


BAE43557
73993088
putative lycopene beta cyclase [Taxodium distichum var.




imbricarium]


BAE43558
73993090
putative lycopene beta cyclase [Taxodium distichum var.




imbricarium]


BAE43553
73993080
putative lycopene beta cyclase [Taxodium distichum var.




imbricarium]


BAE43545
73993064
putative lycopene beta cyclase [Taxodium distichum var.




distichum]


BAE43556
73993086
putative lycopene beta cyclase [Taxodium distichum var.




imbricarium]


BAE43552
73993078
putative lycopene beta cyclase [Taxodium distichum var.




distichum]


BAE43560
73993094
putative lycopene beta cyclase [Taxodium distichum var.




imbricarium]


BAE43554
73993082
putative lycopene beta cyclase [Taxodium distichum var.




imbricarium]


BAE43551
73993076
putative lycopene beta cyclase [Taxodium distichum var.




distichum]


BAE43519
73993012
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43535
73993044
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43541
73993056
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43542
73993058
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43517
73993008
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43534
73993042
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43537
73993048
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43533
73993040
putative lycopene beta cyclase [Cryptomeria japonica]


BAD02774
38603277
putative lycopene beta cyclase [Cryptomeria japonica]


BAD02766
38603261
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43540
73993054
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43514
73993002
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43544
73993062
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43538
73993050
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43528
73993030
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43546
73993066
putative lycopene beta cyclase [Taxodium distichum var.




distichum]


BAE43526
73993026
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43543
73993060
putative lycopene beta cyclase [Cryptomeria japonica]


BAD02742
38603213
putative lycopene beta cyclase [Cryptomeria japonica]


BAD02770
38603269
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43522
73993018
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43559
73993092
putative lycopene beta cyclase [Taxodium distichum var.




imbricarium]


BAE43527
73993028
putative lycopene beta cyclase [Cryptomeria japonica]


BAE43548
73993070
putative lycopene beta cyclase [Taxodium distichum var.




distichum]


AAF44700
14550425
lycopene beta-cyclase [Citrus sinensis]


BAE43555
73993084
putative lycopene beta cyclase [Taxodium distichum var.




imbricarium]


BAE43549
73993072
putative lycopene beta cyclase [Taxodium distichum var.




distichum]


AAU14144
51922063
lycopene beta-cyclase [Citrus sinensis]


AAN86060
27261727
lycopene cyclase [Citrus unshiu]


AAR89632
40756518
lycopene-beta-cyclase [Citrus maxima]


AAM21152
20530862
lycopene beta-cyclase [Citrus sinensis]


AAD38049
13959731
lycopene cyclase [Citrus x paradisi]


AAU05146
51511939
lycopene beta-cyclase [Citrus sinensis]


AAU05145
51511937
lycopene beta-cyclase [Citrus sinensis]


AAK07430
12746305
lycopene beta-cyclase [Adonis palaestina]


ABB72443
82394885
lycopene beta-cyclase [Citrus sinensis]


BAE79544
87299423
lycopene beta-cyclase [Chrysanthemum x morifolium]


BAE78471
85717882
lycopene beta cyclase [Taraxacum officinale]


Q43415
11133019
Lycopene beta cyclase, chloroplast precursor


AAF23013
6665782
lycopene epsilon-cyclase [Daucus carota]


ABB52071
79154899
putative lycopene beta cyclase [Daucus carota subsp. sativus]


AAW88382
59665024
lycopene beta-cyclase [Lycium barbarum]


AAG10429
9971818
beta cyclase [Tagetes erecta]


AAM45381
21360357
beta cyclase [Tagetes erecta]


AAM14335
20259239
putative lycopene beta cyclase [Arabidopsis thaliana]


AAO18661
27728515
lycopene beta-cyclase [Zea mays]


AAA81880
735882
lycopene cyclase


Q43503
11133022
Lycopene beta cyclase, chloroplast precursor


S66350
2129931
lycopene beta-cyclase (EC 5.5.1.—) - tomato


XP_464409
50905841
putative lycopene beta-cyclase [Oryza sativa (japonica




cultivar-group)]


CAD70565
45237491
lycopene cyclase [Bixa orellana]


Q43578
11133025
Lycopene beta cyclase, chloroplast precursor


AAL92175
19569782
beta-lycopene cyclase [Sandersonia aurantiaca]


AAX54906
61742130
putative chloroplast lycopene beta cyclase precursor




[Chlamydomonas reinhardtii]


S66349
2129954
lycopene beta-cyclase (EC 5.5.1.—) - common tobacco


AAG21133
10644119
chromoplast-specific lycopene beta-cyclase [Lycopersicon




esculentum]


CAB92977
8247354
neoxanthin synthase [Solanum tuberosum]


CAB93342
8249885
neoxanthin synthase [Lycopersicon esculentum]


Q9SEA0
11131528
Capsanthin/capsorubin synthase, chloroplast precursor


Q42435
12643508
Capsanthin/capsorubin synthase, chloroplast precursor


AAO64977
37730608
lycopene beta cyclase [Haematococcus pluvialis]


Q40424
11133011
Lycopene beta cyclase, chloroplast precursor


ABB52072
79154940
putative capsanthin-capsorubin synthase [Daucus carota




subsp. sativus]


AAQ02668
33304511
lycopene cyclase [Setaria italica]


CAA54961
840729
putative chromoplastic oxydo-reductase [Capsicum annuum]


EAJ62838
44489136
unknown [environmental sequence]


YP_401079
81300871
Lycopene cyclase, beta and epsilon [Synechococcus




elongatus PCC 7942]


YP_172741
56752040
lycopene cyclase [Synechococcus elongatus PCC 6301]


ZP_011 . . .
88808972
lycopene beta cyclase [Synechococcus sp. WH 7805]


EAK50052
44615956
unknown [environmental sequence]


NP_892751
33861190
putative lycopene epsilon cyclase [Prochlorococcus marinus




subsp. pastoris str. CCMP1986]


NP_875182
33240240
Lycopene epsilon cyclase [Prochlorococcus marinus subsp.




marinus str. CCMP1375]


YP_382237
78213458
Lycopene cyclase, beta and epsilon [Synechococcus sp.




CC9605]


YP_397130
78779018
Lycopene cyclase, beta and epsilon [Prochlorococcus marinus




str. MIT 9312]


NP_896821
33865262
lycopene beta cyclase [Synechococcus sp. WH 8102]


YP_397570
78779458
Lycopene cyclase, beta and epsilon [Prochlorococcus marinus




str. MIT 9312]


ZP_010 . . .
87302144
lycopene cyclase [Synechococcus sp. WH 5701]


EAK17149
44569190
unknown [environmental sequence]


YP_291882
72382527
lycopene cyclase, beta and epsilon [Prochlorococcus marinus




str. NATL2A]


NP_875528
33240586
Lycopene beta cyclase related dehydrogenase




[Prochlorococcus marinus subsp. marinus str. CCMP1375]


NP_893181
33861620
putative lycopene beta cyclase [Prochlorococcus marinus




subsp. pastoris str. CCMP1986]


NP_895600
33864040
putative lycopene epsilon cyclase [Prochlorococcus marinus




str. MIT 9313]


EAI47456
44325573
unknown [environmental sequence]


YP_291268
72381913
lycopene cyclase, beta and epsilon [Prochlorococcus marinus




str. NATL2A]


ZP_010 . . .
84517806
Lycopene beta cyclase related dehydrogenase




[Prochlorococcus marinus str. MIT 9211]


AAF34191
6970079
lycopene epsilon cyclase [Daucus carota]


ZP_010 . . .
84518202
Lycopene epsilon cyclase [Prochlorococcus marinus str. MIT




9211]


YP_376736
78184301
Lycopene cyclase, beta and epsilon [Synechococcus sp.




CC9902]


ZP_003 . . .
66796756
Lycopene cyclase, beta and epsilon [Deinococcus




geothermalis DSM 11300]


NP_894954
33863394
putative lycopene beta cyclase [Prochlorococcus marinus str.




MIT 9313]


AAT76051
50365502
lycopene cyclase [Citrus clementina]


EAK22047
44576122
unknown [environmental sequence]


NP_294525
15805827
lycopene cyclase [Deinococcus radiodurans R1]
















TABLE 24







Examples of carotenoid glucosyltransferase polypeptides.









ACCESSION
GI
PROTEIN DESCRIPTION












AAA21261
148395
CrtX [Pantoea agglomerans]


AAN85597
27228291
Zeaxanthin Glucosyl Transferase [Pantoea stewartii]


BAB79601
18143446
crtX [Pantoea agglomerans pv. milletiae]


AAZ73147
72536082
zeaxanthin glucosyl transferase [Enterobacteriaceae bacterium




DC413]


AAZ73128
72536060
zeaxanthin glucosyl transferase [Enterobacteriaceae bacterium




DC260]


AAZ73140
72536074
zeaxanthin glucosyl transferase [Enterobacteriaceae bacterium




DC416]


Q01330
231911
Zeaxanthin glucosyl transferase


ZP_006...
71674312
UDP-glycosyltransferase, MGT [Trichodesmium erythraeum




IMS101]


NP_439972
16329244
zeaxanthin glucosyl transferase [Synechocystis sp. PCC 6803]


EAH29368
44130903
unknown [environmental sequence]


ZP_005...
67926135
zeaxanthin glucosyl transferase, hypothetical protein




[Crocosphaera watsonii WH 8501]


YP_378763
78188425
hypothetical protein Cag_0447 [Chlorobium chlorochromatii




CaD3]


ZP_005...
68549418
Glycosyl transferase, group 1 [Pelodictyon phaeoclathratiforme




BU-1]


ZP_010...
85713606
glycosyl transferase, group 1 [Nitrobacter sp. Nb-311A]


YP_317171
75674750
glycosyl transferase, group 1 [Nitrobacter winogradskyi Nb-




255]


ZP_006...
69929171
Glycosyl transferase, group 1 [Nitrobacter hamburgensis X14]


ZP_009...
84500589
hypothetical protein OB2597_11541 [Oceanicola batsensis




HTCC2597]


ZP_009...
83953176
hypothetical protein NAS141_12746 [Sulfitobacter sp. NAS-




14.1]


ZP_009...
83942121
hypothetical protein EE36_07793 [Sulfitobacter sp. EE-36]


YP_508020
89052569
glycosyl transferase, group 1 [Jannaschia sp. CCS1]


ZP_010...
85704103
hypothetical protein ROS217_13931 [Roseovarius sp. 217]


ZP_009...
83370850
probable glycosyltransferase [Rhodobacter sphaeroides ATCC




17025]


ZP_006...
69934465
Glycosyl transferase, group 1 [Paracoccus denitrificans




PD1222]


ZP_009...
83949880
probable glycosyltransferase [Roseovarius nubinhibens ISM]


YP_376237
78183803
putative glycosyltransferase [Synechococcus sp. CC9902]


YP_376129
78183695
probable glycosyltransferase [Synechococcus sp. CC9902]


YP_374296
78186253
hypothetical protein Plut_0365 [Pelodictyon luteolum DSM




273]


ZP_010...
87301651
Putative glycosyltransferase [Synechococcus sp. WH 5701]


ZP_011...
88809938
Putative glycosyltransferase [Synechococcus sp. WH 7805]


BAE47471
78483937
carotenoid glucosyltransferase [Paracoccus sp. N81106]


ZP_010...
87303273
probable glycosyltransferase [Synechococcus sp. WH 5701]


YP_376127
78183693
probable glycosyltransferase [Synechococcus sp. CC9902]


YP_501334
88196509
hypothetical protein SAOUHSC_02880 [Staphylococcus




aureus subsp. aureus NCTC 8325]


YP_187370
57652300
glycosyl transferase, group 2 family protein [Staphylococcus




aureus subsp. aureus COL]


CAA66627
1340131u
nnamed protein product [Staphylococcus aureus]


YP_041987
49484763
putative glycosyl transferase [Staphylococcus aureus subsp.




aureus MRSA252]


YP_417885
82752144
hypothetical protein SAB2436c [Staphylococcus aureus




RF122]


YP_252404
70725490
hypothetical protein SH0489 [Staphylococcus haemolyticus




JCSC1435]


NP_693379
23099913
hypothetical protein OB2458 [Oceanobacillus iheyensis




HTE831]


ZP_008...
82501285
conserved hypothetical protein [Caldicellulosiruptor




saccharolyticus DSM 8903]


ZP_010...
87303565
hypothetical protein WH5701_09900 [Synechococcus sp. WH




5701]
















TABLE 25







Examples of acyl CoA:diacyglycerol acyltransferase (DGAT) polypeptides.









ACCESSION
GI
PROTEIN DESCRIPTION












XP_957022
85082953
hypothetical protein [Neurospora crassa N150]


XP_386864
46124621
hypothetical protein FG06688.1 [Gibberella zeae PH-1]


XP_755172
71000982
diacylglycerol O-acyltransferase DGAT [Aspergillus




fumigatus Af293]


XP_663763
67539978
hypothetical protein AN6159.2 [Aspergillus nidulans FGSC




A4]


BAE65302
83775179
unnamed protein product [Aspergillus oryzae]


XP_502557
50550169
hypothetical protein [Yarrowia lipolytica]


AAS78662
56199782
diacylglycerol acyltransferase [Glycine max]


ABB84383
82582915
diacylglycerol acyltransferase [Jatropha curcas]


AAV31083
54145459
1,2-diacyl-sn-glycerol:acyl-CoA acyltransferase [Euonymus




alatus]


AAG23696
10803053
diacylglycerol acyltransferase [Perilla frutescens]


AAF64065
7576941
putative diacylglycerol acyltransferase [Brassica napus]


AAS01606
41387497
acyl-CoA:diacylglycerol acyltransferase 1 [Olea europaea]


AAT73629
50299542
acyl CoA:diacylglycerol acyltransferase [Glycine max]


AAM03340
67043496
putative diacylglycerol acyltransferase [Tropaeolum majus]


XP_645633
66824557
hypothetical protein DDB0202877 [Dictyostelium




discoideum]


AAF19345
6625653
diacylglycerol acylCoA acyltransferase [Nicotiana tabacum]


AAY40785
63376239
diacylglycerol acyltransferase DGAT2 [Brassica juncea]


AAW47581
57231736
diacylglycerol acyltransferase [Oryza sativa (japonica




cultivar-group)]


AAR11479
38146080
diacylglycerol acyltransferase [Ricinus communis]


AAY40784
63376226
diacylglycerol acyltransferase DGAT1 [Brassica juncea]


AAP68322
31711932
At2g19450 [Arabidopsis thaliana]


AAW51456
57545061
diacylglycerol acyltransferase [Lotus corniculatus var.




japonicus]


AAD45536
5579408
putative diacylglycerol acyltransferase [Brassica napus]


BAD53762
53791817
putative acyl-CoA:diacylglycerol acyltransferase [Oryza




sativa (japonica cultivar-group)]


NP_956024
41054343
hypothetical protein LOC325875 [Danio rerio]


AAL49962
18642598
diacylglycerol acyltransferase 1 [Bos taurus]


XP_930884
89028385
similar to Diacylglycerol O-acyltransferase 1 (Diglyceride




acyltransferase) (ACAT-related gene) [Homo sapiens]


NP_777118
27819636
diacylglycerol O-acyltransferase 1 [Bos taurus]


Q9GMF1
18202926
Diacylglycerol O-acyltransferase 1 (Diglyceride




acyltransferase)


NP_036211
6912332
diacylglycerol O-acyltransferase 1 [Homo sapiens]


AAH06263
34782946
DGAT1 protein [Homo sapiens]


XP_780515
72006039
similar to Diacylglycerol O-acyltransferase 1




[Strongylocentrotus purpuratus]


AAD40881
5225382
putative diacylglycerol acyltransferase [Brassica napus]


XP_539214
73974769
similar to Diacylglycerol O-acyltransferase 1 (ACAT related




gene product 1) isoform 1 [Canis familiaris]


AAZ22403
71063860
diacylglycerol O-acyltransferase 1 [Bubalus bubalis]


NP_999216
47522918
diacylglycerol acyltransferase [Sus scrofa]


NP_001...
50539976
hypothetical protein LOC436731 [Danio rerio]


XP_849176
73974767
similar to Diacylglycerol O-acyltransferase 1 (ACAT related




gene product 1) isoform 2 [Canis familiaris]


NP_505828
71997360
H19N07.4 [Caenorhabditis elegans]


AAF82410
9049538
diacylglycerol acyltransferase [Caenorhabditis elegans]


CAE75170
39591950
Hypothetical protein CBG23107 [Caenorhabditis briggsae]


XP_626337
66358318
diacylglycerol acyltransferase 1 [Cryptosporidium parvum




Iowa II]


XP_668402
67624239
acyl-CoA:diacylglycerol acyltransferase 1-related enzyme




[Cryptosporidium hominis TU502]


AAP94208
33113253
acyl-CoA:diacylglycerol acyltransferase 1-related enzyme




[Toxoplasma gondii]


AAP94209
33113255
acyl-CoA:diacylglycerol acyltransferase 1-related enzyme




[Toxoplasma gondii]


XP_579557
62652535
PREDICTED: diacylglycerol O-acyltransferase 1 [Rattus




norvegicus]


BAC66171
29170489
diacylglycerol acyltransferase [Mus musculus]


Q9ERM3
18202872
Diacylglycerol O-acyltransferase 1 (Diglyceride




acyltransferase)


AAL78366
18698659
acyl coenzyme A:diacylglycerol acyltransferase [Drosophila




melanogaster]


NP_995724
45552403
CG31991-PD, isoform D [Drosophila melanogaster]


NP_724017
24584734
CG31991-PC, isoform C [Drosophila melanogaster]


XP_858062
73974765
similar to Diacylglycerol O-acyltransferase 1 (ACAT related




gene product 1) isoform 3 [Canis familiaris]


XP_728984
82915156
hypothetical protein PY01256 [Plasmodium yoelii yoelii str.




17XNL]


CAG11944
47225461
unnamed protein product [Tetraodon nigroviridis]


BAD27526
50199438
acyl-CoA:diacylglycerol acyltransferase [eukaryotic synthetic




construct]


XP_317656
31226099
ENSANGP00000002281 [Anopheles gambiae str. PEST]


AAV59457
55733950
putative diacylglycerol acyltransferase [Oryza sativa




(japonica cultivar-group)]


EAL33593
54644853
GA16599-PA [Drosophila pseudoobscura]


XP_678753
68073677
diacylglycerol O-acyltransferase [Plasmodium berghei strain




ANKA]


XP_520014
55631434
PREDICTED: similar to Diacylglycerol O-acyltransferase 1




(Diglyceride acyltransferase) [Pan troglodytes]


CAG10815
47219451
unnamed protein product [Tetraodon nigroviridis]


XP_624754
66522700
PREDICTED: similar to ENSANGP00000002281 [Apis




mellifera]


CAC69884
15620769
diacylglycerol acyltransferase I [Rattus norvegicus]


XP_686181
68363630
PREDICTED: similar to Diacylglycerol O-acyltransferase 1




(Diglyceride acyltransferase) [Danio rerio]


XP_734008
70921323
diacylglycerol O-acyltransferase [Plasmodium chabaudi




chabaudi]


XP_673128
68062248
hypothetical protein PB300300.00.0 [Plasmodium berghei




strain ANKA]


AAS72376
45642963
acyl-CoA:cholesterol acyltransferase beta [Toxoplasma




gondii]


AAS72375
45642961
acyl-CoA:cholesterol acyltransferase alpha [Toxoplasma




gondii]


NP_586145
19074639
STEROL O-ACYLTRANSFERASE [Encephalitozoon




cuniculi GB-M1]


XP_640280
66812202
hypothetical protein DDB0205259 [Dictyostelium




discoideum]


AAY40783
63376221
diacylglycerol acyltransferase [Brassica juncea]


XP_765774
71032265
diacylglycerol O-acyltransferase [Theileria parva strain




Muguga]


Q876L2
34582301
Sterol O-acyltransferase 2 (Sterol-ester synthase 2)


XP_571260
58268208
sterol O-acyltransferase [Cryptococcus neoformans var.




neoformans JEC21]


EAL20032
50257323
hypothetical protein CNBF3580 [Cryptococcus neoformans




var. neoformans B-3501A]


XP_954478
84999514
acyl transferase [Theileria annulata strain Ankara]


XP_505086
50555355
hypothetical protein [Yarrowia lipolytica]


NP_588558
19076058
hypothetical protein SPCP1E11.05c [Schizosaccharomyces




pombe 972h-]


AAC49441
1389739
acyl-CoA:sterol acyltransferase


NP_014416
6324346
Acyl-CoA:sterol acyltransferase, isozyme of Are1p; Are2p




[Saccharomyces cerevisiae]


XP_750354
70991010
sterol o-acyltransferase APE2 [Aspergillus fumigatus Af293]


XP_382192
46110268
hypothetical protein FG02016.1 [Gibberella zeae PH-1]


BAE54934
83764790
unnamed protein product [Aspergillus oryzae]


XP_885914
76617939
similar to Sterol O-acyltransferase 2 (Cholesterol




acyltransferase 2) (ACAT-2) isoform 2 [Bos taurus]


XP_591251
76617937
similar to Sterol O-acyltransferase 2 (Cholesterol




acyltransferase 2) (ACAT-2) isoform 1 [Bos taurus]


BAC00846
21392392
AcylCoA:Cholesterol Acyltransferase 2 [Rattus norvegicus]


NP_649816
28571583
CG8112-PA [Drosophila melanogaster]


NP_666176
22122547
sterol O-acyltransferase 2 [Mus musculus]


O88908
18202245
Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2)




(ACAT-2)


XP_761502
71022545
hypothetical protein UM05355.1 [Ustilago maydis 521]


NP_714950
40254723
sterol O-acyltransferase 2 [Rattus norvegicus]


EAQ86094
88178626
hypothetical protein CHGG_07347 [Chaetomium globosum




CBS 148.51]


XP_461395
50425599
hypothetical protein DEHA0F25652g [Debaryomyces




hansenii CBS767]


XP_661812
67527926
hypothetical protein AN4208.2 [Aspergillus nidulans FGSC




A4]


AAH96091
64654094
Sterol O-acyltransferase 2 [Homo sapiens]


O75908
18202149
Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2)




(ACAT-2)


AAH96090
64652990
Sterol O-acyltransferase 2 [Homo sapiens]


AAK48829
13898623
acyl coenzyme A: cholesterol acyltransferase-2 [Homo




sapiens]


XP_543637
73996435
PREDICTED: similar to sterol O-acyltransferase 2 [Canis




familiaris]


O77759
18202176
Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2)




(ACAT-2)


AAO32474
28564191
ARE2 [Saccharomyces castellii]


XP_323485
32405744
hypothetical protein [Neurospora crassa]


NP_982606
45184888
AAR065Cp [Eremothecium gossypii]


NP_593708
19114620
hypothetical protein SPAC13G7.06 [Schizosaccharomyces




pombe 972h-]


AAO32554
28564940
ARE2 [Saccharomyces kluyveri]


EAL28962
54639560
GA20833-PA [Drosophila pseudoobscura]


XP_449806
50294790
hypothetical protein CAGL0M10571g [Candida glabrata




CBS138]


NP_033256
84619697
sterol O-acyltransferase 1 [Mus musculus]


Q61263
18202591
Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1)




(ACAT-1)


BAC34925
26342537
unnamed protein product [Mus musculus]


XP_452607
50305295
unnamed protein product [Kluyveromyces lactis]


NP_001...
77735363
hypothetical protein LOC504287 [Bos taurus]


Q60457
18202585
Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1)




(ACAT-1)


XP_320321
58393811
ENSANGP00000016512 [Anopheles gambiae str. PEST]


XP_320320
58393809
ENSANGP00000016486 [Anopheles gambiae str. PEST]


O70536
18202126
Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1)




(ACAT-1)


XP_714776
68482533
acyl-CoA cholesterol acyltransferase [Candida albicans




SC5314]


P84285
56404462
Sterol O-acyltransferase 2 (Sterol-ester synthase) (ASAT)


AAH77916
50416229
Soat1-prov protein [Xenopus laevis]


XP_692855
68364838
PREDICTED: similar to Soat1-prov protein [Danio rerio]


CAI13574
55960156
sterol O-acyltransferase (acyl-Coenzyme A: cholesterol




acyltransferase) 1 [Homo sapiens]


AAL56227
18028942
cholesterol acyltransferase 1 [Gorilla gorilla]


AAL56228
18028944
cholesterol acyltransferase 1 [Pongo pygmaeus]


AAC37532
4878022
acyl-coenzyme A: cholesterol acyltransferase [Homo sapiens]


2201440A
1585676
acyl-CoA/cholesterol acyltransferase


Q876L3
34582302
Sterol O-acyltransferase 1 (Sterol-ester synthase 1)


BAE01048
67969393
unnamed protein product [Macaca fascicularis]


XP_514030
55588858
PREDICTED: hypothetical protein XP_514030 [Pan




troglodytes]


XP_547445
73961286
similar to Sterol O-acyltransferase 1 (Cholesterol




acyltransferase 1) (ACAT-1) [Canis familiaris]


EAQ84619
88177151
hypothetical protein CHGG_08633 [Chaetomium globosum




CBS 148.51]


O77761
18202178
Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1)




(ACAT-1)


XP_422267
50751122
PREDICTED: similar to Sterol O-acyltransferase 1




(Cholesterol acyltransferase 1) (ACAT-1) [Gallus gallus]


XP_693284
68392980
PREDICTED: similar to Sterol O-acyltransferase 1




(Cholesterol acyltransferase 1) (ACAT-1) [Danio rerio]


AAT92940
51013293
YCR048W [Saccharomyces cerevisiae]


XP_956576
85080625
hypothetical protein [Neurospora crassa N150]


XP_624691
66564061
PREDICTED: similar to ENSANGP00000016486 [Apis




mellifera]


CAF96514
47222847
unnamed protein product [Tetraodon nigroviridis]


XP_788209
72085563
PREDICTED: similar to sterol O-acyltransferase 1




[Strongylocentrotus purpuratus]


XP_445307
50285757
unnamed protein product [Candida glabrata]


CAE70002
39596364
Hypothetical protein CBG16409 [Caenorhabditis briggsae]


CAG07990
47225647
unnamed protein product [Tetraodon nigroviridis]


NP_510623
17549960
B0395.2 [Caenorhabditis elegans]


AAX28331
76157393
SJCHGC04421 protein [Schistosoma japonicum]


CAI96158
66347204
Diacylglycerol O-acyltransferase [Bubalus bubalis]


XP_390039
46136695
hypothetical protein FG09863.1 [Gibberella zeae PH-1]


XP_643169
66819019
hypothetical protein DDB0203882 [Dictyostelium




discoideum]


AAO53095
28850306
hypothetical protein [Dictyostelium discoideum]


AAB06959
1515472
acyl-CoA:cholesterol acyltransferase [Oryctolagus cuniculus]


NP_945619
39933343
putative alginate o-acetyltransferase AlgI




[Rhodopseudomonas palustris CGA009]


ZP_008...
77691302
Membrane bound O-acyl transferase, MBOAT




[Rhodopseudomonas palustris BisB5]


XP_465546
50908115
putative wax synthase [Oryza sativa (japonica cultivar-




group)]
















TABLE 29







Examples of Prenyldiphosphate synthase polypeptides









Accession
GI
Description










29A: Bacteria Proteins that require a mitochondrial targeting sequence









ZP_009 . . .
83373595
Trans-hexaprenyltranstransferase [Rhodobacter sphaeroides ATCC




17029]


ZP_009 . . .
83371280
Trans-hexaprenyltranstransferase [Rhodobacter sphaeroides ATCC




17025]


CAD24417
20429105
decaprenyl diphosphate synthase [Paracoccus zeaxanthinifaciens]


ZP_010 . . .
85705714
Geranylgeranyl pyrophosphate synthase/Polyprenyl synthetase




[Roseovarius sp. 217]


ZP_010 . . .
84515724
decaprenyl diphosphate synthase [Loktanella vestfoldensis SKA53]


YP_165582
56695234
decaprenyl diphosphate synthase [Silicibacter pomeroyi DSS-3]


ZP_010 . . .
86139019
decaprenyl diphosphate synthase [Roseobacter sp. MED193]


ZP_009 . . .
83941379
decaprenyl diphosphate synthase [Sulfitobacter sp. EE-36]


ZP_009 . . .
83854856
decaprenyl diphosphate synthase [Sulfitobacter sp. NAS-14.1]


ZP_006 . . .
69299873
Farnesyltranstransferase [Silicibacter sp. TM1040]


ZP_010 . . .
84683979
Geranylgeranyl pyrophosphate synthase/Polyprenyl synthetase




[Rhodobacterales bacterium HTCC2654]


ZP_009 . . .
84500217
decaprenyl diphosphate synthase [Oceanicola batsensis HTCC2597]


ZP_009 . . .
83952381
decaprenyl diphosphate synthase [Roseovarius nubinhibens ISM]


ZP_006 . . .
69937106
Trans-hexaprenyltranstransferase [Paracoccus denitrificans PD1222]


ZP_005 . . .
68180845
Trans-hexaprenyltranstransferase [Jannaschia sp. CCS1]


ZP_008 . . .
78495595
Polyprenyl synthetase [Rhodopseudomonas palustris BisB18]


AAY82368
67866738
decaprenyl diphosphate synthase [Agrobacterium tumefaciens]


NP_353656
15887975
hypothetical protein AGR_C_1125 [Agrobacterium tumefaciens str.




C58]


ZP_008 . . .
77688465
Farnesyltranstransferase [Rhodopseudomonas palustris BisB5]


NP_531334
17934544
octaprenyl-diphosphate synthase [Agrobacterium tumefaciens str.




C58]


YP_484709
86748213
Farnesyltranstransferase [Rhodopseudomonas palustris HaA2]


AAP56240
37903500
decaprenyl diphosphate synthase [Agrobacterium tumefaciens]


YP_192388
58040424
Decaprenyl diphosphate synthase [Gluconobacter oxydans 621H]







29B: Subunit 1- Proteins that contain mitochondrial targeting sequence









T43193
11279237
trans-pentaprenyltranstransferase homolog - fission yeast




(Schizosaccharomyces pombe)


AAD28559
4732024
trans-prenyltransferase [Homo sapiens]


AAI07275
78070698
Trans-prenyltransferase [Mus musculus]


BAE48216
81157931
subunit 1 of decaprenyl diphosphate synthase [Homo sapiens]


AAH49211
29165656
PDSS1 protein [Homo sapiens]


Q33DR2
85700953
Decaprenyl-diphosphate synthase subunit 1 (Solanesyl-diphosphate




synthase subunit 1) (Trans-prenyltransferase)


XP_507706
55633583
PREDICTED: similar to TPRT protein [Pan troglodytes]


XP_586717
76632198
PREDICTED: similar to trans-prenyltransferase [Bos taurus]


XP_849908
73948851
PREDICTED: similar to trans-prenyltransferase [Canis familiaris]







29C: Subunit 2- Proteins that contain mitochondrial targeting sequence









O13851
60389474
Decaprenyl-diphosphate synthase subunit 2 (Decaprenyl




pyrophosphate synthetase subunit 2)


BAE48218
81157935
subunit 2 of solanesyl diphosphate synthase [Mus musculus]


BAE48217
81157933
subunit 2 of decaprenyl diphosphate synthase [Homo sapiens]
















TABLE 30







Examples of PHB-Polyprenyltransferase polypeptides








GI
PROTEIN DESCRIPTION





51013645
YNR041C [Saccharomyces cerevisiae]


50285815
unnamed protein product [Candida glabrata]


50311051
unnamed protein product [Kluyveromyces lactis]


45200866
AGL231Wp [Eremothecium gossypii]


50555263
hypothetical protein [Yarrowia lipolytica]


68473193
para-hydroxybenzoate: polyprenyl transferase [Candida albicans SC5314]


50410039
hypothetical protein DEHA0A14212g [Debaryomyces hansenii CBS767]


83769349
unnamed protein product [Aspergillus oryzae]


70994900
para-hydroxybenzoate-polyprenyltransferase precursor [Aspergillus fumigatus



Af293]


19114131
hypothetical protein SPAC56F8.04c [Schizosaccharomyces pombe 972h-]


39973573
hypothetical protein MG01067.4 [Magnaporthe grisea 70-15]


85078920
protein related to para-hydroxybenzoate polyprenyltransferase precursor



[Neurospora crassa N150]


76660839
PREDICTED: similar to para-hydroxybenzoate-polyprenyltransferase,



mitochondrial [Bos taurus]


52138578
para-hydroxybenzoate-polyprenyltransferase, mitochondrial [Homo sapiens]


18088424
COQ2 protein [Homo sapiens]


47221448
unnamed protein product [Tetraodon nigroviridis]


58385249
ENSANGP00000012220 [Anopheles gambiae str. PEST]


50746583
PREDICTED: similar to hypothetical protein CL640 [Gallus gallus]


54638587
GA21912-PA [Drosophila pseudoobscura]


21355567
CG9613-PA [Drosophila melanogaster]


71005862
hypothetical protein UM01450.1 [Ustilago maydis 521]









Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

## Claims

1. A method of producing a carotenoid, the method comprising cultivating a genetically engineered Yarrowia fungus under conditions that allow production of the carotenoid;
wherein the genetically engineered Yarrowia fungus is characterized by:
i. the fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and
ii. as a result of the genetic engineering, the Yarrowia fungus produces at least one carotenoid, and can accumulate the produced carotenoid to at least about 1% of its dry cell weight.

2. The method of claim 1, wherein the method comprises a second step of isolating the produced carotenoid.

3. The method of claim 2 wherein the step of isolating comprises fractionating the cultivation medium to obtain at least one carotenoid-enriched fraction.

4. The method of claim 1 wherein: the step of cultivating comprises cultivating the fungus under conditions that allow accumulation of the carotenoid in cytoplasmic oil bodies; and the step of isolating comprises isolating oil derived from the cytoplasmic oil bodies.

5. The method of claim 1 wherein the carotenoid is selected from the group consisting of astaxanthin, β-carotene, canthaxanthin, zeaxanthin, lutein, lycopene, and combinations thereof.

6. The method of claim 1 wherein the carotenoid comprises astaxanthin.

7. The method of claim 1 wherein the carotenoid comprises β-carotene.

8. The method of claim 1 wherein the carotenoid comprises canthaxanthin.

9. The method of claim 1 wherein the carotenoid comprises zeaxanthin.

10. The carotenoid produced by the method of claim 1.