Production Of Quinone Derived Compounds In Oleaginous Yeast And Fungi

Abstract

The present invention provides systems for producing engineered oleaginous yeast or fungi that express quinone derived compounds.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. application Ser. No. 12/293,237, filed Sep. 16, 2008, which is the National Stage under 35 U.S.C. .sctn.371 of International Application No. PCT/US2007/006834, filed Mar. 20, 2007, which claims the benefit under 35 U.S.C. .sctn.119(e) of U.S. provisional patent application Ser. No. 60/784,499, filed Mar. 20, 2006, and claims the benefit under 35U.S.C. .sctn.119(e) of U.S. provisional patent application Ser. No. 60/848,064, filed Sep. 28, 2006; the entire contents of these applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Compounds containing or derived from quinone structures (i.e., "quinone derived compounds") play a variety of important roles in biological systems. Certain vitamins, for example, have quinone moieties (e.g., vitamin K) or are derived from such structures (e.g., vitamin E). Ubiquinones also represent an important class of quinone derived compounds. Ubiquinones play a critical function in the production of cellular ATP, serving as electron carriers in the respiratory chain. Furthermore, ubiquinones are lipophilic antioxidants that are capable of regenerating other antioxidants such as ascorbate and tocopherols.

[0003] Although many quinone derived compounds are naturally produced by the human body, they are often produced at levels lower than are required for optimal cell function. Accordingly, they must be obtained through the diet or by other means. There is a rapidly growing market for nutritional supplements and other products containing quinone derived compounds. Improved systems for enabling cost effective production, isolation, and/or formulation of such compounds are needed to effectively meet the growing demand.

SUMMARY OF THE INVENTION

[0004] The present invention provides improved systems for the biological production of quinone derived compounds. In particular, the present invention provides improved systems for the biological production of ubiquinone(s) (e.g., CoQ10 and/or C.sub.5-9 quinone compounds), vitamin K compounds, and vitamin E compounds.

[0005] In one aspect, the invention encompasses the discovery that it is desirable to produce such quinone derived compounds in oleaginous organisms. The present invention thus provides biological systems able to accumulate one or more quinone derived compounds (e.g., ubiquinones, vitamin K compounds, and/or vitamin E compounds) in lipid bodies. In some embodiments, the biological systems may produce higher levels of quinone derived compounds when the compound(s) is/are sequestered in lipid bodies. Regardless of whether absolute levels are higher; however, compounds that are accumulated within lipid bodies in oleaginous organisms are readily isolatable through isolation of the lipid bodies.

[0006] The present invention therefore provides oleaginous fungi (including, for example, yeast or other unicellular fungi) that produce one or more quinone derived compounds (e.g., ubiquinones, vitamin K compounds, and/or vitamin E compounds). The present invention also provides methods of constructing such yeast and fungi and methods of using such yeast and fungi to produce the quinone derived compounds. The present invention further provides methods of preparing quinone derived compounds, as well as compositions containing them, such as food or feed additives, nutritional supplements, or compositions for nutraceutical, pharmaceutical and/or cosmetic applications. In particular, the present invention provides systems and methods for generating yeast and fungi containing one or more oleaginic and/or quinonogenic modifications that increase the oleaginicity and/or alter their ability to produce one or more quinone derived compounds as compared with otherwise identical organisms that lack the modification(s). In some embodiments, the present invention provides a recombinant fungus. In certain embodiments, the recombinant fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and produces at least one quinone derived compound, and can accumulate the produced quinone derived compound to at least about 1% of its dry cell weight; wherein the recombinant fungus comprises at least one modification as compared with a parental fungus, which parental fungus both is not oleaginous and does not accumulate the quinone derived compound to at least about 1% of its dry cell weight, the at least one modification being selected from the group consisting of quinonogenic modifications, oleaginic modifications, and combinations thereof, and wherein the at least one modification alters oleaginicity of the recombinant fungus, confers to the recombinant fungus oleaginy, confers to the recombinant fungus the ability to produce the at least one quinone derived compound to a level at least about 1% of its dry cell weight, or confers to the recombinant fungus the ability to produce at least one quinone derived compound which the parental fungus does not produce.

[0007] In other embodiments, the recombinant fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and the recombinant fungus produces at least one quinone derived compound selected from the group consisting of: a ubiquinone (including, but not limited to, coenzyme Q10 and/or a C.sub.5-9 quinone compound), a vitamin K compound, a vitamin E compound, and combinations thereof, and can accumulate the produced quinone derived compound to at least about 1% of its dry cell weight; wherein the recombinant fungus comprises at least one modification as compared with a parental fungus, the at least one modification being selected from the group consisting of quinonogenic modifications, oleaginic modifications, and combinations thereof, and wherein the at least one modification alters oleaginicity of the recombinant fungus, confers to the recombinant fungus oleaginy, confers to the recombinant fungus the ability to produce the at least one quinone derived compound to a level at least about 1% of its dry cell weight, or confers to the recombinant fungus the ability to produce at least one quinone derived compound which the parental fungus does not naturally produce.

[0008] In some embodiments, the recombinant fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and the recombinant fungus produces at least one quinone derived compound, and can accumulate the produced quinone derived compound to at least about 1% of its dry cell weight; wherein the recombinant fungus is a member of a genus selected from the group consisting of: Aspergillus, Blakeslea, Botrytis, Candida, Cercospora, Cryptococcus, Cunninghamella, Fusarium (Gibberella), Kluyveromyces, Lipomyces, Mortierella, Mucor, Neurospora, Penicillium, Phycomyces, Pichia (Hansenula), Puccinia, Pythium, Rhodosporidium, Rhodotorula, Saccharomyces, Sclerotium, Trichoderma, Trichosporon, Xanthophyllomyces (Phaffia), and Yarrowia; or is a species selected from the group consisting of: Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, Blakeslea trispora, Botrytis cinerea, Candida japonica, Candida pulcherrima, Candida revkaufi, Candida tropicalis, Candida utilis, Cercospora nicotianae, Cryptococcus curvatus, Cunninghamella echinulata, Cunninghamella elegans, Fusarium fujikuroi (Gibberella zeae), Kluyveromyces lactis, Lipomyces starkeyi, Lipomyces lipoferus, Mortierella alpina, Mortierella ramanniana, Mortierella isabellina, Mortierella vinacea, Mucor circinelloides, Neurospora crassa, Phycomyces blakesleanus, Pichia pastoris, Puccinia distincta, Pythium irregulare, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorula pinicola, Rhodotorula gracilis, Saccharomyces cerevisiae, Sclerotium rolfsii, Trichoderma reesei, Trichosporon cutaneum, Trichosporon pullans, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), and Yarrowia lipolytica; wherein the recombinant fungus comprises at least one modification as compared with a parental fungus, the at least one modification being selected from the group consisting of quinonogenic modifications, oleaginic modifications, and combinations thereof, and wherein the at least one modification alters oleaginicity of the recombinant fungus, confers to the recombinant fungus oleaginy, confers to the recombinant fungus the ability to produce the at least one quinone derived compound to a level at least about 1% of its dry cell weight, or confers to the recombinant fungus the ability to produce at least one quinone derived compound which the parental fungus does not naturally produce.

[0009] In some embodiments, the recombinant fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and the recombinant fungus produces at least one quinone derived compound selected from the group consisting of: a ubiquinone (including, but not limited to, coenzyme Q10 and/or a C.sub.5-9 quinone compound), a vitamin K compound, a vitamin E compound, and combinations thereof, and can accumulate the produced quinone derived compound to at least about 1% of its dry cell weight; wherein the recombinant fungus is a member of a genus selected from the group consisting of: Aspergillus, Blakeslea, Botrytis, Candida, Cercospora, Cryptococcus, Cunninghamella, Fusarium (Gibberella), Kluyveromyces, Lipomyces, Mortierella, Mucor, Neurospora, Penicillium, Phycomyces, Pichia (Hansenula), Puccinia, Pythium, Rhodosporidium, Rhodotorula, Saccharomyces, Sclerotium, Trichoderma, Trichosporon, Xanthophyllomyces (Phaffia), and Yarrowia; or is of a species selected from the group consisting of: Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, Blakeslea trispora, Botrytis cinerea, Candida japonica, Candida pulcherrima, Candida revkaufi, Candida tropicalis, Candida utilis, Cercospora nicotianae, Cryptococcus curvatus, Cunninghamella echinulata, Cunninghamella elegans, Fusarium fujikuroi (Gibberella zeae), Kluyveromyces lactis, Lipomyces starkeyi, Lipomyces lipoferus, Mortierella alpina, Mortierella ramanniana, Mortierella isabellina, Mortierella vinacea, Mucor circinelloides, Neurospora crassa, Phycomyces blakesleanus, Pichia pastoris, Puccinia distincta, Pythium irregulare, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorula pinicola, Rhodotorula gracilis, Saccharomyces cerevisiae, Sclerotium rolfsii, Trichoderma reesei, Trichosporon cutaneum, Trichosporon pullans, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), and Yarrowia lipolytica; wherein the recombinant fungus comprises at least one modification as compared with a parental fungus, the at least one modification being selected from the group consisting of quinonogenic modifications, oleaginic modifications, and combinations thereof, and wherein the at least one modification alters oleaginicity of the recombinant fungus, confers to the recombinant fungus oleaginy, confers to the recombinant fungus the ability to produce the at least one quinone derived compound to a level at least about 1% of its dry cell weight, or confers to the recombinant fungus the ability to produce at least one quinone derived compound which the parental fungus does not naturally produce.

[0010] In some embodiments, the recombinant fungus accumulates the produced at least one quinone derived compound to a level selected from the group consisting of: above about 1%, above about 2%, above about 3%, above about 5%, and above about 10% of the fungus' dry cell weight.

[0011] In some embodiments, the present invention provides an engineered S. cerevisiae strain, comprising one or more quinonogenic modifications, wherein the one or more quinonogenic modifications are selected from the group consisting of: increased expression or activity of a decaprenyl diphosphate synthase polypeptide; increased expression or activity of an 4-hydroxybenzoate polyprenyl polypeptide; increased expression or activity of a GGPP synthase polypeptide; increased expression or activity of an FPP synthase polypeptide; increased expression or activity of an HMG CoA reductase polypeptide; decreased expression or activity of a squalene synthase polypeptide; decreased expression of activity of a prenyldiphosphate synthase polypeptide; increased expression or activity of a DAHP synthase polypeptide; increased expression or activity of a chorismate lyase polypeptide; increased expression or activity of a chorismate synthase polypeptide; increased expression or activity of an ATP-citrate lyase polypeptide; increased expression or activity of an AMP deaminase polypeptide; increased expression or activity of a cytosolic malic enzyme polypeptide; and combinations thereof.

[0012] In some embodiments, the present invention provides an engineered Y. lipolytica strain containing a truncated HMG CoA reductase polypeptide.

[0013] In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of a decaprenyl diphosphate synthase gene. In some embodiments, the present invention provides an engineered Y. lipolytica strain having decreased expression or activity of a squalene synthase polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having decreased expression or activity of a squalene synthase polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of a 4-hydroxybenzoate polyprenyl polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of a DAHP synthase polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of a chorismate lyase polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of an ATP-citrate lyase polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of a AMP deaminase polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of a cytosolic malic polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of a GGPP synthase polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of an FPP synthase polypeptide. In some embodiments, the present invention provides an engineered Y. lipolytica strain having increased expression or activity of a chorismate synthase polypeptide.

[0014] In some embodiments, the present invention provides a strain of Y. lipolytica comprising one or more modifications selected from the group consisting of an oleaginic modification, a quinonogenic modification, and combinations thereof, such that the strain accumulates from 1% to 15% of its dry cell weight as at least one quinone derived compound.

[0015] In certain embodiments, the present invention provides an engineered Y. lipolytica strain that produces a ubiquinone, the strain containing one or more quinonogenic modifications selected from the group consisting of: increased expression or activity of a Y. lipolytica GGPP synthase polypeptide; expression or activity of a truncated HMG CoA reductase polypeptide; increased expression or activity of a 4-hydroxybenzoate polyprenyl transferase polypeptide; increased expression or activity of a decaprenyl diphosphate synthase polypeptide; increased expression or activity of a DAHP synthase polypeptide; increased expression or activity of a chorismate lyase polypeptide; increased expression or activity of a shikimate pathway polypeptide; increased expression or activity of a chorismate mutase polypeptide; increased expression or activity of a transketolase polypeptide; increased expression or activity of an FPP synthase polypeptide; increased expression or activity of an IPP isomerase polypeptide; increased expression or activity of an HMG-CoA synthase polypeptide; increased expression or activity of a mevalonate kinase polypeptide; increased expression or activity of a phosphomevalonate kinase polypeptide; increased expression or activity of a mevalonate pyrophosphate decarboxylate polypeptide; increased expression or activity of a cytosolic malic enzyme polypeptide; increased expression or activity of a malate dehydrogenase polypeptide; increased expression or activity of an AMP deaminase polypeptide; increased expression or activity of a glucose 6 phosphate dehydrogenase polypeptide; increased expression or activity of a malate dehydrogenase homolog 2 polypeptide; increased expression or activity of a GND1-6-phosphogluconate dehydrogenase polypeptide; increased expression or activity of a isocitrate dehydrogenase polypeptide; increased expression or activity of a IDH2-isocitrate dehydrogenase polypeptide; increased expression or activity of a fructose 1,6 bisphosphatase polypeptide; increased expression or activity of a Erg10-acetoacetyl CoA thiolase polypeptide; increased expression or activity of a ATP citrate lyase subunit 2 polypeptide; increased expression or activity of a ATP citrate lyase subunit 1 polypeptide; decreased expression or activity of a squalene synthase polypeptide; decreased expression or activity of a prenyldiphosphate synthase polypeptide; or decreased expression or activity of a PHB polyprenyltransferase polypeptide; and combinations thereof.

[0016] In certain embodiments, the present invention provides a recombinant fungus characterized in that the fungus accumulates lipid in the form of cytoplasmic oil bodies.

[0017] In some embodiments, the present invention provides a composition comprising: lipid bodies; at least one quinone derived compound; and intact fungal cells.

[0018] In some embodiments, the present invention provides a method of producing a quinone derived compound, the method comprising steps of cultivating a fungus under conditions that allow production of the quinone derived compound; and isolating the produced quinone derived compound.

[0019] In some embodiments, the present invention provides a composition comprising: an oil suspension comprising: lipid bodies; at least one quinone derived compound; and intact fungal cells; and a binder or filler.

[0020] In some embodiments, the present invention provides a composition comprising: an oil suspension comprising: lipid bodies; at least one quinone derived compound; and intact fungal cells; and one or more other agents selected from the group consisting of chelating agents, pigments, salts, surfactants, moisturizers, viscosity modifiers, thickeners, emollients, fragrances, preservatives, and combinations thereof.

[0021] In some embodiments, the present invention provides an isolated quinone derived compound composition, prepared by a method comprising steps of cultivating a fungus under conditions that allow production of a quinone derived compound; and isolating the produced quinone derived compound.

[0022] In some embodiments, the present invention provides a quinone derived compound composition comprising a Y. lipolytica cell containing at least 1% quinone derived compounds by weight.

[0023] In some embodiments, the present invention provides a quinone derived compound comprising Y. lipolytica lipid bodies; and at least one quinone derived compound, wherein the at least one quinone derived compound is present at a level that is at least 1% by weight of the lipid bodies. In some embodiments, the present invention provides a composition comprising a quinone derived compound and one or more additional compounds (e.g., binders, fillers, chelating agents, pigments, salts, surfactants, moisturizers, viscosity modifiers, thickeners, emollients, fragrances, preservatives, etc. and combinations thereof).

[0024] In some embodiments, the present invention provides a feedstuff comprising a quinone derived compound in lipid bodies.

[0025] In some embodiments, the present invention provides methods comprising steps of cultivating the recombinant fungus under conditions that allow production of a quinone derived compound (e.g., ubiquinones, vitamin K compounds, and/or vitamin E compounds) and isolating the produced quinone derived compound (e.g., extracting with an organic or non-organic solvent). In certain embodiments, the recombinant fungus is cultured by growing under conditions of limiting nutrient (e.g., one or more of carbon, nitrogen, phosphate, magnesium, etc.) and/or by controlling at least one environmental parameter (e.g. one or more of nutrients, pH, temperature, pressure, oxygen concentration, timing of feeds, content of feeds, etc.) for at least a portion of the cultivation. In certain embodiments, the recombinant fungus is cultivated at a temperature of 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30.degree. C. or above, and/or at one or more ranges within these temperatures. In certain embodiments, the recombinant fungus is cultivated at a pH of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or above, and/or at one or more ranges within these pH's. In certain embodiments, the temperature, the pH, or both is varied during the culture period. In certain embodiments, the recombinant fungus is cultivated at an oxygen concentration within the range of about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or more, and/or at one or more ranges within these concentrations. In certain embodiments, the produced quinone derived compound is isolated by crystallization.

[0026] Additional aspects of the present invention will be apparent to those of ordinary skill in the art from the present description, including the appended claims. Various polypeptides are listed in Tables 1-101; as one of ordinary skill in the art will understand, the order in which these polypeptides are listed is not indicative of their importance to the present invention. Various patent and non-patent publications are referenced throughout the present application. Unless otherwise indicated, each of these references is hereby incorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWING

[0027] FIGS. 1A-1C depict various quinone derived compounds including ubiquinone/Coenzyme Q10 in its various oxidated forms (Panel A); vitamin K (Panel B); and vitamin E (Panel C).

[0028] 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.

[0029] FIG. 3 illustrates biosynthetic pathways of aromatic amino acids, and the shikimate pathway for production of chorismate. A depiction of how these pathways feed into ubiquinone biosynthesis is depicted. Boxed numerical references are IUBMB Enzyme Nomenclature EC numbers for enzymes catalyzing the relevant reaction.

[0030] FIG. 4 depicts ubiquinone biosynthesis pathways. FIG. 4A shows the mevalonate isoprenoid biosynthesis pathway, which typically operates in eukaryotes, including fungi; as well as the mevalonate-independent isoprenoid biosynthesis pathway, also known as the DXP pathway, which typically operates in bacteria and in the plastids of plants and production of isoprenoid precursors; FIG. 4B depicts production of para-hydroxybenzoate (23) from precursor chorismate or tyrosine, and condensation with isoprene, resulting in formation of polyprenylhydroxybenzoate (24), followed by subsequent oxygenation, decarboxylation, and methylation reactions involved in production of quinine compound, polyprenyl-6-methoxy-1,4-benzoquinol (30). FIG. 4C depicts the final steps of methylation and oxygenation for generation of the ubiquinone CoQ10.

[0031] FIG. 5, illustrates how intermediates in the isoprenoid biosynthesis pathway can be processed into biomolecules, including ubiquinones, carotenoids, sterols, steroids, and vitamins, such as vitamin E or vitamin K.

[0032] FIG. 6 illustrates how intermediates in ubiquinone biosynthesis feed into the biosynthetic pathway, and how some intermediates can be processed into other molecules.

[0033] FIGS. 7A-7C 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.

[0034] FIG. 8 depicts the steroid biosynthesis pathways, including the isoprenoid biosynthesis pathway, it indicates where certain quinone derived compounds (e.g., a ubiquinone, vitamin E and vitamin K) are produced via geranylgeranyl pyrophosphate.

[0035] FIG. 9 depicts ubiquinone and vitamin K biosynthesis pathways.

[0036] FIG. 10, Panels A-C, depict vitamin E biosynthesis pathways. Panel A shows synthesis of .gamma.- and .alpha.-tocopherols from isopentenyl pyrophosphate and p-hydroxyphenpyruvate through action of geranylgeranylpyrophosphate synthse, geranylgeranyl reductase, p-hydroxyphenpyruvate dioxygenase, prenyl transferase, methyltransferase I, cyclase, and .gamma.-methyltransferase enzyme activities. Panel B depicts the tocopherol biosynthetic pathway in plants. Dashed arrows represent multiple steps. Enzymes are indicated by circled numbers as follows: 1) HGA phytyltransferase; 2) p-hydroxyphenyl pyruvate dioxygenase; 3) HGA dioxygenase; 4) geranylgeranyl diphosphate reductase, 5) geranylgeranyl diphsophate synthase; 6) 1-deoxy-D-xylulose-5-phosphate sunthase; 7) 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase; 8) tocopherol cyclase; and 9) .gamma.-tocopherol methyltransferase. Panel C depicts synthesis of .alpha., .beta., .gamma., and .delta. tocopherols.

[0037] FIGS. 11A-11C are a Table listing certain Y. lipolytica genes representing various polypeptides useful in engineering cells in accordance with the present invention.

[0038] FIG. 12, Panels A-M depict schematic representations of plasmids generated and described in detail in the exemplification

DEFINITIONS

[0039] Aromatic amino acid biosynthesis polypeptide: The term "aromatic amino acid biosynthesis polypeptide" refers to any polypeptide that is involved in the synthesis of aromatic amino acids in yeast and/or bacteria through chorismate and the shikimate pathway. For example, as discussed herein, anthranilate synthase, enzymes of the shikimate pathway, chorismate mutase, chorismate synthase, DAHP synthase, and transketolase are all aromatic amino acid biosynthesis polypeptides. Each of these polypeptides is also a ubiquinone biosynthesis polypeptide or a ubiquinone biosynthesis competitor for purposes of the present invention, as production of chorismate is a precursor in the synthesis of para-hydroxybenzoate for the biosynthesis of a ubiquinone. Representative examples of some of these enzymes are provided in Tables 32-37.

[0040] Aromatic amino acid pathway: The "aromatic amino acid pathway" is understood in the art to refer to a metabolic pathway that produces or utilizes shikimate pathway enzymes and chorismate in the production of phenylalanine, tryptophan or tyrosine. As discussed herein, two different pathways can produce the ubiquinoid precursor para-hydroxybenzoate--the first, the "shikimate pathway" is utilized in prokaryotes and induces conversion of chorismate to para-hydroxybenzoate through the action of chorismate pyruvate lyase; the second is utilized in mammalian systems and induces induction of para-hydroxybenzoate by derivation of tyrosine or phenylalanine. The term "aromatic amino acid pathway" encompasses both of these pathways. Lower eukaryotes such as yeast can utilize either method for production of para-hydroxybenzoate.

[0041] Biosynthesis polypeptide: The term "biosynthesis polypeptide" as used herein (typically in reference to a particular compound or class of compounds), refers to polypeptides involved in the production of the compound or class of compounds. In some embodiments of the invention, biosynthesis polypeptides are synthetic enzymes that catalyze particular steps in a synthesis pathway that ultimately produces a relevant compound. In some embodiments, the term "biosynthesis polypeptide" may also encompass polypeptides that do not themselves catalyze synthetic reactions, but that regulate expression and/or activity of other polypeptides that do so.

[0042] C.sub.5-9 quinone biosynthesis polypeptide: The term "C.sub.5-9 quinone biosynthesis polypeptide" refers to any polypeptide that is involved in the synthesis of a C.sub.5-9 quinone, for example a polyprenyldiphosphate synthase polypeptide. To mention but a few, these include, for example, pentaprenyl, hexaprenyl, heptaprenyl, octaprenyl, and/or solanesyl(nonaprenyl) diphosphate synthase polypeptides (i.e., polypeptides that perform the chemical reactions performed by the pentaprenyl, hexaprenyl, heptaprenyl, octaprenyl, and solanesyl(nonaprenyl) polypeptides, respectively, listed in Tables 61-65 (see also Okada et al., Biochim. Biophys. Acta 1302:217, 1996; Okada et al., J. Bacteriol. 179:5992, 1997). As will be appreciated by those of ordinary skill in the art, in some embodiments of the invention, C.sub.5-9 quinone biosynthesis polypeptides include polypeptides that affect the expression and/or activity of one or more other C.sub.5-9 quinone biosynthesis polypeptides.

[0043] C.sub.5-9 quinone compound: The term "C.sub.5-9 quinone compound", as used herein, refers to a family of ubiquinone compounds having 5-9 isoprenoid units in the side chain.

[0044] C.sub.5-9 quinone production modification: The term "C.sub.5-9 quinone production modification", as used herein, refers to a modification of a host organism that adjusts production of one or more C.sub.5-9 quinones, as described herein. For example, a C.sub.5-9 quinone production modification may increase the production level of one or more C.sub.5-9 quinones, and/or may alter relative production levels of different C.sub.5-9 quinones. In principle, an inventive C.sub.5-9 quinone production modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of one or more C.sub.5-9 quinones 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 C.sub.5-9 quinone production modification will comprise a genetic modification, typically resulting in increased production of one or more selected C.sub.5-9 quinones. In some embodiments, the C.sub.5-9 quinone production modification comprises at least one chemical, physiological, genetic, or other modification; in other embodiments, the C.sub.5-9 quinone production 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, chemical and/or physiological modification(s)).

[0045] 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 carotenogenic modification comprises at least one chemical, physiological, genetic, or other modification; in other embodiments, the carotenogenic 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, chemical and/or physiological modification(s)). In some embodiments, the selected carotenoid is one or more of astaxanthin, .beta.-carotene, canthaxanthin, lutein, lycopene, phytoene, zeaxanthin, and/or modifications of zeaxanthin or astaxanthin (e.g., glucoside, or other ester of zeaxanthin or astaxanthin). In some embodiments, the selected carotenoid is astaxanthin. In some embodiments, the selected carotenoid is other than .beta.-carotene.

[0046] 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.

[0047] 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 (C.sub.30 diapocarotenoids) and tetraterpenes (C.sub.40 carotenoids) as well as their oxygenated derivatives and other compounds that are, for example, C.sub.35, C.sub.50, C.sub.60, C.sub.70, C.sub.80 in length or other lengths. Many carotenoids have strong light absorbing properties and may range in length in excess of C.sub.200. C.sub.30 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 C.sub.30 carotenoids may be formally derived from the acyclic C.sub.30H.sub.42 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. C.sub.40 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 C.sub.40 carotenoids may be formally derived from the acyclic C.sub.40H.sub.56 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 C.sub.40 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; carotenoids include but are not limited to: antheraxanthin, adonirubin, adonixanthin, astaxanthin, canthaxanthin, capsorubrin, .beta.-cryptoxanthin, .alpha.-carotene, .beta.-carotene, .beta.,.psi.-carotene, .delta.-carotene, .epsilon.-carotene, echinenone, 3-hydroxyechinenone, 3'-hydroxyechinenone, .gamma.-carotene, .psi.-carotene, 4-keto-.gamma.-carotene, .zeta.-carotene, .alpha.-cryptoxanthin, deoxyflexixanthin, diatoxanthin, 7,8-didehydroastaxanthin, didehydrolycopene, fucoxanthin, fucoxanthinol, isorenieratene, .beta.-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-.beta.-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).

[0048] 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. In some instances, a single gene may encode a protein with multiple carotenoid biosynthesis polypeptide activities. Representative examples of carotenoid biosynthesis polypeptide sequences are presented in Tables 17-21 and Tables 38-41. As will be appreciated by those of ordinary skill in the art, in some embodiments of the invention, carotenoid biosynthesis polypeptides include polypeptides that affect the expression and/or activity of one or more other carotenoid biosynthesis polypeptides.

[0049] Effective amount. The term "effective amount" is used herein to describe concentrations or amounts of compounds and/or compositions that, when administered to a subject, achieve a desired therapeutic or physiological effect.

[0050] FPP biosynthesis polypeptides: The term "FPP biosynthesis polypeptide" refers to any polypeptide that is involved in the synthesis of farnesyl pyrophosphate. As discussed herein, farnesyl pyrophosphate represents the branchpoint between the sterol biosynthesis pathway and the carotenoid and other biosynthesis pathways. One specific example of an FPP biosynthesis polypeptide is FPP synthase. Representative examples of FPP synthase polypeptide sequences are presented in Table 14. As will be appreciated by those of ordinary skill in the art, in some embodiments of the invention, FPP biosynthesis polypeptides include polypeptides that affect the expression and/or activity of one or more other FPP biosynthesis polypeptides.

[0051] 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).

[0052] 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. Where a plurality of different heterologous polypeptides are to be introduced into and/or expressed by a host cell, different polypeptides may be from different source organisms, or from the same source organism. To give but one example, in some cases, individual polypeptides may represent individual subunits of a complex protein activity and/or may be required to work in concert with other polypeptides in order to achieve the goals of the present invention. In some embodiments, it will often be desirable for such polypeptides to be from the same source organism, and/or to be sufficiently related to function appropriately when expressed together in a host cell. In some embodiments, such polypeptides may be from different, even unrelated source organisms. It will further be understood that, where a heterologous polypeptide is to be expressed in a host cell, it will often be desirable to utilize nucleic acid sequences encoding the polypeptide that have been adjusted to accommodate codon preferences of the host cell and/or to link the encoding sequences with regulatory elements active in the host cell.

[0053] Host cell: As used herein, the "host cell" is a fungal cell or yeast cell that is manipulated according to the present invention to accumulate lipid and/or to express one or more quinone derived compounds as described herein. A "modified host cell", as used herein, is any host cell which has been modified, engineered, or manipulated in accordance with the present invention as compared with a parental cell. In some embodiments, the modified host cell has at least one quinonogenic and/or at least one oleagenic modification. In some embodiments, the modified host cell containing at least one oleaginic modification and/or one quinonogenic modification further has at least one sterologenic modification and/or or at least one carotenogenic modification. In some embodiments, the parental cell is a naturally occurring parental cell.

[0054] 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" or "concentrated". Isolation and/or purification and/or concentration may be performed using any techniques known in the art including, for example, fractionation, extraction, precipitation, or other separation.

[0055] 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. As will be appreciated by those of ordinary skill in the art, in some embodiments of the invention, isoprenoid biosynthesis polypeptides include polypeptides that affect the expression and/or activity of one or more other isoprenoid biosynthesis polypeptides (e.g., of one or more enzymes that participate(s) in isoprenoid synthesis). Thus, for instance, transcription factors that regulate expression of isoprenoid biosynthesis enzymes can be isoprenoid biosynthesis polypeptides for purposes of the present invention. To give but a couple of examples, the S. cerevisiae Upc2 and YLR228c genes, and the Y. lipolytica YALI0B00660g gene encode transcription factors that are isoprenoid biosynthesis polypeptides according to certain embodiments of the present invention. For instance, the semidominant upc2-1 point mutation (G888D) exhibits increased sterol levels (Crowley et al., J. Bacteriol 180:4177-4183, 1998). Corresponding YLR228c mutants have been made and tested (Shianna et al., J Bacteriol 183:830, 2001); such mutants may be useful in accordance with the present invention, as may be YALI0B00660g derivatives with corresponding upc2-1 mutation(s).

[0056] 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.

[0057] 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/or 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, chemical and/or physiological modification(s)).

[0058] 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, malate dehydrogenase, 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, malate dehydrogenase, and/or AMP deaminase is expected to promote oleaginy. Each of these proteins is an oleaginic peptide for the purposes of the present invention, and sequences of representative examples of these enzymes are provided in Tables 1-6, 69. Other peptides that can be involved in regenerating NADPH may include, for example, 6-phosphogluconate dehydrogenase (gnd); Fructose 1,6 bisphosphatase (fbp); Glucose 6 phosphate dehydrogenase (g6pd); NADH kinase (EC 2.7.1.86); and/or transhydrogenase (EC 1.6.1.1 and 1.6.1.2). Alternative or additional strategies to promote oleaginy may include one or more of the following: (1) increased or heterologous expression of one or more of acyl-CoA:diacylglycerol acyltransferase (e.g., DGA1; YALI0E32769g); phospholipid: diacylglycerol acyltransferase (e.g., LRO1; YALI0E16797g); and acyl-CoA:cholesterol acyltransferase (e.g., ARE genes such as ARE1, ARE2, YALI0F06578g), which are involved in triglyceride synthesis (Kalscheuer et al. Appl Environ Microbiol p. 7119-7125, 2004; Oelkers et al. J Biol Chem 277:8877-8881, 2002; and Sorger et al. J Biol Chem 279:31190-31196, 2004), (2) decreased expression of triglyceride lipases (e.g., TGL3 and/or TGL4; YALI0D17534g and/or YALI0F10010g (Kurat et al. J Biol Chem 281:491-500, 2006); and (3) decreased expression of one or more acyl-coenzyme A oxidase activities, for example encoded by POX genes (e.g. POX1, POX2, POX3, POX4, POX5; YALI0C23859g , YALI0D24750g, YALI0E06567g, YALI0E27654g, YALI0E32835g, YALI0F10857g; see, for example, Mlickova et al. Appl Environ Microbiol 70: 3918-3924, 2004; Binns et al. J Cell Biol 173:719, 2006). Each of these proteins is an oleaginic peptide for the purposes of the present invention, and sequences of representative examples of these enzymes are provided in Tables 70-85 and Tables 100-101.

[0059] Oleaginous: The term "oleaginous", as used herein, refers to 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% (e.g., about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or more) 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. For example, for the purposes of the present invention, Yarrowia lipolytica is a naturally oleaginous fungi. 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 example, for the purposes of the present invention, Saccharomyces cerevisiae, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), and Candida utilis are not naturally oleaginous fungi.

[0060] PHB polypeptide or PHB biosynthesis polypeptide: The terms "PHB polypeptide" or "PHB biosynthesis polypeptide" as used herein refers to a polypeptide that is involved in the synthesis of para-hydroxybenzoate from chorismate. In prokaryotes and lower eukaryotes, synthesis of para-hydroxybenzoate occurs by the action of chorismate pyruvate lyase. Biosynthesis of para-hydroxybenzoate from tyrosine or phenylalanine occurs through a five-step process in mammalian cells. Lower eukaryotes such as yeast can utilize either method for production of para-hydroxybenzoate. For example, enzymes of the shikimate pathway, chorismate synthase, DAHP synthase, and transketolase are all PHB biosynthesis polypeptides. Each of these polypeptides is also a ubiquinone biosynthesis polypeptide or a ubiquinone biosynthesis competitor polypeptide for purposes of the present invention. Exemplary PHB polypeptides are provided in Tables 33 and 35-37.

[0061] 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, aromatic amino acid biosynthesis polypeptides, biosynthesis polypeptides, C.sub.5-9 quinone biosynthesis polypeptides, carotenogenic polypeptides, carotenoid biosynthesis polypeptides, isoprenoid biosynthesis polypeptides, oleaginic polypeptides, PHB polypeptides, PHB biosynthesis polypeptides, quinone biosynthesis polypeptides, quinonogenic polypeptides, ubiquinone biosynthesis polypeptides, ubiquinone biosynthesis competitor polypeptides, ubiquinogenic polypeptides, Vitamin E biosynthesis polypeptides, Vitamin K biosynthesis polypeptides, etc. 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. Other regions of similarity and/or identity can be determined by those of ordinary skill in the art by analysis of the sequences of various polypeptides presented in the Tables herein.

[0062] Quinone biosynthesis polypeptide: A "quinone biosynthesis polypeptide", as that term is used herein, refers to any polypeptide involved in the synthesis of one or more quinone derived compound, as described herein. In particular, quinone biosynthesis polypeptides include ubiquinone biosynthesis polypeptides (e.g., biosynthesis polypeptides involved in production of coenzyme Q10 and/or a C.sub.5-9 quinone compound), vitamin K biosynthesis polypeptides, and vitamin E biosynthesis polypeptides.

[0063] Quinone derived compounds: The term "quinone derived compounds" is used herein to refer to certain compounds that either contain a quinone moiety or are derived from a precursor that contains a quinone moiety. In particular, quinone derived compounds according to the present invention are ubiquinones (e.g., coenzyme Q10, C.sub.5-9 quinone compounds, etc.), vitamin E compounds, and/or vitamin K compounds. Structures of representative quinone derived compounds are presented in FIG. 1.

[0064] Quinonogenic modification: The term "quinonogenic modification", as used herein, refers to refers to a modification of a host organism that adjusts production of one or more quinone derived compounds (e.g., ubiquinones, vitamin K compounds, vitamin E compounds, etc.), as described herein. For example, a quinonogenic modification may increase the production level of a particular quinone derived compound, or of a variety of different quinone derived compounds. In some embodiments of the invention, production of a particular quinone derived compound may be increased while production of other quinone derived compounds is decreased. In some embodiments of the invention, production of a plurality of different quinone derived compounds is increased. In principle, an inventive quinonogenic modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of one or more quinone derived compounds 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 quinonogenic modification will comprise a genetic modification, typically resulting in increased production of one or more quinone derived compounds (e.g., ubiquinones, vitamin K compounds, vitamin E compounds). In some embodiments, the quinonogenic modification comprises at least one chemical, physiological, genetic, or other modification; in other embodiments, the quinonogenic 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, chemical and/or physiological modification(s)).

[0065] Quinonogenic polypeptide: The term "quinonogenic polypeptide", as used herein, refers to any polypeptide whose activity in a cell increases production of one or more quinone derived compounds (e.g., ubiquinones, vitamin K compounds, vitamin E compounds) in that cell. The term encompasses both polypeptides that are directly involved in quinone derived compound synthesis and those whose expression or activity affects the extent or level of production of one or more quinone derived compounds, for example by scavenging a substrate or reactant utilized by a quinone biosynthetic polypeptide that is directly involved in quinone derived compound production. Quinonogenic polypeptides include isoprenoid biosynthesis polypeptides, ubiquinone biosynthesis polypeptides (e.g., polypeptides involved in production of coenzyme Q10 and/or a C.sub.5-9 quinone compound), vitamin E biosynthesis polypeptides, and vitamin K biosynthesis polypeptides. Quinonogenic polypeptides may also include ubiquinogenic polypeptides, etc. The term also encompasses polypeptides that may affect the extent to which one or more quinone derived compounds is accumulated in lipid bodies.

[0066] Small Molecule: In general, a small molecule is understood in the art to be an organic molecule that is less than about 5 kilodaltons (Kd) in size. In some embodiments, the small molecule is less than about 3 Kd, 2 Kd, or 1 Kd. In some embodiments, the small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, small molecules are non-polymeric. In some embodiments, small molecules are not proteins, peptides, or amino acids. In some embodiments, small molecules are not nucleic acids or nucleotides. In some embodiments, small molecules are not saccharides or polysaccharides.

[0067] 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 multiple 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, archaebacterial, cyanobacterial, and protozoal source organisms.

[0068] Sterol biosynthesis polypeptide: The term "sterol biosynthesis polypeptide", as used herein, refers to any polypeptide that is involved in the synthesis of one or more sterol compounds. Thus, sterol biosynthesis polypeptides can include isoprenoid biosynthesis polypeptides to the extent that they are involved in production of isopentyl pyrophosphate. Moreover, the term refers to any polypeptide that acts downstream of farnesyl pyrophosphate and in involved in the production of one or more sterol compounds. For example, sterol biosynthesis polypeptides include squalene synthase, which catalyses conversion of farnesyl pyrophosphate to presqualene pyrophosphate, and further catalyzes conversion of presqualene pyrophosphate to squalene (i.e., enzyme 2.5.1.21 in FIG. 8). In some embodiments of the invention, sterol biosynthesis polypeptides further include one or more polypeptides involved in metabolizing squalene into a vitamin D compound. Thus, sterol biosynthesis polypeptides can include one or more of the 1.14.99.7, 5.4.99.7, 5.4.99.8, 5.3.3.5, 1.14.21.6, 1.14.15.-, 1.14.13.13 enzyme polypeptides depicted in FIG. 8, as well as other enzyme polypeptides involved in the illustrated pathways. Furthermore, sterol biosynthesis polypeptides can include one or more enzyme polypeptides including, for example, C-14 demethylase (ERG9), squalene monooxygenase (ERG1), 2,3-oxidosqualene-lanosterol synthase (ERG7), C-1 demethylase (ERG11), C-14 reductase (ERG24), C-4 methyloxidase (ERG25), C-4 decarboxylase (ERG26), 3-ketoreductase (ERG27), C-24 methyltransferase (ERG6), .DELTA.8-7 isomerase (ERG2), C-5 desaturase (ERG3), C-22 desaturase (ERG5) and/or C-24 reductase (ERG4) polypeptides, and/or other polypeptides involved in producing one or more vitamin D compounds (e.g., vitamin D2, vitamin D3, or a precursor thereof). As will be appreciated by those of ordinary skill in the art, in some embodiments of the invention, sterol biosynthesis polypeptides include polypeptides that affect the expression and/or activity of one or more other sterol biosynthesis polypeptides. Thus, for instance, transcription factors that regulate expression of sterol biosynthesis enzymes can be sterol biosynthesis polypeptides for purposes of the present invention. To give but a couple of examples, the S. cerevisiae Upc2 and YLR228c genes, and the Y. lipolytica YALI0B00660g gene encode transcription factors that are sterol biosynthesis polypeptides according to certain embodiments of the present invention. For instance, the semidominant upc2-1 point mutation (G888D) exhibits increased sterol levels (Crowley et al., J. Bacteriol 180:4177-4183, 1998). Corresponding YLR228c mutants have been made and tested (Shianna et al., J Bacteriol 183:830, 2001); such mutants may be useful in accordance with the present invention, as may be YALI0B00660g derivatives with corresponding upc2-1 mutation(s). Representative examples of certain sterol biosynthesis polypeptide sequences are presented in Table 16 and 86-99.

[0069] Sterologenic modification: The term "sterologenic modification", as used herein, refers to a modification of a host organism that adjusts production of one or more sterol compounds (e.g., squalene, lanosterol, zymosterol, ergosterol, 7-dehydrocholesterol (provitamin D3), vitamin D compound(s), etc.), as described herein. For example, a sterologenic modification may increase the production level of a particular sterol compound, or of a variety of different sterol compounds. In some embodiments of the invention, production of a particular sterol compound may be increased while production of other sterol compounds is decreased. In some embodiments of the invention, production of a plurality of different sterol compounds is increased. In principle, an inventive sterologenic modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of one or more sterol compounds 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 sterologenic modification will comprise a genetic modification, typically resulting in increased production of one or more sterol compounds (e.g., squalene, lanosterol, zymosterol, ergosterol, 7-dehydrocholesterol (provitamin D3) or vitamin D compound(s)). 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).

[0070] Subject: The term "subject" is used throughout the present specification to describe an animal, in most instances a human, to whom inventive compositions are administered.

[0071] Ubiquinone: The term "ubiquinone" is understood in the art to refer to a structural class of quinone derivatives with or without isoprenoid side chains. Ubiquinones are described in the Merck Index, 11th Edition, Merck & Co., Inc. Rahway, N.Y., USA, Abstr. 9751 (1989), which is incorporated herein by reference. A dual nomenclature exists for these compounds and is based upon the length of the terpenoid side chain. Those which contain an isoprene side chain are also referred to by the term coenzymes Q. A benzoquinone of this family is therefore properly referred to as either "Coenzyme Qn," where n is an integer from one to twelve and designates the number of isoprenoid units in the side chain, or alternatively, "ubiquinone (x)" where x designates the total number of carbon atoms in the side chain and is a multiple of five. Typically, n is an integer ranging from 0 to 12, in particular from 1 to 12, and more particularly 6, 7, 8, 9, or 10. For example, the most common ubiquinone in animals has a ten isoprenoid side chain and is referred to as either Coenzyme Q10, ubiquinone, or ubidecarenone. In other organisms (e.g. fungi, bacteria), other ubiquinones, for example C.sub.5 (CoQ5), C.sub.6 (CoQ6), C.sub.7 (CoQ7), C.sub.8 (CoQ8), or C.sub.9 (CoQ9) (collectively C.sub.5-9) quinones are more prevalent than Coenzyme Q10 (CoQ10). As mentioned, ubiquinones may lack an isoprene side chain, and may be selected from alkylubiquinones in which the alkyl group may contain from 1 to 20 and preferably from 1 to 12 carbon atoms, such as, for example, decylubiquinones such as 6-decylubiquinone or 2,3-dimethoxy-5-decyl-1,4-ubiquinone, derivatives thereof, and mixtures thereof. Ubiquinones may exist in reduced (ubiquinol), oxidized or superoxidized states. For example, the oxidation states of the ubiquinone coenzyme Q10 are depicted in FIG. 1A.

[0072] Ubiquinone biosynthesis competitor: The term "ubiquinone biosynthesis competitor", as used herein, refers to an agent whose presence or activity in a cell reduces the level of farnesyl pyrophosphate (FPP), geranylgeranyl diphosphate (GGPP), chorismate, or any combination thereof that is available to enter the ubiquinogenic biosynthesis pathway. The term "ubiquinone biosynthesis competitor" encompasses both polypeptide (e.g. ubiquinone biosynthesis competitor polypeptides) and non-polypeptide (e.g., small molecule) inhibitor agents. Those of ordinary skill in the art will appreciate that certain competitor agents that do not act as inhibitors of ubiquinone biosynthesis generally can nonetheless act as inhibitors of biosynthesis of a particular ubiquinone compound (e.g. CoQ10 or a C.sub.5-9 quinone compound). Particular examples of ubiquinone biosynthesis competitor agents act on isoprenoid intermediates prior to FPP or GGPP, such that less FPP or GGPP is generated (see, for example, FIG. 5, FIG. 6). Squalene synthase (also called squalene synthetase) is a ubiquinone biosynthesis competitor according to the present invention; representative squalene synthase sequences are presented in Table 16. In another example, ubiquinone biosynthesis competitors include aromatic amino acid polypeptide enzymes that act on PHB precursors at or prior to chorismate, such that less chorismate is available for synthesis of para-hydroxybenzoate (see, for example FIG. 3). Anthranilate synthase is one ubiquinone biosynthesis competitor according to the present invention, and chorismate mutase another; representative anthranilate synthase and chorismate mutase polypeptides are presented in Table 32, 32B and Table 34, respectively. Those of ordinary skill in the art, considering the known metabolic pathways relating to ubiquinone production and/or metabolism (see, for example, FIG. 1 and other Figures and references herein) will readily appreciate a variety of other particular ubiquinone biosynthesis competitors, including ubiquinone biosynthesis polypeptides.

[0073] Ubiquinone biosynthesis polypeptide: The term "ubiquinone biosynthesis polypeptide" refers to any polypeptide that is involved in the synthesis of a ubiquinone (e.g., CoQ10 or a C.sub.5-9 quinone compound). To mention but a few, these ubiquinone biosynthesis polypeptides include, for example, polypeptides of polyprenyldiphosphate synthase (e.g. penta-, hexa-, hepta, octa-, nona-, deca-prenyldiphosphate synthase, PHB-polyprenyltransferase, and O-methyltransferase. Representative examples of ubiquinone biosynthesis polypeptide sequences are presented in Tables 23 (including 23b and 23c), 24-31, Table 33 as well as 35-37 (see also above PHB biosynthesis, C.sub.5-9 quinone biosynthesis polypeptides (e.g., Tables 61-65), and isoprenoid biosynthesis polypeptides which are ubiquinone biosynthesis polypeptides).

[0074] Ubiquinogenic modification: The term "ubiquinogenic modification", as used herein, refers to a modification of a host organism that adjusts production of a ubiquinone (e.g., CoQ10), as described herein. For example, a ubiquinogenic modification may increase the production level of a ubiquinone (e.g., CoQ 10 and/or a C.sub.5-9 quinone compound), and/or may alter relative levels of a ubiquinone and/or a ubiquinol. In principle, an inventive ubiquinogenic modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of a ubiquinone (e.g., CoQ10 and/or a C.sub.5-9 quinone compound) 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 ubiquinogenic modification will comprise a genetic modification, typically resulting in increased production of a ubiquinone (e.g., CoQ10 and/or a C.sub.5-9 quinone compound). In some embodiments, the ubiquinogenic modification comprises at least one chemical, physiological, genetic, or other modification; in other embodiments, the ubiquinogenic 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, chemical and/or physiological modification(s)).

[0075] Ubiquinogenic polypeptide: The term "ubiquinogenic polypeptide," as used herein, refers to any polypeptide that is involved in the process of producing a ubiquinone (e.g., CoQ10 and/or a C.sub.5-9 quinone compound) in a cell, and may include polypeptides that are involved in processes other than ubiquinone production but whose expression or activity affects the extent or level of production of a ubiquinone and/or a ubiquinol, for example by scavenging a substrate or reactant utilized by a ubiquinone biosynthetic polypeptide that is directly involved in ubiquinone production. Ubiquinogenic polypeptides include isoprenoid biosynthesis polypeptides, ubiquinone biosynthesis polypeptides, and ubiquinone biosynthesis competitor polypeptides, as those terms are defined herein. The term also encompasses polypeptides that may affect the extent to which a given ubiquinone is accumulated in lipid bodies.

[0076] Vitamin D biosynthesis polypeptide: The term "vitamin D biosynthesis polypeptide" refers to any polypeptide that is involved in the synthesis of one or more vitamin D compounds. To mention but a few, these include, for example, the 1.14.99.7, 5.4.99.7, 5.4.99.8, 5.3.3.5, and/or 1.14.21.6, polypeptides depicted in FIG. 8. They further can include the hydroxylases that convert vitamin D.sub.3 to calcitriol (e.g., the 1.14.15.- and 1.14.13.13 polypeptides depicted in FIG. 8). As will be appreciated by those of ordinary skill in the art, in some embodiments of the invention, vitamin D biosynthesis polypeptides include polypeptides that affect the expression and/or activity of one or more other vitamin D biosynthesis polypeptides. Particular examples of certain vitamin D biosynthesis polypeptides are presented in Tables 86-99.

[0077] Vitamin E biosynthesis polypeptide: The term "vitamin E biosynthesis polypeptide" refers to any polypeptide that is involved in the synthesis of vitamin K. To mention but a few, these include, for example, tyrA, pds1(hppd), VTE1, HPT1(VTE2), VTE3, VTE4, and/or GGH polypeptides (i.e., polypeptides that perform the chemical reactions performed by tyrA, pds1(hppd), VTE1, HPT1(VTE2), VTE3, VTE4, and/or GGH, respectively). Particular examples of such vitamin E biosynthesis polypeptides are presented in Tables 54-60.

[0078] Vitamin E compound: The term "vitamin E compound", as used herein, refers to members of a family of structurally related compounds that have a 6-chromanol ring, an isoprenoid side chain, and the biologic activity of .alpha.-tocopherol. The term encompasses the eight known naturally occurring vitamin E compounds, the four tocopherols (.alpha., .beta., .gamma., .delta.) and four tocotrienols (.alpha., .beta., .gamma., .delta.), which all contain a hydrophilic chromanol ring and a hydrophobic side chain. The .alpha., .beta., .gamma., and .delta. forms differ from one another in the number of methyl groups on the chromanol ring. Several synthetic vitamin E compounds have also been prepared, and still others are possible (see, for example, Bramley et al., J. Sci Food Agric 80:913, 2000). .alpha.-tocopherol is a potent antioxidant, and is generally considered to be the most active vitamin E compound in humans.

[0079] Vitamin E production modification: The term "Vitamin E production modification", as used herein, refers to a modification of a host organism that adjusts production of Vitamin E. For example, a Vitamin E production modification may increase the production level of one or more vitamin E compounds, and/or may alter relative production levels of different vitamin E compounds. In principle, an inventive vitamin E production modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of one or more vitamin E compounds 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 vitamin E production modification will comprise a genetic modification, typically resulting in increased production of one or more selected vitamin E compounds. In some embodiments, the vitamin E production modification comprises at least one chemical, physiological, genetic, or other modification; in other embodiments, the vitamin E production 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, chemical and/or physiological modification(s)).

[0080] Vitamin K biosynthesis polypeptide: The term "vitamin K biosynthesis polypeptide" refers to any polypeptide that is involved in the synthesis of vitamin K. To mention but a few, these include, for example, MenF, MenD, MenC, MenE, MenB, MenA, UbiE, and/or MenG polypeptides (i.e., polypeptides that perform the chemical reactions performed by MenF, MenD, MenC, MenE, MenB, MenA, UbiE, and/or MenG, respectively). Particular examples of such vitamin K biosynthesis polypeptides are presented in Tables 46-53.

[0081] Vitamin K compounds: The term "vitamin K compounds", as used herein, refers to members of a family of structurally related compounds that share a common biologic activity. In particular, vitamin K compounds are derivatives of 2-methyl-1,4-naphthoquinone that have coagulation activity. The two natural forms of vitamin K differ in the identity of their side chains at position 3. Vitamin K.sub.1, also known as phylloquinone (based on its presence in plants), has a phytyl side chain in position 3; vitamin K2, also known as menaquinone, has an isoprenyl side chain at position 3. Different forms of menaquinone, having side chains with different numbers of isoprene units (typically 4-13) are found in different types of cells.

[0082] Vitamin K production modification: The term "Vitamin K production modification", as used herein, refers to a modification of a host organism that adjusts production of Vitamin K. For example, a Vitamin K production modification may increase the production level of one or more vitamin K compounds, and/or may alter relative production levels of different vitamin K compounds. In principle, an inventive vitamin K production modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of one or more vitamin K compounds 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 vitamin K production modification will comprise a genetic modification, typically resulting in increased production of one or more selected vitamin K compounds. In some embodiments, the vitamin K production modification comprises at least one chemical, physiological, genetic, or other modification; in other embodiments, the vitamin K production 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, chemical and/or physiological modification(s)).

Detailed Description of Certain Preferred Embodiments of the Invention

[0083] The present invention embraces the reasoning that quinone derived compound(s) (e.g., ubiquinones, vitamin K compounds, and vitamin E compounds) can effectively 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 (ii) produce one or more quinone derived compound(s) at a level at least about 1%, of their dry cell weight, are generated through manipulation of host cells (i.e., strains, including, e.g., naturally-occurring strains and strains which have been previously modified). In certain embodiments, strains can accumulate lipid typically to at least about 20% of their dry cell weight. In some embodiments quinone derived compound(s) can be produced in the strains to 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%, or at least about 20% of fungus' dry cell weight. Thus, the provided manipulated strains can then be used to produce the quinone derived compound(s). In some embodiments, the compound(s) that partition(s) into the lipid bodies can readily be isolated. In some embodiments the quinone derived compound is Coenzyme Q10 (CoQ10; Ubiquinone 10; Ubiquinone 50). In some embodiments, the quinone derived compound is C.sub.5 (CoQ5), C.sub.6 (CoQ6), C.sub.7 (CoQ7), C.sub.8 (CoQ8), or C.sub.9 (CoQ9) quinone. In some embodiments, the quinone derived compound is a tocopherol or a tocotrienols. In some embodiments, the quinone derived compound is phylloquinone (vitamin K.sub.1), or menaquinone (vitamin K.sub.2).

[0084] In some embodiments, it will be desirable to balance oleaginy and production of quinone derived compound(s) in cells such that, as soon as a minimum desirable level of oleaginy is achieved, substantially all further carbon is diverted into a metabolic pathway that results in production of one or more quinone derived compounds. 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 do 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 ubiquinone production.

[0085] To give but one example of adjustments that could be made to achieve a desired balance between oleaginy and production of quinone derived compound(s), we note that, while increasing acetyl CoA carboxylase expression (and/or activity) promotes oleaginy, decreasing its expression and/or activity may promote production of quinone derived compound(s). Those of ordinary skill in the art will appreciate that the expression and/or activity of acetyl CoA carboxylase, or of other polypeptides, may be adjusted up or down as desired according to the characteristics of a particular host cell of interest.

[0086] We note that engineered cells and processes of using them as described herein may provide one or more advantages as compared with unmodified cells. Such advantages may include, but are not limited to: increased yield (e.g., quinone derived compound expressed as either % dry cell weight (mg/mg) or parts per million), titer (g quinone derived compound/L), specific productivity (mg quinone derived compound g.sup.-1 biomass hour.sup.-1), and/or volumetric productivity (g quinone derived compound liter.sup.-1 hour.sup.-1)) of the desired quinone derived compound (and/or intermediates thereof), and/or decreased formation of undesirable side products (for example, undesirable intermediates).

[0087] Thus, for example, the specific productivity for one or more quinone derived compounds (e.g., ubiquinones, vitamin K compounds, and/or vitamin E compounds) or total quinone derived compounds may be at or about 0.1, at or about 0.11, at or about 0.12, at or about 0.13, at or about 0.14, at or about 0.15, at or about 0.16, at or about 0.17, at or about 0.18, at or about 0.19, at or about 0.2, at or about 0.21, at or about 0.22, at or about 0.23, at or about 0.24, at or about 0.25, at or about 0.26, at or about 0.27, at or about 0.28, at or about 0.29, at or about 0.3, at or about 0.31, at or about 0.32, at or about 0.33, at or about 0.34, at or about 0.35, at or about 0.36, at or about 0.37, at or about 0.38, at or about 0.39, at or about 0.4, at or about 0.41, at or about 0.42, at or about 0.43, at or about 0.44, at or about 0.45, at or about 0.46, at or about 0.47, at or about 0.48, at or about 0.49, at or about 0.5, at or about 0.51, at or about 0.52, at or about 0.53, at or about 0.54, at or about 0.55, at or about 0.56, at or about 0.57, at or about 0.58, at or about 0.59, at or about 0.6, at or about 0.61, at or about 0.62, at or about 0.63, at or about 0.64, at or about 0.65, at or about 0.66, at or about 0.67, at or about 0.68, at or about 0.69, at or about 0.7, at or about 0.71, at or about 0.72, at or about 0.73, at or about 0.74, at or about 0.75, at or about 0.76, at or about 0.77, at or about 0.78, at or about 0.79, at or about 0.8, at or about 0.81, at or about 0.82, at or about 0.83, at or about 0.84, at or about 0.85, at or about 0.86, at or about 0.87, at or about 0.88, at or about 0.89, at or about 0.9, at or about 0.91, at or about 0.92, at or about 0.93, at or about 0.94, at or about 0.95, at or about 0.96, at or about 0.97, at or about 0.98, at or about 0.99, at or about 1, 1.05, at or about 1.1, at or about 1.15, at or about 1.2, at or about 1.25, at or about 1.3, at or about 1.35, at or about 1.4, at or about 1.45, at or about 1.5, at or about 1.55, at or about 1.6, at or about 1.65, at or about 1.7, at or about 1.75, at or about 1.8, at or about 1.85, at or about 1.9, at or about 1.95, at or about 2 mg g.sup.-1 hour.sup.-1 or more.

[0088] Thus, for example, the volumetric productivity for one or more quinone derived compounds (e.g., ubiquinones, vitamin K compounds, and/or vitamin E compounds) or total quinone derived compounds may be at or about 0.01, at or about 0.011, at or about 0.012, at or about 0.013, at or about 0.014, at or about 0.015, at or about 0.016, at or about 0.017, at or about 0.018, at or about 0.019, at or about 0.02, at or about 0.021, at or about 0.022, at or about 0.023, at or about 0.024, at or about 0.025, at or about 0.026, at or about 0.027, at or about 0.028, at or about 0.029, at or about 0.03, at or about 0.031, at or about 0.032, at or about 0.033, at or about 0.034, at or about 0.035, at or about 0.036, at or about 0.037, at or about 0.038, at or about 0.039, at or about 0.04, at or about 0.041, at or about 0.042, at or about 0.043, at or about 0.044, at or about 0.045, at or about 0.046, at or about 0.047, at or about 0.048, at or about 0.049, at or about 0.05, at or about 0.051, at or about 0.052, at or about 0.053, at or about 0.054, at or about 0.055, at or about 0.056, at or about 0.057, at or about 0.058, at or about 0.059, at or about 0.06, at or about 0.061, at or about 0.062, at or about 0.063, at or about 0.064, at or about 0.065, at or about 0.066, at or about 0.067, at or about 0.068, at or about 0.069, at or about 0.07, at or about 0.071, at or about 0.072, at or about 0.073, at or about 0.074, at or about 0.075, at or about 0.076, at or about 0.077, at or about 0.078, at or about 0.079, at or about 0.08, at or about 0.081, at or about 0.082, at or about 0.083, at or about 0.084, at or about 0.085, at or about 0.086, at or about 0.087, at or about 0.088, at or about 0.089, at or about 0.09, at or about 0.091, at or about 0.092, at or about 0.093, at or about 0.094, at or about 0.095, at or about 0.096, at or about 0.097, at or about 0.098, at or about 0.099, at or about 0.1, 0.105, at or about 0.110, at or about 0.115, at or about 0.120, at or about 0.125, at or about 0.130, at or about 0.135, at or about 0.14, at or about 0.145, at or about 0.15, at or about 0.155, at or about 0.16, at or about 0.165, at or about 0.17, at or about 0.175, at or about 0.18, at or about 0.185, at or about 0.19, at or about 0.195, at or about 0.20 grams liter.sup.-1 hour.sup.-1 or more.

Host Cells

[0089] 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 one or more quinone derived compounds (e.g., a ubiquinone, a vitamin K compound and/or a vitamin E compound). 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, quinone derived-compound-producing strains. Alternatively, strains that naturally are neither oleaginous nor quinone derived-compound-producing may be employed. Furthermore, even when a particular strain has a natural capacity for oleaginy or for production of one or more quinone derived compounds, its natural capabilities may be adjusted as described herein for optimal production of one or more particular desired compounds.

[0090] In certain embodiments, engineering or manipulation of a strain results in modification of a type of lipid and/or quinone derived compound produced. For example, a strain may be naturally oleaginous and/or may naturally produce one or more quinone derived compounds (e.g., a ubiquinone, including, e.g., CoQ5, CoQ6, CoQ8, CoQ9, CoQ10; vitamin K, including phylloquinone and/or one or more menaquinones; and/or vitamin E, including one or more tocopherols and/or one or more tocotrienols). However, engineering or modification of the strain may be employed so as to change the type or amount of lipid that is accumulated and/or to adjust production of one or more quinone derived compounds (including, for example, modulating relative amounts of particular quinone derived compounds). In some embodiments, production of CoQ10; phylloquinone; menaquinone; and/or .alpha.-tocopherol production will be optimized

[0091] 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. 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 pharmaceutical compositions, food or feed additives, or into nutritional supplements, as desired. 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 within the range of about 20-32.degree. C.) (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32.degree. 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, for example, as mycelia.

[0092] 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, Thraustochytrium, 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 toruloides, Rhodotorula glutinis, R. gracilis, R. graminis, R. mucilaginosa, R. pinicola, Thraustochytrium sp, Trichosporon pullans, T cutaneum, and Yarrowia lipolytica are used

[0093] Of these naturally oleaginous strains, some also naturally produce one or more quinone derived compounds and some do not. In most cases, only low levels (less than about 0.05% dry cell weight) of quinone derived compounds are produced by naturally-occurring oleaginous yeast or fungi.

[0094] In general, any organism that is naturally oleaginous but does not naturally produce one or more particular quinone derived compounds of interest (e.g., does not produce a ubiquinone such as CoQ10, vitamin K compounds, vitamin E compounds 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, Mortierella, and Yarrowia; in some embodiments, the organism is of a species including, but not limited to, Mortierella alpina and Yarrowia lipolytica. At least some fungal strains are known not to produce vitamin K.

[0095] Comparably, the present invention may utilize any naturally oleaginous organism that does naturally produce one or more quinone derived compounds as a host cell. In general, the present invention may be utilized to increase carbon flow into the isoprenoid pathway organisms that naturally produce a particular quinone derived compounds of interest and/or to shift production of one or more different quinone derived compounds. For example, the present invention may be utilized to shift production from one ubiquinone (e.g., CoQ5, CoQ6, CoQ8, CoQ9) to another (e.g., CoQ10). Introduction of one or more quinonogenic modifications (e.g., one or more ubiquinogenic modifications), such as but not limited to increased expression of one or more endogenous or heterologous quinonogenic polypeptides), in accordance with the present invention, can achieve these goals.

[0096] In certain embodiments of the invention, the utilized oleaginous, quinone derived compound-producing organism is a yeast or fungus, for example of a genus such as, but not limited to, Rhodotorula; in some embodiments, the organism is of a species such as Rhodotorula glutinis.

[0097] 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, Schizosaccharomyces, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia); in some embodiments, the organism is of a species including, but not limited to, Candida utilis, 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, Schizosaccharomyces pombe, Sclerotium rolfsii, Trichoderma reesei, and Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

[0098] 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. In some embodiments, the cell will be modified to accumulate at least about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% dry cell weight as lipid, so long as the accumulation level is more than that observed in the unmodified parental cell.

[0099] As with the naturally oleaginous organisms, some of the naturally non-oleaginous fungi naturally produce one or more quinone derived compounds, whereas others do not. Genera of naturally non-oleaginous fungi that do not naturally produce quinone derived compounds (such as CoQ10 or other ubiquinone, vitamin K compounds, vitamin E compounds) for instance, but that 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, Candida utilis, Aspergillus niger, Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and Saccharomyces cerevisiae. Saccharomyces cerevisiae, in particular, is known not to produce vitamin E or vitamin K compounds; other fungi likely also do not produce these compounds. Genera of naturally non-oleaginous fungi that do naturally produce one or more quinone derived compounds (e.g., CoQ10) and that may desirably be used as host cells in accordance with the present invention include, but are not limited to, yeasts of such genera as Schizosaccharomyces and Basidiomycetes; fungi of such genera as Tremella are capable of producing certain quinone derived compounds, and in particular are capable of producing CoQ10.

[0100] 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 Y. 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, 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, ubiquinone-producing (e.g., CoQ10) Y. lipolytica strain than might be required for other organisms. Furthermore, there is already extensive commercial experience with Y. lipolytica.

[0101] 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.

[0102] The edible fungus, Candida utilis is also a useful host cell in accordance with the present invention. Molecular biology tools and techniques are available in C. utilis (for example, see Iwakiri et al. (2006) Yeast 23:23-34, Iwakiri et al. (2005) Yeast 2005 22:1079-87, Iwakiri et al. (2005) Yeast 22:1049-60, Rodriquez et al. (1998) Yeast 14:1399-406, Rodriquez et al. (1998) FEMS Microbiol Lett. 165:335-40, and Kondo et al. (1995) J. Bacteriol. 177:7171-7).

[0103] Additional useful hosts include Xanthophyllomyces dendrorhous (Phaffia rhodozyma), which is experimentally tractable and naturally produces isoprenoid compounds.

[0104] Aspergillus niger and Mortierella alpina accumulate large amounts of citric acid and fatty acid, respectively; Mortierella alpina is also oleaginous.

[0105] Neurospora or Gibberella are also useful. They are not naturally oleaginous and may require extensive modification to be used 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.

[0106] 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 isoprenoids and may require less modification than other species available.

[0107] 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 may be less amenable to manipulate than several other potential host organisms.

[0108] Additional useful hosts include strains, such as Schizosaccharomyces pombe, Saitoella complicata, and Sporidiobolus ruineniae, which can produce certain quinone derived compounds including, for example, CoQ10.

[0109] 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).

[0110] To give but a few specific examples, of promoters and/or regulatory sequences that may be employed in expression of polypeptides according to the present invention, useful promoters include, but are not limited to, the Leu2 promoter and variants thereof (see, for example, see U.S. Pat. No. 5,786,212); the EF1alpha protein and ribosomal protein S7 gene promoters (see, for example, PCT Application WO 97/44470); the Gpm (see US20050014270), Xpr2 (see U.S. Pat. No. 4,937,189), Tef1, Gpd1 (see, for example, US Application 2005-0014270A1), Cam1 (YALIOC24420g), YALI0D16467g, Tef4 (YALI0B12562g), Yef3 (YALI0E13277g), Pox2, Yat1 (see, for example US Application 2005-0130280; PCT Application WO 06/052754), Fba1 (see, for example WO05049805), and/or Gpat (see WO06031937) promoters; the sequences represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, subsequences thereof, and hybrid and tandem derivatives thereof (e.g., as disclosed in US Application 2004-0146975); the sequences represented by SEQ ID NO: 1, 2, or 3 including fragments (e.g. by 462-1016 and bp 917-1016 of SEQ ID NO: 1; bp 5-523 of SEQ ID NO:3) and complements thereof (e.g., as disclosed in US patent number http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1& Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&1=50&s1- =5952195.PN.&OS=PN/5952195&RS=PN/5952195-h0#h0http://patft.uspto.gov/netac- gi/nph-Parser?Sect1=PTO1& Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&1=50&s1- =5952195.PN.&OS=PN/5952195&RS=PN/5952195-h2#h25,952,195); CYP52A2A (see, for example, US Application 2002-0034788); promoter sequences from fungal (e.g., C. tropicalis) catalase, citrate synthase, 3-ketoacyl-CoA thiolase A, citrate synthase, O-acetylhornserine sulphydrylase, protease, carnitine O-acetyltransferase, hydratase-dehydrogenase, epimerase genes; promoter sequences from Pox4 genes (see, for example, US application 2004-0265980); and/or promoter sequences from Met2, Met3, Met6, Met25 and YALI0D12903g genes. Any such promoters can be used in conjunction with endogenous genes and/or heterologous genes for modification of expression patterns of endogenous polypeptides and/or heterologous polypeptides in accordance with the present invention.

[0111] Alternatively or additionally, regulatory sequences useful in accordance with the present invention may include one or more Xpr2 promoter fragments, for example as described in US patent http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&- p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&1=50&s1=6083717.PN.&OS=PN/60- 83717&RS=PN/6083717-h0#h0http://patft.uspto.gov/netacgi/nph-Parser?Sect1=P- TO1& Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1 &f=G&1=50&s1=6083717.PN.&OS=PN/6083717&RS=PN/6083717-h2#h26,083,717 (e.g. SEQ ID NOS: 1-4 also including sequences with 80% or more identity to these SEQ ID NOs) in one or more copies either in single or in tandem. Similarly, exemplary terminator sequences include, but are not limited to, Y. lipolytica Xpr2 (see U.S. Pat. No. 4,937,189) and Pox2 (YALI0F10857g) terminator sequences and those listed in example 11 herein.

Engineering Oleaginy

[0112] 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.

[0113] 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, there only have been a limited number of 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 accumulate lipid only when grown under conditions of carbon excess and nitrogen limitation. The present invention further establishes that the limitation of other nutrients (e.g. phosphate or magnesium) can also induce lipid accumulation. The present invention establishes, for example, that limitation of nutrients such as phosphate and/or magnesium can induce lipid accumulation, much as is observed under conditions of nitrogen 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.

[0114] 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.fwdarw.acetyl-CoA+oxaloacetate+ADP+P.sub.i. (1)

[0115] 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.

[0116] 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.

[0117] 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 the absence of nitrogen or other nutrient (e.g., phosphate or magnesium), the activity of AMP deaminase, which catalyzes the reaction:

AMP.fwdarw.inosine 5'-monophosphate+NH.sub.3 (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.

[0118] 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. Appi. Microbiol. 51:1, 2002, each of which is incorporated herein by reference in its entirety).

[0119] Other activities which can be involved in regenerating NADPH include, for example, 6-phosphogluconate dehydrogenase (gnd); Fructose 1,6 bisphosphatase (fbp); Glucose 6 phosphate dehydrogenase (g6pd); NADH kinase (EC 2.7.1.86); and/or transhydrogenase (EC 1.6.1.1 and 1.6.1.2).

[0120] Gnd is part of the pentose phosphate pathway and catalyses the reaction:

6-phospho-D-gluconate+NADP+.fwdarw.D-ribulose 5-phosphate+CO.sub.2+NADPH

[0121] Fbp is a hydrolase that catalyses the gluconeogenic reaction:

D-fructose 1,6-bisphosphate+H.sub.2O.fwdarw.D-fructose 6-phosphate+phosphate

Fbp redirects carbon flow from glycolysis towards the pentose phosphate pathway. The oxidative portion of the pentose phosphate pathway, which includes glucose 6 phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, enables the regeneration of NADPH.

[0122] G6pd is part of the pentose phosphate pathway and catalyses the reaction:

D-glucose 6-phosphate+NADP.sup.+.fwdarw.D-glucono-1,5-lactone 6-phosphate+NADPH+H.sup.+

[0123] NADH kinase catalyzes the reaction:

ATP+NADH.fwdarw.ADP+NADPH

[0124] Transhydrogenase catalyzes the reaction:

NADPH+NAD.sup.+.revreaction.NADP.sup.++NADH

[0125] Thus, enhancing the expression and/or activity of any of these enzymes can increase NADPH levels and promote anabolic pathways requiring NADPH.

[0126] Alternative or additional strategies to promote oleaginy may include one or more of the following: (1) increased or heterologous expression of one or more of acyl-CoA:diacylglycerol acyltransferase (e.g., DGA1; YALI0E32769g); phospholipid:diacylglycerol acyltransferase (e.g., LRO1; YALI0E16797g); and acyl-CoA:cholesterol acyltransferase (e.g., ARE genes such as ARE1, ARE2, YALI0F06578g), which are involved in triglyceride synthesis (Kalscheuer et al. Appl Environ Microbiol p. 7119-7125, 2004; Oelkers et al. J Biol Chem 277:8877-8881, 2002; and Sorger et al. J Biol Chem 279:31190-31196, 2004), (2) decreased expression of triglyceride lipases (e.g., TGL3 and/or TGL4; YALI0D17534g and/or YALI0F10010g (Kurat et al. J Biol Chem 281:491-500, 2006); and (3) decreased expression of one or more acyl-coenzyme A oxidase activities, for example encoded by POX genes (e.g. POX1, POX2, POX3, POX4, POX5; YALI0C23859g, YALI0D24750g, YALI0E06567g, YALI0E27654g, YALI0E32835g, YALI0F10857g; see for example Mlickova et al. Appl Environ Microbiol 70: 3918-3924, 2004; Binns et al. J Cell Biol 173:719, 2006).

[0127] 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 and/or altering lipid levels through other mechanisms. 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, AMP-deaminase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fructose 1,6 bisphosphatase, NADH kinase, transhydrogenase, acyl-CoA: diacylglycerol acyltransferase, phospholipid: diacylglycerol acyltransferase, acyl-CoA:cholesterol acyltransferase, triglyceride lipase, acyl-coenzyme A oxidase 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, AMP-deaminase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fructose 1,6 bisphosphatase, NADH kinase, transhydrogenase, acyl-CoA: diacylglycerol acyltransferase, phospholipid: diacylglycerol acyltransferase, acyl-CoA:cholesterol acyltransferase, triglyceride lipase, acyl-coenzyme A oxidase polypeptides provided in Tables 1-6 and Tables 69-85 respectively.

[0128] 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.

[0129] 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) and/or regulatory factors (e.g., polypeptides that modulate transcription, splicing, translation, modification, etc.) 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)).

[0130] 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.

[0131] 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, and Tables 69-85. 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.

[0132] 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, malic 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 lipolytica.

[0133] Aspergillus niger accumulates large amounts of citric acid, whereas Mortierella alpina and Thraustochytrium sp. accumulate large amounts of fatty acid, respectively; Mortierella alpina and Thraustochytrium are also oleaginous. To give but one particular example of a host cell engineered to be oleaginous (or at least to accumulate increased levels of lipid) in accordance with the present invention, S. cerevisiae can be engineered to express one or more oleaginic polypeptides, e.g., from heterologous source organisms.

[0134] In some embodiments, a plurality of different oleaginic polypeptides are expressed, optionally from different source organisms. For instance, in some embodiments, S. cerevisiae cells are engineered to express (and/or to increase expression of) ATP-citrate lyase (e.g., from N. crassa), AMP deaminase (e.g., from S. cerevisiae), and/or malic enzyme (e.g., from M. circinelloides). In other embodiments, Candida utilis and Phaffia rhodozyma can be similarly modified. Modified S. cerevisiae, C. utilis, and P. rhodozyma strains can be further modified as described herein to increase production of one or more quinone derived compounds.

Engineering Production of Quinone Derived Compounds

[0135] The present invention encompasses the recognition that lipid-accumulating systems are useful for the production and/or isolation of certain quinone derived compounds, and particularly of a ubiquinone (e.g., CoQ10 and/or one or more C.sub.5-9 quinone compounds such as CoQ5, CoQ6, CoQ7, CoQ8, CoQ9), one or more vitamin K compounds (e.g., phylloquinone and/or one or more menaquinones), and/or one or more vitamin E compounds (e.g., one or more tocopherols and/or tocotrienols). Without wishing to be bound by theory, the present inventors propose that the higher intracellular membrane content may facilitate increased quinone derived compound production and/or accumulation. The present invention therefore encompasses the discovery that certain quinone derived compounds can desirably be produced in oleaginous yeast and fungi.

[0136] According to the present invention, strains that both (i) accumulate lipid, often in the form of cytoplasmic lipid bodies and typically to at least about 20% of their dry cell weight; and (ii) produce one or more quinone derived compounds 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 generally available strains (e.g., naturally-occurring strains and strains which have been previously genetically modified, whether via recombinant DNA techniques or mutagenesis directed modification). These manipulated strains are then used to produce one or more quinone derived compounds, so that compound(s) that partitions into the lipid bodies can readily be isolated.

[0137] In certain embodiments of the invention, host cells are Yarrowia lipolytica cells. Advantages of Y. lipolytica include, for example, tractable genetics and molecular biology, availability of genomic sequence, 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, quinone derived compound-producing Y. lipolytica strain than might be required for other organisms. Furthermore, there is already extensive commercial experience with Y. lipolytica. In certain embodiments, host cells are Saccharomyces cerevisiae cells. In other embodiments, host cells are Candida utilis cells.

[0138] As mentioned, quinone derived compounds are produced from the isoprenoid compound isopentyl pyrophosphate (IPP), via geranylgeranyl pyrophosphate (see, for example, FIG. 8. IPP can be generated through one of two different isoprenoid biosynthesis pathways. The most common isoprenoid biosynthesis pathway, sometimes referred to as the "mevalonate pathway", is generally depicted in FIG. 4A. 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).

[0139] An alternative isoprenoid biosynthesis pathway, that is utilized by some organisms (particularly bacteria) and is sometimes called the "mevalonate-independent pathway", is also depicted in FIG. 4A. 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. 4B, into IPP.

[0140] Various proteins involved in isoprenoid biosynthesis have been identified and characterized in a number of organisms. Moreover, isoprenoids are synthesized in many, if not most, organisms. Thus, 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 FIGS. 4-6 and 8 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.

[0141] Alternatively or additionally, modified mevalonate kinase polypeptides that exhibit decreased feedback inhibition properties (e.g., to farnesyl pyrophosphate (FPP)) may be utilized in accordance with the present invention. Such modified mevalonate kinase polypeptides may be of eukaryotic or prokaryotic origin. For example, modified versions of mevalonate kinase polypeptides from animals (including humans), plants, algae, fungi (including yeast), and/or bacteria may be employed; for instance, modified versions of mevalonate kinase polypeptides disclosed in Table 10 herein may be utilized.

[0142] Particular examples of modified mevalonate kinase polypeptides include "feedback-resistant mevalonate kinases" disclosed in PCT Application WO 06/063,752. Thus, for example, a modified mevalonate kinase polypeptide may include one or more mutation(s) at one or more amino acid position(s) selected from the group consisting of amino acid positions corresponding to positions 17, 47, 93, 94, 132, 167, 169, 204, and 266 of the amino acid sequence of Paracoccus zeaxanthinifaciens mevalonate kinase as shown in SEQ ID NO:1 of PCT Application WO 04/111,214. For example, the modified mevalonate kinase polypeptide may contain one or more substitutions at positions corresponding to one or more of I 17T, G47D, K93E, V94I, R204H and C266S.

[0143] To give but a few specific examples, when a modified mevalonate kinase polypeptide comprises 2 amino acid changes as compared with a parent mevalonate kinase polypeptide, it may comprise changes at positions corresponding to the following positions 132/375,167/169, 17/47 and/or 17/93 of SEQ ID NO:1 of WO 04/111,214 (e.g. P132A/P375R, R167W/K169Q, 117T/G47D or I17T/K93E); when a modified mevalonate kinase polypeptide comprises 3 amino acid changes as compared with a parent mevalonate kinase, it may comprise changes at positions corresponding to the following positions 17/167/169, 17/132/375, 93/132/375, and/or 17/47/93 of SEQ ID NO: 1 of WO/2004/111214 (e.g., I17T/R167W/K169Q, I17T/P132A/P375R, K93E/P132A/P375R, I17T/R167W/K169H, I17T/R167T/K169M, I17T/R167T/K169Y, I17T/R167F/K169Q, I17T/R167I/K169N, I17T/R167H/K169Y, I17T/G47D/K93E or I17T/G47D/K93Q).

[0144] Thus, for example, a modified mevalonate kinase polypeptide may include one or more mutation(s) (particularly substitutions), as compared with a parent mevalonate kinase polypeptide, at one or more amino acid position(s) selected from the group consisting of amino acid positions corresponding to positions 55, 59, 66, 83, 106, 111, 117, 142, 152, 158, 218, 231, 249, 367 and 375 of the amino acid sequence of Saccharomyces cerevisiae mevalonate kinase as shown in SEQ ID NO:1 of PCT application WO 06/063,752. For example, such corresponding substitutions may comprise one or more of P55L, F59S, N66K, C117S, or I152M. A modified mevalonate kinase may comprise a substitution corresponding to F59S substitution. A modified mevalonate kinase polypeptide comprising 2 amino acid changes as compared with its parent mevalonate kinase polypeptide may, for example, comprise changes at positions corresponding to the following positions 55/117, 66/152, 83/249, 111/375 or 106/218 of to SEQ ID NO: 1 of WO 06/063,752 (e.g. P55L/C117S, N66K/I152M, K83E/S249P, H111N/K375N or L106P/S218P). A modified mevalonate kinase may comprise a substitution corresponding to N66K/I152M. A modified mevalonate kinase polypeptide comprising 4 amino acid changes as compared with its parent mevalonate kinase polypeptide may have changes at positions corresponding to one or more of the following positions 42/158/231/367 of SEQ ID NO:1 of WO 06/063,752 (e.g., 1142N/L158S/L2311/T367S).

[0145] According to the present invention, quinone derived compound 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 well even in significantly divergent organisms. Furthermore, should it be desirable to introduce more than one heterologous isoprenoid biosynthesis polypeptide (e.g., more than one version of the same polypeptide and/or more than one different polypeptides), in many cases polypeptides from different source organisms may function well 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; in other embodiments the plurality includes polypeptides from different source organisms.

[0146] In certain 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.

Ubiquinone-10/Coenzyme Q10

[0147] Ubiquinone-10 or coenzyme Q10 (CoQ10), is synthesized de novo in all mammalian tissues, and serves a critical function in mediating electron transport in the mitochondrial respiratory chain. The reduced form of CoQ10 is also thought to function as an antioxidant and to protect against lipid peroxidation as well as against other free radical-induced oxidative damage. Recent studies have confirmed the role of CoQ10 in certain disease states, and additional roles in certain disorders continue to be elucidated. For example, administration of cholesterol lowering drugs, including HMG-CoA reductase inhibitors (e.g., the statins) to patients can result in diminished CoQ10 levels since cholesterol biosynthesis and CoQ10 biosynthesis share many of the same biosynthetic steps. Additionally, several clinical studies have been conducted to examine whether supplemental CoQ10 administration provides protection against, for example, deterioration in cardiac function (e.g., congestive heart failure), slows deterioration in Parkinson's patients, or protects the heart from damage caused by certain chemotherapeutic drugs.

[0148] While cells are capable of endogenously producing CoQ10, only about half of the levels required for optimum human cell function are produced in the body; the other half is acquired through dietary sources. Also, cellular levels of CoQ10 decrease under stress, with increasing age, and after certain pharmacological treatments, further increasing the need for exogenous CoQ10 to maintain optimum cell function.

[0149] CoQ10 has been used commercially in dietary and nutritional supplements as well as in functional foods, cosmetics and in personal care items. A rapidly growing market exists for CoQ10. The 2005 market for CoQ10 as an ingredient is estimated to be approximate $500 million, which represented approximately 250 metric tonnes of CoQ10. Further market expansion for CoQ10 is expected. Improved systems for enabling cost effective production, isolation, and/or formulation of CoQ10 are needed to effectively meet the growing demand for CoQ10.

[0150] 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 (see FIG. 9). Lower eukaryotes, such as yeast, can synthesize PHB from either tyrosine or chorismate. The 3-decaprenyl-4-hydroxybenzoic acid resulting from the condensation reaction undergoes further modifications, which include hydroxylation, methylation and decarboxylation, in order to form ubiquinone. Ubiquinone biosynthetic enzymes and genes encoding these proteins have been characterized in several organisms. The most extensive analysis has been performed in bacterial systems such as Escherichia coli and Rhodobacter sphaeroides as well as the yeast, Saccharomyces cerevisiae. At least 8 enzymes are required for the synthesis of CoQ10 from PHB and the isoprene precursors.

[0151] Biomanufacturing processes for CoQ10 have been developed or attempted using several genera of bacteria and yeast. Bacterial hosts have included R. sphaeroides, Agrobacterium tumefaciens, Paracoccus denitrificans, Rhodopseudomonas spheroides, and recombinant strains of E. coli, which naturally produces CoQ8. Yeast hosts have included Saitoella complicata, Sporidiobolus ruineniae, Schizosaccharomyces pombe, and several species of Rhodotorula and Candida. In addition, a semi-synthetic process for producing CoQ10 has been developed using solanesol extracted from tobacco leaves as a starting material; this process has economic and environmental disadvantages.

[0152] Despite the early identification of several microorganisms as potential hosts for CoQ10 production, only limited progress has been made towards the development of a cost-effective CoQ10 process using fermentation. Without wishing to be bound by any particular theory, the present inventors note that the hydrophobic isoprenoid side chain on CoQ10 causes it to localize to cell membranes, and offer that enhanced CoQ 10 concentrations in the membranes might be detrimental to cell growth or viability. Efforts to engineer strains with increased production of CoQ10 have focused primarily in prokaryotes such as E. coli. While E. coli is a host with facile molecular genetic tools, this organism's fundamental physiology, may limit its ultimate utility as a CoQ10 production system.

[0153] The present invention encompasses the recognition that lipid-accumulating systems are useful for the production and/or isolation of ubiquinone(s). Indeed, several of the more productive natural CoQ10 strains (e.g., P. denitrificans, R. sphaeroides) display a relatively high intracellular membrane content. Without wishing to be bound by theory, the present inventors propose that the higher intracellular membrane content may facilitate increased CoQ10 production and/or accumulation. The present invention therefore encompasses the discovery that CoQ10 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 lipid bodies and typically to at least about 20% of their dry cell weight; and (ii) produce CoQ10 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 generally available strains (e.g., naturally-occurring strains and strains which have been previously genetically modified, whether via recombinant DNA techniques or mutagenesis directed modification). These manipulated strains are then used to produce CoQ10, so that the CoQ10 that partitions into the lipid bodies can readily be isolated.

[0154] In certain embodiments of the invention, host cells are Yarrowia lipolytica cells. Advantages of Y. lipolytica include, for example, tractable genetics and molecular biology, availability of genomic sequence, 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, CoQ10-producing Y. lipolytica strain than might be required for other organisms. Furthermore, there is already extensive commercial experience with Y. lipolytica.

[0155] As mentioned, ubiquinone is formed by the combination of para-hydroxybenzoate and isoprenoid chains produced, for example, via the isoprenoid biosynthesis pathways discussed above. Once IPP is formed according to that pathway, it isomerizes into dimethylallyl pyrophophate (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), which can be used to form the twenty-carbon compound geranylgeranyl pyrophosphate (GGPP). In many instances, FPP appears to be the predominant substrate used by polyprenyldiphosphate synthases (e.g., Coq1 polypeptides) during ubiquinone biosynthesis. According to the present invention, ubiquinone production in a host organism may be adjusted by modifying the expression or activity of one or more proteins involved in isoprenoid biosynthesis as discussed above.

[0156] As discussed herein, two different pathways can produce the ubiquinoid precursor para-hydroxybenzoate--the first, the "shikimate pathway" is utilized in prokaryotes and yeast, involves synthesis of para-hydroxybenzoate (PHB) through chorismate. Biosynthesis of para-hydroxybenzoate from chorismate occurs by the action of chorismate pyruvate lyase. For example, as discussed herein, enzymes of the shikimate pathway, chorismate synthase, DAHP synthase, and transketolase are involved in this process. Representative examples of these enzymes are provided in Table 33 and Tables 35 through 37. In the second possible pathway, para-hydroxybenzoate is produced by derivation of tyrosine or phenylalanine. Biosynthesis of para-hydroxybenzate from tyrosine or phenylalanine occurs through a five step process in mammalian cells (see, for example FIG. 3).

[0157] Accordingly, ubiquinone (e.g., CoQ10 and/or C.sub.5-9 quinone compounds) production in a host organism may be adjusted by modifying the expression or activity of one or more proteins involved in PHB biosynthesis. In some embodiments, such modification involves introduction of one or more heterologous PHB polypeptides into the host cell; alternatively or additionally, modifications may be made to the expression or activity of one or more endogenous or heterologous additional PHB polypeptide, and/or isoprenoid biosynthesis polypeptides. Given the considerable conservation of components of the PHB biosynthesis polypeptides, it is expected that heterologous PHB biosynthesis polypeptides will often function well even in significantly divergent organisms. Further, given the conservation of the pathways among organism, it is anticipated that heterologous polypeptides throughout the ubiquinone biosynthetic pathway will function together effectively. Furthermore, should it be desirable to introduce more than one heterologous PHB polypeptide and/or isoprenoid biosynthesis polypeptide, in many cases polypeptides from different source organisms will function well together. In some embodiments of the invention, a plurality of different heterologous PHB and/or isoprenoid biosynthesis polypeptides is introduced into the same host cell. In some embodiments, this plurality contains only polypeptides from the same source organism; in other embodiments the plurality includes polypeptides from different source organisms. In still other embodiments, modification of endogenous PHB and/or isoprenoid biosynthesis polypeptides are also utilized, either alone or in combination with heterologous polypeptides as discussed herein.

[0158] As noted herein, the isoprenoid biosynthesis pathway is also involved in the production of non-ubiquinone compounds, such as carotenoids, sterols, steroids, and vitamins, such as vitamin K or vitamin E. Proteins that act on isoprenoid biosynthesis pathway intermediates, and divert them into biosynthesis of non-ubiquinone compounds are therefore indirect inhibitors of ubiquinone biosynthesis (see, for example, FIG. 5, which illustrates points at which isoprenoid intermediates are channeled into other biosynthesis pathways). Such proteins are therefore considered ubiquinone biosynthesis competitor polypeptides. Reductions of the level or activity of such ubiquinone biosynthesis competitor polypeptides are expected to increase ubiquinone (e.g., CoQ10 and/or C.sub.5-9 quinone compounds) production in host cells according to the present invention.

[0159] In some embodiments of the present invention, production or activity of endogenous ubiquinone biosynthesis competitor polypeptides may be reduced or eliminated in host cells. In some embodiments, this reduction or elimination of the activity of a ubiquinone 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 (Erg9), squalene epoxidase (Erg1), 2,3-oxidosqualene-lanosterol cyclase (Erg7), cytochrome P450 lanosterol 14.alpha.-demethylase (Erg11), C-14 sterol reductase (Erg24), C-4 sterol methyl oxidase (Erg25), SAM:C-24 sterol methyltransferase (Erg6), C-8 sterol isomerase (Erg2), C-5 sterol desaturase (Erg3), C-22 sterol desaturase (Erg5), and C-24 sterol reductase (Erg4) polypeptides. Each of these enzymes is considered a ubiquinone biosynthesis competitor polypeptide. Regulators of these enzymes may also be considered ubiquinone biosynthesis competitor polypeptides (e.g., the yeast proteins Sut1 (Genbank Accession JC4374 GI:2133159) and Mot3 (Genbank Accession NP.sub.--013786 GI:6323715), which may or may not have homologs in other organisms.

[0160] In some embodiments of the invention, production or activity of endogenous ubiquinone biosynthesis competitor polypeptides may be reduced or eliminated in host cells. In some embodiments, this reduction or elimination of the activity of a ubiquinone 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 14.alpha.-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 a ubiquinone biosynthesis competitor polypeptide. Regulators of these enzymes may also be considered ubiquinone biosynthesis competitor polypeptides (e.g., the yeast proteins Sut1 (Genbank Accession JC4374 GI:2133159) and Mot3 (Genbank Accession NP.sub.--013786 GI:6323715), which may or may not have homologs in other organisms).

[0161] Known small molecule inhibitors of some ubiquinone 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,5S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-1,2,3,5-tetrahyd- ro-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 (e.g., LAMISIL.RTM.), naftifine (NAFTIN.RTM.), S-allylcysteine, garlic, resveratrol, NB-598 (e.g., from Banyu Pharmaceutical Co), and/or green tea phenols that inhibit squalene epoxidase (see, for example, J. Biol Chem 265:18075, 1990; Biochem. Biophys. Res. Commun. 268:767, 2000); various azoles that inhibit cytochrome P450 lanosterol 14.alpha.-demethylase; and fenpropimorph that inhibits the C-14 sterol reductase and the C-8 sterol isomerase. In other embodiments, heterologous ubiquinone biosynthesis competitor polypeptides may be utilized (whether functional or non-functional; in some embodiments, dominant negative mutants are employed).

[0162] One ubiquinone 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

[0163] Another ubiquinone biosynthesis competitor polypeptide useful according to the present invention is anthranilate synthase, which has also been identified in a variety of organisms; representative anthranilate synthase polypeptides are provided in Table 32 and 32B. In some embodiments of the invention, anthranilate synthase polypeptide, or modifications thereof are utilized and adapted from source organisms including, but not limited to: Kluyveromyces lactis, Candida glabrata, Saccharomyces cerevisiae, Yarrowia lipolytica, Debaryomyces hansenii, Candida albicans, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus nidulans, Ustilago maydis, Neurospora crassa, Schizosaccharomyces pombe, Gibberella zeae, and Cryptococcus neoformans var.

[0164] Similarly, yet another ubiquinone biosynthesis competitor polypeptide useful according to the invention is chorismate mutase, which has also been identified in a variety of organisms; representative chorismate mutase polypeptides are provided in Table 34. In some embodiments of the invention, chorismate mutase polypeptide, or modifications thereof are utilized and adapted from source organisms including, but not limited to: Kluyveromyces lactis, Candida glabrata, Arabidopsis thaliana, Yarrowia lipolytica, Candida albicans, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus nidulans, Ustilago maydis, Neurospora crassa, Schizosaccharomyces pombe, Gibberella zeae, and Pichia pastoris.

[0165] Particularly for embodiments of the present invention directed toward production of CoQ10, it will often be desirable to utilize one or more genes from a natural CoQ 10-producing organism. In general, 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.

[0166] Bacterial ubiquinogenic genes have already been demonstrated to be transferable to other organisms, and are therefore useful in accordance with the present invention (see, for example, Okada et al., FEMS Lett. 431:241-244 (1998)). In some embodiments of this invention, it may be desirable to fused sequences encoding specific targeting signals to bacterial ubiquinogenic genes. For example, in certain embodiments mitochondrial signal sequences are useful in conjunction with, e.g., bacterial ubiquinogenic polypeptides for effective targeting of polypeptides for proper functioning. Mitochondrial signal sequences are known in the art, and include, but are not limited to example, mitochondrial signal sequences provided in Table 22. In other embodiments, it may be desirable to utilize genes from other source organisms such as animals, plants, alga, or microalgae, fungi, yeast, insect, protozoa, and mammals.

[0167] The present invention contemplates not only introduction of heterologous ubiquinogenic polypeptides, but also adjustment of expression or activity levels of heterologous or endogenous ubiquinogenic polypeptides, including, for example, alteration of constitutive or inducible expression patterns so as to increase activity of ubiquinogenic polypeptides. 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 ubiquinogenic polypeptide(s)); alternatively, such genetic modifications may be included so as to confer regulation of expression of heterologous polypeptides (e.g., ubiquinogenic polypeptide(s)).

[0168] To give but a few specific examples of strains engineered to produce CoQ10 (and optionally to be oleaginic) according to the present invention, in some embodiments, Yarrowia lipolytica cells are engineered to express a decaprenyl diphosphate synthase polypeptide(s) from a source organism. In some embodiments, the source organism is selected from the group consisting of Silibacter pomeroyi and Loktanella vestfoldensis. As is discussed herein, where the source organism is other than Y. lipolytica, nucleic acid sequences encoding the polypeptide(s) can be established with Y. lipolytica codon preferences and/or targeting sequences (e.g., mitochondrial targeting sequences). In some embodiments, one or more endogenous polyprenyl synthase genes (e.g., nonaprenyl diphosphate synthase) is/are disrupted or inactivated, and a decaprenyl diphosphate synthase gene from a different source organism is introduced. In some embodiments, the different source organism is selected from the group consisting of Silibacter pomeroyi and Loktanella vestfoldensis.

[0169] Alternatively or additionally, in some embodiments of the invention, host cells are engineered to produce CoQ10 by introducing or increasing expression or activity of one or more ubiquinone biosynthesis polypeptides, and in particular of one or more polypeptides involved in converting para-hydroxybenzoic acid to CoQ10 (e.g., via a ubiquinol pathway). To give but one specific example, Yarrowia lipolytica cells are engineered to express a 4-hydroxybenzoate polyprenyltransferase polypeptide from source organism, for example selected from the group consisting of Silibacter pomeroyi and Bos taurus. Sequences encoding the polypeptide(s) can optionally be established with Y. lipolytica codon preferences and/or targeting sequences (e.g., mitochondrial targeting sequences).

[0170] In some embodiments, CoQ10 production in cells, e.g., in Yarrowia lipolytica cells is enhanced by engineering the cells to increase production of para-hydroxybenzoic acid (PHB), for example by increasing expression or activity of one or more PHB polypeptides. In some embodiments, Y. lipolytica cells are engineered to express one or more of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) and chorismate lyase polypeptides from a source organism. Where the source organism is other than Y. lipolytica, nucleic acid sequences encoding the polypeptide(s) can be established with Y. lipolytica codon preferences and/or targeting sequences (e.g., mitochondrial targeting sequences). In some embodiments, one or both of the DAHP and chorismate lyase polypeptides are from heterologous source organisms, which may be the same or different. In some embodiments, at least one source organism is Erwinia carotovora.

[0171] In some embodiments, host cells (e.g., Yarrowia lipolytica) engineered to increase production of para-hydroxybenzoic acid are further engineered to increase expression and/or activity of one or more ubiquinone biosynthesis polypeptides involved in converting para-hydroxybenzoic acid to a ubiquinone (e.g., via a ubiquinol pathway), and/or of one or more decaprenyl diphosphate synthase. To give but a couple of particular examples, such strains may be engineered to express a 4-hydroxybenzoate polyprenyltransferase polypeptide from source organism, for example selected from the group consisting of Silibacter pomeroyi and Bos taurus and/or to express a decaprenyl diphosphate synthase polypeptide(s) from a source organism, for example selected from the group consisting of Silibacter pomeroyi and Loktanella vestfoldensis.

[0172] Alternatively or additionally, in some specific embodiments, CoQ10 production is enhanced by engineering cells to reduce carbon flow into competing metabolic pathways such as, for example, the sterol pathway. To give but one specific example, in some embodiments of the invention, Yarrowia lipolytica cells are engineered to inactivate (e.g., partially) an endogenous gene (e.g., ERG9) encoding a squalene synthase polypeptide.

[0173] Yet further, in some particular embodiments, CoQ10 production is enhanced by engineering cells by expression of a truncated HMG CoA reductase polypeptide (e.g., from a source organism such as Y. lipolytica).

[0174] In some embodiments of the present invention, host cells are engineered to produce one or more ubiquinones other than CoQ10 in addition or as an alternative to CoQ10. Specifically, in some embodiments of the invention, host cells are engineered to produce one or more C.sub.5-9 quinones.

[0175] That is, the present invention provides engineered host cells, e.g., fungal cells, that produce C.sub.5-9 quinones as a result of the engineering. The present invention therefore provides engineered host cells containing a C.sub.5-9 quinones production modification. Such a modification may comprise, for instance, introduction or activation of one or more C.sub.5-9 quinones biosynthetic polypeptides within a host cell. Exemplary such polypeptides include, for example, pentaprenyl, hexaprenyl, heptaprenyl, octaprenyl, and/or solanesyl(nonaprenyl) biosynthesis polypeptides. Specific examples of each of these can be found, for example, in Tables 61-65.

[0176] To give but a few specific examples, in some embodiments of the present invention, Yarrowia lipolytica cells are engineered to express one or more polyprenyl diphosphate synthase genes (e.g., nonaprenyl diphosphate synthase, octaprenyl diphosphate synthase, heptaprenyl diphosphate synthase, hexaprenyl diphosphate synthase, pentaprenyl disphosphate synthase, etc.). In some embodiments, one or more endogenous polyprenyl synthase genes (e.g., nonaprenyl diphosphate synthase) is/are disrupted or inactivated, and a polyprenyl synthase gene from a different source organism is introduced. In some embodiments, the different source organism is selected from the group consisting of Silibacter pomeroyi and Loktanella vestfoldensis.

Vitamin K

[0177] Vitamin K is a generic term that refers to derivatives of 2-methyl-1,4-naphthoquinone that have coagulation activity. The two natural forms of vitamin K differ in the identity of their side chains at position 3. Vitamin K.sub.1, also known as phylloquinone (based on its presence in plants), has a phytyl side chain in position 3; vitamin K.sub.2, also known as menaquinone, has an isoprenyl side chain at position 3. Different forms of menaquinone, having side chains with different numbers of isoprene units (typically 4-13) are found in different types of cells.

[0178] Vitamin K has been found to be an important vitamin involved in the blood coagulation system, and is utilized as a hemostatic agent. Recently it has been suggested that vitamin K is involved in osteo-metabolism, and vitamin K is expected to be applied to the treatment of osteoporosis. Both phylloquinone and menaquinone have been approved as pharmaceuticals.

[0179] The daily requirement for vitamin K is about 1 ug/kg; an average diet typically contains about 75-150 ug/day. Vitamin K deficiency results in hypoprothrombinemia and defective coagulation. Primary vitamin K deficiency is uncommon in adults because of its general availability in the food chain, and potentially also because it can be produced by microbes inhabiting the human gastrointestinal tract. Vitamin K deficiency is observed in newborns, as breast milk contains very little and the newborn gut is sterile (i.e., not inhabited by vitamin K-producing organisms) for the first several days of life.

[0180] Vitamin K is considered an essential nutrient since mammals do not produce it and it is a dietary requirement. As depicted for example in FIG. 9, vitamin K is produced through precursor molecules chorismate and isoprenoids, similar to ubiquinone production, though biosynthesis proceeds through production of isochorismate, and prenyl addition does not occur until the final or next to last step in synthesis. Details of production of vitamin K, and the relevant enzymes involved are known in the art and can be adapted for use in combination with the provided methods and compositions. See, e.g., Meganathan, Vitamins and Hormones 61: 173-219, 2001.

[0181] The present invention provides engineered host cells, e.g., fungal cells, that produce vitamin K as a result of the engineering. That is, the present invention provides engineered host cells containing a Vitamin K production modification. Such a modification may comprise, for instance, introduction or activation of one or more Vitamin K biosynthetic polypeptides within a host cell. Exemplary such polypeptides include, for example, MenF, MenD, MenC, MenE, MenB, MenA, UbiE, and/or MenG polypeptides. Specific examples of each of these can be found, for example, in Tables 46-53.

Vitamin E

[0182] Vitamin E is a generic term for a family of structurally related compounds that have a 6-chromanol ring, an isoprenoid side chain, and the biologic activity of .alpha.-tocopherol. The term encompasses the eight known naturally occurring vitamin E compounds, the four tocopherols (.alpha., .beta., .gamma., .delta.) and four tocotrienols (.alpha., .beta., .gamma., .delta.), which all contain a hydrophilic chromanol ring and a hydrophobic side chain. The .alpha., .beta., .gamma., and .delta. forms differ from one another in the number of methyl groups on the chromanol ring. Several synthetic vitamin E compounds have also been prepared, and still others are possible (see, for example, Bramley et al., J. Sci Food Agric 80:913, 2000). .alpha.-tocopherol is a potent antioxidant, and is generally considered to be the most active vitamin E compound in humans.

[0183] Vitamin E is commonly included in nutritional supplements and also in skin creams and lotions (based on a reported role in wound healing). Vitamin E deficiency is manifested differently in different species and in different individual cases, but can include reproductive disorders; abnormalities of muscle, liver, bone marrow, and/or brain function; red blood cell hemolysis; defective embryogenesis; exudative diathesis, a disorder of capillary permeability; and/or skeletal muscular dystrophy (with or without cardiomyopathy). Vitamin E is used to treat vitamin E deficiency, and is also suspected to be useful in the treatment of cancer (thanks to its antioxidant properties), cataracts and macular degeneration (particularly age-related), heart disease, neurological disorders (e.g., Alzheimer's Disease, Parkinson's Disease, etc.), immune disorders, inflammatory diseases, among other things.

[0184] The U.S. Dietary Reference Intake (DRI) Recommended Daily Amount (RDA) for a 25-year old male for Vitamin E is 15 mg/day. This is approximately 15 IU/day. Specifically, The natural form of alpha-tocopherol: RRR-alpha-tocopherol maintain 1.5 IU/mg.

[0185] Vitamin E compounds are synthesized by higher plants and cyanobacteria by two pathways: the isoprenoid pathway and the homogentisic acid formation pathway. The overall synthesis is depicted in FIG. 10, Panels A-C. As can be seen, the first step is formation of the homogentisic head group (HGA), which is produced from p-hydroxyphenylpyruvic acid (HPP) by the enzyme p-hydroxyphenylpyruvic acid dioxygenase (HPPDase). This is a complex reaction involving the addition of two oxygen atoms as well as the decarboxylation and rearrangement of the HPP side chain.

[0186] In the next step, HGS is prenylated and decarboxylated to form 2-methyl-6-phytylplastoquinol. This step also represents the commitment step for production of tocopherols and/or tocotrienols, as 2-methyl-6-phytylplastoquinol represents the common intermediate in the synthesis of all tocopherols. The present invention provides host cells that have been engineered to accumulate 2-methyl-6-phytylplastoquinol. In some embodiments, the host cells are (or have been engineered to be) oleaginous or lipid-accumulating. In some embodiments, produced 2-methyl-6-phytylplastoquinol accumulates in lipid bodies within the engineered host cells.

[0187] In the final steps of tocopherol synthesis, methylation and ring cyclization reactions convert the 2-methyl-6-phytylplastoquinol into various tocopherols.

[0188] It is expected in accordance with the present invention that availability of tocopherol precursors and/or intermediates may well affect the rate and/or extent of tocopherol (or other vitamin E compound) production and/or accumulation by and/or within cells. The present invention therefore encompasses engineering host cells to adjust the rate or amount of one or more tocopherol precursors and/or intermediates.

[0189] The present invention provides engineered host cells, e.g., fungal cells, that produce vitamin E as a result of the engineering. The present invention specifically provides engineered host cells, e.g., fungal cells, that produce larger amounts of vitamin E that an otherwise identical non-engineered host cell. That is, the present invention provides engineered host cells containing a Vitamin E production modification. Such a modification may comprise, for instance, introduction or activation of one or more Vitamin E biosynthetic polypeptides within a host cell. Exemplary such polypeptides include, for example, tyrA, pds1(hppd), VTE1, HPT1(VTE2), VTE3, VTE4, and/or GGH polypeptides. Specific examples of each of these can be found, for example, in Tables 54-60.

Production and Isolation of Quinone Derived Compounds

[0190] Accumulation of lipid bodies in oleaginous organisms is generally induced by growing the relevant organism under conditions of carbon excess and nitrogen or other nutrient (e.g., phosphate and/or magnesium) limitation. 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. Microbiol. Biotechnol. 30(1):75, 2003; Bioresour Technol. 95(3):287, 2004, each of which is incorporated herein by reference in its entirety).

[0191] 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% 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.

[0192] 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 also be useful for inducing lipid accumulation in a host cell into which a 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.

[0193] It will also be appreciated by those of ordinary skill in the art that it will often be desirable to ensure that production of a desired quinone derived compound by an inventive modified host cell occurs at an appropriate time in relation to the induction of oleaginy such that the compound(s) accumulate(s) in the lipid bodies. In some embodiments, it will be desirable to induce production of a particular quinone derived compound (e.g., a ubiquinone, .alpha.-tocopherol, phylloquinone and/or menaquinone) in a host cell which does not naturally the particular compound, such that detectable levels of the compound are produced. In certain embodiments, host cells that do not naturally produce a particular quinone derived compound are capable of producing one or more other quinone derived compounds (e.g., cells that do not produce CoQ10 may produce one or more of CoQ5, CoQ6, CoQ7, CoQ8, CoQ9, etc). In additional embodiments, it will be desirable to increase production levels of a particular quinone derived compound in a host cell which does naturally produce low levels of that compound, such that increased detectable levels of the compound are produced. In certain aspects, the host cells which do naturally produce a particular compound (e.g., CoQ10) also produce additional quinone derived compound(s) (e.g., CoQ6, CoQ8); in other aspects, the cells which naturally produce the particular compound do not produce additional quinone derived compound(s).

[0194] In certain embodiments of the invention, it will be desirable to accumulate one or more quinone derived compounds (I.e., considering the total amount of all produced quinone derived compounds together or considering a particular quinone derived compound) to levels that are greater than at least about 1% of the dry weight of the cells. In some embodiments, the total quinone derived compound accumulation will be to a level at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, at least about 10%, at least about 10.5%, at least about 11%, at least about 11.5%, at least about 12%, at least about 12.5%, at least about 13%, at least about 13.5%, at least about 14%, at least about 14.5%, at least about 15%, at least about 15.5%, at least about 16%, at least about 16.5%, at least about 17%, at least about 17.5%, at least about 18%, at least about 18.5%, at least about 19%, at least about 19.5%, at least about 20% or more of the total dry weight of the cells.

[0195] In some embodiments of the invention, a particular quinone derived compound may not accumulate to a level as high as 1% of the total dry weight of the cells; appropriately engineered cells according to the present invention, and any lipid bodies and/or quinone derived compound(s) they produce, remain within the scope of the present invention. Thus, in some embodiments, the cells accumulate a given quinone derived compound to a level below about 1% of the dry weight of the cells. In some embodiments, the quinone derived compound accumulates to a level below about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or lower, of the dry cell weight of the cells.

[0196] In some embodiments of the invention, one or more quinone derived compound(s) accumulate both within lipid bodies and elsewhere in the cells. In some embodiments, quinone derived compound (s) accumulate primarily within lipid bodies. In some embodiments, quinone derived compound (s) accumulate substantially exclusively within lipid bodies. In some embodiments, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of a desired produced quinone derived compound (s) accumulates in lipid bodies.

[0197] In some embodiments of the invention, modified host cells are engineered to produce one or more quinone derived compound(s) characterized by negligible solubility in water (whether hot or cold) and detectable solubility in one or more oils. In some embodiments, such compounds have a solubility in oil below about 0.2%. In some embodiments, such compounds have a solubility in oil within the range of about <0.001%-0.2%.

[0198] The present invention therefore provides engineered host cells (and methods of making and using them) that contain lipid bodies and that further contain one or more quinone derived compounds accumulated in the lipid bodies, where the compounds are characterized by a negligible solubility in water and a solubility in oil within the range of about <0.001%-0.2%; 0.004%-0.15%; 0.005-0.1%; or 0.005-0.5%. For example, in some embodiments, such compounds have a solubility in oil below about 0.15%, 0.14%, 0.13%, 0.12%, 0.11%, 0.10%. 0.09, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.05%, or less. In some embodiments, the compounds show such solubility in an oil selected from the group consisting of sesame; soybean; apricot kernel; palm; peanut; safflower; coconut; olive; cocoa butter; palm kernel; shea butter; sunflower; almond; avocado; borage; carnauba; hazel nut; castor; cotton seed; evening primrose; orange roughy; rapeseed; rice bran; walnut; wheat germ; peach kernel; babassu; mango seed; black current seed; jojoba; macadamia nut; sea buckthorn; sasquana; tsubaki; mallow; meadowfoam seed; coffee; emu; mink; grape seed; thistle; tea tree; pumpkin seed; kukui nut; and mixtures thereof.

[0199] Also, it should be noted that, the absolute and/or relative amounts of quinone derived compounds produced in accordance with the present invention can sometimes be altered by adjustment of growth conditions, for example to modulate the isoprenoid biosynthesis pathway and/or one or more downstream pathways. For example, controlling the concentration of dissolved oxygen in a culture during cultivation may regulate relative production levels of CoQ10.

[0200] Inventive modified cells, that have been engineered to produce one or more quinone derived compounds and/or to accumulate lipid (including to be oleaginous), can be cultured under conditions that achieve ubiquinone (e.g. CoQ10 and/or C.sub.5-C.sub.9 quinone compounds) production and/or oleaginy.

[0201] In some embodiments, it will be desirable to control growth conditions in order to maximize production of a particular quinone derived compound or quinone derived compounds and/or to optimize accumulation of the particular quinone derived compound(s) in lipid bodies. In some embodiments it will be desirable to control growth conditions to adjust the relative amounts of different quinone derived compound products produced.

[0202] In some embodiments, it will be desirable to limit accumulation of a particular intermediate, for example ensuring that substantially all of a particular intermediate compound is converted so that accumulation is limited. For example, particularly in situations where a downstream enzyme may be less efficient than an upstream enzyme and it is desirable to limit accumulation of the product of the upstream enzyme (e.g., to avoid its being metabolized via a competitive pathway and/or converted into an undesirable product), it may be desirable to grow cells under conditions that control (e.g., slow) activity of the upstream enzyme so that the downstream enzyme can keep pace.

[0203] Those of ordinary skill in the art will appreciate that any of a variety of growth parameters, including for example amount of a particular nutrient, pH, temperature, pressure, oxygen concentration, timing of feeds, content of feeds, etc can be adjusted as is known in the art to control growth conditions as desired.

[0204] To give but a few examples, in some embodiments, growth and/or metabolism is/are limited by limiting the amount of biomass accumulation. For example, growth and/or metabolism can be limited by growing cells under conditions that are limiting for a selected nutrient. The selected limiting nutrient can then be added in a regulated fashion, as desired. In some embodiments, the limiting nutrient is carbon, nitrogen (e.g., via limiting ammonium or protein), phosphate, magnesium, or combinations thereof. In some embodiments, the limiting nutrient is carbon.

[0205] In some embodiments, use of a limiting nutrient can be utilized to control metabolism of a particular intermediate and/or to adjust relative production of particular quinone derived compounds. In some embodiments, this result can be achieved by controlling metabolism of a particular intermediate as discussed above; in some embodiments, it can be achieved, for example, by limiting progress through the quinone biosynthesis pathway so that a desired quinone derived compound product is not converted to a downstream compound. For example, nutrient limitation (e.g. phosphate limitation) may slow the overall rate of flux through the quinone derived compound biosynthesis pathway and may be utilized to change the ratio of one quinone derived compound versus another.

[0206] In some embodiments, cells are grown in the presence of excess carbon source and limiting nitrogen, phosphate, and/or magnesium to induce oleaginy. In some embodiments cells are grown in the presence of excess carbon source and limiting nitrogen. In some embodiments, the carbon:nitrogen ratio is within the range of about 200:1, 150:1, 125:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, or less. Those of ordinary skill in the art are aware of a wide variety of carbon sources, including, for example, glycerol, glucose, galactose, dextrose, any of a variety of oils (e.g., olive, canola, corn, sunflower, soybean, cottonseed, rapeseed, etc., and combinations thereof) that may be utilized in accordance with the present invention. Combinations of such may also be utilized. For example, common carbon source compositions contain oil:glucose in a ratios within the range of about 5:95 to 50:50 (e.g. about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50).

[0207] Those of ordinary skill in the art are also aware of a variety of different nitrogen sources (e.g., ammonium sulfate, proline, sodium glutamate, soy acid hydrolysate, yeast extract-peptone, yeast nitrogen base, corn steep liquor, etc, and combinations thereof) that can be utilized in accordance with the present invention.

[0208] In some embodiments, cultures are grown at a selected oxygen concentration (e.g., within a selected range of oxygen concentrations). In some embodiments, oxygen concentration may be varied during culture. In some embodiments, oxygen concentration may be controlled during some periods of culture and not controlled, or controlled at a different point, during others. In some embodiments, oxygen concentration is not controlled. In some embodiments, cultures are grown at an oxygen concentration within the rage of about 5-30%, 5-20%, 10-25%, 10-30%, 15-25%, 15-30%, including at about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or more. In some embodiments, oxygen concentration is maintained above about 20%, at least for some period of the culture.

[0209] In some embodiments, cells are grown via fed-batch fermentation. In some embodiments, feed is continued until feed exhaustion and/or the feed is controlled to initiate or increase once a certain level of dissolved oxygen is detected in the culture medium (e.g., about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or more dissolved oxygen). The feed rate can be modulated to maintain the dissolved oxygen at a specific level (e.g., about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or more dissolved oxygen).

[0210] In some embodiments, inventive modified cells are grown in a two-phase feeding protocol in which the first phase is designed to maintain conditions of excess carbon and limiting oxygen, and the second phase results in conditions of excess oxygen and limiting carbon.

[0211] In some embodiments, inventive modified cells are cultivated at constant temperature (e.g., between about 20-40, or 20-30.degree. C., including for example at about 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30.degree. C. or above) and/or pH (e.g., within a range of about 4-7.5, or 4-6.5, 3.5-7, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, etc., including at about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or above); in other embodiments, temperature and/or pH may be varied during the culture period, either gradually or in a stepwise fashion.

[0212] In some embodiments, the temperature at which inventive cells are cultivated is selected so that production of one or more particular carotenoid compound(s) is adjusted (e.g., so that production of one or more particular compound(s) is increased and/or production of one or more other compound(s) is decreased). In some embodiments, the temperature at which inventive cells are cultivated is selected so that the ratio of one quinone derived compound to another, is adjusted. To give but one example, in some embodiments, a temperature is selected to be sufficiently low that levels of one quinone derived compound are reduced and the level of at least one other quinone derived compound(s) is increased.

[0213] In some embodiments, cultures are grown at about pH 5.5 and or at a temperature between about 28-30.degree. C. In some embodiments, it may be desirable to grow inventive modified cells under low pH conditions, in order to minimize growth of other cells. In some embodiments, it will be desirable to grow inventive modified cells under relatively higher temperature conditions in order to slow growth rate and/or increase the ultimate dry cell weight output of quinone derived compounds (e.g. CoQ10, C.sub.5-C.sub.9 quinones, vitamin K compounds, vitamin E compounds).

[0214] One advantage provided by the present invention is that, in addition to allowing the production of high levels of one or more quinone derived compounds, certain embodiments of the present invention allow 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.

[0215] Of course, it is not essential that lipid bodies be specifically isolated in order to collect quinone derived compounds produced according to the present invention. Any of a variety of approaches can be utilized to isolate and/or purify quinone derived compounds. Many useful extraction and/or purification procedures for lipophilic agents generally, are known in the art (see, for example, EP670306, EP719866, U.S. Pat. No. 4,439,629, U.S. Pat. No. 4,680,314, U.S. Pat. No. 5,310,554, U.S. Pat. No. 5,328,845, U.S. Pat. No. 5,356,810, U.S. Pat. No. 5,422,247, U.S. Pat. No. 5,591,343, U.S. Pat. No. 6,166,231, U.S. Pat. No. 6,750,048, U.S. Pat. No. 6,812,001, U.S. Pat. No. 6,818,239, U.S. Pat. No. 7,015,014, US2003/0054070, US2005/0266132, each of which is incorporated herein by reference).

[0216] In many typical isolation procedures, cells are disrupted (e.g., mechanically, chemically [e.g., by exposure to a mild caustic agent such as a detergent or 0.1 N NaOH, for example at room temperature or at elevated temperature], etc.) to allow access of intracellular quinone derived compound(s) to an extraction solvent, and are then extracted one or more times. Cells may optionally be concentrated (e.g., to at least about 100 g/L or more, including to at least about 120 g/l, 150 g/l, 175 g/L, 200 g/L or more) and/or dried (e.g., with a spray dryer, Blaw Knox double drum dryer, single drum vacuum dryer, etc.), prior to exposure to extraction solvent (and/or prior to disruption or homogenization). Disruption can, of course, be performed prior to and/or during exposure to extraction solvent. After extraction, solvent is typically removed (e.g., by evaporation, for example by application of vacuum, change of temperature, etc.).

[0217] In some instances, cells are homogenized and then subjected to supercritical liquid extraction or solvent extraction. Typical liquids or solvents utilized in such extractions include, for example, organic or non-organic liquids or solvents. To give but a few specific examples, such liquids or solvents may include acetone, carbon dioxide (e.g., supercritical carbon dioxide, acetone, heptane, octane, ethanol), chloroform, ethanol, ethyl acetate, heptane, hexane, hexane:ethyl acetate, isopropanol, methanol, methylene chloride, isopropanol, ethyl acetateoctane, tetrahydrofuran (THF), different types of oils (e.g., soybeen, rapeseed, etc.), and combinations thereof. Particular solvents may be selected, for example, based on their ability to solubilize particular quinone derived compounds or sets of quinone derived compounds (e.g., all quinone derived compounds), and/or based on regulatory or other considerations (e.g., toxicity, cost, ease of handling, ease of removal, ease of disposal, etc.). For example, more polar quinone derived compounds may be extracted more efficiently into extraction solvents with increased polarity. In some embodiments, hexane is used as a solvent. In other embodiments, hexane:ethyl acetate is utilized.

[0218] In some embodiments, combinations of solvents may be utilized. In some embodiments, combinations of a relatively polar solvent (e.g., alcohols, acetone, chloroform, methylene chloride, ethyl acetate, etc.) and a relatively non-polar solvent (e.g., hexane, oils, etc.) are utilized for extraction. Those of ordinary skill in the art will readily appreciate that different ratios of polar to non-polar solvent may be employed as appropriate in a particular situation. Just to give a few examples, common ratios include 1:1, 2:1, 3:1, 3:2, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:45, 60:40, 55:45, and 50:50. It will be appreciated that solvents or solvent mixtures of different polarities may be more effective at extracting particular quinone derived compounds (e.g., based on their polarities and/or as a function of other attributes of the host cell material from which they are being extracted). Those of ordinary skill in the art are well able to adjust the overall polarity of the extracting solvent, for instance by adjusting the relative amounts of polar and non-polar solvents in a solvent blend, in order to achieve more efficient extraction.

[0219] Extraction may be performed under any of a variety of environmental conditions, including any of a variety of temperatures. For example, extraction may be performed on ice, at room temperature, or at any of a variety of other temperatures. For example, a solvent may be maintained at a selected temperature (e.g., about 4, 25, 28, 30, 37, 68, 70, 75, 80, 85, 90, 95, or 100.degree. C.) in order to improve or adjust extraction of a particular desired quinone derived compound.

[0220] Extraction typically yields a crude oil suspension. In some embodiments, the crude oil suspension contains some intact host cells but is at least about 95% free of intact host cells. In some embodiments, the crude oil suspension is at least about 96%, 97%, 98%, or 99% or more free of intact host cells. In some embodiments, the suspension is substantially free of water-soluble cell components (e.g., nucleic acids, cell wall or storage carbohydrates, etc.). In some embodiments, the suspension contains less than about 5%, 4%, 3%, 2%, or 1% or less water-soluble cell components.

[0221] Extraction conditions that yield a crude oil suspension will enrich for lipophilic components that accumulate in the lipid bodies within oleaginous organisms. In general, the major components of the lipid bodies consist of triacylglycerols, ergosteryl esters, other steryl esters, free ergosterol, phospholipids, and some proteins, which often function in the synthesis or regulation of the levels of other lipid body components. C16 and C18 (e.g. C16:0, C16:1, C18:0, C18:1, and C18:2) are generally the major fatty acids present in lipid bodies, mainly as components of triacylglycerol and steryl esters.

[0222] In some embodiments of the invention, the crude oil suspension contains at least about 2.5% by weight quinone derived compound(s); in some embodiments, the crude oil suspension contains at least about 5% by weight quinone derived compound(s), at least about 10% by weight quinone derived compound(s), at least about 20% by weight quinone derived compound(s), at least about 30% by weight quinone derived compound(s), at least about 40% by weight quinone derived compound(s), or at least about 50% by weight quinone derived compound(s).

[0223] The crude 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, quinone derived compounds (e.g. CoQ 10) can be isolated from the oil using conventional techniques.

[0224] Given the sensitivity of quinones 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 quinone derived compound isolation. When the desired product is the reduced ubiquinol form, then it may be advantageous to further add conventional reducing agents (e.g. sodium dithionite, ascorbic acid). 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).

[0225] Extracted quinone derived compounds may be further isolated and/or purified, for example, by crystallization, washing, recrystallization, and/or other purification strategies. In some embodiments, carotenoid crystals are collected by filtration and/or centrifugation. Isolated or purified quinone derived compounds may be dried and/or formulated for storage, transport, sale, and/or ultimate use. To give but a few specific examples, quinone derived compounds may be prepared as a cold-water dispersible powder, as a suspension of crystals in oil (e.g., vegetable oil, e.g., about 5%-30% w/w), etc.

Uses

[0226] Quinone derived compounds (e.g., ubiquinones, vitamin K compounds, and/or vitamin E compounds) produced according to the present invention can be utilized in any of a variety of applications, for example exploiting biological or nutritional (e.g., metabolic, anti-oxidant, anti-proliferative, etc.) properties.

[0227] For example, according to the present invention, one or more quinone derived compounds may be used in pharmaceutical and/or nutraceutical applications for treatment and/or prevention of disorders such as cardiovascular disorders (including, congestive heart failure, myocardial infarction and cardiac surgery patients, angina, hypertension, etc), metabolic disorders, diabetes, pain, aging, neurodegenerative disorders (e.g., Parkinson's and Huntington's disorders), inflammatory disorders and cancers. Additionally, supplements of certain quinone derived compounds (including, e.g., CoQ10) have been proposed to improve performance (e.g., athletic performance). See, for example, Choi, et al., Appi. Microbiol., Biotechnol, 68: 9-15, 2005; Kawamukai, J. Biosci. Bioeng, 94:511-517, 2002; Ernster and Dallner, Biochim. Biophys. Acta., 1271: 195-204, 1995; U.S. Pat. No. 6,806,076; U.S. Pat. No. 6,867,024; U.S. Pat. No. 6,686,485; U.S. Pat. No. 6,417,233; U.S. Patent Publication No. 2006/0010519; U.S. Patent Publication No. 2004/0034107; U.S. Patent Publication No. 2003/0236239; U.S. Patent Publication No. 2003/0167556; U.S. Patent Publication No. 2002/0058712), nutritional supplement (see for example, U.S. Pat. No. 6,686,485; U.S. Pat. No. 6,080,788, U.S. Patent Publication No. 2004/0082536) food supplements (see, for example, U.S. Patent Publication Nos. 2005/011226 and 2004/0115309, cosmetics (as anti-oxidants and/or as cosmetics, including fragrances; see for example U.S. Patent Publication No. 2005/0084505; U.S. Patent Publication No. 2003/0167556), etc. The contents of each of the foregoing journal and patent publications are hereby incorporated by reference. Quinone derived compound(s) produced herein can also be co-administered with an HMG CoA reductase inhibitor (e.g. a statin such as atorvastatin, simvastatin, rosuvastatin, etc.)

[0228] It will be appreciated that, in some embodiments of the invention, one or more quinone derived compound(s) produced by manipulated host cells as described herein are incorporated into a final product (e.g., food or feed supplement, pharmaceutical, cosmetic, 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 cells may also be processed prior to incorporation in the product to, e.g., increase bioavailability (e.g., via lysis). Alternatively or additionally, a final product may incorporate only a portion of a host cell (e.g., fractionated by size, solubility), separated from the whole. For example, in some embodiments of the invention, lipid bodies are isolated from host cells and are incorporated into or used as the final product.

[0229] For instance, inventive compound-containing lipid bodies (e.g., from engineered cells, and particularly from engineered fungal cells) may be substituted for the plant oil bodies described in U.S. Pat. No. 6,599,513 (the entire contents of which are hereby incorporated by reference) and incorporated into emulsions or emulsion formulations, as described thereon. In other embodiments, a ubiquinone itself is isolated and reformulated into a final product.

[0230] Preparations of one or more quinone derived compounds, including formulations, dosing, supplements (e.g., dietary supplements), have been described and are known in the art. For example, see citations relating to pharmaceutical preparations, nutritional supplement, and food additives described above. Additionally, see, for example, U.S. Pat. No. 6,906,106, U.S. Pat. No. 6,867,024; U.S. Pat. No. 6,740,338; U.S. Pat. No. 6,300,377; U.S. Patent Publication No. 2003/0105168, U.S. Patent Publication No. 2004/0014817; U.S. Patent Publication No. 2005/0019268; U.S. Patent Publication No. 2004/0152612; U.S. Patent Publication No. 2003/0167556; U.S. Patent Publication No. 2005/0153406; U.S. Patent Publication No. 2005/0181109; U.S. Patent Publication No. 2006/0010519; International Patent Publication No. WO05069916A2, and International Patent Publication No. WO05092123A1, each of which is incorporated herein by reference.

[0231] The amount of any particular quinone derived compound incorporated into a given product may vary dramatically depending on the product, and the particular compound(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 compound may comprise 100% of the product. Thus, the amount of quinone derived compound incorporated into a given product may be, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0232] In some embodiments of the invention, one or more produced quinone derived compound is incorporated into a component of food or feed (e.g., a food supplement, food additive). Types of food products into which quinone derived compound (s) 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 quinone derived compound within bodies of edible lipids as it may facilitate incorporation into certain fat-containing food products.

[0233] Examples of feedstuffs into which one or more quinone derived compounds 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 or cultured fish, etc., feed for farm-raised animals (including livestock and further including fish raised in aquaculture). Food or feed material into which the quinone derived compound (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).

[0234] In some embodiments of the invention, produced quinone derived compound is incorporated into a cosmetic product. Examples of such cosmetics include, for instance, skin cosmetics (e.g., lotions, emulsions, liquids, 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.

[0235] In some embodiments, produced quinone derived compound 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 capsulae, 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 compound-containing lipid droplets may be readily incorporated into oil-based formulations.

[0236] 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.

[0237] In still other embodiments, produced quinone derived compound is incorporated into a nutritional supplement or nutraceutical. Examples of such nutraceuticals, include, for instance, various types of tablets, capsules, drinkable agents, troches, gargles, etc. In some embodiments, the nutraceutical is suitable for topical application. Dosage forms are, as in pharmaceutical products, not particularly limited and include any of the same types of dosages as pharmaceuticals.

[0238] Quinone derived compound(s) produced according to the present invention (whether isolated or in the context of lipid droplets or of cells, e.g., fungal cells) may be incorporated into products as described herein by combination with any of a variety of agents. For instance, such quinone derived compound (s) may be combined with one or more binders or fillers. In some embodiments, inventive products will include one or more chelating agents, pigments, salts, surfactants, moisturizers, viscosity modifiers, thickeners, emollients, fragrances, preservatives, etc., and combinations thereof.

[0239] Useful surfactants include, for example, anionic surfactants such as branched and unbranched alkyl and acyl hydrocarbon compounds, sodium dodecyl sulfate (SDS); sodium lauryl sulfate (SLS); sodium lauryl ether sulfate (SLES); sarconisate; fatty alcohol sulfates, including sodium, potassium, ammonium or triethanolamine salts of C.sub.10 to C.sub.18 saturated or unsaturated forms thereof; ethoxylated fatty alcohol sulfates, including alkyl ether sulfates; alkyl glyceryl ether sulfonate, alpha sulpho fatty acids and esters; fatty acid esters of isethionic acid, including Igepon A; acyl (fatty) N-methyltaurides, including Igepon T; dialkylsulfo succinate esters, including C.sub.8, C.sub.10 and C.sub.12 forms thereof; Miranot BT also referred to as lauroamphocarboxyglycinate and sodium tridecath sulfate; N-acylated amino acids, such as sodium N-lauroyl sarconisate or gluconate; sodium coconut monoglyceride sulfonate; and fatty acid soaps, including sodium, potassium, DEA or TEA soaps.

[0240] Among the cationic surfactants that are useful are monoalkyl trimethyl quartenary salts; dialkyl dimethyl quartenary salts; ethoxylated or propoxylated alkyl quaternary ammonium salts, also referred to in the art as ethoquats and propoquats; cetyl benzylmethylalkyl ammonium chloride; quaternized imidazolines, which are generally prepared by reacting a fat or fatty acid with diethylenetriamine followed by quaternization, and non-fat derived cationic polymers such as the cellulosic polymer, Polymer JR (Union Carbide).

[0241] Further useful cationic surfactants include lauryl trimethyl ammonium chloride; cetyl pyridinium chloride; and alkyltrimethylammonium bromide. Cationic surfactants are particularly useful in the formulation of hair care products, such as shampoos, rinses and conditioners.

[0242] Useful nonionic surfactants include polyethoxylated compounds and polypropoxylated products. Examples of ethoxylated and propoxylated non-ionic surfactants include ethoxylated anhydrohexitol fatty esters, for example Tween 20; mono- and diethanolamides; Steareth-20, also known as Volpo20; polyethylene glycol fatty esters (PEGs), such as PEG-8-stearate, PEG-8 distearate; block co-polymers, which are essentially combinations of hydrophylic polyethoxy chains and lipophilic polypropoxy chains and generically known as Poloaxamers.

[0243] Still other useful non-ionic surfactants include fatty esters of polyglycols or polyhydric alcohols, such as mono and diglyceride esters; mono- and di-ethylene glycol esters; diethylene glycol esters; sorbitol esters also referred to as Spans; sucrose esters; glucose esters; sorbitan monooleate, also referred to as Span80; glyceryl monostearate; and sorbitan monolaurate, Span20 or Arlacel 20.

[0244] Yet other useful nonionic surfactants include polyethylene oxide condensates of alkyl phenols and polyhydroxy fatty acid amide surfactants which may be prepared as for example disclosed in U.S. Pat. No. 2,965,576.

[0245] Examples of amphoteric surfactants which can be used in accordance with the present invention include betaines, which can be prepared by reacting an alkyldimethyl tertiary amine, for example lauryl dimethylamine with chloroacetic acid. Betaines and betaine derivatives include higher alkyl betaine derivatives including coco dimethyl carboxymethyl betaine; sulfopropyl betaine; alkyl amido betaines; and cocoamido propyl betaine. Sulfosultaines which may be used include for example, cocoamidopropyl hydroxy sultaine. Still other amphoteric surfactants include imidazoline derivatives and include the products sold under the trade name "Miranol" described in U.S. Pat. No. 2,528,378 which is incorporated herein by reference in its entirety. Still other amphoterics include phosphates for example, cocamidopropyl PG-dimonium chloride phosphate and alkyldimethyl amine oxides.

[0246] Suitable moisturizers include, for example, polyhydroxy alcohols, including butylene glycol, hexylene glycol, propylene glycol, sorbitol and the like; lactic acid and lactate salts, such as sodium or ammonium salts; C.sub.3 and C.sub.6 diols and triols including hexylene glycol, 1,4 dihydroxyhexane, 1,2,6-hexane triol; aloe vera in any of its forms, for example aloe vera gel; sugars and starches; sugar and starch derivatives, for example alkoxylated glucose; hyaluronic acid; lactamide monoethanolamine; acetamide monoethanolamine; glycolic acid; alpha and beta hydroxy acids (e.g. lactic, glycolic salicylic acid); glycerine; pantheol; urea; vaseline; natural oils; oils and waxes (see: the emollients section herein) and mixtures thereof.)

[0247] Viscosity modifiers that may be used in accordance with the present invention include, for example, cetyl alcohol; glycerol, polyethylene glycol (PEG); PEG-stearate; and/or Keltrol.

[0248] Appropriate thickeners for use in inventive products include, for example, gelling agents such as cellulose and derivatives; Carbopol and derivatives; carob; carregeenans and derivatives; xanthane gum; sclerane gum; long chain alkanolamides; bentone and derivatives; Kaolin USP; Veegum Ultra; Green Clay; Bentonite NFBC; etc.

[0249] Suitable emollients include, for example, natural oils, esters, silicone oils, polyunsaturated fatty acids (PUFAs), lanoline and its derivatives and petrochemicals.

[0250] Natural oils which may be used in accordance with the present invention may be obtained from sesame; soybean; apricot kernel; palm; peanut; safflower; coconut; olive; cocoa butter; palm kernel; shea butter; sunflower; almond; avocado; borage; carnauba; hazel nut; castor; cotton seed; evening primrose; orange roughy; rapeseed; rice bran; walnut; wheat germ; peach kernel; babassu; mango seed; black current seed; jojoba; macadamia nut; sea buckthorn; sasquana; tsubaki; mallow; meadowfoam seed; coffee; emu; mink; grape seed; thistle; tea tree; pumpkin seed; kukui nut; and mixtures thereof.

[0251] Esters which may be used include, for example, C.sub.8-C.sub.30 alklyl esters of C.sub.8-C.sub.30 carboxylic acids; C.sub.1-C.sub.6 diol monoesters and diesters of C.sub.8-C.sub.30 carboxylic acids; C.sub.10-C.sub.20 alcohol monosorbitan esters, C.sub.10-C.sub.20 alcohol sorbitan di- and tri-esters; C.sub.10-C.sub.20 alcohol sucrose mono-, di-, and tri-esters and C.sub.10-C.sub.20 fatty alcohol esters of C.sub.2-C.sub.6 2-hydroxy acids and mixtures thereof. Examples of these materials include isopropyl palmitate; isopropyl myristate; isopropyl isononate; C.sub.12/C.sub.14 benzoate ester (also known as Finesolve); sorbitan palmitate, sorbitan oleate; sucrose palmitate; sucrose oleate; isostearyl lactate; sorbitan laurate; lauryl pyrrolidone carboxylic acid; panthenyl triacetate; and mixtures thereof.

[0252] Further useful emollients include silicone oils, including non-volatile and volatile silicones. Examples of silicone oils that may be used in the compositions of the present invention are dimethicone; cyclomethycone; dimethycone-copolyol; aminofunctional silicones; phenyl modified silicones; alkyl modified silicones; dimethyl and diethyl polysiloxane; mixed C.sub.1-C.sub.30 alkyl polysiloxane; and mixtures thereof. Additionally useful silicones are described in U.S. Pat. No. 5,011,681 to Ciotti et al., incorporated by reference herein.

[0253] A yet further useful group of emollients includes lanoline and lanoline derivatives, for example lanoline esters.

[0254] Petrochemicals which may be used as emollients in the compositions of the present invention include mineral oil; petrolatum; isohexdecane; permethyl 101; isododecanol; C.sub.11-C.sub.12 Isoparrafin, also known as Isopar H.

[0255] Among the waxes which may be included in inventive products are animal waxes such as beeswax; plant waxes such as carnauba wax, candelilla wax, ouricurry wax, Japan wax or waxes from cork fibres or sugar cane. Mineral waxes, for example paraffin wax, lignite wax, microcrystalline waxes or ozokerites and synthetic waxes may also be included.

[0256] Exemplary fragrances for use in inventive products include, for instance, linear and cyclic alkenes (i.e. terpenes); primary, secondary and tertiary alcohols; ethers; esters; ketones; nitrites; and saturated and unsaturated aldehydes; etc.

[0257] Examples of synthetic fragrances that may be used in accordance with the present invention include without limitation acetanisole; acetophenone; acetyl cedrene; methyl nonyl acetaldehyde; musk anbrette; heliotropin; citronellol; sandella; methoxycitranellal; hydroxycitranellal; phenyl ethyl acetate; phenylethylisobutarate; gamma methyl ionone; geraniol; anethole; benzaldehyde; benzyl acetate; benzyl salicate; linalool; cinnamic alcohol; phenyl acetaldehyde; amyl cinnamic aldehyde; caphore; p-tertiairy butyl cyclohexyl acetate; citral; cinnamyl acetate; citral diethyl acetal; coumarin; ethylene brasslate; eugenol; 1-menthol; vanillin; etc.

[0258] Examples of natural fragrances of use herein include without limitation lavandin; heliotropin; sandlewood oil; oak moss; pathouly; ambergris tincture; ambrette seed absolute; angelic root oil; bergamont oil; benzoin Siam resin; buchu leaf oil; cassia oil; cedarwood oil; cassia oil; castoreum; civet absolute; chamomile oil; geranium oil; lemon oil; lavender oil; Ylang Ylang oil; etc.

[0259] A list of generally used fragrance materials can be found in various reference sources, for example, "Perfume and Flavor Chemicals", Vols. I and II; Steffen Arctander Allured Pub. Co. (1994) and "Perfumes: Art, Science and Technology"; Muller, P. M. and Lamparsky, D., Blackie Academic and Professional (1994) both incorporated herein by reference.

[0260] Suitable preservatives include, among others, (e.g., sodium metabisulfite; Glydant Plus; Phenonip; methylparaben; Germall 115; Germaben II; phytic acid; sodium lauryl sulfate (SLS); sodium lauryl ether sulfate (SLES); Neolone; Kathon; Euxyl and combinations thereof), anti-oxidants (e.g., butylated hydroxytoluened (BHT); butylated hydroxyanisol (BHA); ascorbic acid (vitamin C); tocopherol; tocopherol acetate; phytic acid; citric acid; pro-vitamin A.

[0261] In some embodiments, inventive products will comprise an emulsion (e.g., containing inventive lipid bodies), and may include one or more emulsifying agents (e.g., Arlacel, such as Alacel 165; Glucamate; and combinations thereof) and/or emulsion stabilizing agents. In some embodiments, inventive products will include one or more biologically active agents other than the ubiquinone(s). To give but a few examples, inventive cosmetic or pharmaceutical products may include one or more biologically active agents such as, for example, sunscreen actives, anti-wrinkle actives, anti-aging actives, whitening actives, bleaching actives, sunless tanning actives, anti-microbial actives, anti-acne actives, anti-psoriasis actices, anti-eczema actives, antioxidants, anesthetics, vitamins, protein actives, etc.

Engineering Production of Multiple Isoprenoid Compounds

[0262] In certain embodiments of the invention, it may be desirable to generate engineered organisms that accumulate one or more other compounds in addition to the quinone derived compound described herein, and further to accumulate such other compound(s), optionally together with the quinone derived compound(s), in lipid bodies. For example, certain inventive engineered organisms may accumulate quinone derived compound together with at least one other compound derived from an isoprenoid precursor. In some embodiments, the other compound derived from an isoprenoid precursor will be one or more quinone derived compound discussed herein (e.g., a ubiquinone, vitamin K, vitamin E). Alternatively or additionally, in some embodiments the other compound derived from an isoprenoid precursor will be one or more carotenoids. Production of carotenoids in oleaginous organisms is described in U.S. Provisional Application No. 60/663,621, filed Mar. 18, 2005, and is also described in U.S. patent application Ser. No. 11/385,580, entitled Production of Carotenoids in Oleaginous Yeast and Fungi, filed Mar. 20, 2006.

[0263] In some embodiments of the invention, host cells are engineered to produce at least two compounds selected from the group consisting of a ubiquinone (e.g. CoQ10, a C.sub.5-9 quinone), vitamin K, vitamin E, and carotenoids. In some such embodiments, host cells are engineered to produce a least one compound selected from the group consisting of a ubiquinone (e.g. CoQ10, a C.sub.5-9 quinone), vitamin K, vitamin E, and at least one other compound. Such host cells are particularly useful for producing mixtures or combination products that contain a ubiquinone and/or vitamin K. Such host cells are also useful for producing combination products including one or more carotenoids.

[0264] In some embodiments of the invention, host cells are engineered to produce at least one quinone derived compound and at least one carotenoid. Carotenoids, which have an isoprene backbone consisting of 40 carbon atoms, have antioxidant effects as well as use in coloring agents. Carotenoids such as .beta.-carotene, astaxanthin, and cryptoxanthin are believed to possess cancer preventing and immunopotentiating activity.

[0265] The carotenoid biosynthesis pathway branches off from the isoprenoid biosynthesis pathway at the point where GGPP is formed. Up to and including formation of FPP, and potentially GGPP, is as described above for production of a ubiquinone. 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). 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 .gamma.-carotene; action of a desaturase instead produces 3,4-didehydrolycopene. .gamma.-carotene is converted to .beta.-carotene through the action of a cyclase. .beta.-carotene can be processed into any of a number of products, including astaxanthin (via echinone, hydroxyechinone, and phoenicoxanthin).

[0266] Carotenoid production in a host organism may be adjusted by modifying the expression or activity of one or more proteins involved in carotenoid biosynthesis. In some embodiments of the invention, 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.

[0267] In some embodiments of the invention, it will be desirable to introduce one or more carotenogenic modifications into a host cell. In certain embodiments the carotenogenic modification may confer expression of 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. Still further, selection will consider the complementation and/or ability of the selected polypeptide to function in conjunction with additional oleaginic and/or quinonogenic modifications of a cell such that each of oleaginy, ubiquinone biosynthesis and carotenoid biosynthesis are effectuated to the desired extent.

[0268] 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 carotenoid biosynthesis polypeptides are provided in Tables 17-21 and Tables 38-41.

[0269] Alternatively or additionally, modified carotenoid ketolase polypeptides that exhibit improved carotenoid production activity may be utilized in accordance with the present invention. For example, carotenoid ketolase polypeptides comprising one more mutations to corresponding to those identified Sphingomonassp. DC18 which exhibited improved astaxanthin production (Tao et al 2006 Metab Eng. 2006 Jun 27) and Paracoccus sp. strain N81106 which exhibited altered carotenoid production (Ye et al. Appl Environ Microbiol 72:5829, 2006).

[0270] 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.

[0271] 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. Carotenoid ketolase activity is particularly relevant for production of canthaxanthin, as carotenoid hydroxylase activity is for production of lutein and zeaxanthin, among others. Both carotenoid hydroxylase and ketolase activities (and/or astaxanthin synthase) are particularly useful for production of astaxanthin.

[0272] Bacterial carotenogenic genes have already been demonstrated to be transferable to other organisms, and are therefore particularly useful in accordance with the present invention (see, for example, Miura et al., Appi. 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.

[0273] In certain embodiments of the present invention, cells are engineered to produce at least one sterol compound together with the at least one quinone derived compound. The commitment step in sterol biosynthesis is the conversion of farnesyl pyrophosphate into presqualene pyrophosphate. Farnesyl pyrophosphate (FPP) is produced from isopentenyl pyrophosphate (IPP), for example in a process that involves isomerization of IPP into dimethylallyl pyrophosphate (DMAPP), followed by 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). FPP can either enter the sterol biosynthesis pathway by conversion into presqualene puyrophosphate, or alternatively can be diverted toward biosynthesis of carotenoids and other compounds (e.g., a ubiquinone, vitamin E, vitamin K, etc.) by conversion into the twenty-carbon compound geranylgeranyl pyrophosphate (GGPP). In many instances, FPP appears to be the predominant substrate used by polyprenyldiphosphate synthases (Coq1 polypeptides) during biosynthesis of ubiquinones.

[0274] Once the sterol biosynthesis pathway has been entered, presqualene pyrophosphate is then converted to squalene by the same enzyme that performed the farnesyl pyrophosphate.fwdarw.presqualene pyrophosphate conversion. Squalene is then converted into a variety of different sterol compounds, including, but not limited to, lanosterol, zymosterol, ergosterol, 7-dehydrocholesterol (provitamin D3), and vitamin D compounds. The vitamin D.sub.2 biosynthetic pathway and the vitamin D.sub.3 biosynthetic pathway share some common reactions, and there can be multiple points at which a vitamin D.sub.2 intermediate can be converted into a vitamin D.sub.3 intermediate (see, for example, FIG. 1).

[0275] In some embodiments, the sterol compound whose production is engineered is selected from the group consisting of squalene, lanosterol, zymosterol, ergosterol, and vitamin D compounds (e.g., 7-dehydrocholesterol (provitamin D3)).

[0276] In some embodiments of the present invention, host cells are engineered to produce squalene in addition to one or more quinone derived compounds. In some embodiments, squalene production is enhanced in a cell by introduction of one or more sterologenic modifications that increases levels of IPP, FPP and/or squalene itself. In some embodiments, squalene production is enhanced in a cell by increasing the level and/or activity of one or more squalene biosynthesis polypeptides (e.g., one or more isoprenoid biosynthesis polypeptides, an FPP synthase polypeptide, and/or a squalene synthase polypeptide). Alternatively or additionally, in some embodiments, squalene production is enhanced in a cell by decreasing the level and/or activity of one or more sterol biosynthesis competitor polypeptides that diverts one or more intermediates away from squalene production and/or that metabolizes squalene itself.

[0277] For example, in some embodiments of the present invention, squalene production in a host cell is increased by introducing or increasing expression and/or activity of one or more squalene synthase polypeptides in the cell. 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, Aspergillus niger, Saccharomyces cerevisiae, Mucor circinelloides, Candida utilis, Mortierella alpina, Phaffia rhodozyma, and Yarrowia lipolytica.

[0278] In some embodiments of the invention, squalene production in a host cell is increased by reducing the level or activity of one or more squalene biosynthesis competitor polypeptides, including for example one or more vitamin D biosynthesis polypeptides (e.g., which act to metabolize squalene). For instance, in some embodiments, the level or activity of one or more polypeptides active in the ergosterol biosynthetic pathway (see below) is reduced or eliminated.

[0279] In some embodiments, the sterol compound produced in addition to one or more quinone derived compounds according to the present invention is lanosterol. In some embodiments, lanosterol production is enhanced in a cell by introduction of one or more sterologenic modifications that increases levels of IPP, FPP and/or lanosterol itself. In some embodiments, lanosterol production is enhanced in a cell by increasing the level and/or activity of one or more lanosterol biosynthesis polypeptides (e.g., one or more isoprenoid biosynthesis polypeptides, an FPP synthase polypeptide, squalene synthase polypeptide, squalene epoxidase polypeptide, or 2,3-oxidosqualene-lanosterol cyclase polypeptide). Alternatively or additionally, in some embodiments, lanosterol production is enhanced in a cell by decreasing the level and/or activity of one or more sterol biosynthesis competitor polypeptides that diverts one or more intermediates away from lanosterol production and/or that metabolizes lanosterol itself.

[0280] For example, in some embodiments of the present invention, lanosterol production in a host cell is increased by introducing or increasing expression and/or activity of one or more squalene synthase polypeptide, squalene epoxidase polypeptide, or 2,3-oxidosqualene-lanosterol cyclase polypeptide in the cell. Representative examples of squalene synthase polypeptide, squalene epoxidase polypeptide, and 2,3-oxidosqualene-lanosterol cyclase polypeptide sequences are included in Table 16, 83, and 85. In some embodiments of the invention that utilize squalene synthase polypeptide, squalene epoxidase polypeptide, or 2,3-oxidosqualene-lanosterol cyclase polypeptide (or modifications of these polypeptides) source organisms include, but are not limited to, Neurospora crassa, Aspergillus niger, Saccharomyces cerevisiae, Phaffia rhodozyma, Mucor circinelloides, Candida utilis, Mortierella alpina, and Yarrowia lipolytica.

[0281] In some embodiments of the invention, lanosterol production in a host cell is increased by reducing the level or activity of one or more vitamin D biosynthesis polypeptides (e.g., which act to metabolize squalene). For instance, in some embodiments, the level or activity of one or more polypeptides active in the ergosterol biosynthetic pathway (see below).

[0282] In some embodiments of the present invention, host cells are engineered to produce zymosterol in addition to one or more quinone derived compounds. In some embodiments, zymosterol production is enhanced in a cell by introduction of one or more sterologenic modifications that increases levels of IPP, FPP and/or zymosterol itself. In some embodiments, zymosterol production is enhanced in a cell by increasing the level and/or activity of one or more zymosterol biosynthesis polypeptides (e.g., one or more isoprenoid biosynthesis polypeptides, an FPP synthase polypeptide, squalene synthase polypeptide, squalene epoxidase polypeptide, 2,3-oxidosqualene-lanosterol cyclase polypeptide, cytochrome P450 lanosterol 14.alpha.-demethylase polypeptide, C-14 sterol reductase polypeptide, or C-4 sterol methyl oxidase polypeptide). Alternatively or additionally, in some embodiments, zymosterol production is enhanced in a cell by decreasing the level and/or activity of one or more sterol biosynthesis competitor polypeptides that diverts one or more intermediates away from lanosterol production and/or that metabolizes lanosterol itself.

[0283] For example, in some embodiments of the present invention, zymosterol production in a host cell is increased by introducing or increasing expression and/or activity of one or more squalene synthase polypeptide, squalene epoxidase polypeptide, 2,3-oxidosqualene-lanosterol cyclase polypeptide, cytochrome P450 lanosterol 14.alpha.-demethylase polypeptide, C-14 sterol reductase polypeptide, or C-4 sterol methyl oxidase polypeptide in the cell. Representative examples of squalene synthase polypeptide, squalene epoxidase polypeptide, 2,3-oxidosqualene-lanosterol cyclase polypeptide, cytochrome P450 lanosterol 14.alpha.-demethylase polypeptide, C-14 sterol reductase polypeptide, or C-4 sterol methyl oxidase polypeptide sequences are included in Tables 16, and tables 86-99. In some embodiments of the invention that utilize squalene synthase polypeptide, squalene epoxidase polypeptide, 2,3-oxidosqualene-lanosterol cyclase polypeptide, cytochrome P450 lanosterol 14.alpha.-demethylase polypeptide, C-14 sterol reductase polypeptide, or C-4 sterol methyl oxidase polypeptide (or modifications of these polypeptides) source organisms include, but are not limited to, Neurospora crassa, Aspergillus niger, Saccharomyces cerevisiae, Phaffia rhodozyma, Mucor circinelloides, Candida utilis, Mortierella alpina, and Yarrowia

[0284] In some embodiments of the invention, zymosterol production in a host cell is increased by reducing the level or activity of one or more vitamin D biosynthesis polypeptides (e.g., which act to metabolize squalene). For instance, in some embodiments, the level or activity of one or more polypeptides active in the ergosterol biosynthetic pathway (see below).

[0285] In some embodiments of the present invention, host cells are engineered to produce ergosterol in addition to one or more quinone derived compounds. In some embodiments, ergosterol production is enhanced in a cell by introduction of one or more sterologenic modifications that increases levels of IPP, FPP and/or ergosterol itself. In some embodiments, ergosterol production is enhanced in a cell by increasing the level and/or activity of one or more ergosterol biosynthesis polypeptides (e.g., one or more isoprenoid biosynthesis polypeptides, an FPP synthase polypeptide, squalene synthase polypeptide, squalene epoxidase polypeptide, 2,3-oxidosqualene-lanosterol cyclase polypeptide, cytochrome P450 lanosterol 14.alpha.-demethylase polypeptide, C-14 sterol reductase polypeptide, C-4 sterol methyl oxidase polypeptide, SAM:C-24 sterol methyltransferase polypeptide, C-8 sterol isomerase polypeptide, C-5 sterol desaturase polypeptide, C-22 sterol desaturase polypeptide, or C-24 sterol reductase polypeptide. Alternatively or additionally, in some embodiments, ergosterol production is enhanced in a cell by decreasing the level and/or activity of one or more sterol biosynthesis competitor polypeptides that diverts one or more intermediates away from ergosterol production and/or that metabolizes ergosterol itself.

[0286] For example, in some embodiments of the present invention, ergosterol production in a host cell is increased by introducing or increasing expression and/or activity of one or more squalene synthase polypeptide, squalene epoxidase polypeptide, 2,3-oxidosqualene-lanosterol cyclase polypeptide, cytochrome P450 lanosterol 14.alpha.-demethylase polypeptide, C-14 sterol reductase polypeptide, C-4 sterol methyl oxidase polypeptide, SAM:C-24 sterol methyltransferase polypeptide, C-8 sterol isomerase polypeptide, C-5 sterol desaturase polypeptide, C-22 sterol desaturase polypeptide, or C-24 sterol reductase polypeptide in the cell. Representative examples of squalene synthase polypeptide, squalene epoxidase polypeptide, 2,3-oxidosqualene-lanosterol cyclase polypeptide, cytochrome P450 lanosterol 14.alpha.-demethylase polypeptide, C-14 sterol reductase polypeptide, C-4 sterol methyl oxidase polypeptide, SAM:C-24 sterol methyltransferase polypeptide, C-8 sterol isomerase polypeptide, C-5 sterol desaturase polypeptide, C-22 sterol desaturase polypeptide, or C-24 sterol reductase polypeptide sequences are included in Table 16, and tables 86-99. In some embodiments of the invention that utilize squalene synthase polypeptide, squalene epoxidase polypeptide, 2,3-oxidosqualene-lanosterol cyclase polypeptide, cytochrome P450 lanosterol 14.alpha.-demethylase polypeptide, C-14 sterol reductase polypeptide, C-4 sterol methyl oxidase polypeptide, SAM:C-24 sterol methyltransferase polypeptide, C-8 sterol isomerase polypeptide, C-5 sterol desaturase polypeptide, C-22 sterol desaturase polypeptide, or C-24 sterol reductase polypeptide (or modifications of these polypeptides) source organisms include, but are not limited to, Neurospora crassa, Aspergillus niger, Saccharomyces cerevisiae, Mucor circinelloides, Candida utilis, Mortierella alpina, and Yarrowia lipolytica.

[0287] In some embodiments of the invention, ergosterol production in a host cell is increased by reducing the level or activity of one or more vitamin D biosynthesis polypeptides (e.g., which act to metabolize squalene). For instance, in some embodiments, the level or activity of one or more polypeptides (see below).

[0288] In some embodiments, the sterol compound produced in addition to one or more quinone derived compounds in accordance with the present invention is a vitamin D compound. Vitamin D compounds are a group of steroid compounds including vitamin D.sub.3 (cholecalciferol), vitamin D.sub.2 (ergocalciferol), their provitamins, and certain metabolites (see, for example, FIG. 10A-B). Vitamins D.sub.3 and D.sub.2 can be produced from their respective provitamins (e.g., 7-dehydrocholesterol and ergosterol) by ultraviolet irradiation (e.g., by the action of sunlight). The most biologically active form of vitamin D is 1,25-dihydroxy vitamin D.sub.3, which is also known as calcitriol. Calcitriol is produced by hydroxylation of vitamin D.sub.3 at the 25 position, followed by hydroxylation to generate calcitriol.

[0289] In some embodiments, vitamin D production is enhanced in a cell by introduction of one or more sterologenic modifications that increases levels of IPP, FPP and/or squalene. In some embodiments, vitamin D production is enhanced in a cell by increasing the level and/or activity of one or more vitamin D biosynthesis polypeptides (e.g., one or more isoprenoid biosynthesis polypeptides, an FPP synthase polypeptide, a squalene synthase polypeptide, and/or one or more polypeptides involved in converting squalene into a particular vitamin D compound of interest [e.g., 7-dehydrocholesterol and/or calcitriol]). Alternatively or additionally, in some embodiments, vitamin D production is enhanced in a cell by decreasing the level and/or activity of one or more sterol biosynthesis competitor polypeptides that diverts one or more intermediates away from vitamin D production. In some embodiments of the invention, production of a particular vitamin D compound (e.g., 7-dehydrocholesterol and/or calcitriol) is enhanced by decreasing the level and/or activity of one or more polypeptides that diverts a relevant intermediate toward an alternative vitamin D compound (e.g., ergosterol, vitamin D.sub.2).

[0290] To give but one particular example of a sterologenic modification that can be employed in accordance with the present invention to increase production of one or more vitamin D.sub.3 compounds (e.g., 7-dehydrocholesterol and/or calcitriol), in accordance with some embodiments of the present invention, the level and/or activity of one or more polypeptides that diverts a relevant intermediate toward a vitamin D.sub.2 compound (e.g., ergosterol, vitamin D.sub.2), and away from vitamin D.sub.3 compounds, can be inhibited or destroyed. In some embodiments of the invention, where multiple heterologous polypeptides are to be expressed (e.g., because one or more activities of interest require two or more polypeptide chains and/or because multiple activities of interest are being engineered), it may be desirable to utilize the same source organism for all, or to utilize closely related source organisms; in other embodiments, heterologous polypeptides may be from different source organisms. In some embodiments, two or more versions of a particular heterologous polypeptide, optionally from different source organisms, may be introduced into and/or engineered within, a single host cell.

[0291] Having now generally described the invention, the same will be more readily understood through reference to the following exemplification which is provided by way of illustration, and is not intended to be limiting of the present invention.

EXEMPLIFICATION

[0292] Table 42 describes the Yarrowia lipolytica strains used in the following exemplification:

TABLE-US-00001 TABLE 42 Yarrowia lipolytica strains. NRRL Y-1095 Wild type diploid Derived from strain ATCC76861 MATB ura2-21 lyc1-5 LYS1-5B ATCC76982 MATB ade1 leu2-35 lyc1-5 xpr2 ATCC201249 MATA ura3-302 leu2-270 lys8-11 PEX17-HA MF346 MATA ura2-21 ATCC76861 .times. ATCC201249 MF350 MATB ura2-21 leu2-35 ade1 ATCC76982 .times. MF346 MF358 MATB adel ATCC76982 .times. MF346 MF454 MATB ura3-302 ade1 leu2-270 ATCC201249 .times. MF358

[0293] (The genotypes at LYC1, LYS1, XPR2, and PEX17 were not determined in crosses nor verified for ATCC strains.)

[0294] 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). The GPD1 and TEF1 promoters are from Y. lipolytica as is the XPR2 terminator.

[0295] Plasmids were generated for construction of CoQ10 producing strains. The following subparts describe production of plasmids encoding ubiquinogenic polypeptides. General plasmid construction for expression vectors are described in Table 43, and further constructions in the text. 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 in Table 42.

TABLE-US-00002 TABLE 43 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 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 MO4568 & MO4659 (KpnI + NheI); pMB4637 pMB4629 (NheI + MluI) 1.5 kb hmg1.sup.trunc ORF (XbaI + MO4657 & MO4658 MluI) pMB4662 pMB4631 (SpeI + XhoI) 1.8 kb URA3 fragment MO4684 & MO4685 (SpeI + BsaI pMB4691 pMB4662 (Acc65I + MluI) 0.4 kb TEF1 promoter From pMB4629 (Acc65I + MluI)

TABLE-US-00003 TABLE 44 Oligonucleotides referenced in Table 43 MO4471 5'CTGGGTGACCTGGAAGCCTT MO4472 5'AAGATCAATCCGTAGAAGTTCAG MO4475 5'AAGCGATTACAATCTTCCTTTGG MO4476 5'CCAGTCCATCAACTCAGTCTCA MO4477 5'GCATTGCTTATTACGAAGACTAC MO4478 5'CCACTGTCCTCCACTACAAACAC MO4566 5'CACAAACTAGTTTGCCACCTACAAGCCAGAT MO4568 5'CACAAGGTACCAATGTGAAAGTGCGCGTGAT MO4571 5'CACAAGGTACCAGAGACCGGGTTGGCGG MO4591 5'CACAAGCGGCCGCGCTAGCATGGGGATCGATCTCTTATAT MO4592 5'CACAAGCGGCCGCGCTAGCGAATGATTCTTATACTCAGAAG MO4593 5'CACAAGCGGCCGCACGCGTGCAATTAACAGATAGTTTGCC MO4659 5'CACAAGCTAGCTGGGGATGCGATCTCTTATATC MO4684 5'CATTCACTAGTGGTGTGTTCTGTGGAGCATTC MO4685 5'CACACGGTCTCATCGAGGTGTAGTGGTAGTGCAGTG MO4657 5'CACACTCTAGACACAAAAATGACCCAGTCTGTGAAGGTGG MO4658 5'CACACACGCGTACACCTATGACCGTATGCAAAT

Example 1

Biosynthesis of CoQ10 in Yarrowia lipolytica

[0296] Decaprenyl diphosphate synthases ("dpdpS") based on the amino acid sequence of those found in Silicibacter pomeroyi and in Loktanella vestfoldensis are expressed in Yarrowia lipolytica, using nucleotide sequences with the appropriate Yarrowia codon bias, and with Yarrowia mitochondrial targeting sequences. Insertion of the expression cassette results in the disruption of the native COQ1 gene, encoding nonaprenyl diphosphate synthase.

[0297] To construct the mitochondrial leader sequence encoding DNA of NADH:ubiquinone oxidoreductases (complex I) ["NUAM"], four oligos were annealed and treated sequentially with Klenow fragment and ligase:

TABLE-US-00004 MO4859: 5'-TCTAGACACAAAAATGCTCTCGAGAAACCTCAGCAAGTTTG MO4860: 5'-GTGGTTGCTGGCCGGATGAGACCGGCTCGAGCAAACTTGCT MO4861: 5'-CAGCAACCACATCCACACACACCCGACTATTCTCTGTCTCC MO4862: 5'-GGATCCCGTCTCGGACAGACGTCGGGCGGAGACAGAG.

[0298] The resulting fragment was subsequently amplified with MO4859 and MO4862 using Pfu polymerase and the product was phosphorylated and ligated to pBluescriptSKII.sup.- cut with EcoRV to create pMB4776. The mitochondrial leader sequence of NUAM is thereby encoded within the resultant XbaI-BsmBI (underline) fragment:

TABLE-US-00005 (NUAMLdr): tctagacacaaaaatgctctcgagaaacctcagcaagtttgctcgag ccggtctcatccggccagcaaccacatccacacacacccgactattc tctgtctccgcccgacgtctgtccgagacgggatcc

[0299] To construct the mitochondrial leader sequence encoding DNA of the native Y. lipolytica Coq1 protein, Y. lipolytica genomic DNA was amplified with MO4857 (5'-CGGATCCCGTCTCGGACATTTCTTGCGACACAATG) and MO4858 (5'-CTCTAGACACAAAAATGCTGAGAGTCGGACGAAT) and the 0.25 kb product was phosphorylated and ligated to pBluescriptSKII.sup.- cut with EcoRV to create pMB4750. The mitochondrial leader sequence of Coq1 is thereby encoded within the resultant XbaI-BsmBI (underline) fragment:

TABLE-US-00006 (Coq1ldr): ctctagacacaaaaatgctgagagtcggacgaattggcaccaagacc ctagccagcagcagcctgcgtttcgtggcaggtgctcggcccaaatc cacgctcaccgaggccgtgctggagaccacagggctgctgaaaacca cgccccaaaaccccgagtggtctggagccgtcaagcaggcatctcgt ctggtggagaccgacactccgatccgagacccgttttccattgtgtc gcaagaaatgtccgagacgggatccg.

[0300] To construct a cassette encoding the decaprenyl diphosphate synthase (dpdpS) from Silicibacter pomeroyi, the following sequence was synthesized de novo:

TABLE-US-00007 (Sp-dpdpS): GGATCCCGTCTCTTGTCTGATGCCAAAGTCTCTACAAAGCCTCACGAGATGCTCGCTGCCACACT CTCTCAGGAAATGGCCGCTGTCAACGCCCTTATTCGAACCCGTATGGCTTCTGAACACGCCCCTA GAATCCCCGAAGTTACAGCCCACCTCGTCGAAGCCGGCGGAAAACGATTGCGACCTATGCTGAC ACTTGCTGCTGCCCGACTGTGTGGATACCAGGGCGAAGATCATGTCAAACTGGCTGCCACTGTT GAATTTATTCACACTGCTACACTTTTGCACGATGATGTCGTGGACGAGTCTGGACAAAGACGTG GACGACCTACTGCTAATCTTCTTTGGGATAACAAGTCCTCTGTTCTTGTGGGCGACTATCTTTTTG CACGATCGTTCCAGCTGATGGTCGAAACCGGTTCCCTTCGAGTGCTTGACATCCTCGCTAACGCT GCCGCGACAATTGCCGAAGGTGAAGTGCTCCAGATGACCGCTGCTTCCGATCTTAGAACTGATG AATCCGTTTACCTTCAGGTCGTTCGAGGTAAAACTGCCGCTCTTTTTTCTGCTGCTACTGAAGTTG GTGGTGTTATTGCCGGAGTCCCCGAAGCCCAAGTTCGAGCACTTTTTGAATACGGTGATGCGCTG GGAATTGCATTTCAGATTGCTGATGACCTCCTCGACTACCAAGGTGATGCTAAGGCCACTGGAA AGAATGTTGGAGATGACTTCAGAGAAAGAAAACTCACATTGCCTGTTATTAAAGCCGTCGCTCA AGCTACTGATGAGGAACGAGCGTTTTGGGTTAGAACTATTGAAAAGGGAAAGCAAGCTGAAGG AGATCTTGAACAGGCTCTCGCTCTCATGGAAAAATACGGAACACTTGCCGCAACCAGAGCCGAT GCGCATGCTTGGGCCGAAAAAGCACGAACCGCCCTCGAACTGTTGCCGAATCACGAAATCAGAA CAATGCTCTCCGACCTCGCCGATTATGTCGTTGCTAGATTGTCTTAAACGCGT.

[0301] To construct a cassette encoding the decaprenyl diphosphate synthase (dpdpS) from Loktanella vestfoldensis, the following sequence was synthesized de novo:

TABLE-US-00008 (Lv-dpdpS): GGATCCCGTCTCTTGTCTCTTGATCACGCTGCCACCAAACCTCATGAACAACTTGCCGCCGCCCT TGCCGACGACCTTACCGCAGTTAACGCTATGATTAGAGACCGAATGGCCAGCGAACATGCCCCC AGAATTCCACAAGTTACCGCACACCTCGTTGAAGCCGGAGGTAAAAGACTTCGTCCAATGCTGA CCCTTGCCGCCGCCCGTATGTGTGGCTACGACGGACCTTACCACATCCACCTTGCAGCCACCGTT GAGTTCATTCACACCGCCACCTTGCTCCATGACGATGTTGTTGATGAGTCCTCCCAACGTCGTGG TCGACCTACCGCTAATCTTCTGTGGGATAATACCTCTTCTGTTTTGGTTGGAGACTACCTCTTTGC ACGATCTTTTCAGTTGATGGTTGAGACCGGATCGCTTCGAGTTCTTGACATCCTGGCCAATGCTT CTGCTACCATCGCCGAAGGTGAGGTTCTGCAACTTACCGCCGCCGCTGACCTTGCAACTACCGA GGACATTTACATCAAAGTTGTTCGAGGAAAGACTGCTGCACTCTTTTCTGCTGCTATGGAAGTTG GTGGAGAAATCGCCGGTCAGGACCCGGCTATTAAACAGGCTCTCTTTGACTACGGCGACGCTCT CGGAATTTCTTTTCAGATTGTTGATGACCTGTTGGATTACGGTGGAACAAAAGCTACAGGAAAG AACGTTGGTGATGACTTCCGAGAAAGAAAGCTCACCCTCCCTGTTATTCGTGCCGTTGCTGCTGC TGATGCTGATGAAAGAGCTTTTTGGGAACGAACAATTCAAAAAGGAAGACAACAAGATGGAGA CCTGGACCATGCAATTGCCCTTCTTCATAGACACGGAACCCTTGAATCCACACGACAAGACGCA ATCCTTTGGGCCGCTAAAGCTAAAGAAGCACTCGGAGTTATCCCGGACCATGCTCTTAAAACCA TGCTCGTTGACCTTGCTGACTACGTTGTTGCTCGACTCACCTAAACGCGT.

[0302] A NUAMldr-Sp-dpdpS fusion capable of being expressed in Y. lipolytica was constructed by ligating the XbaI-BsmBI NUAMldr fragment together with the BsmBI-MluI Sp-dpdpS fragment into pMB4691 cut with NheI and MluI, to create pMB4799.

[0303] A NUAMldr-Lv-dpdpS fusion capable of being expressed in Y. lipolytica was constructed by ligating the XbaI-BsmBI NUAMldr fragment together with the BsmBI-MluI Lv-dpdpS fragment into pMB4691 cut with NheI and MluI, to create pMB4797.

[0304] A Coq1ldr-Sp-dpdpS fusion capable of being expressed in Y. lipolytica was constructed by ligating the XbaI-BsmBI Coq1ldr fragment together with the BsmBI-MluI Sp-dpdpS fragment into pMB4691 cut with NheI and MluI, to create pMB4798.

[0305] A Coq1ldr-Lv-dpdpS fusion capable of being expressed in Y. lipolytica was constructed by ligating the XbaI-BsmBI Coq1ldr fragment together with the BsmBI-MluI Lv-dpdpS fragment into pMB4691 cut with NheI and MluI, to create pMB4796.

[0306] To effect the disruption of the native COQ1 gene and its replacement by the fusions described above, Y. lipolytica genomic DNA was amplified with two pairs of primers:

TABLE-US-00009 5'- CACACGGTACCGGTATAGGCACAAGTGC [MO4848] with 5'- CACTCGGATCCGTACGTCTGTGGTGCTGTGGT [MO4856], and 5'- CACACGGATCCGCTAGCTCTGAGAAACCTCACCATG [MO4850] with 5'- CACACTCTAGATTTTCTTGGCCATGAACGGT [MO4851],

yielding a fragment of approximately 0.7 kb and 0.85 kb in length, respectively. Fragment MO4848/4856 was cleaved with Acc65I and BamHI, fragment MO4850/4851 was cleaved with BamHI and XbaI, and pBluescriptSKII.sup.- was cleaved with XbaI and Acc65I. These three segments were ligated together to yield plasmid pMB4747.

[0307] The 3.6 (or 3.7) kb Acc65I-XbaI fragments from pMB4796, pMB4797, pMB4798, and pMB4799 containing various tef-dpdpS constructs and the URA3 marker were each ligated to pMB4747 cleaved with BsiWI and NheI. The resulting plasmids, pMB4800 (Coq1ldr-Lv-dpdpS), pMB4801 (NUAMldr-Lv-dpdpS), pMB4802 (Coq1ldr-Sp-dpdpS), pMB4803 (NUAMldr-Sp-dpdpS), are cleaved with Acc65I and XbaI and the 5.1 (or 5.3) kb fragment containing various coq1-tef1p-dpdpS-URA3-coq1 sequences is used to transform Y. lipolytica strain MF454 (MATB ura3 leu2 ade1) to uracil prototrophy. Transformants were checked by PCR for confirmation of homologous integration based gene replacement which confirmed the presence of the heterologous dpdpS and the absence of the wild type Y. lypolytica COQ1 allele. The strains were designated MF975 (MATB ura3 leu2 ade1 coq1.DELTA.NUAMldr-Lv-dpdpS-URA3), MFL976 (MATB ura3 leu2 ade1 coq1.DELTA.NUAMldr-Lv-dpdpS-URA3) as duplicate and MF977 (MATB ura3 leu2 ade1 coq1.DELTA.NUAMldr-Sp-dpdpS-URA3). Strains were analyzed for ubiquinone by TLC and HPLC analysis as described in subsequent examples.

Example 2

Increased CoQ10 Production Resulting from Enhanced PHB-Polyprenyltransferase Activity

[0308] 4-hydroxybenzoate polyprenyl transferases ("phbPPt") based on the amino acid sequence of those found in Silicibacter pomeroyi and in Bos taurus are expressed in Yarrowia lipolytica, using nucleotide sequences with the appropriate Yarrowia codon bias, and in the case of the S. pomeroyi sequence, with Yarrowia mitochondrial targeting sequences.

[0309] The following sequences are synthesized de novo:

TABLE-US-00010 G: Yarrowia lipolytica Coq1 signal sequence. + Silicibacter pomeroyi phbPPt 5'- TTCTAGACACAAAAATGCTGCGAGTGGGCCGAATCGGCACCAAGACCCTGGCCTCTTCTTC TCTGCGATTCGTGGCCGGCGCCCGACCCAAGTCTACCCTGACCGAGGCCGTGCTGGAGAC CACCGGCCTGCTGAAGACCACCCCCCAGAACCCCGAGTGGTCTGGCGCCGTGAAGCAGGC CTCTCGACTGGTGGAGACCGACACCCCCATCCGAGACCCCTTCTCTATCGTGTCTCAGGAG ATGCAGGGCCAGGCCCCCACCCCCGACGGCCAGGTGGCCGACGCCGTGACCGGCAACTGGGTG GACATCCACGCCCCCGCCTGGTCTCGACCCTACCTGCGACTGTCTCGAGCCGACCGACCCATCGG CACCTGGCTGCTGCTGATCCCCTGTTGGTGGGGCCTGGCCCTGGCCATGCTGGACGGCCAGGAC GCCCGATGGGGCGACCTGTGGATCGCCCTGGGCTGTGCCATCGGCGCCTTCCTGATGCGAGGCG CCGGCTGTACCTGGAACGACATCACCGACCGAGAGTTCGACGGCCGAGTGGAGCGAACCCGATC TCGACCCATCCCCTCTGGCCAGGTGTCTGTGCGAATGGCCGTGGTGTGGATGATCGCCCAGGCCC TGCTGGCCCTGATGATCCTGCTGACCTTCAACCGAATGGCCATCGCCATGGGCGTGCTGTCTCTG CTGCCCGTGGCCGTGTACCCCTTCGCCAAGCGATTCACCTGGTGGCCCCAGGTGTTCCTGGGCCT GGCCTTCAACTGGGGCGCCCTGCTGGCCTGGACCGCCCACTCTGGCTCTCTGGGCTGGGGCGCCC TGTTCCTGTACCTGGCCGGCATCGCCTGGACCCTGTTCTACGACACCATCTACGCCCACCAGGAC ACCGAGGACGACGCCCTGATCGGCGTGAAGTCTACCGCCCGACTGTTCGGCGCCCAGACCCCCC GATGGATGTCTTACTTCCTGGTGGCCACCGTGTCTCTGATGGGCATCGCCGTGTTCGAGGCCGCC CTGCCCGACGCCTCTATCCTGGCCCTGGTGCTGGCCCTGGCCGGCCCCTGGGCCATGGGCTGGCA CATGGCCTGGCAGCTGCGAGGCCTGGACCTGGACGACAACGGCAAGCTGCTGCAGCTGTTCCGA GTGAACCGAGACACCGGCCTGATCCCCCTGATCTTCTTCGTGATCGCCCTGTTCGCCTAACGCGT H: Bos taurus phpPPt 5'- TTCTAGACACAAAAATGCTGGGCTCTTGTGGCGCCGGCCTGGTGCGAGGCCTGCGAGCCGAGAC CCAGGCCTGGCTGTGGGGCACCCGAGGCCGATCTCTGGCCCTGGTGCACGCCGCCCGAGGCCTG CACGCCGCCAACTGGCAGCCCTCTCCCGGCCAGGGCCCCCGAGGCCGACCCCTGTCTCTGTCTGC CGCCGCCGTGGTGAACTCTGCCCCCCGACCCCTGCAGCCCTACCTGCGACTGATGCGACTGGAC AAGCCCATCGGCACCTGGCTGCTGTACCTGCCCTGTACCTGGTCTATCGGCCTGGCCGCCGACCC CGGCTGTCTGCCCGACTGGTACATGCTGTCTCTGTTCGGCACCGGCGCCGTGCTGATGCGAGGCG CCGGCTGTACCATCAACGACATGTGGGACCGAGACTACGACAAGAAGGTGACCCGAACCGCCTC TCGACCCATCGCCGCCGGCGACATCTCTACCTTCCGATCTTTCGTGTTCCTGGGCGGCCAGCTGA CCCTGGCCCTGGGCGTGCTGCTGTGTCTGAACTACTACTCTATCGCCCTGGGCGCCGCCTCTCTG CTGCTGGTGACCACCTACCCCCTGATGAAGCGAATCACCTACTGGCCCCAGCTGGCCCTGGGCCT GACCTTCAACTGGGGCGCCCTGCTGGGCTGGTCTGCCGTGAAGGGCTCTTGTGACCCCTCTGTGT GTCTGCCCCTGTACTTCTCTGGCATCATGTGGACCCTGATCTACGACACCATCTACGCCCACCAG GACAAGAAGGACGACGCCCTGATCGGCCTGAAGTCTACCGCCCTGCTGTTCCGAGAGGACACCA AGAAGTGGCTGTCTGGCTTCTCTGTGGCCATGCTGGGCGCCCTGTCTCTGGTGGGCGTGAACTCT GGCCAGACCATGCCCTACTACACCGCCCTGGCCGCCGTGGGCGCCCACCTGGCCCACCAGATCT ACACCCTGGACATCAACCGACCCGAGGACTGTTGGGAGAAGTTCACCTCTAACCGAACCATCGG CCTGATCATCTTCCTGGGCATCGTGCTGGGCAACCTGTGTAAGGCCAAGGAGACCGACAAGACC CGAAAGAACATCGAGAACCGAATGGAGAACTAACGCGT

[0310] The above sequences are cleaved with MluI and XbaI, and inserted into NheI- and MluI-cleaved pMB4603, placing the genes under the control of the Y. lipolytica TEF1 promoter, and adjacent to a functional Y. lipolytica LEU2 gene. These plasmids are designated tef-transS (from sequence G) and tef-transB (from sequence H).

[0311] These plasmids can be used to transform Y. lipolytica strain MF454, ML975, MF976, or MF977 to leucine prototrophy by cleaving with enzymes that lie within plasmid backbone sequences to favor nonhomologous integration events. Transformants can be checked by Southern analysis for confirmation of ectopic integration. Production of CoQ10 can be assessed using HPLC or TLC analysis.

Example 3

Extraction of Ubiquinone from Yarrowia Lipolytica Cells

[0312] Shake-flask testing was conducted using 20 ml cultures containing YPD medium (1% yeast extract, 2% peptone, 2% glucose) in 125 ml flasks grown at 30.degree. C. Y. lipolytica cells were harvested from 24 hour cultures, and extractions are performed to determine ubiquinone form and quantity. Entire culture was pelleted and washed with 50 ml H.sub.2O. Pelleted cells may be frozen at -80.degree. C. and stored. 0.5 ml of cubic zirconium 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.degree. C. The mixture was then spun at maximum speed for 2 minutes, and the supernatant 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 zirconium beads; all supernatants were pooled in the 16 ml glass vial. A Speed Vac was used to concentrate the supernatant (room temperature in dark), and the samples were stored at -20.degree. C. or -80.degree. C. until immediately before TLC and HPLC analysis. Prior to TLC analysis, the samples were resuspended in 50 .mu.l ice-cold solvent and then transferred to a cold amber vial. In an alternative similar protocol, Y. lipolytica cells can be harvested from 72-96 hour 20 ml cultures grown in 125 ml flasks grown at 30.degree. C. 1.8 ml of culture is placed into a pre-weighed tube with a hole poked in the top. Cells are pelleted and washed with 1 ml H.sub.2O. The washing procedure is repeated and then cells are lyophilized overnight. After drying to completion, the tube is weighed in order to calculate dry cell weight. 1 ml from the same shake flask culture is placed into a screw-cap tube for ubiquinone extraction. Cells are pelleted and washed once with 1 ml H.sub.2O and once with 1 M potassium phosphate solution, pH 7.1. Pelleted cells may be frozen at -80.degree. C. and stored. Cubic zirconium beads addition and extraction is performed as describe above. Following extraction, the glass vial is spun for 5 minutes at 2000 rpm at 4.degree. C. in a Sorvall tabletop centrifuge, and the supernatant is transferred to a new cold 16 ml glass vial. A Speed Vac is used to concentrate the supernatant (room temperature in dark), and the samples are stored at -20.degree. C. or -80.degree. C. until immediately before HPLC analysis. Prior to HPLC analysis, the samples are resuspended in 1 ml ice-cold solvent and then transferred to a cold amber vial. Throughout the extraction protocols, care is taken to avoid contact with oxygen, light, heat, and acids.

Example 4a

Purification of Ubiquinones from Crude Extract by Thin Layer Chromatography (TLC)

[0313] Ubiquinone containing samples from the primary extraction in example 3 were spotted to immobilized silica gel plates next to the primary standards (Coenzyme Q9 (Sigma 275970) and Coenzyme Q10 (Sigma 27595)) and resolved in a 80:40:1 hexanes:diethyl ether:acetic acid solvent mixture. Ubiquinone containing bands were identified by UV shadowing and migration with the purified ubiquinone standard and excised from the TLC plate. Samples were extracted from the silica gel with 50 .mu.l acetone followed by mixing, centrifugation, decanting and drying before HPLC analysis.

Example 4b

Quantification of Ubiquinone Production by HPLC

[0314] Ubiquinone-containing samples are 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 .mu.m, 2.1.times.50 mm) and Thermo Basic 8 guard column (2.1.times.10 mm) is used to resolve ubiquinone at 25.degree. C.; authentic ubiquinone samples are used as standards. Ubiquinone molecules were detected at 275 nm (alternatively, they can also be detected at 405 nm), and retention times include CoQ10 (4.272 min) and CoQ9 (4.164 min) The mobile phases and flow rates are shown in Table 45 (Solvent A=Ethyl Acetate; Solvent B=Water; Solvent C=Methanol; Solvent D=Acetonitrile). The injection volume was 10 .mu.L. The detector was a Waters 996 photodiode array detector. A peak corresponding to Coenzyme Q10 was observed in strains MF975, MF976, and MF977.

[0315] The retention times for additional lipophilic molecules include astaxanthin (1.159 min), zeaxanthin (1.335), .beta.-apo-8'-carotenal (2.86 min), ergosterol (3.11 min), lycopene (3.69 min), 13-Carotene (4.02 min), and phytoene (4.13 min) Astaxanthin, zeaxanthin, .beta.-apo-8'-carotenal, lycopene and .beta.-Carotene are detected at 475 nm, whereas ergosterol and phytoene are detected at 286 nm.

TABLE-US-00011 TABLE 45 Mobile Phases and Flow Rates for Ubiquinone Resolution 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

Engineering of PHB Biosynthetic Pathway Genes Results in Increased Para-Hydroxybenzoic Acid Production

[0316] Production of para-hydroxybenzoic acid (PHB) is increased by overexpression of a heterologous 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and a heterologous chorismate lyase (UbiC). Both genes are codon-optimized variants of the Erwinia carotovora coding sequences. The DAHP synthase is based on the amino acid sequence of that found in Erwinia is expressed in Yarrowia lipolytica, using nucleotide sequences with the appropriate Yarrowia codon bias, and containing a P148L substitution. The P 148L substitution can be employed to alleviate tyrosine-mediated allosteric regulation of the Erwinia DAHP synthase. The following sequences are synthesized:

TABLE-US-00012 J: DAHP E. carotovora (optimized) (AroF with P148L substitution) 5'- TTCTAGACACAAAAATGCAGAAGGACTCTCTGAACAACATCAACATCTCTGCCGAGCAGGTGCT GATCACCCCCGACGAGCTGAAGGCCAAGTTCCCCCTGAACGACGTGGAGCAGCGAGACATCGCC CAGGCCCGAGCCACCATCGCCGACATCATCCACGGCCGAGACGACCGACTGCTGATCGTGTGTG GCCCCTGTTCTATCCACGACACCGACGCCGCCCTGGAGTACGCCCGACGACTGCAGCTGCTGGC CGCCGAGCTGAACGACCGACTGTACATCGTGATGCGAGTGTACTTCGAGAAGCCCCGAACCACC GTGGGCTGGAAGGGCCTGATCAACGACCCCTTCATGGACGGCTCTTTCGACGTGGAGTCTGGCC TGCACATCGCCCGAGGCCTGCTGCTGCAGCTGGTGAACATGGGCCTGCCCCTGGCCACCGAGGC CCTGGACCTGAACTCTCCCCAGTACCTGGGCGACCTGTTCTCTTGGTCTGCCATCGGCGCCCGAA CCACCGAGTCTCAGACCCACCGAGAGATGGCCTCTGGCCTGTCTATGCCCGTGGGCTTCAAGAA CGGCACCGACGGCTCTCTGGGCACCGCCATCAACGCCATGCGAGCCGCCGCCATGCCCCACCGA TTCGTGGGCATCAACCAGACCGGCCAGGTGTGTCTGCTGCAGACCCAGGGCAACGGCGACGGCC ACGTGATCCTGCGAGGCGGCAAGACCCCCAACTACTCTGCCCAGGACGTGGCCGAGTGTGAGAA GCAGATGCAGGAGGCCGGCCTGCGACCCGCCCTGATGATCGACTGTTCTCACGGCAACTCTAAC AAGGACTACCGACGACAGCCCCTGGTGGTGGAGTCTGCCATCGAGCAGATCAAGGCCGGCAAC CGATCTATCATCGGCCTGATGCTGGAGTCTCACCTGAACGAGGGCTCTCAGTCTTCTGAGCAGCC CCGATCTGACATGCGATACGGCGTGTCTGTGACCGACGCCTGTATCTCTTGGGAGTCTACCGAGA CCCTGCTGCGATCTGTGCACCAGGACCTGTCTGCCGCCCGAGTGAAGCACTCTGGCGAGTAACG CGT K: Chorismate lyase E. carotovora (optimized) (UbiC) 5'- TTCTAGACACAAAAATGTCTGACGACGCCTCTACCCTGCTGCGAACCATCTCTTGGTTCACCGAG CCCCCCTCTGTGCTGCCCGAGCACATCGGCGACTGGCTGATGGAGACCTCTTCTATGACCCAGCG ACTGGAGAAGTACTGTGCCCAGCTGCGAGTGACCCTGTGTCGAGAGGGCTTCATCACCCCCCAG ATGCTGGGCGAGGAGCGAGACCAGCTGCCCGCCGACGAGCGATACTGGCTGCGAGAGGTGGTG CTGTACGGCGACGACCGACCCTGGCTGTTCGGCCGAACCATCGTGCCCCAGCAGACCCTGGAGG GCTCTGGCGCCGCCCTGACCAAGATCGGCAACCAGCCCCTGGGCCGATACCTGTTCGAGCAGAA GTCTCTGACCCGAGACTACATCCACACCGGCTGTTGTGAGGGCCTGTGGGCCCGACGATCTCGA CTGTGTCTGTCTGGCCACCCCCTGCTGCTGACCGAGCTGTTCCTGCCCGAGTCTCCCGTGTACTA CACCCCCGGCGACGAGGGCTGGCAGGTGATCTAACGCGT

[0317] The above sequences are cleaved with MluI and XbaI. Sequence J is inserted into NheI- and MluI-cleaved pMB4603, and sequence K is inserted into NheI- and MluI-cleaved pMB4691, placing the genes under the control of the Y. lipolytica TEF1 promoter, and adjacent to a functional Y. lipolytica LEU2 (J) or URA3 (K) gene. These plasmids are designated tef-aroF P148L (from sequence J) and tef-ubiC (from sequence K). They can be integrated sequentially, by cleavage of tef-aroF P148L with SspI and of tef-ubiC with SalI, into the genome of Y. lipolytica strain ATCC201249 (MATA ura3 leu2 lys8), and screened for confirmation of homologous integration as well as for increased PHB and ubiquinone production.

Example 6

Production of CoQ10 in Yarrowia lipolytica Strains Harboring Multiple Exogenous Ubiquinogenic pathway genes

[0318] The PHB overproducing strain produced in Example 5 is mated with CoQ10-producing strains described in Example 1 or Example 2, and diploids are selected on minimal medium. Upon meiosis and sporulation, haploid recombinants may be selected that harbor all four heterologous genes: AroF P148L, ubiC, dpdpS, and phbPPt. Genotypes of selected segregants can be confirmed by Southern blotting and polymerase chain reaction (PCR) analysis. Furthermore, increased production of CoQ10 in the selected recombinants is assessed as described in prior examples. In addition, segregants can be chosen that also harbor an ade1 auxotrophy to facilitate further manipulation.

Example 7

Decreasing Carbon Flow to the Competing Sterol Pathway Results in Enhanced Ubiquinone Production

[0319] 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 Fol.sup.+ transformants are screened for decreased squalene synthase activity.

[0320] The following oligonucleotides are synthesized:

TABLE-US-00013 5'-CCTTCTAGTCGTACGTAGTCAGC [L]; 5'-CCACTGATCTAGAATCTCTTTCTGG [M].

[0321] They are used to amplify a 2.3 kb fragment from Y. lipolytica genomic DNA spanning most of the FOL3 gene, using Pfu polymerase. The fragment is cleaved with XbaI and phosphorylated, and ligated into pBluescriptSK.sup.- (GenBank accession number X52330) 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 BS-fol3, is then cleaved with Acc65I and EcoRI, treated with T4pol as above, and ligated to the 3.4 kb EcoRV-SpeI ADE1 fragment (treated with T4pol) from pMB4529.

[0322] The resulting plasmid, pBSfol3.DELTA.ade, can be cleaved with BsiWI and XbaI to liberate a 5.5 kb fragment that is used to transform the ade1 CoQ10-overproducing strains described above to adenine prototrophy. Ade.sup.+ transformants are screened for a folinic acid requirement, and for homologous integration by PCR analysis.

[0323] Strains that harbor the resultant fol3.DELTA.ADE1 allele can be transformed with a 3.5 kb DNA fragment generated by mutagenic PCR amplification using the primers:

TABLE-US-00014 5'-GGCTCATTGCGCATGCTAACATCG [N]; 5'-CGACGATGCTATGAGCTTCTAGACG [P],

and Y. lipolytica genomic DNA as template. This fragment contains the N-terminal three-quarters of the FOL3 ORF and the C-terminal nine-tenths of the ERG9 ORF. Fol.sup.+ Ade.sup.- transformants are screened for decreased squalene synthase activity by sensitivity to agents such as zaragozic acid, itraconazole, or fluconazole, or by resistance to reactive oxygen species-generating agents such as antimycin.

[0324] In addition, the above fragment could be cloned, and altered in such a way as to remove the 3'-untranslated region of ERG9. Replacement of the fol3.DELTA.ADE1 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 described above. This approach may also be used in a Fol.sup.+ Ade.sup.- strain, using the ADE1 marker to disrupt the ERG9 3'-UTR.

[0325] Partially defective ERG9 alleles can also 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

Expression of a Truncated Form of HMG-CoA Reductase to Elevate CoQ10 Production

[0326] In order to further increase CoQ10 production, carbon flow through the isoprenoid pathway is enhanced by introducing a truncated variant of the Y. lipolytica HMG-CoA reductase gene, which also encodes a 77 amino acid leader sequence derived from S. cerevisiae Hmg1.

[0327] The following oligonucleotides are synthesized:

TABLE-US-00015 5'-TTCTAGACACAAAAATGGCTGCAGACCAATTGGTGA [Q]; 5'-CATTAATTCTTCTAAAGGACGTATTTTCTTATC [R]; 5'-GTTCTCTGGACGACCTAGAGG [S]; 5'-CACACACGCGTACACCTATGACCGTATGCAAAT [T].

Q and R 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. S and T 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 pQR and pST, are verified by sequencing. The QR fragment is liberated with XbaI and AseI, and the ST fragment is liberated with MaeI and MluI. These fragments are then ligated to the ADE1 tef1p expression vector pMB4629 cut with NheI and MluI.

[0328] The resulting plasmid, pTefHMG, can be cleaved with SnaBI, BbvCI, or Bsu36I to direct integration at the ade1 locus, or with BamHI to direct integration at the HMG1 locus, of the CoQ10-producing strains described above, restoring them to adenine prototrophy. Ade transformants are screened for increased CoQ10 production.

[0329] Alternatively, the native HMG1 gene from Y. lipolytica may be modified through truncation, and without S. cerevisiae sequences, by amplifying Y. lipolytica genomic DNA with primer T above and

TABLE-US-00016 T2 5'-CACACTCTAGACACAAAAATGACCCAGTCTGTGAAGGTGG

[0330] yielding a 1.5 kb fragment that is cleaved with XbaI and MluI and ligated to pMB4629 cut with NheI and MluI to create pMB4637. pMB4637 may be cut with SnaBI, Bsu36I, or BbvCI to direct integration at the ade1 locus, or with BamHI to direct integration at the HMG1 locus, of the CoQ10-producing strains described above, restoring them to adenine prototrophy. Ade transformants are screened for increased CoQ10 production.

Example 9

Constructing an Oleaginous Strain of Saccharomyces cerevisiae

[0331] 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.

[0332] 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.

TABLE-US-00017 acl1: 1fwd: 5' CACACGGATCCTATAatgccttccgcaacgaccg 1rev: 5' CACACACTAGttaaatttggacctcaacacgaccc acl2: 2fwd: 5' CACACGGATCCAATATAAatgtctgcgaagagcatcctcg 2rev: 5' CACACGCATGCttaagcttggaactccaccgcac

[0333] 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.

TABLE-US-00018 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 "p1gal2" and "p2gal1", containing GALL-acl1/GAL10-acl2 and GAL10-acl1/GAL1-acl2, respectively (Genbank accession: acl1: CAB91740.2; acl2: CAB91741.2).

[0334] 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:

TABLE-US-00019 AMD1 ORF: AMD1FWD: 5' CACACGAGCTCAAAAatggacaatcaggctacacagag AMD1rev: 5' CACACCCTAGGtcacttttcttcaatggttctcttgaaattg GAL7p: gal7prox: 5' CACACGAGCTCggaatattcaactgtttttttttatcatgttgatg gal7dist: 5' CACACGGAtccttcttgaaaatatgcactctatatcttttag,

The resulting fragment from the AMD1 reaction (2.4 kb) is cleaved with Sad 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, AMD1, encoding AMP deaminase, under the control of the galactose-inducible GAL7 promoter.

[0335] 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:

TABLE-US-00020 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.sup.+-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.

[0336] The plasmids "p1gal2", "pAMPD", and "pTEFMAE" are sequentially transformed into a strain of S. cereviseae to restore prototrophy for uracil ("p1gal2"), 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 (e.g., 20 ml cultures in 125 ml flasks grown at 30.degree. C. for 72-96 hour cultures) in either synthetic complete (SC) medium lacking uracil, leucine and histidine 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 NH.sub.4C1, 7 g/L KH.sub.2PO.sub.4, 5 g/L Na.sub.2HPO.sub.4-12H.sub.2O, 1.5 g/L MgSO.sub.4.7H.sub.2O, 0.08 g/L FeCl.sub.3-6H.sub.2O, 0.01 g/L ZnSO.sub.4.7H.sub.2O, 0.1 g/L CaCl.sub.2-2H.sub.2O, 0.1 mg/L MnSO.sub.4.5H.sub.2O, 0.1 mg/L CuSO.sub.4.5H.sub.2O, 0.1 mg/L Co(NO.sub.3).sub.2-6H.sub.2O; pH 5.5 (J Am Oil Chem Soc 70:891-894 (1993)).

[0337] 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). Yeast strains expressing multiple oleaginic polypeptides produce elevated total lipid (for example, in the range of 17% and 25% dry cell weight basis) following growth in YPD and lipid accumulation medium when compared to non-transformed S. cerevisiae strains which may, for example, produce in the range of 6% and 10% total lipid after growth in YPD and lipid accumulation medium.

Example 10

Y. lipolytica Oleaginic and Isoprenoid Biosynthesis Genes

[0338] FIG. 11 is a list of Y. lipolytica genes representing various polypeptides (e.g. oleaginic and isoprenoid biosynthesis peptides) useful in the fungal strains and methods described herein. The Genbank accession number and GI number is given for each polypeptide in addition to oligo pairs which can be used to amplify the coding region for each gene from Y. lipolytica genomic DNA or cDNA. Resulting PCR fragments can be cleaved with restriction enzyme pairs (e.g. depending on what site is present within the oligo sequence, XbaI/MluI or NheI/MluI or XbaI/AscI or NheI/AscI) and inserted into expression vectors (e.g. fungal expression vectors including Y. lipolytica expression vectors such as MB4629 and MB4691 described herein).

[0339] The DNA and proteins they encode of the Y. lipolytica genes represented in FIG. 11 are as follows (intron sequence is underlined):

TABLE-US-00021 YALI0F30481g DNA: atgtcgcaaccccagaacgttggaatcaaagccctcgagatctacgtgccttctcgaattgtcaaccaggctga- gc tcgagaagcacgacggtgtcgctgctggcaagtacaccattggtcttggtcagaccaacatggcctttgtcgac- ga cagagaggacatctattcctttgccctgaccgccgtctctcgactgctcaagaacaacaacatcgaccctgcat- ct attggtcgaatcgaggttggtactgaaacccttctggacaagtccaagtccgtcaagtctgtgctcatgcagct- ct ttggcgagaacagcaacattgagggtgtggacaacgtcaacgcctgctacggaggaaccaacgccctgttcaac- gc tatcaactgggttgagggtcgatcttgggacggccgaaacgccatcgtcgttgccggtgacattgccctctacg- ca aagggcgctgcccgacccaccggaggtgccggctgtgttgccatgctcattggccccgacgctcccctggttct- tg acaacgtccacggatcttacttcgagcatgcctacgatttctacaagcctgatctgacctccgagtacccctat- gt tgatggccactactccctgacctgttacacaaaggccctcgacaaggcctacgctgcctacaacgcccgagccg- ag aaggtcggtctgttcaaggactccgacaagaagggtgctgaccgatttgactactctgccttccacgtgcccac- ct gcaagcttgtcaccaagtcttacgctcgacttctctacaacgactacctcaacgacaagagcctgtacgagggc- ca ggtccccgaggaggttgctgccgtctcctacgatgcctctctcaccgacaagaccgtcgagaagaccttccttg- gt attgccaaggctcagtccgccgagcgaatggctccttctctccagggacccaccaacaccggtaacatgtacac- cg cctctgtgtacgcttctctcatctctctgctgacttttgtccccgctgagcagctgcagggcaagcgaatctct- ct cttctcttacggatctggtcttgcttccactcttttctctctgaccgtcaagggagacatttctcccatcgtca- ag gcctgcgacttcaaggctaagctcgatgaccgatccaccgagactcccgtcgactacgaggctgccaccgatct- cc gagagaaggcccacctcaagaagaactttgagccccagggagacatcaagcacatcaagtctggcgtctactac- ct caccaacatcgatgacatgttccgacgaaagtacgagatcaagcagtag Protein: Msqpqnvgikaleiyvpsrivnqaelekhdgvaagkytiglgqtnmafvddrediysfaltavsrllknnnidp- as igrievgtetlldksksyksylmqlfgensniegvdnvnacyggtnalfnainwvegrswdgrnaivvagdial- ya kgaarptggagcvamligpdaplvldnvhgsyfehaydfykpdltseypyvdghysltcytkaldkayaaynar- ae kvglfkdsdkkgadrfdysafhvptcklvtksyarllyndylndkslyegqvpeevaavsydasltdktvektf- lg iakaqsaermapslqgptntgnmytasvyaslislltfvpaeqlqgkrislfsygsglastlfsltvkgdispi- vk acdfkaklddrstetpvdyeaatdlrekahlkknfepqgdikhiksgvyyltniddmfrrkyeikq YALI0B16038g DNA: atggactacatcatttcggcgccaggcaaagtgattctatttggtgaacatgccgctgtgtttggtaagcctgc- ga ttgcagcagccatcgacttgcgaacatacctgcttgtcgaaaccacaacatccgacaccccgacagtcacgttg- ga gtttccagacatccacttgaacttcaaggtccaggtggacaagctggcatctctcacagcccagaccaaggccg- ac catctcaattggtcgactcccaaaactctggataagcacattttcgacagcttgtctagcttggcgcttctgga- ag aacctgggctcactaaggtccagcaggccgctgttgtgtcgttcttgtacctctacatccacctatgtccccct- tc tgtgtgcgaagattcatcaaactgggtagttcgatcaacgctgcctatcggcgcgggcctgggctcttccgcat- cc atttgtgtctgtttggctgcaggtcttctggttctcaacggccagctgagcattgaccaggcaagagatttcaa- gt ccctgaccgagaagcagctgtctctggtggacgactggtccttcgtcggtgaaatgtgcattcacggcaacccg- tc gggcatcgacaatgctgtggctactcagggaggtgctctgttgttccagcgacctaacaaccgagtccctcttg- tt gacattcccgagatgaagctgctgcttaccaatacgaagcatcctcgatctaccgcagacctggttggtggagt- cg gagttctcactaaagagtttggctccatcatggatcccatcatgacttcagtaggcgagatttccaaccaggcc- at ggagatcatttctagaggcaagaagatggtggaccagtctaaccttgagattgagcagggtatcttgcctcaac- cc acctctgaggatgcctgcaacgtgatggaagatggagctactcttcaaaagttgagagatatcggttcggaaat- gc agcatctagtgagaatcaatcacggcctgcttatcgctatgggtgtttcccacccgaagctcgaaatcattcga- ac tgcctccattgtccacaacctgggtgagaccaagctcactggtgctggaggaggaggttgcgccatcactctag- tc acttctaaagacaagactgcgacccagctggaggaaaatgtcattgctttcacagaggagatggctacccatgg- ct tcgaggtgcacgagactactattggtgccagaggagttggtatgtgcattgaccatccctctctcaagactgtt- ga agccttcaagaaggtggagcgggcggatctcaaaaacatcggtccctggacccattag Protein: mdyiisapgkvilfgehaavfgkpaiaaaidlrtyllvetttsdtptvtlefpdihlnfkvqvdklasltaqtk- ad hlnwstpktldkhifdslsslalleepgltkvqqaavvsflylyihlcppsvcedssnwvvrstlpigaglgss- as icvclaagllvlngqlsidqardfksltekqlslvddwsfygemcihgnpsgidnavatqggallfqrpnnrvp- lv dipemkllltntkhprstadlvggvgvltkefgsimdpimtsvgeisnqameiisrgkkmvdqsnleieqgilp- qp tsedacnvmedgatlqklrdigsemqhlvrinhglliamgvshpkleiirtasivhnlgetkltgaggggcait- lv tskdktatqleenviafteemathgfevhettigargvgmcidhpslktveafkkveradlknigpwth YALI0E06193g DNA: atgaccacctattcggctccgggaaaggccctcctttgcggcggttatttggttattgatccggcgtattcagc- at acgtcgtgggcctctcggcgcgtatttacgcgacagtttcggcttccgaggcctccaccacctctgtccatgtc- gt ctctccgcagtttgacaagggtgaatggacctacaactacacgaacggccagctgacggccatcggacacaacc- ca tttgctcacgcggccgtcaacaccgttctgcattacgttcctcctcgaaacctccacatcaacatcagcatcaa- aa gtgacaacgcgtaccactcgcaaattgacagcacgcagagaggccagtttgcataccacaaaaaggcgatccac- ga ggtgcctaaaacgggcctcggtagctccgctgctcttaccaccgttcttgtggcagctttgctcaagtcatacg- gc attgatcccttgcataacacccacctcgttcacaacctgtcccaggttgcacactgctcggcacagaagaagat- tg ggtctggatttgacgtggcttcggccgtttgtggctctctagtctatagacgtttcccggcggagtccgtgaac- at ggtcattgcagctgaagggacctccgaatacggggctctgttgagaactaccgttaatcaaaagtggaaggtga- ct ctggaaccatccttcttgccgccgggaatcagcctgcttatgggagacgtccagggaggatctgagactccagg- ta tggtggccaaggtgatggcatggcgaaaagcaaagccccgagaagccgagatggtgtggagagatctcaacgct- gc caacatgctcatggtcaagttgttcaacgacctgcgcaagctctctctcactaacaacgaggcctacgaacaac- tt ttggccgaggctgctcctctcaacgctctaaagatgataatgttgcagaaccctctcggagaactagcacgatg- ca ttatcactattcgaaagcatctcaagaagatgacacgggagactggtgctgctattgagccggatgagcagtct- gc attgctcaacaagtgcaacacttatagtggagtcattggaggtgttgtgcctggagcaggaggctacgatgcta- tt tctcttctggtgatcagctctacggtgaacaatgtcaagcgagagagccagggagtccaatggatggagctcaa- gg aggagaacgagggtctgcggctcgagaaggggttcaagtag Protein: mttysapgkallcggylvidpaysayvvglsariyatvsaeseasttsvhvvspqfdkgewtynytngqltaig- hn pfahaavntvlhyvpprnlhinisiksdnayhsqidstqrgqfayhkkaihcvpktglgssaalttvlvaallk- sy gidplhnthlvhnlsqvahcsaqkkigsgfdvasavcgslvyrrfpaesvnmviaaegtseygallrttvnqkw- kv tlepsflppgisllmgdvqggsetpgmvakvmawrkakpreaemvwrdlnaanmlmvklfndlrklsltnneay- eq llaeaaplnalkmimlqnplgelarciitirkhlkkmtretgaaiepdeqsallnkcntysgviggvvpgaggy- da isllvisstvnnvkresqgvqwmelkeeneglrlekgfk YALI0F05632g DNA: atgatccaccaggcctccaccaccgctccggtgaacattgcgacactcaagtactggggcaagcgagaccctgc- tc tcaatctgcccactaacaactccatctcctgtgactttgtcgcaggatgatctgcggaccctcaccacagcctc- gt gttcccctgatttcacccaggacgagctgtggctcaatggcaagcaggaggacgtgagcggcaaacgtctggtt- gc gtgtttccgagagctgcgggctctgcgacacaaaatggaggactccgactcttctctgcctaagctggccgatc- ag aagctcaagatcgtgtccgagaacaacttccccaccgccgctggtctcgcctcatcggctgctggctttgccgc- cc tgatccgagccgttgcaaatctctacgagctccaggagacccccgagcagctgtccattgtggctcgacagggc- tc tggatccgcctgtcgatctctctacggaggctacgtggcatgggaaatgggcaccgagtctgacggaagcgact- cg cgagcggtccagatcgccaccgccgaccactggcccgagatgcgagccgccatcctcgttgtctctgccgacaa- ga aggacacgtcgtccactaccggtatgcaggtgactgtgcacacttctcccctcttcaaggagcgagtcaccact- gt ggttcccgagcggtttgcccagatgaagaagtcgattctggaccgagacttccccacctttgccgagctcacca- tg cgagactcaaaccagttccacgccacctgtctggactcgtatcctcccattttctacctcaacgacgtgtcgcg- ag

cctccattcgggtagttgaggccatcaacaaggctgccggagccaccattgccgcctacacctttgatgctgga- cc caactgtgtcatctactacgaggacaagaacgaggagctggttctgggtgctctcaaggccattctgggccgtg- tg gagggatgggagaagcaccagtctgtggacgccaagaagattgatgttgacgagcggtgggagtccgagctggc- ca acggaattcagcgggtgatccttaccaaggttggaggagatcccgtgaagaccgctgagtcgcttatcaacgag- ga tggttctctgaagaacagcaagtag Protein: mihqasttapvniatlkywgkrdpalnlptnnsisvtlsqddlrtlttascspdftqdelwlngkqedvsgkrl- va cfrelralrhkmedsdsslpkladqklkivsennfptaaglassaagfaaliravanlyelqetpeqlsivarq- gs gsacrslyggyvawemgtesdgsdsravqiatadhwpemraailvvsadkkdtssttgmqvtvhtsplfkervt- tv vperfaqmkksildrdfptfaeltmrdsnqfhatcldsyppifylndvsrasirvveainkaagatiaaytfda- gp ncviyyedkneelvlgalkailgrvegwekhqsvdakkidvderweselangiqrviltkvggdpvktaeslin- ed gslknsk YALI0F04015 DNA: Atgacgacgtcttacagcgacaaaatcaagagtatcagcgtgagctctgtggctcagcagtttcctgaggtggc- gc cgattgcggacgtgtccaaggctagccggcccagcacggagtcgtcggactcgtcggccaagctatttgatggc- ca cgacgaggagcagatcaagctgatggacgagatctgtgtggtgctggactgggacgacaagccgattggcggcg- cg tccaaaaagtgtctgtcatctgatggacaacatcaacgacggactggtgcatcgggccttttccgtgttcatgt- tc aacgaccgcggtgagctgcttctgcagcagcgggcggcggaaaaaatcacctttgccaacatgtggaccaacac- gt gctgctcgcatcctctggcggtgcccagcgagatgggcgggctggatctggagtcccggatccagggcgccaaa- aa cgccgcggtccggaagcttgagcacgagctgggaatcgaccccaaggccgttccggcagacaagttccatttcc- tc acccggatccactacgccgcgccctcctcgggcccctggggcgagcacgagattgactacattctgtttgtccg- gg gcgaccccgagctcaaggtggtggccaacgaggtccgcgataccgtgtgggtgtcgcagcagggactcaaggac- at gatggccgatcccaagctggttttcaccccttggttccggctcatttgtgagcaggcgctgtttccctggtggg- ac cagttggacaatctgcccgcgggcgatgacgagattcggcggtggatcaagtag Protein: mttsysdkiksisvssvaqqfpevapiadvskasrpstessdssaklfdghdeeqiklmdeicvvldwddkpig- ga skkcchlmdnindglvhrafsvfmfndrgelllqqraaekitfanmwtntccshplavpsemggldlesriqga- kn aavrklehelgidpkavpadkfhfltrihyaapssgpwgeheidyilfvrgdpelkvvanevrdtvwvsqqglk- dm madpklvftpwfrliceqalfpwwdqldnlpagddeirrwik YALI0E05753 DNA: atgtccaaggcgaaattcgaaagcgtgttcccccgaatctccgaggagctggtgcagctgctgcgagacgaggg- tc tgccccaggatgccgtgcagtggttttccgactcacttcagtacaactgtgtgggtggaaagctcaaccgaggc- ct gtctgtggtcgacacctaccagctactgaccggcaagaaggagctcgatgacgaggagtactaccgactcgcgc- tg ctcggctggctgattgagctgctgcaggcgtttttcctcgtgtcggacgacattatggatgagtccaagacccg- ac gaggccagccctgctggtacctcaagcccaaggtcggcatgattgccatcaacgatgctttcatgctagagagt- gg catctacattctgcttaagaagcatttccgacaggagaagtactacattgaccttgtcgagctgttccacgaca- tt tcgttcaagaccgagctgggccagctggtggatcttctgactgcccccgaggatgaggttgatctcaaccggtt- ct ctctggacaagcactcctttattgtgcgatacaagactgcttactactccttctacctgcccgttgttctagcc- at gtacgtggccggcattaccaaccccaaggacctgcagcaggccatggatgtgctgatccctctcggagagtact- tc caggtccaggacgactaccttgacaactttggagaccccgagttcattggtaagatcggcaccgacatccagga- ca acaagtgctcctggctcgttaacaaagcccttcagaaggccacccccgagcagcgacagatcctcgaggacaac- ta cggcgtcaaggacaagtccaaggagctcgtcatcaagaaactgtatgatgacatgaagattgagcaggactacc- tt gactacgaggaggaggttgttggcgacatcaagaagaagatcgagcaggttgacgagagccgaggcttcaagaa- gg aggtgctcaacgctttcctcgccaagatttacaagcgacagaagtag Protein: mskakfesvfpriseelvqllrdeglpqdavqwfsdslqyncvggklnrglsvvdtyqlltgkkelddeeyyrl- al lgwliellqafflvsddimdesktrrgqpcwylkpkvgmiaindafmlesgiyillkkhfrqekyyidlvelfh- di sfktelgqlvdlltapedevdlnrfsldkhsfivryktayysfylpvvlamyvagitnpkdlqqamdvliplge- yf qvqddyldnfgdpefigkigtdiqdnkcswlvnkalqkatpeqrqilednygvkdkskelvikklyddmkieqd- yl dyeeevvgdikkkieqvdesrgfkkevlnaflakiykrqk YALI0E18634g DNA: atgttacgactacgaaccatgcgacccacacagaccagcgtcagggcggcgcttgggcccaccgccgcggcccg- aa acatgtcctcctccagcccctccagcttcgaatactcgtcctacgtcaagggcacgcgggaaatcggccaccga- aa ggcgcccacaacccgtctgtcggttgagggccccatctacgtgggcttcgacggcattcgtcttctcaacctgc- cg catctcaacaagggctcgggattccccctcaacgagcgacgggaattcagactcagtggtcttctgccctctgc- cg aagccaccctggaggaacaggtcgaccgagcataccaacaattcaaaaagtgtggcactcccttagccaaaaac- gg gttctgcacctcgctcaagttccaaaacgaggtgctctactacgccctgctgctcaagcacgttaaggaggtct- tc cccatcatctatacaccgactcagggagaagccattgaacagtactcgcggctgttccggcggcccgaaggctg- ct tcctcgacatcaccagtccctacgacgtggaggagcgtctgggagcgtttggagaccatgacgacattgactac- at tgtcgtgactgactccgagggtattctcggaattggagaccaaggagtgggcggtattggtatttccatcgcca- ag ctggctctcatgactctatgtgctggagtcaacccctcacgagtcattcctgtggttctggatacgggaaccaa- ca accaggagctgctgcacgaccccctgtatctcggccgacgaatgccccgagtgcgaggaaagcagtacgacgac- tt catcgacaactttgtgcagtctgcccgaaggctgtatcccaaggcggtgatccatttcgaggactttgggctcg- ct aacgcacacaagatcctcgacaagtatcgaccggagatcccctgcttcaacgacgacatccagggcactggagc- cg tcactttggcctccatcacggccgctctcaaggtgctgggcaaaaatatcacagatactcgaattctcgtgtac- gg agctggttcggccggcatgggtattgctgaacaggtctatgataacctggttgcccagggtctcgacgacaaga- ct gcgcgacaaaacatctttctcatggaccgaccgggtctactgaccaccgcacttaccgacgagcagatgagcga- cg tgcagaagccgtttgccaaggacaaggccaattacgagggagtggacaccaagactctggagcacgtggttgct- gc cgtcaagccccatattctcattggatgttccactcagcccggcgcctttaacgagaaggtcgtcaaggagatgc- tc aaacacacccctcgacccatcattctccctctttccaaccccacacgtcttcatgaggctgtccctgcagatct- gt acaagtggaccgacggcaaggctctggttgccaccggctcgccctttgacccagtcaacggcaaggagacgtct- ga gaacaataactgctttgttttccccggaatcgggctgggagccattctgtctcgatcaaagctcatcaccaaca- cc atgattgctgctgccatcgagtgcctcgccgaacaggcccccattctcaagaaccacgacgagggagtacttcc- cg acgtagctctcatccagatcatttcggcccgggtggccactgccgtggttcttcaggccaaggctgagggccta- gc cactgtcgaggaagagctcaagcccggcaccaaggaacatgtgcagattcccgacaactttgacgagtgtctcg- cc tgggtcgagactcagatgtggcggcccgtctaccggcctctcatccatgtgcgggattacgactag Protein: mlrlrtmrptqtsvraalgptaaarnmsssspssfeyssyvkgtreighrkapttrlsvegpiyvgfdgirlln- lp hlnkgsgfplnerrefrlsgllpsaeatleeqvdrayqqfkkcgtplakngfctslkfqnevlyyalllkhvke- vf piiytptqgeaieqysrlfrrpegcflditspydveerlgafgdhddidyivvtdsegilgigdqgvggigisi- ak lalmtlcagvnpsrvipvvldtgtnnqellhdplylgrrmprvrgkqyddfidnfvqsarrlypkavihfedfg- la nahkildkyrpeipcfnddiqgtgavtlasitaalkvlgknitdtrilvygagsagmgiaeqvydnlvaqgldd- kt arqniflmdrpgllttaltdeqmsdvqkpfakdkanyegvdtktlehvvaavkphiligcstqpgafnekvvke- ml khtprpiilplsnptrlheavpadlykwtdgkalvatgspfdpvngketsennncfvfpgiglgailsrsklit- nt miaaaieclaeqapilknhdegvlpdvaliqiisarvatavvlqakaeglatveeelkpgtkehvqipdnfdec- la wvetqmwrpvyrplihvrdyd YALI0E11495g DNA: atgccgcagcaagcaatggatatcaagggcaaggccaagtctgtgcccatgcccgaagaagacgacctggactc- gc attttgtgggtcccatctctccccgacctcacggagcagacgagattgctggctacgtgggctgcgaagacgac- ga agacgagcttgaagaactgggaatgctgggccgatctgcgtccacccacttctcttacgcggaagaacgccacc- tc

atcgaggttgatgccaagtacagagctcttcatggccatctgcctcatcagcactctcagagtcccgtgtccag- at cttcgtcatttgtgcgggccgaaatgaaccacccccctcccccaccctccagccacacccaccaacagccagag- ga cgatgacgcatcttccactcgatctcgatcgtcgtctcgagcttctggacgcaagttcaacagaaacagaacca- ag tctggatcttcgctgagcaagggtctccagcagctcaacatgaccggatcgctcgaagaagagccctacgagag- cg atgacgatgcccgactatctgcggaagacgacattgtctatgatgctacccagaaagacacctgcaagcccata- tc tcctactctcaaacgcacccgcaccaaggacgacatgaagaacatgtccatcaacgacgtcaaaatcaccacca- cc acagaagatcctcttgtggcccaggagctgtccatgatgttcgaaaaggtgcagtactgccgagacctccgaga- ca agtaccaaaccgtgtcgctacagaaggacggagacaaccccaaggatgacaagacacactggaaaatttacccc- ga gcctccaccaccctcctggcacgagaccgaaaagcgattccgaggctcgtccaaaaaggagcaccaaaagaaag- ac ccgacaatggatgaattcaaattcgaggactgcgaaatccccggacccaacgacatggtcttcaagcgagatcc- ta cctgtgtctatcaggtctatgaggatgaaagctctctcaacgaaaataagccgtttgttgccatcccctcaatc- cg agattactacatggatctggaggatctcattgtggcttcgtctgacggacctgccaagtcttttgctttccgac- ga ctgcaatatctagaagccaagtggaacctctactacctgctcaacgagtacacggagacaaccgagtccaagac- ca acccccatcgagacttttacaacgtacgaaaggtcgacacccacgttcaccactctgcctgcatgaaccagaag- ca tctgctgcgattcatcaaatacaagatgaagaactgccctgatgaagttgtcatccaccgagacggtcgggagc- tg acactctcccaggtgtttgagtcacttaacttgactgcctacgacctgtctatcgatacccttgatatgcatgc- tc acaaggactcgttccatcgatttgacaagttcaacctcaagtacaaccctgtcggtgagtctcgactgcgagaa- at cttcctaaagaccgacaactacatccagggtcgatacctagctgagatcacaaaggaggtgttccaggatctcg- ag aactcgaagtaccagatggcggagtaccgtatttccatctacggtcggtccaaggacgagtgggacaagctggc- tg cctgggtgctggacaacaaactgttttcgcccaatgttcggtggttgatccaggtgcctcgactgtacgacatt- ta caagaaggctggtctggttaacacctttgccgacattgtgcagaacgtctttgagcctcttttcgaggtcacca- ag gatcccagtacccatcccaagctgcacgtgttcctgcagcgagttgtgggctttgactctgtcgatgacgagtc- ga agctggaccgacgtttccaccgaaagttcccaactgcagcatactgggacagcgcacagaaccctccctactcg- ta ctggcagtactatctatacgccaacatggcctccatcaacacctggagacagcgtttgggctataatacttttg- ag ttgcgaccccatgctggagaggctggtgacccagagcatcttctgtgcacttatctggttgctcagggtatcaa- cc acggtattctgttgcgaaaggtgcccttcattcagtacctttactacctggaccagatccccattgccatgtct- cc tgtgtccaacaatgcgctgttcctcacgttcgacaagaaccccttctactcatacttcaagcggggtctcaacg- tg tccttgtcatcggatgatcctctgcagtttgcttacactaaggaggctctgattgaggagtactctgtggctgc- gc tcatttacaagctttccaacgtggatatgtgtgagcttgctcgaaactcggtactgcaatctggctttgagcga- at catcaaggagcattggatcggcgaaaactacgagatccatggccccgagggcaacaccatccagaagacaaacg- tg cccaatgtgcgtctggccttccgagacgagactttgacccacgagcttgctctggtggacaagtacaccaatct- tg aggagtttgagcggctgcatggttaa Protein: mpqqamdikgkaksvpmpeeddldshfvgpisprphgadeiagyvgceddedeleelgmlgrsasthfsyaeer- hl ievdakyralhghlphqhsqspvsrsssfvraemnhpppppsshthqqpedddasstrsrsssrasgrkfnrnr- tk sgsslskglqqlnmtgsleeepyesdddarlsaeddivydatqkdtckpisptlkrtrtkddmknmsindvkit- tt tedplvaqelsmmfekvqycrdlrdkyqtvslqkdgdnpkddkthwkiypeppppswhetekrfrgsskkehqk- kd ptmdefkfedceipgpndmvfkrdptcvyqvyedesslnenkpfvaipsirdyymdledlivassdgpaksfaf- rr lqyleakwnlyyllneytettesktnphrdfynvrkvdthvhhsacmnqkhllrfikykmkncpdevvihrdgr- el tlsqvfeslnitaydlsidtldmhahkdsfhrfdkfnlkynpvgesrlreiflktdnyiqgrylaeitkevfqd- le nskyqmaeyrisiygrskdewdklaawvldnklfspnvrwliqvprlydiykkaglvntfadivqnvfeplfev- tk dpsthpklhvflqrvvgfdsvddeskldrrfhrkfptaaywdsaqnppysywqyylyanmasintwrqrlgynt- fe lrphageagdpehllctylvaqginhgillrkvpfiqylyyldqipiamspvsnnalfltfdknpfysyflagl- nv slssddplqfaytkealieeysvaaliyklsnvdmcelarnsvlqsgferiikehwigenyeihgpegntiqkt- nv pnvrlafrdetlthelalvdkytnleeferlhg YALI0D16753g DNA: atgttccgaacccgagttaccggctccaccctgcgatccttctccacctccgctgcccgacagcacaaggttgt- cg tccttggcgccaacggaggcattggccagcccctgtctctgctgctcaagctcaacaagaacgtgaccgacctc- gg tctgtacgatctgcgaggcgcccccggcgttgctgccgatgtctcccacatccccaccaactccaccgtggccg- gc tactctcccgacaacaacggcattgccgaggccctcaagggcgccaagctggtgctgatccccgccggtgtccc- cc gaaagcccggcatgacccgagacgatctgttcaacaccaacgcctccattgtgcgagacctggccaaggccgtc- gg tgagcacgcccccgacgcctttgtcggagtcattgctaaccccgtcaactccaccgtccccattgtcgccgagg- tg ctcaagtccaagggcaagtacgaccccaagaagctcttcggtgtcaccaccctcgacgtcatccgagccgagcg- at tcgtctcccagctcgagcacaccaaccccaccaaggagtacttccccgttgttggcggccactccggtgtcacc- at tgtccccctcgtgtcccagtccgaccaccccgacattgccggtgaggctcgagacaagcttgtccaccgaatcc- ag tttggcggtgacgaggttgtcaaggccaaggacggtgccggatccgccaccctttccatggcccaggctgccgc- cc gattcgccgactctctcctccgaggtgtcaacggcgagaaggacgttgttgagcccactttcgtcgactctcct- ct gttcaagggtgagggcatcgacttcttctccaccaaggtcactcttggccctaacggtgttgaggagatccacc- cc atcggaaaggtcaacgagtacgaggagaagctcatcgaggctgccaaggccgatctcaagaagaacattgagaa- gg gtgtcaactttgtcaagcagaacccttaa Protein: mfrtrvtgstlrsfstsaarqhkvvvlganggigqplslllklnknvtdlglydlrgapgvaadvshiptnstv- ag yspdnngiaealkgaklvlipagvprkpgmtrddlfntnasivrdlakavgehapdafvgvianpvnstvpiva- ev lkskgkydpkklfgvttldviraerfvsqlehtnptkeyfpvvgghsgvtivplvsqsdhpdiageardklvhr- iq fggdevvkakdgagsatlsmaqaaarfadsllrgvngekdvveptfvdsplfkgegidffstkvtlgpngveei- hp igkvneyeeklieaakadlkkniekgvnfvkqnp YALI0D16247g DNA: atgacacaaacgcacaatctgttttcgccaatcaaagtgggctcttcggagctccagaaccggatcgttctcgc- ac ccttgactcgaaccagagctctgcccggaaacgtgccctcggatcttgccacagagtactacgcacaaagagca- gc atctccaggcactctcctcatcaccgaggccacatacatctcccccggatctgctggagtgcccattccaggag- ac ggaatcgttccgggcatctggagtgacgagcagctcgaagcatggaaaaaggtgttcaaggccgtgcacgaccg- ag gatccaaaatctacgtccagctgtgggacattggacgtgtcgcatggtaccacaagctgcaggaactgggcaac- ta cttccctacaggcccctcagctatccccatgaagggagaggagagcgagcatctcaaggctctgactcactggg- ag atcaagggcaaggtggccctctacgtcaacgctgccaagaacgccattgccgcaggcgctgatggcgtcgagat- cc actcggccaacggctaccttcccgacacatttctgagaagcgcctccaaccaacgaacagacgaatatggagga- ag catcgagaaccgggcccgattctcgctggagattgtcgacgctatcaccgaggccattggagcagacaaaaccg- cc atccgtctgtctccctggtccactttccaggacattgaggtgaatgacaccgagacccccgcacagttcacata- cc tgtttgagcagctgcagaagcgagccgacgagggaaagcagctggcctacgtgcatgtagttgagccccgactg- tt tggtccccccgagccctgggccaccaatgagcctttcagaaaaatttggaagggtaacttcattagagcaggtg- ga tacgatagagagactgctcttgaggatgcagacaagtcagacaacaccctgattgcctttggtcgagacttcat- tg ccaatcctgatctcgtccaacgcctcaagaataacgagcctttggccaagtacgacagaacaaccttctacgtt- cc aggtgccaagggctacactgattaccctgcgtacaagatgtaa Protein: mtqthnlfspikvgsselqnrivlapltrtralpgnvpsdlateyyaqraaspgtlliteatyispgsagvpip- gd givpgiwsdeqleawkkvfkavhdrgskiyvqlwdigrvawyhklqelgnyfptgpsaipmkgeesehlkalth- we ikgkvalyvnaaknaiaagadgveihsangylpdtflrsasnqrtdeyggsienrarfsleivdaiteaigadk- ta irlspwstfqdievndtetpaqftylfeqlqkradegkqlayvhvveprlfgppepwatnepfrkiwkgnfira- gg ydretaledadksdntliafgrdfianpdlvqrlknneplakydrttfyvpgakgytdypaykm YALI0A15972g

DNA: atggaagccaaccccgaagtccagaccgatatcatcacgctgacccggttcattctgcaggaacagaacaaggt- gg gcgcgtcgtccgcaatccccaccggagacttcactctgctgctcaactcgctgcagtttgccttcaagttcatt- gc ccacaacatccgacgatcgaccctggtcaacctgattggcctgtcgggaaccgccaactccaccggcgacgacc- ag aagaagctggacgtgatcggagacgagatcttcatcaacgccatgaaggcctccggtaaggtcaagctggtggt- gt ccgaggagcaggaggacctcattgtgtttgagggcgacggccgatacgccgtggtctgcgaccccatcgacgga- tc ctccaacctcgacgccggcgtctccgtcggcaccattttcggcgtctacaagctccccgagggctcctccggat- cc atcaaggacgtgctccgacccggaaaggagatggttgccgccggctacaccatgtacggtgcctccgccaacct- gg tgctgtccaccggaaacggctgcaacggcttcactctcgatgaccctctgggagagttcatcctgacccacccc- ga tctcaagctccccgatctgcgatccatctactccgtcaacgagggtaactcctccctgtggtccgacaacgtca- ag gactacttcaaggccctcaagttccccgaggacggctccaagccctactcggcccgatacattggctccatggt- cg ccgacgtgcaccgaaccattctctacggaggtatgtttgcctaccccgccgactccaagtccaagaagggcaag- ct ccgacttttgtacgagggtttccccatggcctacatcattgagcaggccggcggtcttgccatcaacgacaacg- gc gagcgaatcctcgatctggtccccaccgagatccacgagcgatccggcgtctggctgggctccaagggcgagat- tg agaaggccaagaagtaccttctgaaatga Protein: meanpevqtdiitltrfilqeqnkvgassaiptgdftlllnslqfafkfiahnirrstlvnliglsgtanstgd- dq kkldvigdeifinamkasgkvklvvseeqedlivfegdgryavvcdpidgssnldagvsvgtifgvyklpegss- gs ikdvlrpgkemvaagytmygasanlvlstgngcngftlddplgefilthpdlklpdlrsiysvnegnsslwsdn- vk dyfkalkfpedgskpysaryigsmvadvhrtilyggmfaypadskskkgklrllyegfpmayiieqagglaind- ng erildlvpteihersgvwlgskgeiekakkyllk YALI0E11099g DNA: atgcgactcactctgccccgacttaacgccgcctacattgtaggagccgcccgaactcctgtcggcaagttcaa- cg gagccctcaagtccgtgtctgccattgacctcggtatcaccgctgccaaggccgctgtccagcgatccaaggtc- cc cgccgaccagattgacgagtttctgtttggccaggtgctgaccgccaactccggccaggcccccgcccgacagg- tg gttatcaagggtggtttccccgagtccgtcgaggccaccaccatcaacaaggtgtgctcttccggcctcaagac- cg tggctctggctgcccaggccatcaaggccggcgaccgaaacgttatcgtggccggtggaatggagtccatgtcc- aa caccccctactactccggtcgaggtcttgttttcggcaaccagaagctcgaggactccatcgtcaaggacggtc- tc tgggacccctacaacaacatccacatgggcaactgctgcgagaacaccaacaagcgagacggcatcacccgaga- gc agcaggacgagtacgccatcgagtcctaccgacgggccaacgagtccatcaagaacggcgccttcaaggatgag- at tgtccccgttgagatcaagacccgaaagggcaccgtgactgtctccgaggacgaggagcccaagggagccaacg- cc gagaagctcaagggcctcaagcctgtctttgacaagcagggctccgtcactgccggtaacgcctcccccatcaa- cg atggtgcttctgccgttgtcgttgcctctggcaccaaggccaaggagctcggtacccccgtgctcgccaagatt- gt ctcttacgcagacgccgccaccgcccccattgactttaccattgctccctactggccattcccgccgccctcaa- ga aggctggccttaccaaggacgacattgccctctgggagatcaacgaggccttctccggtgtcgctctcgccaac- ct catgcgactcggaattgacaagtccaaggtcaacgtcaagggtggagctgttgctctcggccaccccattggtg- cc tccggtaaccgaatctttgtgactttggtcaacgccctcaaggagggcgagtacggagttgccgccatctgcaa- cg gtggaggagcttccaccgccatcgtcatcaagaaggtctcttctgtcgagtag Protein: mrltlprlnaayivgaartpvgkfngalksvsaidlgitaakaavqrskvpadqideflfgqvltansgqapar- qv vikggfpesveattinkvcssglktvalaaqaikagdrnvivaggmesmsntpyysgrglvfgnqkledsivkd- gl wdpynnihmgnccentnkrdgitreqqdeyaiesyrranesikngafkdeivpveiktrkgtvtvsedeepkga- na eklkglkpvfdkqgsvtagnaspindgasavvvasgtkakelgtpvlakivsyadaatapidftiapslaipaa- lk kagltkddialweineafsgvalanlmrlgidkskvnvkggavalghpigasgnrifvtlvnalkegeygvaai- cn gggastaivikkvssve YALI0E34793g DNA: atgtctgccaacgagaacatctcccgattcgacgcccctgtgggcaaggagcaccccgcctacgagctcttcca- ta accacacacgatctttcgtctatggtctccagcctcgagcctgccagggtatgctggacttcgacttcatctgt- aa gcgagagaacccctccgtggccggtgtcatctatcccttcggcggccagttcgtcaccaagatgtactggggca- cc aaggagactcttctccctgtctaccagcaggtcgagaaggccgctgccaagcaccccgaggtcgatgtcgtggt- ca actttgcctcctctcgatccgtctactcctctaccatggagctgctcgagtacccccagttccgaaccatcgcc- at tattgccgagggtgtccccgagcgacgagcccgagagatcctccacaaggcccagaagaagggtgtgaccatca- tt ggtcccgctaccgtcggaggtatcaagcccggttgcttcaaggttggaaacaccggaggtatgatggacaacat- tg tcgcctccaagctctaccgacccggctccgttgcctacgtctccaagtccggaggaatgtccaacgagctgaac- aa cattatctctcacaccaccgacggtgtctacgagggtattgctattggtggtgaccgataccctggtactacct- tc attgaccatatcctgcgatacgaggccgaccccaagtgtaagatcatcgtcctccttggtgaggttggtggtgt- tg aggagtaccgagtcatcgaggctgttaagaacggccagatcaagaagcccatcgtcgcttgggccattggtact- tg tgcctccatgttcaagactgaggttcagttcggccacgccggctccatggccaactccgacctggagactgcca- ag gctaagaacgccgccatgaagtctgctggcttctacgtccccgataccttcgaggacatgcccgaggtccttgc- cg agctctacgagaagatggtcgccaagggcgagctgtctcgaatctctgagcctgaggtccccaagatccccatt- ga ctactcttgggcccaggagcttggtcttatccgaaagcccgctgctttcatctccactatttccgatgaccgag- gc caggagcttctgtacgctggcatgcccatttccgaggttttcaaggaggacattggtatcggcggtgtcatgtc- tc tgctgtggttccgacgacgactccccgactacgcctccaagtttcttgagatggttctcatgcttactgctgac- ca cggtcccgccgtatccggtgccatgaacaccattatcaccacccgagctggtaaggatctcatttcttccctgg- tt gctggtctcctgaccattggtacccgattcggaggtgctcttgacggtgctgccaccgagttcaccactgccta- cg acaagggtctgtccccccgacagttcgttgataccatgcgaaagcagaacaagctgattcctggtattggccat- cg agtcaagtctcgaaacaaccccgatttccgagtcgagcttgtcaaggactttgttaagaagaacttcccctcca- cc cagctgctcgactacgcccttgctgtcgaggaggtcaccacctccaagaaggacaacctgattctgaacgttga- cg gtgctattgctgtttcttttgtcgatctcatgcgatcttgcggtgcctttactgtggaggagactgaggactac- ct caagaacggtgttctcaacggtctgttcgttctcggtcgatccattggtctcattgcccaccatctcgatcaga- ag cgactcaagaccggtctgtaccgacatccttgggacgatatcacctacctggttggccaggaggctatccagaa- ga agcgagtcgagatcagcgccggcgacgtttccaaggccaagactcgatcatag Protein: msanenisrfdapvgkehpayelfhnhtrsfvyglqpracqgmldfdfickrenpsvagviypfggqfvtkmyw- gt ketllpvyqqvekaaakhpevdvvvnfassrsvysstmelleypqfrtiaiiaegvperrareilhkaqkkgvt- ii gpatvggikpgcfkvgntggmmdnivasklyrpgsvayvsksggmsnelnniishttdgvyegiaiggdrypgt- tf idhilryeadpkckiivllgevggveeyrvieavkngqikkpivawaigtcasmfktevqfghagsmansdlet- ak aknaamksagfyvpdtfedmpevlaelyekmvakgelsrisepevpkipidyswaqelglirkpaafistisdd- rg qellyagmpisevfkedigiggvmsllwfrrrlpdyaskflemvlmltadhgpaysgamntiittragkdliss- lv aglltigtrfggaldgaatefttaydkglsprqfvdtmrkqnklipgighrvksrnnpdfrvelvkdfvkknfp- st qlldyalaveevttskkdnlilnvdgaiaysfvdlmrscgaftveetedylkngvlnglfvlgrsigliahhld- qk rlktglyrhpwdditylvgqeaiqkkrveisagdvskaktrs YALI0D24431g DNA: atgtcagcgaaatccattcacgaggccgacggcaaggccctgctcgcacactttctgtccaaggcgcccgtgtg- gg ccgagcagcagcccatcaacacgtttgaaatgggcacacccaagctggcgtctctgacgttcgaggacggcgtg- gc ccccgagcagatcttcgccgccgctgaaaagacctacccctggctgctggagtccggcgccaagtttgtggcca- ag cccgaccagctcatcaagcgacgaggcaaggccggcctgctggtactcaacaagtcgtgggaggagtgcaagcc- ct ggatcgccgagcgggccgccaagcccatcaacgtggagggcattgacggagtgctgcgaacgttcctggtcgag- cc ctttgtgccccacgaccagaagcacgagtactacatcaacatccactccgtgcgagagggcgactggatcctct- tc taccacgagggaggagtcgacgtcggcgacgtggacgccaaggccgccaagatcctcatccccgttgacattga-

ga acgagtacccctccaacgccacgctcaccaaggagctgctggcacacgtgcccgaggaccagcaccagaccctg- ct cgacttcatcaaccggctctacgccgtctacgtcgatctgcagtttacgtatctggagatcaaccccctggtcg- tg atccccaccgcccagggcgtcgaggtccactacctggatcttgccggcaagctcgaccagaccgcagagtttga- gt gcggccccaagtgggctgctgcgcggtcccccgccgctctgggccaggtcgtcaccattgacgccggctccacc- aa ggtgtccatcgacgccggccccgccatggtcttccccgctcctttcggtcgagagctgtccaaggaggaggcgt- ac attgcggagctcgattccaagaccggagcttctctgaagctgactgttctcaatgccaagggccgaatctggac- cc ttgtggctggtggaggagcctccgtcgtctacgccgacgccattgcgtctgccggctttgctgacgagctcgcc- aa ctacggcgagtactctggcgctcccaacgagacccagacctacgagtacgccaaaaccgtactggatctcatga- cc cggggcgacgctcaccccgagggcaaggtactgttcattggcggaggaatcgccaacttcacccaggttggatc- ca ccttcaagggcatcatccgggccttccgggactaccagtcttctctgcacaaccacaaggtgaagatttacgtg- cg acgaggcggtcccaactggcaggagggtctgcggttgatcaagtcggctggcgacgagctgaatctgcccatgg- ag atttacggccccgacatgcacgtgtcgggtattgttcctttggctctgcttggaaagcggcccaagaatgtcaa- gc cttttggcaccggaccttctactgaggcttccactcctctcggagtttaa Protein: Msaksiheadgkallahflskapvwaeqqpintfemgtpklasltfedgvapeqifaaaektypwllesgakfv- ak pdqlikrrgkagllvlnksweeckpwiaeraakpinvegidgvlrtflvepfvphdqkheyyinihsvregdwi- lf yheggvdvgdvdakaakilipvdieneypsnatltkellahvpedqhqtlldfinrlyavyvdlqftyleinpl- vv iptaqgvevhyldlagkldqtaefecgpkwaaarspaalgqvvtidagstkvsidagpamvfpapfgrelskee- ay iaeldsktgaslkltvlnakgriwtlvagggasvvyadaiasagfadelanygeysgapnetqtyeyaktvldl- mt rgdahpegkvlfigggianftqvgstfkgiirafrdyqsslhnhkvkiyvrrggpnwqeglrliksagdelnlp- me iygpdmhvsgivplallgkrpknvkpfgtgpsteastplgv YALI0E14190g DNA: atggttattatgtgtgtgggacctcagcacacgcatcatcccaacacagggtgcagtatatatagacagacgtg- tt ccttcgcaccgttcttcacatatcaaaacactaacaaattcaaaagtgagtatcatggtgggagtcaattgatt- gc tcggggagttgaacaggcaacaatggcatgcacagggccagtgaaggcagactgcagtcgctgcacatggatcg- tg gttctgaggcgttgctatcaaaagggtcaattacctcacgaaacacagctggatgttgtgcaatcgtcaattga- aa aacccgacacaatgcaagatctctttgcgcgcattgccatcgctgttgccatcgctgtcgccatcgccaatgcc- gc tgcggattattatccctaccttgttccccgcttccgcacaaccggcgatgtctttgtatcatgaactctcgaaa- ct aactcagtggttaaagctgtcgttgccggagccgctggtggtattggccagcccctttctcttctcctcaaact- ct ctccttacgtgaccgagcttgctctctctacgatgtcgtcaactcccccggtgttgccgctgacctctcccaca- tc tccaccaaggctaaggtcactggctacctccccaaggatgacggtctcaagaacgctctgaccggcgccaacat- tg tcgttatccccgccggtatcccccgaaagcccggtatgacccgagacgatctgttcaagatcaacgctggtatc- gt ccgagatctcgtcaccggtgtcgcccagtacgcccctgacgcctttgtgctcatcatctccaaccccgtcaact- ct accgtccctattgctgccgaggtcctcaagaagcacaacgtcttcaaccctaagaagctcttcggtgtcaccac- cc ttgacgttgtccgagcccagaccttcaccgccgctgttgttggcgagtctgaccccaccaagctcaacatcccc- gt cgttggtggccactccggagacaccattgtccctctcctgtctctgaccaagcctaaggtcgagatccccgccg- ac aagctcgacgacctcgtcaagcgaatccagtttggtggtgacgaggttgtccaggctaaggacggtcttggatc- cg ctaccctctccatggcccaggctggtttccgatttgccgaggctgtcctcaagggtgccgctggtgagaagggc- at catcgagcccgcctacatctaccttgacggtattgatggcacctccgacatcaagcgagaggtcggtgtcgcct- tc ttctctgtccctgtcgagttcggccctgagggtgccgctaaggcttacaacatccttcccgaggccaacgacta- cg agaagaagcttctcaaggtctccatcgacggtctttacggcaacattgccaagggcgaggagttcattgttaac- cc tcctcctgccaagtaa Protein: vvkavvagaaggigqplslllklspyvtelalydvvnspgvaadlshistkakvtgylpkddglknaltganiv- vi pagiprkpgmtrddlfkinagivrdlvtgvaqyapdafvliisnpvnstvpiaaevlkkhnvfnpkklfgvttl- dv vraqtftaavvgesdptklnipvvgghsgdtivpllsltkpkveipadklddlvkriqfggdevvqakdglgsa- tl smaqagfrfaeavlkgaagekgiiepayiyldgidgtsdikrevgvaffsvpvefgpegaakaynilpeandye- kk llkvsidglygniakgeefivnpppak YALI0E22649g DNA: atgactggcaccttacccaagttcggcgacggaaccaccattgtggttcttggagcctccggcgacctcgctaa- ga agaagaccgtgagtattgaaccagactgaggtcaattgaagagtaggagagtctgagaacattcgacggacctg- at tgtgctctggaccactcaattgactcgttgagagccccaatgggtcttggctagccgagtcgttgacttgttga- ct tgttgagcccagaacccccaacttttgccaccatacaccgccatcaccatgacacccagatgtgcgtgcgtatg- tg agagtcaattgttccgtggcaaggcacagcttattccaccgtgttccttgcacaggtggtctttacgctctccc- ac tctatccgagcaataaaagcggaaaaacagcagcaagtcccaacagacttctgctccgaataaggcgtctagca- ag tgtgcccaaaactcaattcaaaaatgtcagaaacctgatatcaacccgtcttcaaaagctaaccccagttcccc- gc cctcttcggcctttaccgaaacggcctgctgcccaaaaatgttgaaatcatcggctacgcacggtcgaaaatga- ct caggaggagtaccacgagcgaatcagccactacttcaagacccccgacgaccagtccaaggagcaggccaagaa- gt tccttgagaacacctgctacgtccagggcccttacgacggtgccgagggctaccagcgactgaatgaaaagatt- ga ggagtttgagaagaagaagcccgagccccactaccgtcttttctacctggctctgccccccagcgtcttccttg- ag gctgccaacggtctgaagaagtatgtctaccccggcgagggcaaggcccgaatcatcatcgagaagccctttgg- cc acgacctggcctcgtcacgagagctccaggacggccttgctcctctctggaaggagtctgagatcttccgaatc- ga ccactacctcggaaaggagatggtcaagaacctcaacattctgcgatttggcaaccagttcctgtccgccgtgg- gt gacaagaacaccatttccaacgtccagatctccttcaaggagccctttggcactgagggccgaggtggatactt- ca acgacattggaatcatccgagacgttattcagaaccatctgttgcaggttctgtccattctagccatggagcga- cc cgtcactttcggcgccgaggacattcgagatgagaaggtcaaggtgctccgatgtgtcgacattctcaacattg- ac gacgtcattctcggccagtacggcccctctgaagacggaaagaagcccggatacaccgatgacgatggcgttcc- cg atgactcccgagctgtgacctttgctgctctccatctccagatccacaacgacagatgggagggtgttcctttc- at cctccgagccggtaaggctctggacgagggcaaggtcgagatccgagtgcagttccgagacgtgaccaagggcg- tt gtggaccatctgcctcgaaatgagctcgtcatccgaatccagccctccgagtccatctacatgaagatgaactc- ca agctgcctggccttactgccaagaacattgtcaccgacctggatctgacctacaaccgacgatactcggacgtg- cg aatccctgaggcttacgagtctctcattctggactgcctcaagggtgaccacaccaactttgtgcgaaacgacg- ag ctggacatttcctggaagattttcaccgatctgctgcacaagattgacgaggacaagagcattgtgcccgagaa- gt acgcctacggctctcgtggccccgagcgactcaagcagtggctccgagaccgaggctacgtgcgaaacggcacc- ga gctgtaccaatggcctgtcaccaagggctcctcgtga Protein: mtgtlpkfgdgttivvlgasgdlakkktfpalfglyrngllpknveiigyarskmtqeeyherishyfktpddq- sk eqakkflentcyvqgpydgaegyqrlnekieefekkkpephyrlfylalppsvfleaanglkkyvypgegkari- ii ekpfghdlassrelqdglaplwkeseifridhylgkemyknlnilrfgnqflsavwdkntisnvqisfkepfgt- eg rggyfndigiirdviqnhllqvlsilamerpvtfgaedirdekvkvlrcvdilniddvilgqygpsedgkkpgy- td ddgvpddsravtfaalhlqihndrwegvpfilragkaldegkveirvqfrdvtkgvvdhlprnelviriqpses- iy mkmnsklpgltaknivtdldltynrrysdvripeayeslildclkgdhtnfvrndeldiswkiftdllhkided- ks ivpekyaygsrgperlkqwlrdrgyvrngtelyqwpvtkgss YALI0B15598g DNA: atgactgacacttcaaacatcaagtgagtattgccgcacacaattgcaatcaccgccgggctctacctcctcag- ct ctcgacgtcaatgggccagcagccgccatttgaccccaattacactggttgtgtaaaaccctcaaccacaatcg- ct tatgctcaccacagactacgacttaaccaagtcatgtcacaggtcaaagtaaagtcagcgcaacaccccctcaa- tc tcaacacacttttgctaactcaggcctgtcgctgacattgccctcatcggtctcgccgtcatgggccagaacct- ga

tcctcaacatggccgaccacggtaagtatcaattgactcaagacgcaccagcaagatacagagcatacccagca- at cgctcctctgataatcgccattgtaacactacgttggttagattgatctaaggtcgttgctggttccatgcact- tc cacttgctcatatgaagggagtcaaactctattttgatagtgtcctctcccatccccgaaatgtcgcattgttg- ct aacaataggctacgaggttgttgcctacaaccgaaccacctccaaggtcgaccacttcctcgagaacgaggcca- ag ggtgagtatccgtccagctatgctgtttacagccattgaccccaccttcccccacaattgctacgtcaccatta- aa aaacaaaattaccggtatcggcaagctagactttcatgcaacctacgcagggtaacaagttgagtttcagccgt- gc accttacaggaaaaccagtcatacgccgaggcagtgtgaaagcgaaagcacacagcctacggtgattgattgca- tt tttttgacataggagggaaacacgtgacatggcaagtgcccaacacgaatactaacaaacaggaaagtccatta- tt ggtgctcactctatcaaggagctgtgtgctctgctgaagcgaccccgacgaatcattctgctcgttaaggccgg- tg ctgctgtcgattctttcatcgaacagctcctgccctatctcgataagggtgatatcatcattgacggtggtaac- tc ccacttccccgactccaaccgacgatacgaggagcttaacgagaagggaatcctctttgttggttccggtgttt- cc ggcggtgaggagggtgcccgatacggtccctccatcatgcccggtggaaacaaggaggcctggccccacattaa- ga agattttccaggacatctctgctaaggctgatggtgagccctgctgtgactgggtcggtgacgctggtgccggc- ac ctttgtcaagatggttcacaacggtattgagtatggtgacatgcagcttatctgcgaggcttacgacctcatga- ag cgaggtgctggtttcaccaatgaggagattggagacgttttcgccaagtggaacaacggtatcctcgactcctt- cc tcattgagatcacccgagacatcttcaagtacgacgacggctctggaactcctctcgttgagaagatctccgac- ac tgctggccagaagggtactggaaagtggaccgctatcaacgctcttgaccttggtatgcccgtcaccctgatcg- gt gaggccgtcttcgctcgatgcctttctgccctcaagcaggagcgtgtccgagcttccaaggttcttgatggccc- cg agcccgtcaagttcactggtgacaagaaggagtttgtcgaccagctcgagcaggccctttacgcctccaagatc- at ctcttacgcccagggtttcatgcttatccgagaggccgccaagacctacggctgggagctcaacaacgccggta- tt gccctcatgtggcgaggtggttgcatcatccgatccgtcttccttgctgacatcaccaaggcttaccgacagga- cc ccaacctcgagaacctgctgttcaacgacttcttcaagaacgccatctccaaggccaacccctcttggcgagct- ac cgtggccaaggctgtcacctggggtgttcccactcccgcctttgcctcggctctggctttctacgacggttacc- ga tctgccaagctccccgctaacctgctccaggcccagcgagactacttcggcgcccacacctaccagctcctcga- tg gtgatggaaagtggatccacaccaactggaccggccgaggtggtgaggtttcttcttccacttacgatgataa Protein: mtdtsnikpvadialiglavmgqnlilnmadhgyevvaynrttskvdhfleneakgksiigahsikelcallkr- pr riillvkagaavdsfieqllpyldkgdiiidggnshfpdsnrryeelnekgilfvgsgvsggeegarygpsimp- gg nkeawphikkifqdisakadgepccdwygdagaghfvkmvhngieygdmqliceaydlmkrgagftneeigdyf- ak wnngildsflieitrdifkyddgsgtplvekisdtagqkgtgkwtainaldlgmpvtligeavfarclsalkqe- rv raskvldgpepvkftgdkkefvdqleqalyaskiisyaqgfmlireaaktygwelnnagialmwrggciirsvf- la ditkayrqdpnlenllfndffknaiskanpswratvakavtwgvptpafasalafydgyrsaklpanllqaqrd- yf gahtyqlldgdgkwihtnwtgrggevssstyda YALI0D06303g DNA: atgctcaaccttagaaccgcccttcgagctgtgcgacccgtcactctggtgagtatctcggagcccgggacggc- ta ccaacacacaagcaagatgcaacagaaaccggactttttaaatgcggattgcggaaaatttgcatggcggcaac- ga ctcggagaaggagcgggacaattgcaatggcaggatgccattgacgaactgagggtgatgagagaccgggcctc- cg atgacgtggtggtgacgacagcccggctggtgttgccgggactgtctctgaaaagcaatttctctatctccggt- ct caacagactccccttctctagctcaattggcattgtcttcagaaggtgtcttagtggtatccccattgttatct- tc ttttccccaatgtcaatgtcaatgtcaatggctccgacctctttcacattaacacggcgcaaacacagatacca- cg gaaccgactcaaacaaatccaaagagacgcagcggaataattggcatcaacgaacgatttgggatactctggcg- ag aatgccgaaatatttcgcttgtcttgttgtttctcttgagtgagttgtttgtgaagtcgtttggaagaaggttc- cc aatgtcacaaaccataccaactcgttacagccagcttgtaatcccccacctcttcaatacatactaacgcagac- cc gatcctacgccacttccgtggcctctttcaccggccagaagaactccaacggcaagtacactgtgtctctgatt- ga gggagacggtatcggaaccgagatctccaaggctgtcaaggacatctaccatgccgccaaggtccccatcgact- gg gaggttgtcgacgtcacccccactctggtcaacggcaagaccaccatccccgacagcgccattgagtccatcaa- cc gaaacaaggttgccctcaagggtcccctcgccacccccatcggtaagggccacgtttccatgaacctgactctg- cg acgaaccttcaacctgttcgccaacgtccgaccttgcaagtccgtcgtgggctacaagaccccttacgagaacg- tc gacaccctgctcatccgagagaacactgagggtgagtactccggtatcgagcacaccgtcgtccccggtgtcgt- tc agtccatcaagctgatcacccgagaggcttccgagcgagtcatccggtacgcttacgagtacgccctgtcccga- gg catgaagaaggtccttgttgtccacaaggcctctattatgaaggtctccgatggtcttttccttgaggttgctc- ga gagctcgccaaggagtacccctccattgacctttccgtcgagctgatcgacaacacctgtctgcgaatggtcca- gg accccgctctctaccgagatgtcgtcatggtcatgcccaacctttacggtgacattctgtccgatcttgcctcc- gg tcttatcggtggtcttggtctgaccccctccggtaacatgggtgacgaggtctccatcttcgaggccgtccacg- ga tccgctcccgacattgctggcaagggtcttgctaaccccactgctctgctgctctcctccgtgatgatgctgcg- ac acatgggtctcaacgacaacgccaccaacatcgagcaggccgtctttggcaccattgcttccggccccgagaac- cg aaccaaggatcttaagggtaccgccaccacttctcactttgctgagcagattatcaagcgactcaagtag Protein: mlnlrtalravrpvtltrsyatsvasftgqknsngkytvsliegdgigteiskavkdiyhaakvpidwevvdvt- pt lvngkttipdsaiesinrnkvalkgplatpigkghvsmnltlrrtfnlfanvrpcksvvgyktpyenvdtllir- en tegeysgiehtvvpgvvqsiklitreaserviryayeyalsrgmkkvlvvhkasimkvsdglflevarelakey- ps idlsvelidntclrmvqdpalyrdvvmvmpnlygdilsdlasgligglgltpsgnmgdevsifeavhgsapdia- gk glanptalllssvmmlrhmglndnatnieqavfgtiasgpenrtkdlkgtattshfaeqiikrlk

Example 11

Regulatory Sequences

[0340] Sequences which consist of, consist essentially of, or comprise the following regulatory sequences (e.g. promoters and terminator sequences, including functional fragments thereof) may be useful to control expression of endogenous and heterologous genes in recombinant fungi described herein.

TABLE-US-00022 Met2 promoter 5'cctctcactttgtgaatcgtgaaacatgaatcttcaagccaagaatgttaggcaggggaagctttctttcag- ac tttttggaattggtcctcttttggacattattgacgatattattattttttccccgtccaatgttgacccttgt- aa gccattccggttctggagcgcatctcgtctgaaggagtcttcgtgtggctataactacaagcgttgtatggtgg- at cctatgaccgtctatatagggcaacttttgctcttgttcttccccctccttgagggacgtatggcaatggctat- ga caactatcgtagtgagcctctataacccattgaagtacaagtcctccaccttgctgccaaactcgcgagaaaaa- aa gtccaccaactccgccgggaaatactggagaacacctctaagacgtgggcttctgcacctgtgtggcttgggtc- tg ggttttgcgagctctgagccacaacctaaggacggtgtgattgggagataagtagtcgttggttttctaatcgc- ac gtgatatgcaagccacacttataacacaatgaagacaggccgatgaactgcatgtcattgtacaggtgcggaga- gc aagaaactctggggcggaggtgaaagatgagacaaaaagcctcaggtgcaaggtagggagttgatcaacgtcaa- ac acaaataatctaggttgttaggcagctaaacatgtatataactgggctgccaccgagtgttacttgtcattaac- gt cgcattttcgcctacacaaaatttgggttactcgccactacactgctcaaatctttcagctgtgcaacaagatt- ca ggtcacacatagactcgcataaggacccgggtcatctgttattctccactggtaaaccaatagtcctagctgat- tt gggtacagaagctcactttcacatcttttcatcttatctacaaccatc Met3 promoter 5'atctgtgaggagcccctggcgtcactgtcgactgtgccggcatttctgatggtatttccagccccgcagttc- tc gagacccccgaacaaatgtgccacacccttgccaaaatgacgaatacacggcgtcgcggccgggaatcgaactc- tt ggcaccgccacaggagtgaaatttgaaatttgaaatttgaaaaataattcacattttgagtttcaataatatat- cg atgaccctcccaaaagacccaagtcgagacgcaaaaaaacacccagacgacatggatgcggtcacgtgaccgca- aa aaccgccccggaaatccgtttgtgacgtgttcaattccatctctatgtttttctgcggtttctacgatgccgca- at ggtggccaatgtgcgtttcactgccgtagtggctggaacaagccacagggggtcgtcgggccaatcagacggtc- cc tgacatggttctgcgccctaacccgggaactctaacccccgtggtggcgcaatcgctgtcttcatgtgctttat- ct cacgtgacggctggaatctggcagaagacggagtatgtacattttgtcgttggtcacgttatccctaaaacgtg- gt gtttaaactggtcgaatgcttggcccagaacacaagaagaaaaaaacgagacaacttgatcagtttcaacgcca- ca gcaagcttgtcttcactgtggttggtcttctccacgccacaagcaacacgtacatgtcaattacgtcagggtct- tt taagttctgtggcttttgaaccagttataaagaaccaaccacccttttttcaaagctaatcaagacggggaaat- tt tttttttgatatttttcgaca Met6 promoter 5'gatactgcagacggtgcattacttacccgtgtcgactgagagtctacttggtacttggccctgtggctaagc- ag tatttgagcaacaatgcaatgcagttgctgactcggttccagatccccttgccccgatgtgtggaagcgttgtt- tt tggggcaagggcatgtgggggctgcatcatactgtggctggggccgttggaagagccgtcggcagcgagcctga- gt cgcttctcggggccttattccccccgcctctaggtcagcggcggccgaagtgtcgtactcagctcgcctgtaca- gt atgacgtgaccgaatagcctctggaaggttggagaagtacagtgcaaaaaaaagttgcaaaatttcattttagc- gt tcgatccgacgtggcagttggacaatgaatcgatggagacatgatcatgggcagaaatagaaggtaccatgttc- aa tggcagtaccaattgagcaacagacgggtcgacaggcggcgggcacaccatccgccaccacatggcgcaatcgt- ca gtgcagcgattcgtactcggattgcatcatgttgcaccgaaagttggggcccgcacgttggagaggcgaggagc- ca gggttagctttggtggggtcctttgttgtcacgtggcatcagcgaatggcgtcctccaatcagggccgtcagcg- aa gtcggcgtgtgatagtgcgtggggagcgaatagagtttctgggggggggcggcccaaaacgtgaaatccgagta- cg catgtagagtgtaaattgggtgtatagtgacattgtttgactctgaccctgagagtaatatataatgtgtacgt- gt ccccctccgttggtcttctttttttacctttctcctaaccaacacccaaactaatcaatc Met25 promoter 5'aagtcgtattaacataactttccttacatttttttaaagcacgtcactatccacgtgacctagccacgcgat- ac caagtattcatccataatgacacactcatgacgtccggaggacgtcatcatcgtccagtcacgtgccaaggcac- at gactaatcataacaccttatgactagcttctgaatcgctacacagttccaattcgcaaataaactcgaaatgac- ga aatgccataataaaaatgacgaaactcgagattgagagcagcacatgcactgaagtggtggacaaccagcgtat- cc ggagacacgacggatccagcaccatggaagctggccgaaaaagagatccccagcacattgagcaaccaagtcag- ct caattgagtaacatcacacactcagatcgagtctgatggtggtcccatttgttccttcacttgaaaaataattg- aa aataacaataacaataaaaataaaaacaaaataaaaataaaaataaaaataaaaataaaaaaataaaaaaacct- tg ccgcatttagcgtcagccaccccccgcattgacctgagtacgttggattgaccccgatcctgcacgtcgagcgt- gg tcggccaaaaagcgcccgtggctggtgagtcagaaatagcagggttgcaagagagagctgcgcaacgagcaata- aa cggtgtttttttcgcttctgtgctgcttagagtggagagccgaccctcgccatgctcacgtgaccattcacgtg- gt tgcaaactccaccttagtatagccgtgtccctacgctacccattatcgcatcgtactccagccacatttttttg- tt ccccgctaaatccggaaccttatctgggtcacgtgaaattgcaatacgacaggaggttatacttatagagtgag- ac actccacgcaaggtgttgcaagtcaattgacaccacctcacctcagactaacatccaca Pox2 promoter 5'gaatctgcccccacattttataccgcttttgactgtttttctcccccctttcacactctgcttttggctaca- ta aaccccgcaccgtttggaactctgttggtccggggaagccgccgttaggtgtgtcagatggagagcgccagacg- ag cagaaccgagggacagcggatcgggggagggctgtcacgtgacgaagggcactgttgacgtggtgaatgtcgcc- cg ttctcacgtgacccgtctcctctatatgtgtatccgcctctttgtttggttttttttctgcttccccccccccc- cc cccaccccaatcacatgctcagaaagtagacatctgcatcgtcctgcatgccatcccacaagacgaacaagtga- ta ggccgagagccgaggacgaggtggagtgcacaaggggtaggcgaatggtacgattccgccaagtgagactggcg- at cgggagaagggttggtggtcatgggggatagaatttgtacaagtggaaaaaccactacgagtagcggatttgat- ac cacaagtagcagagatatacagcaatggtgggagtgcaagtatcggaatgtactgtacctcctgtactcgtact- cg tacggcactcgtagaaacggggcaatacgggggagaagcgatcgcccgtctgttcaatcgccacaagtccgagt- aa tgctcgagtatcgaagtcttgtacctccctgtcaatcatggcaccactggtcttgacttgtctattcatactgg- ac aagcgccagagttaagcttgtagcgaatttcgccctcggacatcaccccatacgacggacacacatgcccgaca- aa cagcctctcttattgtagctgaaagtatattgaatgtgaacgtgtacaatatcaggtaccagcgggaggttacg- gc caaggtgataccggaataaccctggcttggagatggtcggtccattgtactgaagtgtccgtgtcgtttccgtc- ac tgccccaattggacatgtttgtttttccgatctttcgggcgccctctccttgtctccttgtctgtctcctggac- tg ttgctaccccatttctttggcctccattggttcctccccgtctttcacgtcgtctatggttgcatggtttccct- ta tacttttccccacagtcacatgttatggaggggtctagatggaggcctaattttgacgtgcaaggggcgaattg- gg gcgagaaacacgtcgtggacatggtgcaaggcccgcagggttgattcgacgatttccgcgaaaaaaacaagtcc- aa atacccccgtttattctccctcggctctcggtatttcacatgaaaactataacctagactacacgggcaacctt- aa ccccagagtatacttatataccaaagggatgggtcctcaaaaatcacacaagcaacg Yef3 (YALI0E13277g) promoter 5'cgccattcggttccttccagaccattccagatcaatccacctcttcttatctcaggtgggtgtgctgacatc- ag accccgtagcccttctcccagtggcgaacagcaggcataaaacagggccattgagcagagcaaacaaggtcggt- ga aatcgtcgaaaaagtcggaaaacggttgcaagaaattggagcgtcacctgccaccctccaggctctatataaag- ca ttgccccaattgctaacgcttcatatttacacctttggcaccccagtccatccctccaataaaatgtactacat- gg gacacaacaagagaggatgcgcgcccaaaccctaacctagcacatgcacgatgattctattgtctgtgaaaaaa- tt tttccaccaaaatttccccattgggatgaaaccctaaccgcaaccaaaagtttttaactatcatcttgtacgtc- ac ggtttccgattcttctcttctctttcatcatcatcacttgtgacc Cam1 (YALI0C24420g) promoter 5'aactaccataaagtaccgagaaatataggcaattgtacaaattgtccacctccttcacttacattaccgaac- ca tggccatatcaccaaaataccccgagtgctaaaacacctccctccaaatgttctcttaccttccaccgaaaacc- ga tcttattatcccaacgcttgttgtggcttgacgcgccgcacccgctgggcttgccatttcgataccaatccaag- ag gaaaagctcatgagaaacaatcggaatatcacgagaacggcctggcgaaccaacaggatatttttgaatataat- ta cccctcgaatctagtcatatctatgtctactgtagacttgggcggcatcatgatgtacattattttagcgtctg- ga accctaaagttcacgtacaatcatgtgacaaacgaggctaaaaaatgtcaatttcgtatattagtgttattacg- tg gctcacatttccgaatcatctaccaccccccacctaaaaa YALI0D16467g promoter

5'tttttttaattttcatatttattttcatatttattttcatatttattttcatttatttattcatgtatttat- tt attactttttaagtattttaaactcctcactaaaccgtcgattgcacaatattaaccttcattacacctgcagc- gt ggtttttgtggtcgttagccgaagtcttccaacgtgggtataagtaggaacaattgggccgattttttgagccg- tc taaatctctcgactcaattgatctgctgtcgaaaatccggctctctagctccttttccccgtccgctggagctc- ct cttcattgtgccgtttttccaacatttaactttgccacccaccaccacccccactaccatcacccactcgatct- ct gttcgtgtcaccacgactttgtcttctcacacatactctgtttgtgcaccacacattgcgaa Tef4 (YALI0B12562g) promoter 5'gctacaatagattattggccctattgagcacgctacaattcggtccagtatgtacaacgtctatgcgcacta- ac ggccatacagtgagttacagcacacccaaaagtaaccctgcctgacctgtctgcctgagacaggaagattaact- ct tgtagtgaccgagctcgataagactcaagccacacaatttttttatagccttgcttcaagagtcgccaaaatga- ca ttacacaactccacggaccgtcggttccatgtccacacccttggatgggtaagcgctccacgcacgtaccacgt- gc attgagtttaaccacaaacataggtctgtgtcccagagttaccctgctgcatcagccaagtcttgaaagcaaaa- tt tcttgcacaatttttcctcttcttttcttcactgatcgcagtccaaacacaaaca YALI0D12903g promoter 5'gcgctctgatccacttgtatggctccaagttcagtgtaccaagtagttggtgatgcagggagggatgtctct- at ccaccaataatgaactcatgggcgaaattgtttctgttaaacactccaactgtcgttttaaatctcattctctt- tg catttggactccattcgcttccgttgggccaatataatccatcgtaacgtactttagatggaaatttagttacc- tg ctacttgtctcaacaccccaacaggggctgttcgacagaggtaatagagcgtcaatgggttaataaaaacacac- tg tcgattttcactcattgtctttatgatattacctgttttccgctgttatcaatgccgagcatcgtgttatatct- tc caccccaactacttgcatttacttaactattacctcaactatttacaccccgaattgttacctcccaataagta- ac tttatttcaaccaatgggacgagagcatactgagaacatcgatctatctagtcaatattgcccagaatcgttcg- aa aaaaaacaccaaaaggtttacagcgccattataaatataaattcgttgtcaattcccccgcaatgtctgttgaa- at ctcattttgagaccttccaacattaccactctcccgtctggtcacatgacgtgactgcttcttcccaaaacgaa- ca ctcccaactcttcccccccgtcagtgaaaagtatacatccgacctccaaatcttttcttcactcaac Tef1 (YALI0C09141g) promoter 5'agagacgggttggcggcgtatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgaccc- ag tagcgggcccaaccccggcgagagcccccttcaccccacatatcaaacctcccccggttcccacacttgccgtt- aa gggcgtagggtactgcagtaggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggt- aa cccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaa- ga ccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcat- tt ccttctgagtataagaatcattc Fba1 (YALI0E26004g) promoter 5'gctgcgctgatctggacaccacagaggttccgagcactttaggttgcaccaaatgtcccaccaggtgcaggc- ag aaaacgctggaacagcgtgtacagtttgtcttagcaaaaagtgaaggcgctgaggtcgagcagggtggtgtgac- tt gttatagcctttagagagcgaaagcgcgtatggatttggctcatcaggccagattgagggtctgtggacacatg- tc atgttagtgtacttcaatcgccccctggatatagccccgacaataggccgtggcctcatttttttgccttccgc- ac atttccattgctcggtacccacaccttgatctcctgcacttgccaaccttaatactggtttacattgaccaaca- tc ttacaagcggggggcttgtctagggtatatataaacagtggctctcccaatcggttgccagtctcttttttcat- tc tttccccacagattcgaaatctaaactacacatc Pox2 terminator: 5'gatgaggaatagacaagcgggtatttattgtatgaataaagattatgtattgattgcaaaaaagtgcatttg- ta gatgtggtttattgtagagagtacggtatgtactgtacgaacattaggagctacttctacaagtagattttctt- aa caagggtgaaatttactaggaagtacatgcatatttcgttagtagaatcacaaaagaaatgtacaagcacgtac- ta cttgtactccacaatgtggagtgggagcaaaaaaattggacgacaccggaatcgaaccggggacctcgcgcatg- ct aagcgcatgtgataaccaactacaccagacgcccaagaactttcttggtgattatggaatacgtggtctgctat- at ctcaattttgctgtaatgaatcattagaattaaaaaaaaaaccccatttttgtgtgattgtcggccaagagatg- ga acaggaagaatacgtgaacaagcgagcacgaatgccatatgctcttctgaacaaccgagtccgaatccgatttg- tg ggtatcacatgtacaagtagagaaatgtatttcgctagaataaaataaatgagattaagaattaaaaatattgg- aa tatattttcctagaatagaaactttggattttttttcggctattacagtagaactggacaaacggctgactata- ta taaatattattgggtctgttttcttgtttatgtcgaaattatctgggttttactactgtgtcgtcgagtataga- gt ggcctgactggagaaaatgcagtagtatggacagtaggtactgccagccagagaagtttttggaattgatactt- ga gtcatttttccattccccattccccattccaacacaatcaactgtttctgaacattttccaaaacgcggagatg- ta tgtcacttggcactgcaagtctcgattcaaaatgcatctctttcagaccaaagtgtcatcagctttgtttggcc- cc aaattaccgcaaatacttgtcgaaattgaagtgcaatacggcctcgtctgccatgaaacctgcctattctcttc- aa attggcgtcaggtttcacgtccagcattcctcgcccagacagagttgctatggttgaatcgtgtactgttaata- ta tgtatgtattatactcgtactacgatatactgttcaatagagtctcttataatcgtacgacgattctgggca

Example 12

Cultures Conditions, Such as Limitation for Nitrogen, Magnesium or Phosphate, can Promote Lipid Accumulation in Y. lipolytica

[0341] 12a. Strains Used to Analyze Lipid Accumulation During Growth Under Various Conditions.

[0342] Strains MF760, MF858, and MF921 were grown under an array of culture conditions, and then harvested cells were extracted and analyzed for total lipid content and levels of specific lipophilic metabolites. FIG. 12 depicts schematic representations of certain plasmids generated and described in this example. Strain MF760 has genotype MATB ura2::URA2/tef-GGS1 ChrA-1635618::tef-carB ura3-302 ade1::?ADE1/tef-HMG1trunc leu2::?LEU2/tef-carRP (questions marks denote presumed loci of chromosomal integration). Strain MF760 harbors four insertion plasmids, pMB4637, pMB4591, pMB4705, and pMB4660, which encode native or heterologous genes required for synthesis of either isoprenoid metabolites in general, or carotenoid metabolites specifically. In all insertion plasmids, except pMB4789, described in this example, the Y. lipolytica TEF 1 promoter and XPR2 terminator were the regulatory sequences used to control expression of genes of interest. Also, in some instances multiple URA3-containing plasmids can be utilized in the same strain, since 5-fluoroorotic acid can be used to select for Ura.sup.- segregants following transformation with a URA3 plasmid. pMB4637 is an ADE1 plasmid that encodes a truncated variant of the Y. lipolytica HMG-CoA reductase. pMB4591 is a URA5 plasmid that encodes the Y. lipolytica geranylgeranylpyrophosphate synthase. pMB4705 is a LEU2 plasmid that encodes the phytoene synthase/lycopene cyclase (CarRP) from Mucor circinelloides. pMB4660 is a URA3 plasmid that encodes a phytoene dehydrogenase from M. circinelloides.

[0343] Strain MF858 has genotype MATB ura2::URA2/tef-GGS1 ChrA-1635618::tef-carB ura3-302::?URA3/tef-plasmid ade1::?ADE1/tef-HMG1trunc leu2::?LEU2/tef-carRP. Strain MF858 harbors the same four plasmids as MF760, and an addition control plasmid (pMB4691), which is a URA3 plasmid that contains regulatory sequences but no gene of interest.

[0344] Strain MF921 has genotype MATB erg9-3'UTR::URA3 ura2::URA2/tef-GGS1 ChrA-1635618::tef-carB ura3-302 ade1::?ADE1/tef-HMG1trunc leu2::?LEU2/tef-carRP. Strain MF921 harbors the same four plasmids as MF760, and an addition URA3 plasmid, pMB4789, which contains sequences for insertion into the 3' UTR of the native ERG9. Insertion into 3' UTR of ERG9 is presumed to result in a hypomorphic mutation to decrease squalene synthase activity.

12b. Lipid Accumulation in Media Containing Various Carbon: Nitrogen Ratios

[0345] Shake flask testing was conducted using carbon to nitrogen (C/N) ratios of 160, 80, 60, 40, 30, 20, and 10 with yeast nitrogen base being the base medium providing vitamins, trace elements and salts. Ammonium sulfate (which contains 21% nitrogen) was used as the nitrogen source and glucose (which contains 40% carbon) was used as the carbon source at a concentration of 30 g/L. The concentrations of ammonium sulfate corresponding to these ratios are: 0.36, 0.71, 0.95, 1.43, 1.91, 2.86, and 4.6 g/L, respectively. Uracil was supplemented at 0.2 mM. As controls, strains were also grown in yeast extract-peptone with 50 g/L of glucose (media in which lipids do not accumulate at high levels) and yeast extract-peptone with 5% olive oil (v/v) (media in which lipids accumulate at high levels). Strain MF760 (10-14 ml of culture) was harvested after 4 days of growth at 30.degree. C., during which time the cultures were shaking at 250 rpm. Following harvesting, cells were washed three times with water, with the exception of the oil-grown cells which were washed three times in 0.5% BSA and one time with water before lipid extractions. Lipids were extracted as described in Folch J, Lees, M, and Stanley, G. H. S. J. Biol. Chem. 226: 497-509, 1957. In brief, cell pellets were resuspended in 6 ml of water. A 1 ml aliquot was transferred to a pre-weighed tube with a hole on the lid, spun down and the cell pellet lyophilized overnight to determine the dry cell weight. The remaining 5 ml were placed in a 15 ml Falcon tube and spun down. Cell pellets were frozen at -20.degree. C. until extractions were performed. Two to three volumes of a Zymolyase solution (2 mg/ml Zymolyase 100T in 1M Sorbitol, 50 mM EDTA and 0.01% .beta.-mercaptoethanol) was added to each cell pellet and placed at 37.degree. C. with constant agitation for 1 hr. Two volumes of cubic zirconia beads were added to each tube and vortexed for 15-20 min. Samples were viewed under a microscope to ensure cell breakage before continuing with extractions. After cell breakage was complete, 6 ml of extraction solvent was added (a 2:1 mix of chloroform and methanol) and mixed. The mixture was spun down for 5 min at 3000 rpm and the organic layer was transferred to a clean tube. NaCl was added to the remaining aqueous layer to make it a 0.29% NaCl solution. 6 ml of extraction solvent was added and mixed, and the mixture was spun down for 5 min. The organic layers were pooled and filtered through a 0.2 .mu.m an filter to get rid of any cell debris. The extract was washed with 0.2 volumes of 0.29% NaCl solution and another 6 ml of extraction solvent added and mixed. Mixtures were spun and the organic layer was placed in a pre-weighed glass vial, the solvent was evaporated under a flow on nitrogen and the vial was weighed again to determine the weight of the lipid extracted. The dry cell weight is used to determine the percentage of lipid per dry cell weight. The lipid accumulation results are in the Table 66 below:

TABLE-US-00023 TABLE 66 Lipid accumulation under various carbon:nitrogen ratio growth conditions C/N Ratio % lipid YNB 160 61 3% Glucose 80 49 60 34 40 17 30 16 20 14 10 15 YEP 5% Glucose 22 5% olive oil 38

Other nitrogen sources tested were proline (12% nitrogen), sodium glutamate (7% nitrogen), soy acid hydrolysate (12% nitrogen), and yeast extract-peptone (26.8% nitrogen). All nitrogen sources tested at C/N ratios of 80 (with glucose as a carbon source), had significantly larger lipid bodies than at C/N ratios of 10 (also with glucose as a carbon source).

[0346] Strains MF858 and MF921 were harvested after 4 days of growth at 30.degree. C. (3% glucose was used as the carbon source). Cells were washed three times with water and lipids extracted as described above. Lipid accumulation data for soy hydrolysate, yeast extract-peptone and yeast nitrogen base, used as a control, are listed in Table 67 below.

TABLE-US-00024 TABLE 67 Lipid accumulation under different carbon and nitrogen conditions with various nitrogen sources % lipid C/N Ratio MF858 MF921 Soy hydrolysate 80 36 36 60 36 35 10 14 15 Yeast Extract- 80 37 37 Peptone 10 15 14 Yeast Nitrogen 80 37 38 Base 10 13 11

12c. Determination of Lipid Levels Under High Carbon and Phosphate or Magnesium Limiting Conditions.

[0347] To test whether other nutrient limitations, under high carbon conditions, will allow for higher lipid accumulation, phosphate or magnesium limiting conditions were tested. For phosphate limiting conditions, yeast nitrogen base medium without phosphate was prepared. Shake flask testing was performed using carbon to phosphate ratios ranging from 5376 down to 42. This range corresponds to 7.8 mg/L up to 1 g/L, respectively, and the latter concentration corresponds to that are commonly used in yeast nitrogen base medium. Glucose, at 30 g/L, was used at the carbon source. Potassium phosphate monobasic (containing 28.7% phosphate) was used as the phosphate source.

[0348] For magnesium limiting conditions, yeast nitrogen base medium without magnesium was prepared. Shake flask testing was conducted using carbon to magnesium ratios ranging from 31360 down to 245. This range corresponds to 0.375 mg/L up to 0.5 g/L, and the latter magnesium concentration corresponds to that commonly used in yeast nitrogen base. Glucose, at 30 g/L, was used as the carbon source. Magnesium sulfate (containing 9.8% magnesium) was used as the magnesium source.

[0349] Strains MF858 and MF921 were harvested after 4 days of growth at 30.degree. C., during which time the cultures were shaking at 250 rpm. Cells were washed three times with water before lipid extraction. Lipids were extracted as described above. Lipid accumulation data is listed in the Table 68 below:

TABLE-US-00025 TABLE 68 Lipid accumulation in phosphate or magnesium limiting growth conditions % Lipid g/L MF858 MF921 phosphate 1 14 14 0.0625 18 20 0.0313 34 41 0.0156 62 63 0.0078 83 76 magnesium 0.5 12 11 0.0313 NA 16 0.0156 NA 25 0.0078 NA 42 0.0039 48 48

[0350] The following tables are referenced throughout the description. Each reference and information designated by each of the Genbank Accession and GI numbers are hereby incorporated by reference in their entirety.

TABLE-US-00026 Lengthy table referenced here US20140315279A1-20141023-T00001 Please refer to the end of the specification for access instructions.

TABLE-US-00027 Lengthy table referenced here US20140315279A1-20141023-T00002 Please refer to the end of the specification for access instructions.

TABLE-US-00028 Lengthy table referenced here US20140315279A1-20141023-T00003 Please refer to the end of the specification for access instructions.

TABLE-US-00029 Lengthy table referenced here US20140315279A1-20141023-T00004 Please refer to the end of the specification for access instructions.

TABLE-US-00030 Lengthy table referenced here US20140315279A1-20141023-T00005 Please refer to the end of the specification for access instructions.

TABLE-US-00031 Lengthy table referenced here US20140315279A1-20141023-T00006 Please refer to the end of the specification for access instructions.

TABLE-US-00032 Lengthy table referenced here US20140315279A1-20141023-T00007 Please refer to the end of the specification for access instructions.

TABLE-US-00033 Lengthy table referenced here US20140315279A1-20141023-T00008 Please refer to the end of the specification for access instructions.

TABLE-US-00034 Lengthy table referenced here US20140315279A1-20141023-T00009 Please refer to the end of the specification for access instructions.

TABLE-US-00035 Lengthy table referenced here US20140315279A1-20141023-T00010 Please refer to the end of the specification for access instructions.

TABLE-US-00036 Lengthy table referenced here US20140315279A1-20141023-T00011 Please refer to the end of the specification for access instructions.

TABLE-US-00037 Lengthy table referenced here US20140315279A1-20141023-T00012 Please refer to the end of the specification for access instructions.

TABLE-US-00038 Lengthy table referenced here US20140315279A1-20141023-T00013 Please refer to the end of the specification for access instructions.

TABLE-US-00039 Lengthy table referenced here US20140315279A1-20141023-T00014 Please refer to the end of the specification for access instructions.

TABLE-US-00040 Lengthy table referenced here US20140315279A1-20141023-T00015 Please refer to the end of the specification for access instructions.

TABLE-US-00041 Lengthy table referenced here US20140315279A1-20141023-T00016 Please refer to the end of the specification for access instructions.

TABLE-US-00042 Lengthy table referenced here US20140315279A1-20141023-T00017 Please refer to the end of the specification for access instructions.

TABLE-US-00043 Lengthy table referenced here US20140315279A1-20141023-T00018 Please refer to the end of the specification for access instructions.

TABLE-US-00044 Lengthy table referenced here US20140315279A1-20141023-T00019 Please refer to the end of the specification for access instructions.

TABLE-US-00045 Lengthy table referenced here US20140315279A1-20141023-T00020 Please refer to the end of the specification for access instructions.

TABLE-US-00046 Lengthy table referenced here US20140315279A1-20141023-T00021 Please refer to the end of the specification for access instructions.

TABLE-US-00047 Lengthy table referenced here US20140315279A1-20141023-T00022 Please refer to the end of the specification for access instructions.

TABLE-US-00048 Lengthy table referenced here US20140315279A1-20141023-T00023 Please refer to the end of the specification for access instructions.

TABLE-US-00049 Lengthy table referenced here US20140315279A1-20141023-T00024 Please refer to the end of the specification for access instructions.

TABLE-US-00050 Lengthy table referenced here US20140315279A1-20141023-T00025 Please refer to the end of the specification for access instructions.

TABLE-US-00051 Lengthy table referenced here US20140315279A1-20141023-T00026 Please refer to the end of the specification for access instructions.

TABLE-US-00052 Lengthy table referenced here US20140315279A1-20141023-T00027 Please refer to the end of the specification for access instructions.

TABLE-US-00053 Lengthy table referenced here US20140315279A1-20141023-T00028 Please refer to the end of the specification for access instructions.

TABLE-US-00054 Lengthy table referenced here US20140315279A1-20141023-T00029 Please refer to the end of the specification for access instructions.

TABLE-US-00055 Lengthy table referenced here US20140315279A1-20141023-T00030 Please refer to the end of the specification for access instructions.

TABLE-US-00056 Lengthy table referenced here US20140315279A1-20141023-T00031 Please refer to the end of the specification for access instructions.

TABLE-US-00057 Lengthy table referenced here US20140315279A1-20141023-T00032 Please refer to the end of the specification for access instructions.

TABLE-US-00058 Lengthy table referenced here US20140315279A1-20141023-T00033 Please refer to the end of the specification for access instructions.

TABLE-US-00059 Lengthy table referenced here US20140315279A1-20141023-T00034 Please refer to the end of the specification for access instructions.

TABLE-US-00060 Lengthy table referenced here US20140315279A1-20141023-T00035 Please refer to the end of the specification for access instructions.

TABLE-US-00061 Lengthy table referenced here US20140315279A1-20141023-T00036 Please refer to the end of the specification for access instructions.

TABLE-US-00062 Lengthy table referenced here US20140315279A1-20141023-T00037 Please refer to the end of the specification for access instructions.

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TABLE-US-00068 Lengthy table referenced here US20140315279A1-20141023-T00043 Please refer to the end of the specification for access instructions.

TABLE-US-00069 Lengthy table referenced here US20140315279A1-20141023-T00044 Please refer to the end of the specification for access instructions.

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TABLE-US-00071 Lengthy table referenced here US20140315279A1-20141023-T00046 Please refer to the end of the specification for access instructions.

TABLE-US-00072 Lengthy table referenced here US20140315279A1-20141023-T00047 Please refer to the end of the specification for access instructions.

TABLE-US-00073 Lengthy table referenced here US20140315279A1-20141023-T00048 Please refer to the end of the specification for access instructions.

TABLE-US-00074 Lengthy table referenced here US20140315279A1-20141023-T00049 Please refer to the end of the specification for access instructions.

TABLE-US-00075 Lengthy table referenced here US20140315279A1-20141023-T00050 Please refer to the end of the specification for access instructions.

TABLE-US-00076 Lengthy table referenced here US20140315279A1-20141023-T00051 Please refer to the end of the specification for access instructions.

TABLE-US-00077 Lengthy table referenced here US20140315279A1-20141023-T00052 Please refer to the end of the specification for access instructions.

TABLE-US-00078 Lengthy table referenced here US20140315279A1-20141023-T00053 Please refer to the end of the specification for access instructions.

TABLE-US-00079 Lengthy table referenced here US20140315279A1-20141023-T00054 Please refer to the end of the specification for access instructions.

TABLE-US-00080 Lengthy table referenced here US20140315279A1-20141023-T00055 Please refer to the end of the specification for access instructions.

TABLE-US-00081 Lengthy table referenced here US20140315279A1-20141023-T00056 Please refer to the end of the specification for access instructions.

TABLE-US-00082 Lengthy table referenced here US20140315279A1-20141023-T00057 Please refer to the end of the specification for access instructions.

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TABLE-US-00084 Lengthy table referenced here US20140315279A1-20141023-T00059 Please refer to the end of the specification for access instructions.

TABLE-US-00085 Lengthy table referenced here US20140315279A1-20141023-T00060 Please refer to the end of the specification for access instructions.

TABLE-US-00086 Lengthy table referenced here US20140315279A1-20141023-T00061 Please refer to the end of the specification for access instructions.

TABLE-US-00087 Lengthy table referenced here US20140315279A1-20141023-T00062 Please refer to the end of the specification for access instructions.

TABLE-US-00088 Lengthy table referenced here US20140315279A1-20141023-T00063 Please refer to the end of the specification for access instructions.

TABLE-US-00089 Lengthy table referenced here US20140315279A1-20141023-T00064 Please refer to the end of the specification for access instructions.

TABLE-US-00090 Lengthy table referenced here US20140315279A1-20141023-T00065 Please refer to the end of the specification for access instructions.

TABLE-US-00091 Lengthy table referenced here US20140315279A1-20141023-T00066 Please refer to the end of the specification for access instructions.

TABLE-US-00092 Lengthy table referenced here US20140315279A1-20141023-T00067 Please refer to the end of the specification for access instructions.

TABLE-US-00093 Lengthy table referenced here US20140315279A1-20141023-T00068 Please refer to the end of the specification for access instructions.

TABLE-US-00094 Lengthy table referenced here US20140315279A1-20141023-T00069 Please refer to the end of the specification for access instructions.

TABLE-US-00095 Lengthy table referenced here US20140315279A1-20141023-T00070 Please refer to the end of the specification for access instructions.

TABLE-US-00096 Lengthy table referenced here US20140315279A1-20141023-T00071 Please refer to the end of the specification for access instructions.

TABLE-US-00097 Lengthy table referenced here US20140315279A1-20141023-T00072 Please refer to the end of the specification for access instructions.

TABLE-US-00098 Lengthy table referenced here US20140315279A1-20141023-T00073 Please refer to the end of the specification for access instructions.

TABLE-US-00099 Lengthy table referenced here US20140315279A1-20141023-T00074 Please refer to the end of the specification for access instructions.

TABLE-US-00100 Lengthy table referenced here US20140315279A1-20141023-T00075 Please refer to the end of the specification for access instructions.

TABLE-US-00101 Lengthy table referenced here US20140315279A1-20141023-T00076 Please refer to the end of the specification for access instructions.

TABLE-US-00102 Lengthy table referenced here US20140315279A1-20141023-T00077 Please refer to the end of the specification for access instructions.

TABLE-US-00103 Lengthy table referenced here US20140315279A1-20141023-T00078 Please refer to the end of the specification for access instructions.

TABLE-US-00104 Lengthy table referenced here US20140315279A1-20141023-T00079 Please refer to the end of the specification for access instructions.

TABLE-US-00105 Lengthy table referenced here US20140315279A1-20141023-T00080 Please refer to the end of the specification for access instructions.

TABLE-US-00106 Lengthy table referenced here US20140315279A1-20141023-T00081 Please refer to the end of the specification for access instructions.

TABLE-US-00107 Lengthy table referenced here US20140315279A1-20141023-T00082 Please refer to the end of the specification for access instructions.

TABLE-US-00108 Lengthy table referenced here US20140315279A1-20141023-T00083 Please refer to the end of the specification for access instructions.

TABLE-US-00109 Lengthy table referenced here US20140315279A1-20141023-T00084 Please refer to the end of the specification for access instructions.

TABLE-US-00110 Lengthy table referenced here US20140315279A1-20141023-T00085 Please refer to the end of the specification for access instructions.

TABLE-US-00111 Lengthy table referenced here US20140315279A1-20141023-T00086 Please refer to the end of the specification for access instructions.

TABLE-US-00112 Lengthy table referenced here US20140315279A1-20141023-T00087 Please refer to the end of the specification for access instructions.

TABLE-US-00113 Lengthy table referenced here US20140315279A1-20141023-T00088 Please refer to the end of the specification for access instructions.

TABLE-US-00114 Lengthy table referenced here US20140315279A1-20141023-T00089 Please refer to the end of the specification for access instructions.

TABLE-US-00115 Lengthy table referenced here US20140315279A1-20141023-T00090 Please refer to the end of the specification for access instructions.

TABLE-US-00116 Lengthy table referenced here US20140315279A1-20141023-T00091 Please refer to the end of the specification for access instructions.

TABLE-US-00117 Lengthy table referenced here US20140315279A1-20141023-T00092 Please refer to the end of the specification for access instructions.

TABLE-US-00118 Lengthy table referenced here US20140315279A1-20141023-T00093 Please refer to the end of the specification for access instructions.

TABLE-US-00119 Lengthy table referenced here US20140315279A1-20141023-T00094 Please refer to the end of the specification for access instructions.

EQUIVALENTS

[0351] Those skilled in the art will recognize, or be able to understand that the foregoing description and examples are illustrative of practicing the provided invention. Those skilled in the art will be able to ascertain using no more than routine experimentation, many variations of the detail presented herein may be made to the specific embodiments of the invention described herein without departing from the spirit and scope of the present invention.

TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140315279A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Sequence CWU 1

1

152120DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1ctgggtgacc tggaagcctt 20223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 2aagatcaatc cgtagaagtt cag 23323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 3aagcgattac aatcttcctt tgg 23422DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 4ccagtccatc aactcagtct ca 22523DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 5gcattgctta ttacgaagac tac 23623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 6ccactgtcct ccactacaaa cac 23731DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 7cacaaactag tttgccacct acaagccaga t 31831DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 8cacaaggtac caatgtgaaa gtgcgcgtga t 31928DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 9cacaaggtac cagagaccgg gttggcgg 281040DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 10cacaagcggc cgcgctagca tggggatcga tctcttatat 401141DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 11cacaagcggc cgcgctagcg aatgattctt atactcagaa g 411240DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 12cacaagcggc cgcacgcgtg caattaacag atagtttgcc 401333DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 13cacaagctag ctggggatgc gatctcttat atc 331432DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 14cattcactag tggtgtgttc tgtggagcat tc 321536DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 15cacacggtct catcgaggtg tagtggtagt gcagtg 361640DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 16cacactctag acacaaaaat gacccagtct gtgaaggtgg 401733DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 17cacacacgcg tacacctatg accgtatgca aat 331841DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 18tctagacaca aaaatgctct cgagaaacct cagcaagttt g 411941DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 19gtggttgctg gccggatgag accggctcga gcaaacttgc t 412041DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 20cagcaaccac atccacacac acccgactat tctctgtctc c 412137DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 21ggatcccgtc tcggacagac gtcgggcgga gacagag 3722130DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 22tctagacaca aaaatgctct cgagaaacct cagcaagttt gctcgagccg gtctcatccg 60gccagcaacc acatccacac acacccgact attctctgtc tccgcccgac gtctgtccga 120gacgggatcc 1302335DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 23cggatcccgt ctcggacatt tcttgcgaca caatg 352434DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 24ctctagacac aaaaatgctg agagtcggac gaat 3425261DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 25ctctagacac aaaaatgctg agagtcggac gaattggcac caagacccta gccagcagca 60gcctgcgttt cgtggcaggt gctcggccca aatccacgct caccgaggcc gtgctggaga 120ccacagggct gctgaaaacc acgccccaaa accccgagtg gtctggagcc gtcaagcagg 180catctcgtct ggtggagacc gacactccga tccgagaccc gttttccatt gtgtcgcaag 240aaatgtccga gacgggatcc g 261261020DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 26ggatcccgtc tcttgtctga tgccaaagtc tctacaaagc ctcacgagat gctcgctgcc 60acactctctc aggaaatggc cgctgtcaac gcccttattc gaacccgtat ggcttctgaa 120cacgccccta gaatccccga agttacagcc cacctcgtcg aagccggcgg aaaacgattg 180cgacctatgc tgacacttgc tgctgcccga ctgtgtggat accagggcga agatcatgtc 240aaactggctg ccactgttga atttattcac actgctacac ttttgcacga tgatgtcgtg 300gacgagtctg gacaaagacg tggacgacct actgctaatc ttctttggga taacaagtcc 360tctgttcttg tgggcgacta tctttttgca cgatcgttcc agctgatggt cgaaaccggt 420tcccttcgag tgcttgacat cctcgctaac gctgccgcga caattgccga aggtgaagtg 480ctccagatga ccgctgcttc cgatcttaga actgatgaat ccgtttacct tcaggtcgtt 540cgaggtaaaa ctgccgctct tttttctgct gctactgaag ttggtggtgt tattgccgga 600gtccccgaag cccaagttcg agcacttttt gaatacggtg atgcgctggg aattgcattt 660cagattgctg atgacctcct cgactaccaa ggtgatgcta aggccactgg aaagaatgtt 720ggagatgact tcagagaaag aaaactcaca ttgcctgtta ttaaagccgt cgctcaagct 780actgatgagg aacgagcgtt ttgggttaga actattgaaa agggaaagca agctgaagga 840gatcttgaac aggctctcgc tctcatggaa aaatacggaa cacttgccgc aaccagagcc 900gatgcgcatg cttgggccga aaaagcacga accgccctcg aactgttgcc gaatcacgaa 960atcagaacaa tgctctccga cctcgccgat tatgtcgttg ctagattgtc ttaaacgcgt 1020271017DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 27ggatcccgtc tcttgtctct tgatcacgct gccaccaaac ctcatgaaca acttgccgcc 60gcccttgccg acgaccttac cgcagttaac gctatgatta gagaccgaat ggccagcgaa 120catgccccca gaattccaca agttaccgca cacctcgttg aagccggagg taaaagactt 180cgtccaatgc tgacccttgc cgccgcccgt atgtgtggct acgacggacc ttaccacatc 240caccttgcag ccaccgttga gttcattcac accgccacct tgctccatga cgatgttgtt 300gatgagtcct cccaacgtcg tggtcgacct accgctaatc ttctgtggga taatacctct 360tctgttttgg ttggagacta cctctttgca cgatcttttc agttgatggt tgagaccgga 420tcgcttcgag ttcttgacat cctggccaat gcttctgcta ccatcgccga aggtgaggtt 480ctgcaactta ccgccgccgc tgaccttgca actaccgagg acatttacat caaagttgtt 540cgaggaaaga ctgctgcact cttttctgct gctatggaag ttggtggaga aatcgccggt 600caggacccgg ctattaaaca ggctctcttt gactacggcg acgctctcgg aatttctttt 660cagattgttg atgacctgtt ggattacggt ggaacaaaag ctacaggaaa gaacgttggt 720gatgacttcc gagaaagaaa gctcaccctc cctgttattc gtgccgttgc tgctgctgat 780gctgatgaaa gagctttttg ggaacgaaca attcaaaaag gaagacaaca agatggagac 840ctggaccatg caattgccct tcttcataga cacggaaccc ttgaatccac acgacaagac 900gcaatccttt gggccgctaa agctaaagaa gcactcggag ttatcccgga ccatgctctt 960aaaaccatgc tcgttgacct tgctgactac gttgttgctc gactcaccta aacgcgt 10172828DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28cacacggtac cggtataggc acaagtgc 282932DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29cactcggatc cgtacgtctg tggtgctgtg gt 323036DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30cacacggatc cgctagctct gagaaacctc accatg 363131DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31cacactctag attttcttgg ccatgaacgg t 31321210DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 32ttctagacac aaaaatgctg cgagtgggcc gaatcggcac caagaccctg gcctcttctt 60ctctgcgatt cgtggccggc gcccgaccca agtctaccct gaccgaggcc gtgctggaga 120ccaccggcct gctgaagacc accccccaga accccgagtg gtctggcgcc gtgaagcagg 180cctctcgact ggtggagacc gacaccccca tccgagaccc cttctctatc gtgtctcagg 240agatgcaggg ccaggccccc acccccgacg gccaggtggc cgacgccgtg accggcaact 300gggtggacat ccacgccccc gcctggtctc gaccctacct gcgactgtct cgagccgacc 360gacccatcgg cacctggctg ctgctgatcc cctgttggtg gggcctggcc ctggccatgc 420tggacggcca ggacgcccga tggggcgacc tgtggatcgc cctgggctgt gccatcggcg 480ccttcctgat gcgaggcgcc ggctgtacct ggaacgacat caccgaccga gagttcgacg 540gccgagtgga gcgaacccga tctcgaccca tcccctctgg ccaggtgtct gtgcgaatgg 600ccgtggtgtg gatgatcgcc caggccctgc tggccctgat gatcctgctg accttcaacc 660gaatggccat cgccatgggc gtgctgtctc tgctgcccgt ggccgtgtac cccttcgcca 720agcgattcac ctggtggccc caggtgttcc tgggcctggc cttcaactgg ggcgccctgc 780tggcctggac cgcccactct ggctctctgg gctggggcgc cctgttcctg tacctggccg 840gcatcgcctg gaccctgttc tacgacacca tctacgccca ccaggacacc gaggacgacg 900ccctgatcgg cgtgaagtct accgcccgac tgttcggcgc ccagaccccc cgatggatgt 960cttacttcct ggtggccacc gtgtctctga tgggcatcgc cgtgttcgag gccgccctgc 1020ccgacgcctc tatcctggcc ctggtgctgg ccctggccgg cccctgggcc atgggctggc 1080acatggcctg gcagctgcga ggcctggacc tggacgacaa cggcaagctg ctgcagctgt 1140tccgagtgaa ccgagacacc ggcctgatcc ccctgatctt cttcgtgatc gccctgttcg 1200cctaacgcgt 1210331135DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 33ttctagacac aaaaatgctg ggctcttgtg gcgccggcct ggtgcgaggc ctgcgagccg 60agacccaggc ctggctgtgg ggcacccgag gccgatctct ggccctggtg cacgccgccc 120gaggcctgca cgccgccaac tggcagccct ctcccggcca gggcccccga ggccgacccc 180tgtctctgtc tgccgccgcc gtggtgaact ctgccccccg acccctgcag ccctacctgc 240gactgatgcg actggacaag cccatcggca cctggctgct gtacctgccc tgtacctggt 300ctatcggcct ggccgccgac cccggctgtc tgcccgactg gtacatgctg tctctgttcg 360gcaccggcgc cgtgctgatg cgaggcgccg gctgtaccat caacgacatg tgggaccgag 420actacgacaa gaaggtgacc cgaaccgcct ctcgacccat cgccgccggc gacatctcta 480ccttccgatc tttcgtgttc ctgggcggcc agctgaccct ggccctgggc gtgctgctgt 540gtctgaacta ctactctatc gccctgggcg ccgcctctct gctgctggtg accacctacc 600ccctgatgaa gcgaatcacc tactggcccc agctggccct gggcctgacc ttcaactggg 660gcgccctgct gggctggtct gccgtgaagg gctcttgtga cccctctgtg tgtctgcccc 720tgtacttctc tggcatcatg tggaccctga tctacgacac catctacgcc caccaggaca 780agaaggacga cgccctgatc ggcctgaagt ctaccgccct gctgttccga gaggacacca 840agaagtggct gtctggcttc tctgtggcca tgctgggcgc cctgtctctg gtgggcgtga 900actctggcca gaccatgccc tactacaccg ccctggccgc cgtgggcgcc cacctggccc 960accagatcta caccctggac atcaaccgac ccgaggactg ttgggagaag ttcacctcta 1020accgaaccat cggcctgatc atcttcctgg gcatcgtgct gggcaacctg tgtaaggcca 1080aggagaccga caagacccga aagaacatcg agaaccgaat ggagaactaa cgcgt 1135341093DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 34ttctagacac aaaaatgcag aaggactctc tgaacaacat caacatctct gccgagcagg 60tgctgatcac ccccgacgag ctgaaggcca agttccccct gaacgacgtg gagcagcgag 120acatcgccca ggcccgagcc accatcgccg acatcatcca cggccgagac gaccgactgc 180tgatcgtgtg tggcccctgt tctatccacg acaccgacgc cgccctggag tacgcccgac 240gactgcagct gctggccgcc gagctgaacg accgactgta catcgtgatg cgagtgtact 300tcgagaagcc ccgaaccacc gtgggctgga agggcctgat caacgacccc ttcatggacg 360gctctttcga cgtggagtct ggcctgcaca tcgcccgagg cctgctgctg cagctggtga 420acatgggcct gcccctggcc accgaggccc tggacctgaa ctctccccag tacctgggcg 480acctgttctc ttggtctgcc atcggcgccc gaaccaccga gtctcagacc caccgagaga 540tggcctctgg cctgtctatg cccgtgggct tcaagaacgg caccgacggc tctctgggca 600ccgccatcaa cgccatgcga gccgccgcca tgccccaccg attcgtgggc atcaaccaga 660ccggccaggt gtgtctgctg cagacccagg gcaacggcga cggccacgtg atcctgcgag 720gcggcaagac ccccaactac tctgcccagg acgtggccga gtgtgagaag cagatgcagg 780aggccggcct gcgacccgcc ctgatgatcg actgttctca cggcaactct aacaaggact 840accgacgaca gcccctggtg gtggagtctg ccatcgagca gatcaaggcc ggcaaccgat 900ctatcatcgg cctgatgctg gagtctcacc tgaacgaggg ctctcagtct tctgagcagc 960cccgatctga catgcgatac ggcgtgtctg tgaccgacgc ctgtatctct tgggagtcta 1020ccgagaccct gctgcgatct gtgcaccagg acctgtctgc cgcccgagtg aagcactctg 1080gcgagtaacg cgt 109335553DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 35ttctagacac aaaaatgtct gacgacgcct ctaccctgct gcgaaccatc tcttggttca 60ccgagccccc ctctgtgctg cccgagcaca tcggcgactg gctgatggag acctcttcta 120tgacccagcg actggagaag tactgtgccc agctgcgagt gaccctgtgt cgagagggct 180tcatcacccc ccagatgctg ggcgaggagc gagaccagct gcccgccgac gagcgatact 240ggctgcgaga ggtggtgctg tacggcgacg accgaccctg gctgttcggc cgaaccatcg 300tgccccagca gaccctggag ggctctggcg ccgccctgac caagatcggc aaccagcccc 360tgggccgata cctgttcgag cagaagtctc tgacccgaga ctacatccac accggctgtt 420gtgagggcct gtgggcccga cgatctcgac tgtgtctgtc tggccacccc ctgctgctga 480ccgagctgtt cctgcccgag tctcccgtgt actacacccc cggcgacgag ggctggcagg 540tgatctaacg cgt 5533623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 36ccttctagtc gtacgtagtc agc 233725DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 37ccactgatct agaatctctt tctgg 253824DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38ggctcattgc gcatgctaac atcg 243925DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39cgacgatgct atgagcttct agacg 254036DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 40ttctagacac aaaaatggct gcagaccaat tggtga 364133DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 41cattaattct tctaaaggac gtattttctt atc 334221DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 42gttctctgga cgacctagag g 214333DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 43cacacacgcg tacacctatg accgtatgca aat 334440DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 44cacactctag acacaaaaat gacccagtct gtgaaggtgg 404534DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 45cacacggatc ctataatgcc ttccgcaacg accg 344635DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 46cacacactag ttaaatttgg acctcaacac gaccc 354740DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 47cacacggatc caatataaat gtctgcgaag agcatcctcg 404834DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 48cacacgcatg cttaagcttg gaactccacc gcac 344947DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 49cacacggatc caattttcaa aaattcttac tttttttttg gatggac 475041DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 50cacacggatc cttttttctc cttgacgtta aagtatagag g 415138DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 51cacacgagct caaaaatgga caatcaggct acacagag 385242DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 52cacaccctag gtcacttttc ttcaatggtt ctcttgaaat tg 425346DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 53cacacgagct cggaatattc aactgttttt ttttatcatg ttgatg 465442DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 54cacacggatc cttcttgaaa atatgcactc tatatctttt ag 425543DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 55cacacgctag ctacaaaatg ttgtcactca aacgcatagc aac 435638DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 56cacacgtcga cttaatgatc tcggtatacg agaggaac 38571341DNAYarrowia lipolytica 57atgtcgcaac cccagaacgt tggaatcaaa gccctcgaga tctacgtgcc ttctcgaatt 60gtcaaccagg ctgagctcga gaagcacgac ggtgtcgctg ctggcaagta caccattggt 120cttggtcaga ccaacatggc ctttgtcgac gacagagagg acatctattc ctttgccctg 180accgccgtct ctcgactgct caagaacaac aacatcgacc ctgcatctat tggtcgaatc 240gaggttggta ctgaaaccct tctggacaag tccaagtccg tcaagtctgt gctcatgcag 300ctctttggcg agaacagcaa cattgagggt gtggacaacg tcaacgcctg ctacggagga 360accaacgccc tgttcaacgc tatcaactgg gttgagggtc gatcttggga cggccgaaac 420gccatcgtcg ttgccggtga cattgccctc tacgcaaagg gcgctgcccg acccaccgga 480ggtgccggct gtgttgccat gctcattggc cccgacgctc ccctggttct tgacaacgtc 540cacggatctt acttcgagca tgcctacgat ttctacaagc ctgatctgac ctccgagtac 600ccctatgttg atggccacta ctccctgacc tgttacacaa aggccctcga caaggcctac 660gctgcctaca acgcccgagc cgagaaggtc ggtctgttca aggactccga caagaagggt 720gctgaccgat ttgactactc tgccttccac gtgcccacct gcaagcttgt caccaagtct 780tacgctcgac ttctctacaa cgactacctc aacgacaaga gcctgtacga gggccaggtc 840cccgaggagg ttgctgccgt ctcctacgat gcctctctca ccgacaagac cgtcgagaag 900accttccttg gtattgccaa ggctcagtcc gccgagcgaa tggctccttc tctccaggga 960cccaccaaca ccggtaacat gtacaccgcc tctgtgtacg cttctctcat ctctctgctg 1020acttttgtcc ccgctgagca gctgcagggc

aagcgaatct ctctcttctc ttacggatct 1080ggtcttgctt ccactctttt ctctctgacc gtcaagggag acatttctcc catcgtcaag 1140gcctgcgact tcaaggctaa gctcgatgac cgatccaccg agactcccgt cgactacgag 1200gctgccaccg atctccgaga gaaggcccac ctcaagaaga actttgagcc ccagggagac 1260atcaagcaca tcaagtctgg cgtctactac ctcaccaaca tcgatgacat gttccgacga 1320aagtacgaga tcaagcagta g 134158446PRTYarrowia lipolytica 58Met Ser Gln Pro Gln Asn Val Gly Ile Lys Ala Leu Glu Ile Tyr Val 1 5 10 15 Pro Ser Arg Ile Val Asn Gln Ala Glu Leu Glu Lys His Asp Gly Val 20 25 30 Ala Ala Gly Lys Tyr Thr Ile Gly Leu Gly Gln Thr Asn Met Ala Phe 35 40 45 Val Asp Asp Arg Glu Asp Ile Tyr Ser Phe Ala Leu Thr Ala Val Ser 50 55 60 Arg Leu Leu Lys Asn Asn Asn Ile Asp Pro Ala Ser Ile Gly Arg Ile 65 70 75 80 Glu Val Gly Thr Glu Thr Leu Leu Asp Lys Ser Lys Ser Val Lys Ser 85 90 95 Val Leu Met Gln Leu Phe Gly Glu Asn Ser Asn Ile Glu Gly Val Asp 100 105 110 Asn Val Asn Ala Cys Tyr Gly Gly Thr Asn Ala Leu Phe Asn Ala Ile 115 120 125 Asn Trp Val Glu Gly Arg Ser Trp Asp Gly Arg Asn Ala Ile Val Val 130 135 140 Ala Gly Asp Ile Ala Leu Tyr Ala Lys Gly Ala Ala Arg Pro Thr Gly 145 150 155 160 Gly Ala Gly Cys Val Ala Met Leu Ile Gly Pro Asp Ala Pro Leu Val 165 170 175 Leu Asp Asn Val His Gly Ser Tyr Phe Glu His Ala Tyr Asp Phe Tyr 180 185 190 Lys Pro Asp Leu Thr Ser Glu Tyr Pro Tyr Val Asp Gly His Tyr Ser 195 200 205 Leu Thr Cys Tyr Thr Lys Ala Leu Asp Lys Ala Tyr Ala Ala Tyr Asn 210 215 220 Ala Arg Ala Glu Lys Val Gly Leu Phe Lys Asp Ser Asp Lys Lys Gly 225 230 235 240 Ala Asp Arg Phe Asp Tyr Ser Ala Phe His Val Pro Thr Cys Lys Leu 245 250 255 Val Thr Lys Ser Tyr Ala Arg Leu Leu Tyr Asn Asp Tyr Leu Asn Asp 260 265 270 Lys Ser Leu Tyr Glu Gly Gln Val Pro Glu Glu Val Ala Ala Val Ser 275 280 285 Tyr Asp Ala Ser Leu Thr Asp Lys Thr Val Glu Lys Thr Phe Leu Gly 290 295 300 Ile Ala Lys Ala Gln Ser Ala Glu Arg Met Ala Pro Ser Leu Gln Gly 305 310 315 320 Pro Thr Asn Thr Gly Asn Met Tyr Thr Ala Ser Val Tyr Ala Ser Leu 325 330 335 Ile Ser Leu Leu Thr Phe Val Pro Ala Glu Gln Leu Gln Gly Lys Arg 340 345 350 Ile Ser Leu Phe Ser Tyr Gly Ser Gly Leu Ala Ser Thr Leu Phe Ser 355 360 365 Leu Thr Val Lys Gly Asp Ile Ser Pro Ile Val Lys Ala Cys Asp Phe 370 375 380 Lys Ala Lys Leu Asp Asp Arg Ser Thr Glu Thr Pro Val Asp Tyr Glu 385 390 395 400 Ala Ala Thr Asp Leu Arg Glu Lys Ala His Leu Lys Lys Asn Phe Glu 405 410 415 Pro Gln Gly Asp Ile Lys His Ile Lys Ser Gly Val Tyr Tyr Leu Thr 420 425 430 Asn Ile Asp Asp Met Phe Arg Arg Lys Tyr Glu Ile Lys Gln 435 440 445 591350DNAYarrowia lipolytica 59atggactaca tcatttcggc gccaggcaaa gtgattctat ttggtgaaca tgccgctgtg 60tttggtaagc ctgcgattgc agcagccatc gacttgcgaa catacctgct tgtcgaaacc 120acaacatccg acaccccgac agtcacgttg gagtttccag acatccactt gaacttcaag 180gtccaggtgg acaagctggc atctctcaca gcccagacca aggccgacca tctcaattgg 240tcgactccca aaactctgga taagcacatt ttcgacagct tgtctagctt ggcgcttctg 300gaagaacctg ggctcactaa ggtccagcag gccgctgttg tgtcgttctt gtacctctac 360atccacctat gtcccccttc tgtgtgcgaa gattcatcaa actgggtagt tcgatcaacg 420ctgcctatcg gcgcgggcct gggctcttcc gcatccattt gtgtctgttt ggctgcaggt 480cttctggttc tcaacggcca gctgagcatt gaccaggcaa gagatttcaa gtccctgacc 540gagaagcagc tgtctctggt ggacgactgg tccttcgtcg gtgaaatgtg cattcacggc 600aacccgtcgg gcatcgacaa tgctgtggct actcagggag gtgctctgtt gttccagcga 660cctaacaacc gagtccctct tgttgacatt cccgagatga agctgctgct taccaatacg 720aagcatcctc gatctaccgc agacctggtt ggtggagtcg gagttctcac taaagagttt 780ggctccatca tggatcccat catgacttca gtaggcgaga tttccaacca ggccatggag 840atcatttcta gaggcaagaa gatggtggac cagtctaacc ttgagattga gcagggtatc 900ttgcctcaac ccacctctga ggatgcctgc aacgtgatgg aagatggagc tactcttcaa 960aagttgagag atatcggttc ggaaatgcag catctagtga gaatcaatca cggcctgctt 1020atcgctatgg gtgtttccca cccgaagctc gaaatcattc gaactgcctc cattgtccac 1080aacctgggtg agaccaagct cactggtgct ggaggaggag gttgcgccat cactctagtc 1140acttctaaag acaagactgc gacccagctg gaggaaaatg tcattgcttt cacagaggag 1200atggctaccc atggcttcga ggtgcacgag actactattg gtgccagagg agttggtatg 1260tgcattgacc atccctctct caagactgtt gaagccttca agaaggtgga gcgggcggat 1320ctcaaaaaca tcggtccctg gacccattag 135060449PRTYarrowia lipolytica 60Met Asp Tyr Ile Ile Ser Ala Pro Gly Lys Val Ile Leu Phe Gly Glu 1 5 10 15 His Ala Ala Val Phe Gly Lys Pro Ala Ile Ala Ala Ala Ile Asp Leu 20 25 30 Arg Thr Tyr Leu Leu Val Glu Thr Thr Thr Ser Asp Thr Pro Thr Val 35 40 45 Thr Leu Glu Phe Pro Asp Ile His Leu Asn Phe Lys Val Gln Val Asp 50 55 60 Lys Leu Ala Ser Leu Thr Ala Gln Thr Lys Ala Asp His Leu Asn Trp 65 70 75 80 Ser Thr Pro Lys Thr Leu Asp Lys His Ile Phe Asp Ser Leu Ser Ser 85 90 95 Leu Ala Leu Leu Glu Glu Pro Gly Leu Thr Lys Val Gln Gln Ala Ala 100 105 110 Val Val Ser Phe Leu Tyr Leu Tyr Ile His Leu Cys Pro Pro Ser Val 115 120 125 Cys Glu Asp Ser Ser Asn Trp Val Val Arg Ser Thr Leu Pro Ile Gly 130 135 140 Ala Gly Leu Gly Ser Ser Ala Ser Ile Cys Val Cys Leu Ala Ala Gly 145 150 155 160 Leu Leu Val Leu Asn Gly Gln Leu Ser Ile Asp Gln Ala Arg Asp Phe 165 170 175 Lys Ser Leu Thr Glu Lys Gln Leu Ser Leu Val Asp Asp Trp Ser Phe 180 185 190 Val Gly Glu Met Cys Ile His Gly Asn Pro Ser Gly Ile Asp Asn Ala 195 200 205 Val Ala Thr Gln Gly Gly Ala Leu Leu Phe Gln Arg Pro Asn Asn Arg 210 215 220 Val Pro Leu Val Asp Ile Pro Glu Met Lys Leu Leu Leu Thr Asn Thr 225 230 235 240 Lys His Pro Arg Ser Thr Ala Asp Leu Val Gly Gly Val Gly Val Leu 245 250 255 Thr Lys Glu Phe Gly Ser Ile Met Asp Pro Ile Met Thr Ser Val Gly 260 265 270 Glu Ile Ser Asn Gln Ala Met Glu Ile Ile Ser Arg Gly Lys Lys Met 275 280 285 Val Asp Gln Ser Asn Leu Glu Ile Glu Gln Gly Ile Leu Pro Gln Pro 290 295 300 Thr Ser Glu Asp Ala Cys Asn Val Met Glu Asp Gly Ala Thr Leu Gln 305 310 315 320 Lys Leu Arg Asp Ile Gly Ser Glu Met Gln His Leu Val Arg Ile Asn 325 330 335 His Gly Leu Leu Ile Ala Met Gly Val Ser His Pro Lys Leu Glu Ile 340 345 350 Ile Arg Thr Ala Ser Ile Val His Asn Leu Gly Glu Thr Lys Leu Thr 355 360 365 Gly Ala Gly Gly Gly Gly Cys Ala Ile Thr Leu Val Thr Ser Lys Asp 370 375 380 Lys Thr Ala Thr Gln Leu Glu Glu Asn Val Ile Ala Phe Thr Glu Glu 385 390 395 400 Met Ala Thr His Gly Phe Glu Val His Glu Thr Thr Ile Gly Ala Arg 405 410 415 Gly Val Gly Met Cys Ile Asp His Pro Ser Leu Lys Thr Val Glu Ala 420 425 430 Phe Lys Lys Val Glu Arg Ala Asp Leu Lys Asn Ile Gly Pro Trp Thr 435 440 445 His 611257DNAYarrowia lipolytica 61atgaccacct attcggctcc gggaaaggcc ctcctttgcg gcggttattt ggttattgat 60ccggcgtatt cagcatacgt cgtgggcctc tcggcgcgta tttacgcgac agtttcggct 120tccgaggcct ccaccacctc tgtccatgtc gtctctccgc agtttgacaa gggtgaatgg 180acctacaact acacgaacgg ccagctgacg gccatcggac acaacccatt tgctcacgcg 240gccgtcaaca ccgttctgca ttacgttcct cctcgaaacc tccacatcaa catcagcatc 300aaaagtgaca acgcgtacca ctcgcaaatt gacagcacgc agagaggcca gtttgcatac 360cacaaaaagg cgatccacga ggtgcctaaa acgggcctcg gtagctccgc tgctcttacc 420accgttcttg tggcagcttt gctcaagtca tacggcattg atcccttgca taacacccac 480ctcgttcaca acctgtccca ggttgcacac tgctcggcac agaagaagat tgggtctgga 540tttgacgtgg cttcggccgt ttgtggctct ctagtctata gacgtttccc ggcggagtcc 600gtgaacatgg tcattgcagc tgaagggacc tccgaatacg gggctctgtt gagaactacc 660gttaatcaaa agtggaaggt gactctggaa ccatccttct tgccgccggg aatcagcctg 720cttatgggag acgtccaggg aggatctgag actccaggta tggtggccaa ggtgatggca 780tggcgaaaag caaagccccg agaagccgag atggtgtgga gagatctcaa cgctgccaac 840atgctcatgg tcaagttgtt caacgacctg cgcaagctct ctctcactaa caacgaggcc 900tacgaacaac ttttggccga ggctgctcct ctcaacgctc taaagatgat aatgttgcag 960aaccctctcg gagaactagc acgatgcatt atcactattc gaaagcatct caagaagatg 1020acacgggaga ctggtgctgc tattgagccg gatgagcagt ctgcattgct caacaagtgc 1080aacacttata gtggagtcat tggaggtgtt gtgcctggag caggaggcta cgatgctatt 1140tctcttctgg tgatcagctc tacggtgaac aatgtcaagc gagagagcca gggagtccaa 1200tggatggagc tcaaggagga gaacgagggt ctgcggctcg agaaggggtt caagtag 125762418PRTYarrowia lipolytica 62Met Thr Thr Tyr Ser Ala Pro Gly Lys Ala Leu Leu Cys Gly Gly Tyr 1 5 10 15 Leu Val Ile Asp Pro Ala Tyr Ser Ala Tyr Val Val Gly Leu Ser Ala 20 25 30 Arg Ile Tyr Ala Thr Val Ser Ala Ser Glu Ala Ser Thr Thr Ser Val 35 40 45 His Val Val Ser Pro Gln Phe Asp Lys Gly Glu Trp Thr Tyr Asn Tyr 50 55 60 Thr Asn Gly Gln Leu Thr Ala Ile Gly His Asn Pro Phe Ala His Ala 65 70 75 80 Ala Val Asn Thr Val Leu His Tyr Val Pro Pro Arg Asn Leu His Ile 85 90 95 Asn Ile Ser Ile Lys Ser Asp Asn Ala Tyr His Ser Gln Ile Asp Ser 100 105 110 Thr Gln Arg Gly Gln Phe Ala Tyr His Lys Lys Ala Ile His Glu Val 115 120 125 Pro Lys Thr Gly Leu Gly Ser Ser Ala Ala Leu Thr Thr Val Leu Val 130 135 140 Ala Ala Leu Leu Lys Ser Tyr Gly Ile Asp Pro Leu His Asn Thr His 145 150 155 160 Leu Val His Asn Leu Ser Gln Val Ala His Cys Ser Ala Gln Lys Lys 165 170 175 Ile Gly Ser Gly Phe Asp Val Ala Ser Ala Val Cys Gly Ser Leu Val 180 185 190 Tyr Arg Arg Phe Pro Ala Glu Ser Val Asn Met Val Ile Ala Ala Glu 195 200 205 Gly Thr Ser Glu Tyr Gly Ala Leu Leu Arg Thr Thr Val Asn Gln Lys 210 215 220 Trp Lys Val Thr Leu Glu Pro Ser Phe Leu Pro Pro Gly Ile Ser Leu 225 230 235 240 Leu Met Gly Asp Val Gln Gly Gly Ser Glu Thr Pro Gly Met Val Ala 245 250 255 Lys Val Met Ala Trp Arg Lys Ala Lys Pro Arg Glu Ala Glu Met Val 260 265 270 Trp Arg Asp Leu Asn Ala Ala Asn Met Leu Met Val Lys Leu Phe Asn 275 280 285 Asp Leu Arg Lys Leu Ser Leu Thr Asn Asn Glu Ala Tyr Glu Gln Leu 290 295 300 Leu Ala Glu Ala Ala Pro Leu Asn Ala Leu Lys Met Ile Met Leu Gln 305 310 315 320 Asn Pro Leu Gly Glu Leu Ala Arg Cys Ile Ile Thr Ile Arg Lys His 325 330 335 Leu Lys Lys Met Thr Arg Glu Thr Gly Ala Ala Ile Glu Pro Asp Glu 340 345 350 Gln Ser Ala Leu Leu Asn Lys Cys Asn Thr Tyr Ser Gly Val Ile Gly 355 360 365 Gly Val Val Pro Gly Ala Gly Gly Tyr Asp Ala Ile Ser Leu Leu Val 370 375 380 Ile Ser Ser Thr Val Asn Asn Val Lys Arg Glu Ser Gln Gly Val Gln 385 390 395 400 Trp Met Glu Leu Lys Glu Glu Asn Glu Gly Leu Arg Leu Glu Lys Gly 405 410 415 Phe Lys 631164DNAYarrowia lipolytica 63atgatccacc aggcctccac caccgctccg gtgaacattg cgacactcaa gtactggggc 60aagcgagacc ctgctctcaa tctgcccact aacaactcca tctccgtgac tttgtcgcag 120gatgatctgc ggaccctcac cacagcctcg tgttcccctg atttcaccca ggacgagctg 180tggctcaatg gcaagcagga ggacgtgagc ggcaaacgtc tggttgcgtg tttccgagag 240ctgcgggctc tgcgacacaa aatggaggac tccgactctt ctctgcctaa gctggccgat 300cagaagctca agatcgtgtc cgagaacaac ttccccaccg ccgctggtct cgcctcatcg 360gctgctggct ttgccgccct gatccgagcc gttgcaaatc tctacgagct ccaggagacc 420cccgagcagc tgtccattgt ggctcgacag ggctctggat ccgcctgtcg atctctctac 480ggaggctacg tggcatggga aatgggcacc gagtctgacg gaagcgactc gcgagcggtc 540cagatcgcca ccgccgacca ctggcccgag atgcgagccg ccatcctcgt tgtctctgcc 600gacaagaagg acacgtcgtc cactaccggt atgcaggtga ctgtgcacac ttctcccctc 660ttcaaggagc gagtcaccac tgtggttccc gagcggtttg cccagatgaa gaagtcgatt 720ctggaccgag acttccccac ctttgccgag ctcaccatgc gagactcaaa ccagttccac 780gccacctgtc tggactcgta tcctcccatt ttctacctca acgacgtgtc gcgagcctcc 840attcgggtag ttgaggccat caacaaggct gccggagcca ccattgccgc ctacaccttt 900gatgctggac ccaactgtgt catctactac gaggacaaga acgaggagct ggttctgggt 960gctctcaagg ccattctggg ccgtgtggag ggatgggaga agcaccagtc tgtggacgcc 1020aagaagattg atgttgacga gcggtgggag tccgagctgg ccaacggaat tcagcgggtg 1080atccttacca aggttggagg agatcccgtg aagaccgctg agtcgcttat caacgaggat 1140ggttctctga agaacagcaa gtag 116464387PRTYarrowia lipolytica 64Met Ile His Gln Ala Ser Thr Thr Ala Pro Val Asn Ile Ala Thr Leu 1 5 10 15 Lys Tyr Trp Gly Lys Arg Asp Pro Ala Leu Asn Leu Pro Thr Asn Asn 20 25 30 Ser Ile Ser Val Thr Leu Ser Gln Asp Asp Leu Arg Thr Leu Thr Thr 35 40 45 Ala Ser Cys Ser Pro Asp Phe Thr Gln Asp Glu Leu Trp Leu Asn Gly 50 55 60 Lys Gln Glu Asp Val Ser Gly Lys Arg Leu Val Ala Cys Phe Arg Glu 65 70 75 80 Leu Arg Ala Leu Arg His Lys Met Glu Asp Ser Asp Ser Ser Leu Pro 85 90 95 Lys Leu Ala Asp Gln Lys Leu Lys Ile Val Ser Glu Asn Asn Phe Pro 100 105 110 Thr Ala Ala Gly Leu Ala Ser Ser Ala Ala Gly Phe Ala Ala Leu Ile 115 120 125 Arg Ala Val Ala Asn Leu Tyr Glu Leu Gln Glu Thr Pro Glu Gln Leu 130 135 140 Ser Ile Val Ala Arg Gln Gly Ser Gly Ser Ala Cys Arg Ser Leu Tyr 145 150 155 160 Gly Gly Tyr Val Ala Trp Glu Met Gly Thr Glu Ser Asp Gly Ser Asp 165 170 175 Ser Arg Ala Val Gln Ile Ala Thr Ala Asp His Trp Pro Glu Met Arg 180 185 190 Ala Ala Ile Leu Val Val Ser Ala Asp Lys Lys Asp Thr Ser Ser Thr 195 200 205 Thr Gly Met Gln Val Thr Val His Thr Ser Pro Leu Phe Lys Glu Arg 210 215 220 Val Thr Thr Val Val Pro Glu Arg Phe Ala Gln Met Lys Lys Ser Ile 225 230 235 240 Leu Asp Arg Asp Phe Pro Thr Phe Ala Glu Leu Thr Met Arg Asp Ser 245 250 255 Asn Gln Phe His Ala Thr Cys Leu Asp Ser Tyr Pro Pro Ile Phe Tyr 260 265 270 Leu Asn Asp Val Ser Arg Ala Ser Ile Arg Val Val Glu Ala Ile Asn 275 280 285 Lys Ala Ala Gly Ala Thr Ile Ala Ala Tyr Thr Phe Asp Ala Gly Pro 290 295 300 Asn Cys Val Ile Tyr Tyr Glu Asp Lys Asn Glu Glu Leu Val Leu Gly 305 310 315 320 Ala Leu Lys Ala

Ile Leu Gly Arg Val Glu Gly Trp Glu Lys His Gln 325 330 335 Ser Val Asp Ala Lys Lys Ile Asp Val Asp Glu Arg Trp Glu Ser Glu 340 345 350 Leu Ala Asn Gly Ile Gln Arg Val Ile Leu Thr Lys Val Gly Gly Asp 355 360 365 Pro Val Lys Thr Ala Glu Ser Leu Ile Asn Glu Asp Gly Ser Leu Lys 370 375 380 Asn Ser Lys 385 65813DNAYarrowia lipolytica 65atgacgacgt cttacagcga caaaatcaag agtatcagcg tgagctctgt ggctcagcag 60tttcctgagg tggcgccgat tgcggacgtg tccaaggcta gccggcccag cacggagtcg 120tcggactcgt cggccaagct atttgatggc cacgacgagg agcagatcaa gctgatggac 180gagatctgtg tggtgctgga ctgggacgac aagccgattg gcggcgcgtc caaaaagtgc 240tgtcatctga tggacaacat caacgacgga ctggtgcatc gggccttttc cgtgttcatg 300ttcaacgacc gcggtgagct gcttctgcag cagcgggcgg cggaaaaaat cacctttgcc 360aacatgtgga ccaacacgtg ctgctcgcat cctctggcgg tgcccagcga gatgggcggg 420ctggatctgg agtcccggat ccagggcgcc aaaaacgccg cggtccggaa gcttgagcac 480gagctgggaa tcgaccccaa ggccgttccg gcagacaagt tccatttcct cacccggatc 540cactacgccg cgccctcctc gggcccctgg ggcgagcacg agattgacta cattctgttt 600gtccggggcg accccgagct caaggtggtg gccaacgagg tccgcgatac cgtgtgggtg 660tcgcagcagg gactcaagga catgatggcc gatcccaagc tggttttcac cccttggttc 720cggctcattt gtgagcaggc gctgtttccc tggtgggacc agttggacaa tctgcccgcg 780ggcgatgacg agattcggcg gtggatcaag tag 81366270PRTYarrowia lipolytica 66Met Thr Thr Ser Tyr Ser Asp Lys Ile Lys Ser Ile Ser Val Ser Ser 1 5 10 15 Val Ala Gln Gln Phe Pro Glu Val Ala Pro Ile Ala Asp Val Ser Lys 20 25 30 Ala Ser Arg Pro Ser Thr Glu Ser Ser Asp Ser Ser Ala Lys Leu Phe 35 40 45 Asp Gly His Asp Glu Glu Gln Ile Lys Leu Met Asp Glu Ile Cys Val 50 55 60 Val Leu Asp Trp Asp Asp Lys Pro Ile Gly Gly Ala Ser Lys Lys Cys 65 70 75 80 Cys His Leu Met Asp Asn Ile Asn Asp Gly Leu Val His Arg Ala Phe 85 90 95 Ser Val Phe Met Phe Asn Asp Arg Gly Glu Leu Leu Leu Gln Gln Arg 100 105 110 Ala Ala Glu Lys Ile Thr Phe Ala Asn Met Trp Thr Asn Thr Cys Cys 115 120 125 Ser His Pro Leu Ala Val Pro Ser Glu Met Gly Gly Leu Asp Leu Glu 130 135 140 Ser Arg Ile Gln Gly Ala Lys Asn Ala Ala Val Arg Lys Leu Glu His 145 150 155 160 Glu Leu Gly Ile Asp Pro Lys Ala Val Pro Ala Asp Lys Phe His Phe 165 170 175 Leu Thr Arg Ile His Tyr Ala Ala Pro Ser Ser Gly Pro Trp Gly Glu 180 185 190 His Glu Ile Asp Tyr Ile Leu Phe Val Arg Gly Asp Pro Glu Leu Lys 195 200 205 Val Val Ala Asn Glu Val Arg Asp Thr Val Trp Val Ser Gln Gln Gly 210 215 220 Leu Lys Asp Met Met Ala Asp Pro Lys Leu Val Phe Thr Pro Trp Phe 225 230 235 240 Arg Leu Ile Cys Glu Gln Ala Leu Phe Pro Trp Trp Asp Gln Leu Asp 245 250 255 Asn Leu Pro Ala Gly Asp Asp Glu Ile Arg Arg Trp Ile Lys 260 265 270 671035DNAYarrowia lipolytica 67atgtccaagg cgaaattcga aagcgtgttc ccccgaatct ccgaggagct ggtgcagctg 60ctgcgagacg agggtctgcc ccaggatgcc gtgcagtggt tttccgactc acttcagtac 120aactgtgtgg gtggaaagct caaccgaggc ctgtctgtgg tcgacaccta ccagctactg 180accggcaaga aggagctcga tgacgaggag tactaccgac tcgcgctgct cggctggctg 240attgagctgc tgcaggcgtt tttcctcgtg tcggacgaca ttatggatga gtccaagacc 300cgacgaggcc agccctgctg gtacctcaag cccaaggtcg gcatgattgc catcaacgat 360gctttcatgc tagagagtgg catctacatt ctgcttaaga agcatttccg acaggagaag 420tactacattg accttgtcga gctgttccac gacatttcgt tcaagaccga gctgggccag 480ctggtggatc ttctgactgc ccccgaggat gaggttgatc tcaaccggtt ctctctggac 540aagcactcct ttattgtgcg atacaagact gcttactact ccttctacct gcccgttgtt 600ctagccatgt acgtggccgg cattaccaac cccaaggacc tgcagcaggc catggatgtg 660ctgatccctc tcggagagta cttccaggtc caggacgact accttgacaa ctttggagac 720cccgagttca ttggtaagat cggcaccgac atccaggaca acaagtgctc ctggctcgtt 780aacaaagccc ttcagaaggc cacccccgag cagcgacaga tcctcgagga caactacggc 840gtcaaggaca agtccaagga gctcgtcatc aagaaactgt atgatgacat gaagattgag 900caggactacc ttgactacga ggaggaggtt gttggcgaca tcaagaagaa gatcgagcag 960gttgacgaga gccgaggctt caagaaggag gtgctcaacg ctttcctcgc caagatttac 1020aagcgacaga agtag 103568344PRTYarrowia lipolytica 68Met Ser Lys Ala Lys Phe Glu Ser Val Phe Pro Arg Ile Ser Glu Glu 1 5 10 15 Leu Val Gln Leu Leu Arg Asp Glu Gly Leu Pro Gln Asp Ala Val Gln 20 25 30 Trp Phe Ser Asp Ser Leu Gln Tyr Asn Cys Val Gly Gly Lys Leu Asn 35 40 45 Arg Gly Leu Ser Val Val Asp Thr Tyr Gln Leu Leu Thr Gly Lys Lys 50 55 60 Glu Leu Asp Asp Glu Glu Tyr Tyr Arg Leu Ala Leu Leu Gly Trp Leu 65 70 75 80 Ile Glu Leu Leu Gln Ala Phe Phe Leu Val Ser Asp Asp Ile Met Asp 85 90 95 Glu Ser Lys Thr Arg Arg Gly Gln Pro Cys Trp Tyr Leu Lys Pro Lys 100 105 110 Val Gly Met Ile Ala Ile Asn Asp Ala Phe Met Leu Glu Ser Gly Ile 115 120 125 Tyr Ile Leu Leu Lys Lys His Phe Arg Gln Glu Lys Tyr Tyr Ile Asp 130 135 140 Leu Val Glu Leu Phe His Asp Ile Ser Phe Lys Thr Glu Leu Gly Gln 145 150 155 160 Leu Val Asp Leu Leu Thr Ala Pro Glu Asp Glu Val Asp Leu Asn Arg 165 170 175 Phe Ser Leu Asp Lys His Ser Phe Ile Val Arg Tyr Lys Thr Ala Tyr 180 185 190 Tyr Ser Phe Tyr Leu Pro Val Val Leu Ala Met Tyr Val Ala Gly Ile 195 200 205 Thr Asn Pro Lys Asp Leu Gln Gln Ala Met Asp Val Leu Ile Pro Leu 210 215 220 Gly Glu Tyr Phe Gln Val Gln Asp Asp Tyr Leu Asp Asn Phe Gly Asp 225 230 235 240 Pro Glu Phe Ile Gly Lys Ile Gly Thr Asp Ile Gln Asp Asn Lys Cys 245 250 255 Ser Trp Leu Val Asn Lys Ala Leu Gln Lys Ala Thr Pro Glu Gln Arg 260 265 270 Gln Ile Leu Glu Asp Asn Tyr Gly Val Lys Asp Lys Ser Lys Glu Leu 275 280 285 Val Ile Lys Lys Leu Tyr Asp Asp Met Lys Ile Glu Gln Asp Tyr Leu 290 295 300 Asp Tyr Glu Glu Glu Val Val Gly Asp Ile Lys Lys Lys Ile Glu Gln 305 310 315 320 Val Asp Glu Ser Arg Gly Phe Lys Lys Glu Val Leu Asn Ala Phe Leu 325 330 335 Ala Lys Ile Tyr Lys Arg Gln Lys 340 691890DNAYarrowia lipolytica 69atgttacgac tacgaaccat gcgacccaca cagaccagcg tcagggcggc gcttgggccc 60accgccgcgg cccgaaacat gtcctcctcc agcccctcca gcttcgaata ctcgtcctac 120gtcaagggca cgcgggaaat cggccaccga aaggcgccca caacccgtct gtcggttgag 180ggccccatct acgtgggctt cgacggcatt cgtcttctca acctgccgca tctcaacaag 240ggctcgggat tccccctcaa cgagcgacgg gaattcagac tcagtggtct tctgccctct 300gccgaagcca ccctggagga acaggtcgac cgagcatacc aacaattcaa aaagtgtggc 360actcccttag ccaaaaacgg gttctgcacc tcgctcaagt tccaaaacga ggtgctctac 420tacgccctgc tgctcaagca cgttaaggag gtcttcccca tcatctatac accgactcag 480ggagaagcca ttgaacagta ctcgcggctg ttccggcggc ccgaaggctg cttcctcgac 540atcaccagtc cctacgacgt ggaggagcgt ctgggagcgt ttggagacca tgacgacatt 600gactacattg tcgtgactga ctccgagggt attctcggaa ttggagacca aggagtgggc 660ggtattggta tttccatcgc caagctggct ctcatgactc tatgtgctgg agtcaacccc 720tcacgagtca ttcctgtggt tctggatacg ggaaccaaca accaggagct gctgcacgac 780cccctgtatc tcggccgacg aatgccccga gtgcgaggaa agcagtacga cgacttcatc 840gacaactttg tgcagtctgc ccgaaggctg tatcccaagg cggtgatcca tttcgaggac 900tttgggctcg ctaacgcaca caagatcctc gacaagtatc gaccggagat cccctgcttc 960aacgacgaca tccagggcac tggagccgtc actttggcct ccatcacggc cgctctcaag 1020gtgctgggca aaaatatcac agatactcga attctcgtgt acggagctgg ttcggccggc 1080atgggtattg ctgaacaggt ctatgataac ctggttgccc agggtctcga cgacaagact 1140gcgcgacaaa acatctttct catggaccga ccgggtctac tgaccaccgc acttaccgac 1200gagcagatga gcgacgtgca gaagccgttt gccaaggaca aggccaatta cgagggagtg 1260gacaccaaga ctctggagca cgtggttgct gccgtcaagc cccatattct cattggatgt 1320tccactcagc ccggcgcctt taacgagaag gtcgtcaagg agatgctcaa acacacccct 1380cgacccatca ttctccctct ttccaacccc acacgtcttc atgaggctgt ccctgcagat 1440ctgtacaagt ggaccgacgg caaggctctg gttgccaccg gctcgccctt tgacccagtc 1500aacggcaagg agacgtctga gaacaataac tgctttgttt tccccggaat cgggctggga 1560gccattctgt ctcgatcaaa gctcatcacc aacaccatga ttgctgctgc catcgagtgc 1620ctcgccgaac aggcccccat tctcaagaac cacgacgagg gagtacttcc cgacgtagct 1680ctcatccaga tcatttcggc ccgggtggcc actgccgtgg ttcttcaggc caaggctgag 1740ggcctagcca ctgtcgagga agagctcaag cccggcacca aggaacatgt gcagattccc 1800gacaactttg acgagtgtct cgcctgggtc gagactcaga tgtggcggcc cgtctaccgg 1860cctctcatcc atgtgcggga ttacgactag 189070629PRTYarrowia lipolytica 70Met Leu Arg Leu Arg Thr Met Arg Pro Thr Gln Thr Ser Val Arg Ala 1 5 10 15 Ala Leu Gly Pro Thr Ala Ala Ala Arg Asn Met Ser Ser Ser Ser Pro 20 25 30 Ser Ser Phe Glu Tyr Ser Ser Tyr Val Lys Gly Thr Arg Glu Ile Gly 35 40 45 His Arg Lys Ala Pro Thr Thr Arg Leu Ser Val Glu Gly Pro Ile Tyr 50 55 60 Val Gly Phe Asp Gly Ile Arg Leu Leu Asn Leu Pro His Leu Asn Lys 65 70 75 80 Gly Ser Gly Phe Pro Leu Asn Glu Arg Arg Glu Phe Arg Leu Ser Gly 85 90 95 Leu Leu Pro Ser Ala Glu Ala Thr Leu Glu Glu Gln Val Asp Arg Ala 100 105 110 Tyr Gln Gln Phe Lys Lys Cys Gly Thr Pro Leu Ala Lys Asn Gly Phe 115 120 125 Cys Thr Ser Leu Lys Phe Gln Asn Glu Val Leu Tyr Tyr Ala Leu Leu 130 135 140 Leu Lys His Val Lys Glu Val Phe Pro Ile Ile Tyr Thr Pro Thr Gln 145 150 155 160 Gly Glu Ala Ile Glu Gln Tyr Ser Arg Leu Phe Arg Arg Pro Glu Gly 165 170 175 Cys Phe Leu Asp Ile Thr Ser Pro Tyr Asp Val Glu Glu Arg Leu Gly 180 185 190 Ala Phe Gly Asp His Asp Asp Ile Asp Tyr Ile Val Val Thr Asp Ser 195 200 205 Glu Gly Ile Leu Gly Ile Gly Asp Gln Gly Val Gly Gly Ile Gly Ile 210 215 220 Ser Ile Ala Lys Leu Ala Leu Met Thr Leu Cys Ala Gly Val Asn Pro 225 230 235 240 Ser Arg Val Ile Pro Val Val Leu Asp Thr Gly Thr Asn Asn Gln Glu 245 250 255 Leu Leu His Asp Pro Leu Tyr Leu Gly Arg Arg Met Pro Arg Val Arg 260 265 270 Gly Lys Gln Tyr Asp Asp Phe Ile Asp Asn Phe Val Gln Ser Ala Arg 275 280 285 Arg Leu Tyr Pro Lys Ala Val Ile His Phe Glu Asp Phe Gly Leu Ala 290 295 300 Asn Ala His Lys Ile Leu Asp Lys Tyr Arg Pro Glu Ile Pro Cys Phe 305 310 315 320 Asn Asp Asp Ile Gln Gly Thr Gly Ala Val Thr Leu Ala Ser Ile Thr 325 330 335 Ala Ala Leu Lys Val Leu Gly Lys Asn Ile Thr Asp Thr Arg Ile Leu 340 345 350 Val Tyr Gly Ala Gly Ser Ala Gly Met Gly Ile Ala Glu Gln Val Tyr 355 360 365 Asp Asn Leu Val Ala Gln Gly Leu Asp Asp Lys Thr Ala Arg Gln Asn 370 375 380 Ile Phe Leu Met Asp Arg Pro Gly Leu Leu Thr Thr Ala Leu Thr Asp 385 390 395 400 Glu Gln Met Ser Asp Val Gln Lys Pro Phe Ala Lys Asp Lys Ala Asn 405 410 415 Tyr Glu Gly Val Asp Thr Lys Thr Leu Glu His Val Val Ala Ala Val 420 425 430 Lys Pro His Ile Leu Ile Gly Cys Ser Thr Gln Pro Gly Ala Phe Asn 435 440 445 Glu Lys Val Val Lys Glu Met Leu Lys His Thr Pro Arg Pro Ile Ile 450 455 460 Leu Pro Leu Ser Asn Pro Thr Arg Leu His Glu Ala Val Pro Ala Asp 465 470 475 480 Leu Tyr Lys Trp Thr Asp Gly Lys Ala Leu Val Ala Thr Gly Ser Pro 485 490 495 Phe Asp Pro Val Asn Gly Lys Glu Thr Ser Glu Asn Asn Asn Cys Phe 500 505 510 Val Phe Pro Gly Ile Gly Leu Gly Ala Ile Leu Ser Arg Ser Lys Leu 515 520 525 Ile Thr Asn Thr Met Ile Ala Ala Ala Ile Glu Cys Leu Ala Glu Gln 530 535 540 Ala Pro Ile Leu Lys Asn His Asp Glu Gly Val Leu Pro Asp Val Ala 545 550 555 560 Leu Ile Gln Ile Ile Ser Ala Arg Val Ala Thr Ala Val Val Leu Gln 565 570 575 Ala Lys Ala Glu Gly Leu Ala Thr Val Glu Glu Glu Leu Lys Pro Gly 580 585 590 Thr Lys Glu His Val Gln Ile Pro Asp Asn Phe Asp Glu Cys Leu Ala 595 600 605 Trp Val Glu Thr Gln Met Trp Arg Pro Val Tyr Arg Pro Leu Ile His 610 615 620 Val Arg Asp Tyr Asp 625 712610DNAYarrowia lipolytica 71atgccgcagc aagcaatgga tatcaagggc aaggccaagt ctgtgcccat gcccgaagaa 60gacgacctgg actcgcattt tgtgggtccc atctctcccc gacctcacgg agcagacgag 120attgctggct acgtgggctg cgaagacgac gaagacgagc ttgaagaact gggaatgctg 180ggccgatctg cgtccaccca cttctcttac gcggaagaac gccacctcat cgaggttgat 240gccaagtaca gagctcttca tggccatctg cctcatcagc actctcagag tcccgtgtcc 300agatcttcgt catttgtgcg ggccgaaatg aaccaccccc ctcccccacc ctccagccac 360acccaccaac agccagagga cgatgacgca tcttccactc gatctcgatc gtcgtctcga 420gcttctggac gcaagttcaa cagaaacaga accaagtctg gatcttcgct gagcaagggt 480ctccagcagc tcaacatgac cggatcgctc gaagaagagc cctacgagag cgatgacgat 540gcccgactat ctgcggaaga cgacattgtc tatgatgcta cccagaaaga cacctgcaag 600cccatatctc ctactctcaa acgcacccgc accaaggacg acatgaagaa catgtccatc 660aacgacgtca aaatcaccac caccacagaa gatcctcttg tggcccagga gctgtccatg 720atgttcgaaa aggtgcagta ctgccgagac ctccgagaca agtaccaaac cgtgtcgcta 780cagaaggacg gagacaaccc caaggatgac aagacacact ggaaaattta ccccgagcct 840ccaccaccct cctggcacga gaccgaaaag cgattccgag gctcgtccaa aaaggagcac 900caaaagaaag acccgacaat ggatgaattc aaattcgagg actgcgaaat ccccggaccc 960aacgacatgg tcttcaagcg agatcctacc tgtgtctatc aggtctatga ggatgaaagc 1020tctctcaacg aaaataagcc gtttgttgcc atcccctcaa tccgagatta ctacatggat 1080ctggaggatc tcattgtggc ttcgtctgac ggacctgcca agtcttttgc tttccgacga 1140ctgcaatatc tagaagccaa gtggaacctc tactacctgc tcaacgagta cacggagaca 1200accgagtcca agaccaaccc ccatcgagac ttttacaacg tacgaaaggt cgacacccac 1260gttcaccact ctgcctgcat gaaccagaag catctgctgc gattcatcaa atacaagatg 1320aagaactgcc ctgatgaagt tgtcatccac cgagacggtc gggagctgac actctcccag 1380gtgtttgagt cacttaactt gactgcctac gacctgtcta tcgataccct tgatatgcat 1440gctcacaagg actcgttcca tcgatttgac aagttcaacc tcaagtacaa ccctgtcggt 1500gagtctcgac tgcgagaaat cttcctaaag accgacaact acatccaggg tcgataccta 1560gctgagatca caaaggaggt gttccaggat ctcgagaact cgaagtacca gatggcggag 1620taccgtattt ccatctacgg tcggtccaag gacgagtggg acaagctggc tgcctgggtg 1680ctggacaaca aactgttttc gcccaatgtt cggtggttga tccaggtgcc tcgactgtac 1740gacatttaca agaaggctgg tctggttaac acctttgccg acattgtgca gaacgtcttt 1800gagcctcttt tcgaggtcac caaggatccc agtacccatc ccaagctgca cgtgttcctg 1860cagcgagttg tgggctttga ctctgtcgat gacgagtcga agctggaccg acgtttccac 1920cgaaagttcc caactgcagc atactgggac agcgcacaga accctcccta ctcgtactgg 1980cagtactatc tatacgccaa catggcctcc atcaacacct ggagacagcg tttgggctat 2040aatacttttg agttgcgacc ccatgctgga gaggctggtg acccagagca tcttctgtgc 2100acttatctgg ttgctcaggg tatcaaccac ggtattctgt tgcgaaaggt gcccttcatt 2160cagtaccttt actacctgga ccagatcccc attgccatgt ctcctgtgtc caacaatgcg 2220ctgttcctca cgttcgacaa gaaccccttc tactcatact tcaagcgggg tctcaacgtg 2280tccttgtcat cggatgatcc tctgcagttt gcttacacta aggaggctct gattgaggag 2340tactctgtgg ctgcgctcat ttacaagctt tccaacgtgg atatgtgtga gcttgctcga 2400aactcggtac tgcaatctgg ctttgagcga atcatcaagg agcattggat cggcgaaaac 2460tacgagatcc

atggccccga gggcaacacc atccagaaga caaacgtgcc caatgtgcgt 2520ctggccttcc gagacgagac tttgacccac gagcttgctc tggtggacaa gtacaccaat 2580cttgaggagt ttgagcggct gcatggttaa 261072869PRTYarrowia lipolytica 72Met Pro Gln Gln Ala Met Asp Ile Lys Gly Lys Ala Lys Ser Val Pro 1 5 10 15 Met Pro Glu Glu Asp Asp Leu Asp Ser His Phe Val Gly Pro Ile Ser 20 25 30 Pro Arg Pro His Gly Ala Asp Glu Ile Ala Gly Tyr Val Gly Cys Glu 35 40 45 Asp Asp Glu Asp Glu Leu Glu Glu Leu Gly Met Leu Gly Arg Ser Ala 50 55 60 Ser Thr His Phe Ser Tyr Ala Glu Glu Arg His Leu Ile Glu Val Asp 65 70 75 80 Ala Lys Tyr Arg Ala Leu His Gly His Leu Pro His Gln His Ser Gln 85 90 95 Ser Pro Val Ser Arg Ser Ser Ser Phe Val Arg Ala Glu Met Asn His 100 105 110 Pro Pro Pro Pro Pro Ser Ser His Thr His Gln Gln Pro Glu Asp Asp 115 120 125 Asp Ala Ser Ser Thr Arg Ser Arg Ser Ser Ser Arg Ala Ser Gly Arg 130 135 140 Lys Phe Asn Arg Asn Arg Thr Lys Ser Gly Ser Ser Leu Ser Lys Gly 145 150 155 160 Leu Gln Gln Leu Asn Met Thr Gly Ser Leu Glu Glu Glu Pro Tyr Glu 165 170 175 Ser Asp Asp Asp Ala Arg Leu Ser Ala Glu Asp Asp Ile Val Tyr Asp 180 185 190 Ala Thr Gln Lys Asp Thr Cys Lys Pro Ile Ser Pro Thr Leu Lys Arg 195 200 205 Thr Arg Thr Lys Asp Asp Met Lys Asn Met Ser Ile Asn Asp Val Lys 210 215 220 Ile Thr Thr Thr Thr Glu Asp Pro Leu Val Ala Gln Glu Leu Ser Met 225 230 235 240 Met Phe Glu Lys Val Gln Tyr Cys Arg Asp Leu Arg Asp Lys Tyr Gln 245 250 255 Thr Val Ser Leu Gln Lys Asp Gly Asp Asn Pro Lys Asp Asp Lys Thr 260 265 270 His Trp Lys Ile Tyr Pro Glu Pro Pro Pro Pro Ser Trp His Glu Thr 275 280 285 Glu Lys Arg Phe Arg Gly Ser Ser Lys Lys Glu His Gln Lys Lys Asp 290 295 300 Pro Thr Met Asp Glu Phe Lys Phe Glu Asp Cys Glu Ile Pro Gly Pro 305 310 315 320 Asn Asp Met Val Phe Lys Arg Asp Pro Thr Cys Val Tyr Gln Val Tyr 325 330 335 Glu Asp Glu Ser Ser Leu Asn Glu Asn Lys Pro Phe Val Ala Ile Pro 340 345 350 Ser Ile Arg Asp Tyr Tyr Met Asp Leu Glu Asp Leu Ile Val Ala Ser 355 360 365 Ser Asp Gly Pro Ala Lys Ser Phe Ala Phe Arg Arg Leu Gln Tyr Leu 370 375 380 Glu Ala Lys Trp Asn Leu Tyr Tyr Leu Leu Asn Glu Tyr Thr Glu Thr 385 390 395 400 Thr Glu Ser Lys Thr Asn Pro His Arg Asp Phe Tyr Asn Val Arg Lys 405 410 415 Val Asp Thr His Val His His Ser Ala Cys Met Asn Gln Lys His Leu 420 425 430 Leu Arg Phe Ile Lys Tyr Lys Met Lys Asn Cys Pro Asp Glu Val Val 435 440 445 Ile His Arg Asp Gly Arg Glu Leu Thr Leu Ser Gln Val Phe Glu Ser 450 455 460 Leu Asn Leu Thr Ala Tyr Asp Leu Ser Ile Asp Thr Leu Asp Met His 465 470 475 480 Ala His Lys Asp Ser Phe His Arg Phe Asp Lys Phe Asn Leu Lys Tyr 485 490 495 Asn Pro Val Gly Glu Ser Arg Leu Arg Glu Ile Phe Leu Lys Thr Asp 500 505 510 Asn Tyr Ile Gln Gly Arg Tyr Leu Ala Glu Ile Thr Lys Glu Val Phe 515 520 525 Gln Asp Leu Glu Asn Ser Lys Tyr Gln Met Ala Glu Tyr Arg Ile Ser 530 535 540 Ile Tyr Gly Arg Ser Lys Asp Glu Trp Asp Lys Leu Ala Ala Trp Val 545 550 555 560 Leu Asp Asn Lys Leu Phe Ser Pro Asn Val Arg Trp Leu Ile Gln Val 565 570 575 Pro Arg Leu Tyr Asp Ile Tyr Lys Lys Ala Gly Leu Val Asn Thr Phe 580 585 590 Ala Asp Ile Val Gln Asn Val Phe Glu Pro Leu Phe Glu Val Thr Lys 595 600 605 Asp Pro Ser Thr His Pro Lys Leu His Val Phe Leu Gln Arg Val Val 610 615 620 Gly Phe Asp Ser Val Asp Asp Glu Ser Lys Leu Asp Arg Arg Phe His 625 630 635 640 Arg Lys Phe Pro Thr Ala Ala Tyr Trp Asp Ser Ala Gln Asn Pro Pro 645 650 655 Tyr Ser Tyr Trp Gln Tyr Tyr Leu Tyr Ala Asn Met Ala Ser Ile Asn 660 665 670 Thr Trp Arg Gln Arg Leu Gly Tyr Asn Thr Phe Glu Leu Arg Pro His 675 680 685 Ala Gly Glu Ala Gly Asp Pro Glu His Leu Leu Cys Thr Tyr Leu Val 690 695 700 Ala Gln Gly Ile Asn His Gly Ile Leu Leu Arg Lys Val Pro Phe Ile 705 710 715 720 Gln Tyr Leu Tyr Tyr Leu Asp Gln Ile Pro Ile Ala Met Ser Pro Val 725 730 735 Ser Asn Asn Ala Leu Phe Leu Thr Phe Asp Lys Asn Pro Phe Tyr Ser 740 745 750 Tyr Phe Lys Arg Gly Leu Asn Val Ser Leu Ser Ser Asp Asp Pro Leu 755 760 765 Gln Phe Ala Tyr Thr Lys Glu Ala Leu Ile Glu Glu Tyr Ser Val Ala 770 775 780 Ala Leu Ile Tyr Lys Leu Ser Asn Val Asp Met Cys Glu Leu Ala Arg 785 790 795 800 Asn Ser Val Leu Gln Ser Gly Phe Glu Arg Ile Ile Lys Glu His Trp 805 810 815 Ile Gly Glu Asn Tyr Glu Ile His Gly Pro Glu Gly Asn Thr Ile Gln 820 825 830 Lys Thr Asn Val Pro Asn Val Arg Leu Ala Phe Arg Asp Glu Thr Leu 835 840 845 Thr His Glu Leu Ala Leu Val Asp Lys Tyr Thr Asn Leu Glu Glu Phe 850 855 860 Glu Arg Leu His Gly 865 731017DNAYarrowia lipolytica 73atgttccgaa cccgagttac cggctccacc ctgcgatcct tctccacctc cgctgcccga 60cagcacaagg ttgtcgtcct tggcgccaac ggaggcattg gccagcccct gtctctgctg 120ctcaagctca acaagaacgt gaccgacctc ggtctgtacg atctgcgagg cgcccccggc 180gttgctgccg atgtctccca catccccacc aactccaccg tggccggcta ctctcccgac 240aacaacggca ttgccgaggc cctcaagggc gccaagctgg tgctgatccc cgccggtgtc 300ccccgaaagc ccggcatgac ccgagacgat ctgttcaaca ccaacgcctc cattgtgcga 360gacctggcca aggccgtcgg tgagcacgcc cccgacgcct ttgtcggagt cattgctaac 420cccgtcaact ccaccgtccc cattgtcgcc gaggtgctca agtccaaggg caagtacgac 480cccaagaagc tcttcggtgt caccaccctc gacgtcatcc gagccgagcg attcgtctcc 540cagctcgagc acaccaaccc caccaaggag tacttccccg ttgttggcgg ccactccggt 600gtcaccattg tccccctcgt gtcccagtcc gaccaccccg acattgccgg tgaggctcga 660gacaagcttg tccaccgaat ccagtttggc ggtgacgagg ttgtcaaggc caaggacggt 720gccggatccg ccaccctttc catggcccag gctgccgccc gattcgccga ctctctcctc 780cgaggtgtca acggcgagaa ggacgttgtt gagcccactt tcgtcgactc tcctctgttc 840aagggtgagg gcatcgactt cttctccacc aaggtcactc ttggccctaa cggtgttgag 900gagatccacc ccatcggaaa ggtcaacgag tacgaggaga agctcatcga ggctgccaag 960gccgatctca agaagaacat tgagaagggt gtcaactttg tcaagcagaa cccttaa 101774338PRTYarrowia lipolytica 74Met Phe Arg Thr Arg Val Thr Gly Ser Thr Leu Arg Ser Phe Ser Thr 1 5 10 15 Ser Ala Ala Arg Gln His Lys Val Val Val Leu Gly Ala Asn Gly Gly 20 25 30 Ile Gly Gln Pro Leu Ser Leu Leu Leu Lys Leu Asn Lys Asn Val Thr 35 40 45 Asp Leu Gly Leu Tyr Asp Leu Arg Gly Ala Pro Gly Val Ala Ala Asp 50 55 60 Val Ser His Ile Pro Thr Asn Ser Thr Val Ala Gly Tyr Ser Pro Asp 65 70 75 80 Asn Asn Gly Ile Ala Glu Ala Leu Lys Gly Ala Lys Leu Val Leu Ile 85 90 95 Pro Ala Gly Val Pro Arg Lys Pro Gly Met Thr Arg Asp Asp Leu Phe 100 105 110 Asn Thr Asn Ala Ser Ile Val Arg Asp Leu Ala Lys Ala Val Gly Glu 115 120 125 His Ala Pro Asp Ala Phe Val Gly Val Ile Ala Asn Pro Val Asn Ser 130 135 140 Thr Val Pro Ile Val Ala Glu Val Leu Lys Ser Lys Gly Lys Tyr Asp 145 150 155 160 Pro Lys Lys Leu Phe Gly Val Thr Thr Leu Asp Val Ile Arg Ala Glu 165 170 175 Arg Phe Val Ser Gln Leu Glu His Thr Asn Pro Thr Lys Glu Tyr Phe 180 185 190 Pro Val Val Gly Gly His Ser Gly Val Thr Ile Val Pro Leu Val Ser 195 200 205 Gln Ser Asp His Pro Asp Ile Ala Gly Glu Ala Arg Asp Lys Leu Val 210 215 220 His Arg Ile Gln Phe Gly Gly Asp Glu Val Val Lys Ala Lys Asp Gly 225 230 235 240 Ala Gly Ser Ala Thr Leu Ser Met Ala Gln Ala Ala Ala Arg Phe Ala 245 250 255 Asp Ser Leu Leu Arg Gly Val Asn Gly Glu Lys Asp Val Val Glu Pro 260 265 270 Thr Phe Val Asp Ser Pro Leu Phe Lys Gly Glu Gly Ile Asp Phe Phe 275 280 285 Ser Thr Lys Val Thr Leu Gly Pro Asn Gly Val Glu Glu Ile His Pro 290 295 300 Ile Gly Lys Val Asn Glu Tyr Glu Glu Lys Leu Ile Glu Ala Ala Lys 305 310 315 320 Ala Asp Leu Lys Lys Asn Ile Glu Lys Gly Val Asn Phe Val Lys Gln 325 330 335 Asn Pro 751107DNAYarrowia lipolytica 75atgacacaaa cgcacaatct gttttcgcca atcaaagtgg gctcttcgga gctccagaac 60cggatcgttc tcgcaccctt gactcgaacc agagctctgc ccggaaacgt gccctcggat 120cttgccacag agtactacgc acaaagagca gcatctccag gcactctcct catcaccgag 180gccacataca tctcccccgg atctgctgga gtgcccattc caggagacgg aatcgttccg 240ggcatctgga gtgacgagca gctcgaagca tggaaaaagg tgttcaaggc cgtgcacgac 300cgaggatcca aaatctacgt ccagctgtgg gacattggac gtgtcgcatg gtaccacaag 360ctgcaggaac tgggcaacta cttccctaca ggcccctcag ctatccccat gaagggagag 420gagagcgagc atctcaaggc tctgactcac tgggagatca agggcaaggt ggccctctac 480gtcaacgctg ccaagaacgc cattgccgca ggcgctgatg gcgtcgagat ccactcggcc 540aacggctacc ttcccgacac atttctgaga agcgcctcca accaacgaac agacgaatat 600ggaggaagca tcgagaaccg ggcccgattc tcgctggaga ttgtcgacgc tatcaccgag 660gccattggag cagacaaaac cgccatccgt ctgtctccct ggtccacttt ccaggacatt 720gaggtgaatg acaccgagac ccccgcacag ttcacatacc tgtttgagca gctgcagaag 780cgagccgacg agggaaagca gctggcctac gtgcatgtag ttgagccccg actgtttggt 840ccccccgagc cctgggccac caatgagcct ttcagaaaaa tttggaaggg taacttcatt 900agagcaggtg gatacgatag agagactgct cttgaggatg cagacaagtc agacaacacc 960ctgattgcct ttggtcgaga cttcattgcc aatcctgatc tcgtccaacg cctcaagaat 1020aacgagcctt tggccaagta cgacagaaca accttctacg ttccaggtgc caagggctac 1080actgattacc ctgcgtacaa gatgtaa 110776368PRTYarrowia lipolytica 76Met Thr Gln Thr His Asn Leu Phe Ser Pro Ile Lys Val Gly Ser Ser 1 5 10 15 Glu Leu Gln Asn Arg Ile Val Leu Ala Pro Leu Thr Arg Thr Arg Ala 20 25 30 Leu Pro Gly Asn Val Pro Ser Asp Leu Ala Thr Glu Tyr Tyr Ala Gln 35 40 45 Arg Ala Ala Ser Pro Gly Thr Leu Leu Ile Thr Glu Ala Thr Tyr Ile 50 55 60 Ser Pro Gly Ser Ala Gly Val Pro Ile Pro Gly Asp Gly Ile Val Pro 65 70 75 80 Gly Ile Trp Ser Asp Glu Gln Leu Glu Ala Trp Lys Lys Val Phe Lys 85 90 95 Ala Val His Asp Arg Gly Ser Lys Ile Tyr Val Gln Leu Trp Asp Ile 100 105 110 Gly Arg Val Ala Trp Tyr His Lys Leu Gln Glu Leu Gly Asn Tyr Phe 115 120 125 Pro Thr Gly Pro Ser Ala Ile Pro Met Lys Gly Glu Glu Ser Glu His 130 135 140 Leu Lys Ala Leu Thr His Trp Glu Ile Lys Gly Lys Val Ala Leu Tyr 145 150 155 160 Val Asn Ala Ala Lys Asn Ala Ile Ala Ala Gly Ala Asp Gly Val Glu 165 170 175 Ile His Ser Ala Asn Gly Tyr Leu Pro Asp Thr Phe Leu Arg Ser Ala 180 185 190 Ser Asn Gln Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala 195 200 205 Arg Phe Ser Leu Glu Ile Val Asp Ala Ile Thr Glu Ala Ile Gly Ala 210 215 220 Asp Lys Thr Ala Ile Arg Leu Ser Pro Trp Ser Thr Phe Gln Asp Ile 225 230 235 240 Glu Val Asn Asp Thr Glu Thr Pro Ala Gln Phe Thr Tyr Leu Phe Glu 245 250 255 Gln Leu Gln Lys Arg Ala Asp Glu Gly Lys Gln Leu Ala Tyr Val His 260 265 270 Val Val Glu Pro Arg Leu Phe Gly Pro Pro Glu Pro Trp Ala Thr Asn 275 280 285 Glu Pro Phe Arg Lys Ile Trp Lys Gly Asn Phe Ile Arg Ala Gly Gly 290 295 300 Tyr Asp Arg Glu Thr Ala Leu Glu Asp Ala Asp Lys Ser Asp Asn Thr 305 310 315 320 Leu Ile Ala Phe Gly Arg Asp Phe Ile Ala Asn Pro Asp Leu Val Gln 325 330 335 Arg Leu Lys Asn Asn Glu Pro Leu Ala Lys Tyr Asp Arg Thr Thr Phe 340 345 350 Tyr Val Pro Gly Ala Lys Gly Tyr Thr Asp Tyr Pro Ala Tyr Lys Met 355 360 365 771017DNAYarrowia lipolytica 77atggaagcca accccgaagt ccagaccgat atcatcacgc tgacccggtt cattctgcag 60gaacagaaca aggtgggcgc gtcgtccgca atccccaccg gagacttcac tctgctgctc 120aactcgctgc agtttgcctt caagttcatt gcccacaaca tccgacgatc gaccctggtc 180aacctgattg gcctgtcggg aaccgccaac tccaccggcg acgaccagaa gaagctggac 240gtgatcggag acgagatctt catcaacgcc atgaaggcct ccggtaaggt caagctggtg 300gtgtccgagg agcaggagga cctcattgtg tttgagggcg acggccgata cgccgtggtc 360tgcgacccca tcgacggatc ctccaacctc gacgccggcg tctccgtcgg caccattttc 420ggcgtctaca agctccccga gggctcctcc ggatccatca aggacgtgct ccgacccgga 480aaggagatgg ttgccgccgg ctacaccatg tacggtgcct ccgccaacct ggtgctgtcc 540accggaaacg gctgcaacgg cttcactctc gatgaccctc tgggagagtt catcctgacc 600caccccgatc tcaagctccc cgatctgcga tccatctact ccgtcaacga gggtaactcc 660tccctgtggt ccgacaacgt caaggactac ttcaaggccc tcaagttccc cgaggacggc 720tccaagccct actcggcccg atacattggc tccatggtcg ccgacgtgca ccgaaccatt 780ctctacggag gtatgtttgc ctaccccgcc gactccaagt ccaagaaggg caagctccga 840cttttgtacg agggtttccc catggcctac atcattgagc aggccggcgg tcttgccatc 900aacgacaacg gcgagcgaat cctcgatctg gtccccaccg agatccacga gcgatccggc 960gtctggctgg gctccaaggg cgagattgag aaggccaaga agtaccttct gaaatga 101778338PRTYarrowia lipolytica 78Met Glu Ala Asn Pro Glu Val Gln Thr Asp Ile Ile Thr Leu Thr Arg 1 5 10 15 Phe Ile Leu Gln Glu Gln Asn Lys Val Gly Ala Ser Ser Ala Ile Pro 20 25 30 Thr Gly Asp Phe Thr Leu Leu Leu Asn Ser Leu Gln Phe Ala Phe Lys 35 40 45 Phe Ile Ala His Asn Ile Arg Arg Ser Thr Leu Val Asn Leu Ile Gly 50 55 60 Leu Ser Gly Thr Ala Asn Ser Thr Gly Asp Asp Gln Lys Lys Leu Asp 65 70 75 80 Val Ile Gly Asp Glu Ile Phe Ile Asn Ala Met Lys Ala Ser Gly Lys 85 90 95 Val Lys Leu Val Val Ser Glu Glu Gln Glu Asp Leu Ile Val Phe Glu 100 105 110 Gly Asp Gly Arg Tyr Ala Val Val Cys Asp Pro Ile Asp Gly Ser Ser 115 120 125 Asn Leu Asp Ala Gly Val Ser Val Gly Thr Ile Phe Gly Val Tyr Lys 130 135 140 Leu Pro Glu Gly Ser Ser Gly Ser Ile Lys Asp Val Leu Arg Pro Gly 145 150 155 160 Lys Glu Met Val Ala Ala Gly Tyr Thr Met Tyr Gly Ala Ser Ala Asn 165 170 175 Leu Val Leu Ser Thr Gly Asn Gly Cys Asn Gly Phe Thr Leu Asp Asp 180

185 190 Pro Leu Gly Glu Phe Ile Leu Thr His Pro Asp Leu Lys Leu Pro Asp 195 200 205 Leu Arg Ser Ile Tyr Ser Val Asn Glu Gly Asn Ser Ser Leu Trp Ser 210 215 220 Asp Asn Val Lys Asp Tyr Phe Lys Ala Leu Lys Phe Pro Glu Asp Gly 225 230 235 240 Ser Lys Pro Tyr Ser Ala Arg Tyr Ile Gly Ser Met Val Ala Asp Val 245 250 255 His Arg Thr Ile Leu Tyr Gly Gly Met Phe Ala Tyr Pro Ala Asp Ser 260 265 270 Lys Ser Lys Lys Gly Lys Leu Arg Leu Leu Tyr Glu Gly Phe Pro Met 275 280 285 Ala Tyr Ile Ile Glu Gln Ala Gly Gly Leu Ala Ile Asn Asp Asn Gly 290 295 300 Glu Arg Ile Leu Asp Leu Val Pro Thr Glu Ile His Glu Arg Ser Gly 305 310 315 320 Val Trp Leu Gly Ser Lys Gly Glu Ile Glu Lys Ala Lys Lys Tyr Leu 325 330 335 Leu Lys 791194DNAYarrowia lipolytica 79atgcgactca ctctgccccg acttaacgcc gcctacattg taggagccgc ccgaactcct 60gtcggcaagt tcaacggagc cctcaagtcc gtgtctgcca ttgacctcgg tatcaccgct 120gccaaggccg ctgtccagcg atccaaggtc cccgccgacc agattgacga gtttctgttt 180ggccaggtgc tgaccgccaa ctccggccag gcccccgccc gacaggtggt tatcaagggt 240ggtttccccg agtccgtcga ggccaccacc atcaacaagg tgtgctcttc cggcctcaag 300accgtggctc tggctgccca ggccatcaag gccggcgacc gaaacgttat cgtggccggt 360ggaatggagt ccatgtccaa caccccctac tactccggtc gaggtcttgt tttcggcaac 420cagaagctcg aggactccat cgtcaaggac ggtctctggg acccctacaa caacatccac 480atgggcaact gctgcgagaa caccaacaag cgagacggca tcacccgaga gcagcaggac 540gagtacgcca tcgagtccta ccgacgggcc aacgagtcca tcaagaacgg cgccttcaag 600gatgagattg tccccgttga gatcaagacc cgaaagggca ccgtgactgt ctccgaggac 660gaggagccca agggagccaa cgccgagaag ctcaagggcc tcaagcctgt ctttgacaag 720cagggctccg tcactgccgg taacgcctcc cccatcaacg atggtgcttc tgccgttgtc 780gttgcctctg gcaccaaggc caaggagctc ggtacccccg tgctcgccaa gattgtctct 840tacgcagacg ccgccaccgc ccccattgac tttaccattg ctccctctct ggccattccc 900gccgccctca agaaggctgg ccttaccaag gacgacattg ccctctggga gatcaacgag 960gccttctccg gtgtcgctct cgccaacctc atgcgactcg gaattgacaa gtccaaggtc 1020aacgtcaagg gtggagctgt tgctctcggc caccccattg gtgcctccgg taaccgaatc 1080tttgtgactt tggtcaacgc cctcaaggag ggcgagtacg gagttgccgc catctgcaac 1140ggtggaggag cttccaccgc catcgtcatc aagaaggtct cttctgtcga gtag 119480397PRTYarrowia lipolytica 80Met Arg Leu Thr Leu Pro Arg Leu Asn Ala Ala Tyr Ile Val Gly Ala 1 5 10 15 Ala Arg Thr Pro Val Gly Lys Phe Asn Gly Ala Leu Lys Ser Val Ser 20 25 30 Ala Ile Asp Leu Gly Ile Thr Ala Ala Lys Ala Ala Val Gln Arg Ser 35 40 45 Lys Val Pro Ala Asp Gln Ile Asp Glu Phe Leu Phe Gly Gln Val Leu 50 55 60 Thr Ala Asn Ser Gly Gln Ala Pro Ala Arg Gln Val Val Ile Lys Gly 65 70 75 80 Gly Phe Pro Glu Ser Val Glu Ala Thr Thr Ile Asn Lys Val Cys Ser 85 90 95 Ser Gly Leu Lys Thr Val Ala Leu Ala Ala Gln Ala Ile Lys Ala Gly 100 105 110 Asp Arg Asn Val Ile Val Ala Gly Gly Met Glu Ser Met Ser Asn Thr 115 120 125 Pro Tyr Tyr Ser Gly Arg Gly Leu Val Phe Gly Asn Gln Lys Leu Glu 130 135 140 Asp Ser Ile Val Lys Asp Gly Leu Trp Asp Pro Tyr Asn Asn Ile His 145 150 155 160 Met Gly Asn Cys Cys Glu Asn Thr Asn Lys Arg Asp Gly Ile Thr Arg 165 170 175 Glu Gln Gln Asp Glu Tyr Ala Ile Glu Ser Tyr Arg Arg Ala Asn Glu 180 185 190 Ser Ile Lys Asn Gly Ala Phe Lys Asp Glu Ile Val Pro Val Glu Ile 195 200 205 Lys Thr Arg Lys Gly Thr Val Thr Val Ser Glu Asp Glu Glu Pro Lys 210 215 220 Gly Ala Asn Ala Glu Lys Leu Lys Gly Leu Lys Pro Val Phe Asp Lys 225 230 235 240 Gln Gly Ser Val Thr Ala Gly Asn Ala Ser Pro Ile Asn Asp Gly Ala 245 250 255 Ser Ala Val Val Val Ala Ser Gly Thr Lys Ala Lys Glu Leu Gly Thr 260 265 270 Pro Val Leu Ala Lys Ile Val Ser Tyr Ala Asp Ala Ala Thr Ala Pro 275 280 285 Ile Asp Phe Thr Ile Ala Pro Ser Leu Ala Ile Pro Ala Ala Leu Lys 290 295 300 Lys Ala Gly Leu Thr Lys Asp Asp Ile Ala Leu Trp Glu Ile Asn Glu 305 310 315 320 Ala Phe Ser Gly Val Ala Leu Ala Asn Leu Met Arg Leu Gly Ile Asp 325 330 335 Lys Ser Lys Val Asn Val Lys Gly Gly Ala Val Ala Leu Gly His Pro 340 345 350 Ile Gly Ala Ser Gly Asn Arg Ile Phe Val Thr Leu Val Asn Ala Leu 355 360 365 Lys Glu Gly Glu Tyr Gly Val Ala Ala Ile Cys Asn Gly Gly Gly Ala 370 375 380 Ser Thr Ala Ile Val Ile Lys Lys Val Ser Ser Val Glu 385 390 395 811953DNAYarrowia lipolytica 81atgtctgcca acgagaacat ctcccgattc gacgcccctg tgggcaagga gcaccccgcc 60tacgagctct tccataacca cacacgatct ttcgtctatg gtctccagcc tcgagcctgc 120cagggtatgc tggacttcga cttcatctgt aagcgagaga acccctccgt ggccggtgtc 180atctatccct tcggcggcca gttcgtcacc aagatgtact ggggcaccaa ggagactctt 240ctccctgtct accagcaggt cgagaaggcc gctgccaagc accccgaggt cgatgtcgtg 300gtcaactttg cctcctctcg atccgtctac tcctctacca tggagctgct cgagtacccc 360cagttccgaa ccatcgccat tattgccgag ggtgtccccg agcgacgagc ccgagagatc 420ctccacaagg cccagaagaa gggtgtgacc atcattggtc ccgctaccgt cggaggtatc 480aagcccggtt gcttcaaggt tggaaacacc ggaggtatga tggacaacat tgtcgcctcc 540aagctctacc gacccggctc cgttgcctac gtctccaagt ccggaggaat gtccaacgag 600ctgaacaaca ttatctctca caccaccgac ggtgtctacg agggtattgc tattggtggt 660gaccgatacc ctggtactac cttcattgac catatcctgc gatacgaggc cgaccccaag 720tgtaagatca tcgtcctcct tggtgaggtt ggtggtgttg aggagtaccg agtcatcgag 780gctgttaaga acggccagat caagaagccc atcgtcgctt gggccattgg tacttgtgcc 840tccatgttca agactgaggt tcagttcggc cacgccggct ccatggccaa ctccgacctg 900gagactgcca aggctaagaa cgccgccatg aagtctgctg gcttctacgt ccccgatacc 960ttcgaggaca tgcccgaggt ccttgccgag ctctacgaga agatggtcgc caagggcgag 1020ctgtctcgaa tctctgagcc tgaggtcccc aagatcccca ttgactactc ttgggcccag 1080gagcttggtc ttatccgaaa gcccgctgct ttcatctcca ctatttccga tgaccgaggc 1140caggagcttc tgtacgctgg catgcccatt tccgaggttt tcaaggagga cattggtatc 1200ggcggtgtca tgtctctgct gtggttccga cgacgactcc ccgactacgc ctccaagttt 1260cttgagatgg ttctcatgct tactgctgac cacggtcccg ccgtatccgg tgccatgaac 1320accattatca ccacccgagc tggtaaggat ctcatttctt ccctggttgc tggtctcctg 1380accattggta cccgattcgg aggtgctctt gacggtgctg ccaccgagtt caccactgcc 1440tacgacaagg gtctgtcccc ccgacagttc gttgatacca tgcgaaagca gaacaagctg 1500attcctggta ttggccatcg agtcaagtct cgaaacaacc ccgatttccg agtcgagctt 1560gtcaaggact ttgttaagaa gaacttcccc tccacccagc tgctcgacta cgcccttgct 1620gtcgaggagg tcaccacctc caagaaggac aacctgattc tgaacgttga cggtgctatt 1680gctgtttctt ttgtcgatct catgcgatct tgcggtgcct ttactgtgga ggagactgag 1740gactacctca agaacggtgt tctcaacggt ctgttcgttc tcggtcgatc cattggtctc 1800attgcccacc atctcgatca gaagcgactc aagaccggtc tgtaccgaca tccttgggac 1860gatatcacct acctggttgg ccaggaggct atccagaaga agcgagtcga gatcagcgcc 1920ggcgacgttt ccaaggccaa gactcgatca tag 195382650PRTYarrowia lipolytica 82Met Ser Ala Asn Glu Asn Ile Ser Arg Phe Asp Ala Pro Val Gly Lys 1 5 10 15 Glu His Pro Ala Tyr Glu Leu Phe His Asn His Thr Arg Ser Phe Val 20 25 30 Tyr Gly Leu Gln Pro Arg Ala Cys Gln Gly Met Leu Asp Phe Asp Phe 35 40 45 Ile Cys Lys Arg Glu Asn Pro Ser Val Ala Gly Val Ile Tyr Pro Phe 50 55 60 Gly Gly Gln Phe Val Thr Lys Met Tyr Trp Gly Thr Lys Glu Thr Leu 65 70 75 80 Leu Pro Val Tyr Gln Gln Val Glu Lys Ala Ala Ala Lys His Pro Glu 85 90 95 Val Asp Val Val Val Asn Phe Ala Ser Ser Arg Ser Val Tyr Ser Ser 100 105 110 Thr Met Glu Leu Leu Glu Tyr Pro Gln Phe Arg Thr Ile Ala Ile Ile 115 120 125 Ala Glu Gly Val Pro Glu Arg Arg Ala Arg Glu Ile Leu His Lys Ala 130 135 140 Gln Lys Lys Gly Val Thr Ile Ile Gly Pro Ala Thr Val Gly Gly Ile 145 150 155 160 Lys Pro Gly Cys Phe Lys Val Gly Asn Thr Gly Gly Met Met Asp Asn 165 170 175 Ile Val Ala Ser Lys Leu Tyr Arg Pro Gly Ser Val Ala Tyr Val Ser 180 185 190 Lys Ser Gly Gly Met Ser Asn Glu Leu Asn Asn Ile Ile Ser His Thr 195 200 205 Thr Asp Gly Val Tyr Glu Gly Ile Ala Ile Gly Gly Asp Arg Tyr Pro 210 215 220 Gly Thr Thr Phe Ile Asp His Ile Leu Arg Tyr Glu Ala Asp Pro Lys 225 230 235 240 Cys Lys Ile Ile Val Leu Leu Gly Glu Val Gly Gly Val Glu Glu Tyr 245 250 255 Arg Val Ile Glu Ala Val Lys Asn Gly Gln Ile Lys Lys Pro Ile Val 260 265 270 Ala Trp Ala Ile Gly Thr Cys Ala Ser Met Phe Lys Thr Glu Val Gln 275 280 285 Phe Gly His Ala Gly Ser Met Ala Asn Ser Asp Leu Glu Thr Ala Lys 290 295 300 Ala Lys Asn Ala Ala Met Lys Ser Ala Gly Phe Tyr Val Pro Asp Thr 305 310 315 320 Phe Glu Asp Met Pro Glu Val Leu Ala Glu Leu Tyr Glu Lys Met Val 325 330 335 Ala Lys Gly Glu Leu Ser Arg Ile Ser Glu Pro Glu Val Pro Lys Ile 340 345 350 Pro Ile Asp Tyr Ser Trp Ala Gln Glu Leu Gly Leu Ile Arg Lys Pro 355 360 365 Ala Ala Phe Ile Ser Thr Ile Ser Asp Asp Arg Gly Gln Glu Leu Leu 370 375 380 Tyr Ala Gly Met Pro Ile Ser Glu Val Phe Lys Glu Asp Ile Gly Ile 385 390 395 400 Gly Gly Val Met Ser Leu Leu Trp Phe Arg Arg Arg Leu Pro Asp Tyr 405 410 415 Ala Ser Lys Phe Leu Glu Met Val Leu Met Leu Thr Ala Asp His Gly 420 425 430 Pro Ala Val Ser Gly Ala Met Asn Thr Ile Ile Thr Thr Arg Ala Gly 435 440 445 Lys Asp Leu Ile Ser Ser Leu Val Ala Gly Leu Leu Thr Ile Gly Thr 450 455 460 Arg Phe Gly Gly Ala Leu Asp Gly Ala Ala Thr Glu Phe Thr Thr Ala 465 470 475 480 Tyr Asp Lys Gly Leu Ser Pro Arg Gln Phe Val Asp Thr Met Arg Lys 485 490 495 Gln Asn Lys Leu Ile Pro Gly Ile Gly His Arg Val Lys Ser Arg Asn 500 505 510 Asn Pro Asp Phe Arg Val Glu Leu Val Lys Asp Phe Val Lys Lys Asn 515 520 525 Phe Pro Ser Thr Gln Leu Leu Asp Tyr Ala Leu Ala Val Glu Glu Val 530 535 540 Thr Thr Ser Lys Lys Asp Asn Leu Ile Leu Asn Val Asp Gly Ala Ile 545 550 555 560 Ala Val Ser Phe Val Asp Leu Met Arg Ser Cys Gly Ala Phe Thr Val 565 570 575 Glu Glu Thr Glu Asp Tyr Leu Lys Asn Gly Val Leu Asn Gly Leu Phe 580 585 590 Val Leu Gly Arg Ser Ile Gly Leu Ile Ala His His Leu Asp Gln Lys 595 600 605 Arg Leu Lys Thr Gly Leu Tyr Arg His Pro Trp Asp Asp Ile Thr Tyr 610 615 620 Leu Val Gly Gln Glu Ala Ile Gln Lys Lys Arg Val Glu Ile Ser Ala 625 630 635 640 Gly Asp Val Ser Lys Ala Lys Thr Arg Ser 645 650 831494DNAYarrowia lipolytica 83atgtcagcga aatccattca cgaggccgac ggcaaggccc tgctcgcaca ctttctgtcc 60aaggcgcccg tgtgggccga gcagcagccc atcaacacgt ttgaaatggg cacacccaag 120ctggcgtctc tgacgttcga ggacggcgtg gcccccgagc agatcttcgc cgccgctgaa 180aagacctacc cctggctgct ggagtccggc gccaagtttg tggccaagcc cgaccagctc 240atcaagcgac gaggcaaggc cggcctgctg gtactcaaca agtcgtggga ggagtgcaag 300ccctggatcg ccgagcgggc cgccaagccc atcaacgtgg agggcattga cggagtgctg 360cgaacgttcc tggtcgagcc ctttgtgccc cacgaccaga agcacgagta ctacatcaac 420atccactccg tgcgagaggg cgactggatc ctcttctacc acgagggagg agtcgacgtc 480ggcgacgtgg acgccaaggc cgccaagatc ctcatccccg ttgacattga gaacgagtac 540ccctccaacg ccacgctcac caaggagctg ctggcacacg tgcccgagga ccagcaccag 600accctgctcg acttcatcaa ccggctctac gccgtctacg tcgatctgca gtttacgtat 660ctggagatca accccctggt cgtgatcccc accgcccagg gcgtcgaggt ccactacctg 720gatcttgccg gcaagctcga ccagaccgca gagtttgagt gcggccccaa gtgggctgct 780gcgcggtccc ccgccgctct gggccaggtc gtcaccattg acgccggctc caccaaggtg 840tccatcgacg ccggccccgc catggtcttc cccgctcctt tcggtcgaga gctgtccaag 900gaggaggcgt acattgcgga gctcgattcc aagaccggag cttctctgaa gctgactgtt 960ctcaatgcca agggccgaat ctggaccctt gtggctggtg gaggagcctc cgtcgtctac 1020gccgacgcca ttgcgtctgc cggctttgct gacgagctcg ccaactacgg cgagtactct 1080ggcgctccca acgagaccca gacctacgag tacgccaaaa ccgtactgga tctcatgacc 1140cggggcgacg ctcaccccga gggcaaggta ctgttcattg gcggaggaat cgccaacttc 1200acccaggttg gatccacctt caagggcatc atccgggcct tccgggacta ccagtcttct 1260ctgcacaacc acaaggtgaa gatttacgtg cgacgaggcg gtcccaactg gcaggagggt 1320ctgcggttga tcaagtcggc tggcgacgag ctgaatctgc ccatggagat ttacggcccc 1380gacatgcacg tgtcgggtat tgttcctttg gctctgcttg gaaagcggcc caagaatgtc 1440aagccttttg gcaccggacc ttctactgag gcttccactc ctctcggagt ttaa 149484497PRTYarrowia lipolytica 84Met Ser Ala Lys Ser Ile His Glu Ala Asp Gly Lys Ala Leu Leu Ala 1 5 10 15 His Phe Leu Ser Lys Ala Pro Val Trp Ala Glu Gln Gln Pro Ile Asn 20 25 30 Thr Phe Glu Met Gly Thr Pro Lys Leu Ala Ser Leu Thr Phe Glu Asp 35 40 45 Gly Val Ala Pro Glu Gln Ile Phe Ala Ala Ala Glu Lys Thr Tyr Pro 50 55 60 Trp Leu Leu Glu Ser Gly Ala Lys Phe Val Ala Lys Pro Asp Gln Leu 65 70 75 80 Ile Lys Arg Arg Gly Lys Ala Gly Leu Leu Val Leu Asn Lys Ser Trp 85 90 95 Glu Glu Cys Lys Pro Trp Ile Ala Glu Arg Ala Ala Lys Pro Ile Asn 100 105 110 Val Glu Gly Ile Asp Gly Val Leu Arg Thr Phe Leu Val Glu Pro Phe 115 120 125 Val Pro His Asp Gln Lys His Glu Tyr Tyr Ile Asn Ile His Ser Val 130 135 140 Arg Glu Gly Asp Trp Ile Leu Phe Tyr His Glu Gly Gly Val Asp Val 145 150 155 160 Gly Asp Val Asp Ala Lys Ala Ala Lys Ile Leu Ile Pro Val Asp Ile 165 170 175 Glu Asn Glu Tyr Pro Ser Asn Ala Thr Leu Thr Lys Glu Leu Leu Ala 180 185 190 His Val Pro Glu Asp Gln His Gln Thr Leu Leu Asp Phe Ile Asn Arg 195 200 205 Leu Tyr Ala Val Tyr Val Asp Leu Gln Phe Thr Tyr Leu Glu Ile Asn 210 215 220 Pro Leu Val Val Ile Pro Thr Ala Gln Gly Val Glu Val His Tyr Leu 225 230 235 240 Asp Leu Ala Gly Lys Leu Asp Gln Thr Ala Glu Phe Glu Cys Gly Pro 245 250 255 Lys Trp Ala Ala Ala Arg Ser Pro Ala Ala Leu Gly Gln Val Val Thr 260 265 270 Ile Asp Ala Gly Ser Thr Lys Val Ser Ile Asp Ala Gly Pro Ala Met 275 280 285 Val Phe Pro Ala Pro Phe Gly Arg Glu Leu Ser Lys Glu Glu Ala Tyr 290 295 300 Ile Ala Glu Leu Asp Ser Lys Thr Gly Ala Ser Leu Lys Leu Thr Val 305 310 315 320 Leu Asn Ala Lys Gly Arg Ile Trp Thr Leu Val Ala Gly Gly Gly Ala 325 330 335 Ser Val Val Tyr Ala Asp Ala Ile Ala Ser Ala Gly Phe Ala Asp Glu 340 345 350 Leu Ala Asn Tyr Gly Glu Tyr Ser Gly Ala Pro Asn Glu Thr Gln Thr 355

360 365 Tyr Glu Tyr Ala Lys Thr Val Leu Asp Leu Met Thr Arg Gly Asp Ala 370 375 380 His Pro Glu Gly Lys Val Leu Phe Ile Gly Gly Gly Ile Ala Asn Phe 385 390 395 400 Thr Gln Val Gly Ser Thr Phe Lys Gly Ile Ile Arg Ala Phe Arg Asp 405 410 415 Tyr Gln Ser Ser Leu His Asn His Lys Val Lys Ile Tyr Val Arg Arg 420 425 430 Gly Gly Pro Asn Trp Gln Glu Gly Leu Arg Leu Ile Lys Ser Ala Gly 435 440 445 Asp Glu Leu Asn Leu Pro Met Glu Ile Tyr Gly Pro Asp Met His Val 450 455 460 Ser Gly Ile Val Pro Leu Ala Leu Leu Gly Lys Arg Pro Lys Asn Val 465 470 475 480 Lys Pro Phe Gly Thr Gly Pro Ser Thr Glu Ala Ser Thr Pro Leu Gly 485 490 495 Val 851458DNAYarrowia lipolytica 85atggttatta tgtgtgtggg acctcagcac acgcatcatc ccaacacagg gtgcagtata 60tatagacaga cgtgttcctt cgcaccgttc ttcacatatc aaaacactaa caaattcaaa 120agtgagtatc atggtgggag tcaattgatt gctcggggag ttgaacaggc aacaatggca 180tgcacagggc cagtgaaggc agactgcagt cgctgcacat ggatcgtggt tctgaggcgt 240tgctatcaaa agggtcaatt acctcacgaa acacagctgg atgttgtgca atcgtcaatt 300gaaaaacccg acacaatgca agatctcttt gcgcgcattg ccatcgctgt tgccatcgct 360gtcgccatcg ccaatgccgc tgcggattat tatccctacc ttgttccccg cttccgcaca 420accggcgatg tctttgtatc atgaactctc gaaactaact cagtggttaa agctgtcgtt 480gccggagccg ctggtggtat tggccagccc ctttctcttc tcctcaaact ctctccttac 540gtgaccgagc ttgctctcta cgatgtcgtc aactcccccg gtgttgccgc tgacctctcc 600cacatctcca ccaaggctaa ggtcactggc tacctcccca aggatgacgg tctcaagaac 660gctctgaccg gcgccaacat tgtcgttatc cccgccggta tcccccgaaa gcccggtatg 720acccgagacg atctgttcaa gatcaacgct ggtatcgtcc gagatctcgt caccggtgtc 780gcccagtacg cccctgacgc ctttgtgctc atcatctcca accccgtcaa ctctaccgtc 840cctattgctg ccgaggtcct caagaagcac aacgtcttca accctaagaa gctcttcggt 900gtcaccaccc ttgacgttgt ccgagcccag accttcaccg ccgctgttgt tggcgagtct 960gaccccacca agctcaacat ccccgtcgtt ggtggccact ccggagacac cattgtccct 1020ctcctgtctc tgaccaagcc taaggtcgag atccccgccg acaagctcga cgacctcgtc 1080aagcgaatcc agtttggtgg tgacgaggtt gtccaggcta aggacggtct tggatccgct 1140accctctcca tggcccaggc tggtttccga tttgccgagg ctgtcctcaa gggtgccgct 1200ggtgagaagg gcatcatcga gcccgcctac atctaccttg acggtattga tggcacctcc 1260gacatcaagc gagaggtcgg tgtcgccttc ttctctgtcc ctgtcgagtt cggccctgag 1320ggtgccgcta aggcttacaa catccttccc gaggccaacg actacgagaa gaagcttctc 1380aaggtctcca tcgacggtct ttacggcaac attgccaagg gcgaggagtt cattgttaac 1440cctcctcctg ccaagtaa 145886331PRTYarrowia lipolytica 86Val Val Lys Ala Val Val Ala Gly Ala Ala Gly Gly Ile Gly Gln Pro 1 5 10 15 Leu Ser Leu Leu Leu Lys Leu Ser Pro Tyr Val Thr Glu Leu Ala Leu 20 25 30 Tyr Asp Val Val Asn Ser Pro Gly Val Ala Ala Asp Leu Ser His Ile 35 40 45 Ser Thr Lys Ala Lys Val Thr Gly Tyr Leu Pro Lys Asp Asp Gly Leu 50 55 60 Lys Asn Ala Leu Thr Gly Ala Asn Ile Val Val Ile Pro Ala Gly Ile 65 70 75 80 Pro Arg Lys Pro Gly Met Thr Arg Asp Asp Leu Phe Lys Ile Asn Ala 85 90 95 Gly Ile Val Arg Asp Leu Val Thr Gly Val Ala Gln Tyr Ala Pro Asp 100 105 110 Ala Phe Val Leu Ile Ile Ser Asn Pro Val Asn Ser Thr Val Pro Ile 115 120 125 Ala Ala Glu Val Leu Lys Lys His Asn Val Phe Asn Pro Lys Lys Leu 130 135 140 Phe Gly Val Thr Thr Leu Asp Val Val Arg Ala Gln Thr Phe Thr Ala 145 150 155 160 Ala Val Val Gly Glu Ser Asp Pro Thr Lys Leu Asn Ile Pro Val Val 165 170 175 Gly Gly His Ser Gly Asp Thr Ile Val Pro Leu Leu Ser Leu Thr Lys 180 185 190 Pro Lys Val Glu Ile Pro Ala Asp Lys Leu Asp Asp Leu Val Lys Arg 195 200 205 Ile Gln Phe Gly Gly Asp Glu Val Val Gln Ala Lys Asp Gly Leu Gly 210 215 220 Ser Ala Thr Leu Ser Met Ala Gln Ala Gly Phe Arg Phe Ala Glu Ala 225 230 235 240 Val Leu Lys Gly Ala Ala Gly Glu Lys Gly Ile Ile Glu Pro Ala Tyr 245 250 255 Ile Tyr Leu Asp Gly Ile Asp Gly Thr Ser Asp Ile Lys Arg Glu Val 260 265 270 Gly Val Ala Phe Phe Ser Val Pro Val Glu Phe Gly Pro Glu Gly Ala 275 280 285 Ala Lys Ala Tyr Asn Ile Leu Pro Glu Ala Asn Asp Tyr Glu Lys Lys 290 295 300 Leu Leu Lys Val Ser Ile Asp Gly Leu Tyr Gly Asn Ile Ala Lys Gly 305 310 315 320 Glu Glu Phe Ile Val Asn Pro Pro Pro Ala Lys 325 330 871937DNAYarrowia lipolytica 87atgactggca ccttacccaa gttcggcgac ggaaccacca ttgtggttct tggagcctcc 60ggcgacctcg ctaagaagaa gaccgtgagt attgaaccag actgaggtca attgaagagt 120aggagagtct gagaacattc gacggacctg attgtgctct ggaccactca attgactcgt 180tgagagcccc aatgggtctt ggctagccga gtcgttgact tgttgacttg ttgagcccag 240aacccccaac ttttgccacc atacaccgcc atcaccatga cacccagatg tgcgtgcgta 300tgtgagagtc aattgttccg tggcaaggca cagcttattc caccgtgttc cttgcacagg 360tggtctttac gctctcccac tctatccgag caataaaagc ggaaaaacag cagcaagtcc 420caacagactt ctgctccgaa taaggcgtct agcaagtgtg cccaaaactc aattcaaaaa 480tgtcagaaac ctgatatcaa cccgtcttca aaagctaacc ccagttcccc gccctcttcg 540gcctttaccg aaacggcctg ctgcccaaaa atgttgaaat catcggctac gcacggtcga 600aaatgactca ggaggagtac cacgagcgaa tcagccacta cttcaagacc cccgacgacc 660agtccaagga gcaggccaag aagttccttg agaacacctg ctacgtccag ggcccttacg 720acggtgccga gggctaccag cgactgaatg aaaagattga ggagtttgag aagaagaagc 780ccgagcccca ctaccgtctt ttctacctgg ctctgccccc cagcgtcttc cttgaggctg 840ccaacggtct gaagaagtat gtctaccccg gcgagggcaa ggcccgaatc atcatcgaga 900agccctttgg ccacgacctg gcctcgtcac gagagctcca ggacggcctt gctcctctct 960ggaaggagtc tgagatcttc cgaatcgacc actacctcgg aaaggagatg gtcaagaacc 1020tcaacattct gcgatttggc aaccagttcc tgtccgccgt gtgggacaag aacaccattt 1080ccaacgtcca gatctccttc aaggagccct ttggcactga gggccgaggt ggatacttca 1140acgacattgg aatcatccga gacgttattc agaaccatct gttgcaggtt ctgtccattc 1200tagccatgga gcgacccgtc actttcggcg ccgaggacat tcgagatgag aaggtcaagg 1260tgctccgatg tgtcgacatt ctcaacattg acgacgtcat tctcggccag tacggcccct 1320ctgaagacgg aaagaagccc ggatacaccg atgacgatgg cgttcccgat gactcccgag 1380ctgtgacctt tgctgctctc catctccaga tccacaacga cagatgggag ggtgttcctt 1440tcatcctccg agccggtaag gctctggacg agggcaaggt cgagatccga gtgcagttcc 1500gagacgtgac caagggcgtt gtggaccatc tgcctcgaaa tgagctcgtc atccgaatcc 1560agccctccga gtccatctac atgaagatga actccaagct gcctggcctt actgccaaga 1620acattgtcac cgacctggat ctgacctaca accgacgata ctcggacgtg cgaatccctg 1680aggcttacga gtctctcatt ctggactgcc tcaagggtga ccacaccaac tttgtgcgaa 1740acgacgagct ggacatttcc tggaagattt tcaccgatct gctgcacaag attgacgagg 1800acaagagcat tgtgcccgag aagtacgcct acggctctcg tggccccgag cgactcaagc 1860agtggctccg agaccgaggc tacgtgcgaa acggcaccga gctgtaccaa tggcctgtca 1920ccaagggctc ctcgtga 193788498PRTYarrowia lipolytica 88Met Thr Gly Thr Leu Pro Lys Phe Gly Asp Gly Thr Thr Ile Val Val 1 5 10 15 Leu Gly Ala Ser Gly Asp Leu Ala Lys Lys Lys Thr Phe Pro Ala Leu 20 25 30 Phe Gly Leu Tyr Arg Asn Gly Leu Leu Pro Lys Asn Val Glu Ile Ile 35 40 45 Gly Tyr Ala Arg Ser Lys Met Thr Gln Glu Glu Tyr His Glu Arg Ile 50 55 60 Ser His Tyr Phe Lys Thr Pro Asp Asp Gln Ser Lys Glu Gln Ala Lys 65 70 75 80 Lys Phe Leu Glu Asn Thr Cys Tyr Val Gln Gly Pro Tyr Asp Gly Ala 85 90 95 Glu Gly Tyr Gln Arg Leu Asn Glu Lys Ile Glu Glu Phe Glu Lys Lys 100 105 110 Lys Pro Glu Pro His Tyr Arg Leu Phe Tyr Leu Ala Leu Pro Pro Ser 115 120 125 Val Phe Leu Glu Ala Ala Asn Gly Leu Lys Lys Tyr Val Tyr Pro Gly 130 135 140 Glu Gly Lys Ala Arg Ile Ile Ile Glu Lys Pro Phe Gly His Asp Leu 145 150 155 160 Ala Ser Ser Arg Glu Leu Gln Asp Gly Leu Ala Pro Leu Trp Lys Glu 165 170 175 Ser Glu Ile Phe Arg Ile Asp His Tyr Leu Gly Lys Glu Met Val Lys 180 185 190 Asn Leu Asn Ile Leu Arg Phe Gly Asn Gln Phe Leu Ser Ala Val Trp 195 200 205 Asp Lys Asn Thr Ile Ser Asn Val Gln Ile Ser Phe Lys Glu Pro Phe 210 215 220 Gly Thr Glu Gly Arg Gly Gly Tyr Phe Asn Asp Ile Gly Ile Ile Arg 225 230 235 240 Asp Val Ile Gln Asn His Leu Leu Gln Val Leu Ser Ile Leu Ala Met 245 250 255 Glu Arg Pro Val Thr Phe Gly Ala Glu Asp Ile Arg Asp Glu Lys Val 260 265 270 Lys Val Leu Arg Cys Val Asp Ile Leu Asn Ile Asp Asp Val Ile Leu 275 280 285 Gly Gln Tyr Gly Pro Ser Glu Asp Gly Lys Lys Pro Gly Tyr Thr Asp 290 295 300 Asp Asp Gly Val Pro Asp Asp Ser Arg Ala Val Thr Phe Ala Ala Leu 305 310 315 320 His Leu Gln Ile His Asn Asp Arg Trp Glu Gly Val Pro Phe Ile Leu 325 330 335 Arg Ala Gly Lys Ala Leu Asp Glu Gly Lys Val Glu Ile Arg Val Gln 340 345 350 Phe Arg Asp Val Thr Lys Gly Val Val Asp His Leu Pro Arg Asn Glu 355 360 365 Leu Val Ile Arg Ile Gln Pro Ser Glu Ser Ile Tyr Met Lys Met Asn 370 375 380 Ser Lys Leu Pro Gly Leu Thr Ala Lys Asn Ile Val Thr Asp Leu Asp 385 390 395 400 Leu Thr Tyr Asn Arg Arg Tyr Ser Asp Val Arg Ile Pro Glu Ala Tyr 405 410 415 Glu Ser Leu Ile Leu Asp Cys Leu Lys Gly Asp His Thr Asn Phe Val 420 425 430 Arg Asn Asp Glu Leu Asp Ile Ser Trp Lys Ile Phe Thr Asp Leu Leu 435 440 445 His Lys Ile Asp Glu Asp Lys Ser Ile Val Pro Glu Lys Tyr Ala Tyr 450 455 460 Gly Ser Arg Gly Pro Glu Arg Leu Lys Gln Trp Leu Arg Asp Arg Gly 465 470 475 480 Tyr Val Arg Asn Gly Thr Glu Leu Tyr Gln Trp Pro Val Thr Lys Gly 485 490 495 Ser Ser 892202DNAYarrowia lipolytica 89atgactgaca cttcaaacat caagtgagta ttgccgcaca caattgcaat caccgccggg 60ctctacctcc tcagctctcg acgtcaatgg gccagcagcc gccatttgac cccaattaca 120ctggttgtgt aaaaccctca accacaatcg cttatgctca ccacagacta cgacttaacc 180aagtcatgtc acaggtcaaa gtaaagtcag cgcaacaccc cctcaatctc aacacacttt 240tgctaactca ggcctgtcgc tgacattgcc ctcatcggtc tcgccgtcat gggccagaac 300ctgatcctca acatggccga ccacggtaag tatcaattga ctcaagacgc accagcaaga 360tacagagcat acccagcaat cgctcctctg ataatcgcca ttgtaacact acgttggtta 420gattgatcta aggtcgttgc tggttccatg cacttccact tgctcatatg aagggagtca 480aactctattt tgatagtgtc ctctcccatc cccgaaatgt cgcattgttg ctaacaatag 540gctacgaggt tgttgcctac aaccgaacca cctccaaggt cgaccacttc ctcgagaacg 600aggccaaggg tgagtatccg tccagctatg ctgtttacag ccattgaccc caccttcccc 660cacaattgct acgtcaccat taaaaaacaa aattaccggt atcggcaagc tagactttca 720tgcaacctac gcagggtaac aagttgagtt tcagccgtgc accttacagg aaaaccagtc 780atacgccgag gcagtgtgaa agcgaaagca cacagcctac ggtgattgat tgcatttttt 840tgacatagga gggaaacacg tgacatggca agtgcccaac acgaatacta acaaacagga 900aagtccatta ttggtgctca ctctatcaag gagctgtgtg ctctgctgaa gcgaccccga 960cgaatcattc tgctcgttaa ggccggtgct gctgtcgatt ctttcatcga acagctcctg 1020ccctatctcg ataagggtga tatcatcatt gacggtggta actcccactt ccccgactcc 1080aaccgacgat acgaggagct taacgagaag ggaatcctct ttgttggttc cggtgtttcc 1140ggcggtgagg agggtgcccg atacggtccc tccatcatgc ccggtggaaa caaggaggcc 1200tggccccaca ttaagaagat tttccaggac atctctgcta aggctgatgg tgagccctgc 1260tgtgactggg tcggtgacgc tggtgccggc cactttgtca agatggttca caacggtatt 1320gagtatggtg acatgcagct tatctgcgag gcttacgacc tcatgaagcg aggtgctggt 1380ttcaccaatg aggagattgg agacgttttc gccaagtgga acaacggtat cctcgactcc 1440ttcctcattg agatcacccg agacatcttc aagtacgacg acggctctgg aactcctctc 1500gttgagaaga tctccgacac tgctggccag aagggtactg gaaagtggac cgctatcaac 1560gctcttgacc ttggtatgcc cgtcaccctg atcggtgagg ccgtcttcgc tcgatgcctt 1620tctgccctca agcaggagcg tgtccgagct tccaaggttc ttgatggccc cgagcccgtc 1680aagttcactg gtgacaagaa ggagtttgtc gaccagctcg agcaggccct ttacgcctcc 1740aagatcatct cttacgccca gggtttcatg cttatccgag aggccgccaa gacctacggc 1800tgggagctca acaacgccgg tattgccctc atgtggcgag gtggttgcat catccgatcc 1860gtcttccttg ctgacatcac caaggcttac cgacaggacc ccaacctcga gaacctgctg 1920ttcaacgact tcttcaagaa cgccatctcc aaggccaacc cctcttggcg agctaccgtg 1980gccaaggctg tcacctgggg tgttcccact cccgcctttg cctcggctct ggctttctac 2040gacggttacc gatctgccaa gctccccgct aacctgctcc aggcccagcg agactacttc 2100ggcgcccaca cctaccagct cctcgatggt gatggaaagt ggatccacac caactggacc 2160ggccgaggtg gtgaggtttc ttcttccact tacgatgctt aa 220290489PRTYarrowia lipolytica 90Met Thr Asp Thr Ser Asn Ile Lys Pro Val Ala Asp Ile Ala Leu Ile 1 5 10 15 Gly Leu Ala Val Met Gly Gln Asn Leu Ile Leu Asn Met Ala Asp His 20 25 30 Gly Tyr Glu Val Val Ala Tyr Asn Arg Thr Thr Ser Lys Val Asp His 35 40 45 Phe Leu Glu Asn Glu Ala Lys Gly Lys Ser Ile Ile Gly Ala His Ser 50 55 60 Ile Lys Glu Leu Cys Ala Leu Leu Lys Arg Pro Arg Arg Ile Ile Leu 65 70 75 80 Leu Val Lys Ala Gly Ala Ala Val Asp Ser Phe Ile Glu Gln Leu Leu 85 90 95 Pro Tyr Leu Asp Lys Gly Asp Ile Ile Ile Asp Gly Gly Asn Ser His 100 105 110 Phe Pro Asp Ser Asn Arg Arg Tyr Glu Glu Leu Asn Glu Lys Gly Ile 115 120 125 Leu Phe Val Gly Ser Gly Val Ser Gly Gly Glu Glu Gly Ala Arg Tyr 130 135 140 Gly Pro Ser Ile Met Pro Gly Gly Asn Lys Glu Ala Trp Pro His Ile 145 150 155 160 Lys Lys Ile Phe Gln Asp Ile Ser Ala Lys Ala Asp Gly Glu Pro Cys 165 170 175 Cys Asp Trp Val Gly Asp Ala Gly Ala Gly His Phe Val Lys Met Val 180 185 190 His Asn Gly Ile Glu Tyr Gly Asp Met Gln Leu Ile Cys Glu Ala Tyr 195 200 205 Asp Leu Met Lys Arg Gly Ala Gly Phe Thr Asn Glu Glu Ile Gly Asp 210 215 220 Val Phe Ala Lys Trp Asn Asn Gly Ile Leu Asp Ser Phe Leu Ile Glu 225 230 235 240 Ile Thr Arg Asp Ile Phe Lys Tyr Asp Asp Gly Ser Gly Thr Pro Leu 245 250 255 Val Glu Lys Ile Ser Asp Thr Ala Gly Gln Lys Gly Thr Gly Lys Trp 260 265 270 Thr Ala Ile Asn Ala Leu Asp Leu Gly Met Pro Val Thr Leu Ile Gly 275 280 285 Glu Ala Val Phe Ala Arg Cys Leu Ser Ala Leu Lys Gln Glu Arg Val 290 295 300 Arg Ala Ser Lys Val Leu Asp Gly Pro Glu Pro Val Lys Phe Thr Gly 305 310 315 320 Asp Lys Lys Glu Phe Val Asp Gln Leu Glu Gln Ala Leu Tyr Ala Ser 325 330 335 Lys Ile Ile Ser Tyr Ala Gln Gly Phe Met Leu Ile Arg Glu Ala Ala 340 345 350 Lys Thr Tyr Gly Trp Glu Leu Asn Asn Ala Gly Ile Ala Leu Met Trp 355 360 365 Arg Gly Gly Cys Ile Ile Arg Ser Val Phe Leu Ala Asp Ile Thr Lys 370 375 380 Ala Tyr Arg Gln Asp Pro Asn Leu Glu Asn Leu Leu Phe Asn Asp Phe 385 390 395 400 Phe Lys Asn Ala Ile Ser Lys Ala Asn Pro Ser Trp Arg Ala Thr Val 405 410 415 Ala Lys Ala Val Thr Trp Gly Val Pro Thr Pro Ala Phe Ala Ser Ala 420 425 430 Leu Ala Phe Tyr Asp Gly Tyr Arg Ser Ala Lys Leu Pro Ala Asn Leu 435 440

445 Leu Gln Ala Gln Arg Asp Tyr Phe Gly Ala His Thr Tyr Gln Leu Leu 450 455 460 Asp Gly Asp Gly Lys Trp Ile His Thr Asn Trp Thr Gly Arg Gly Gly 465 470 475 480 Glu Val Ser Ser Ser Thr Tyr Asp Ala 485 911742DNAYarrowia lipolytica 91atgctcaacc ttagaaccgc ccttcgagct gtgcgacccg tcactctggt gagtatctcg 60gagcccggga cggctaccaa cacacaagca agatgcaaca gaaaccggac tttttaaatg 120cggattgcgg aaaatttgca tggcggcaac gactcggaga aggagcggga caattgcaat 180ggcaggatgc cattgacgaa ctgagggtga tgagagaccg ggcctccgat gacgtggtgg 240tgacgacagc ccggctggtg ttgccgggac tgtctctgaa aagcaatttc tctatctccg 300gtctcaacag actccccttc tctagctcaa ttggcattgt cttcagaagg tgtcttagtg 360gtatccccat tgttatcttc ttttccccaa tgtcaatgtc aatgtcaatg gctccgacct 420ctttcacatt aacacggcgc aaacacagat accacggaac cgactcaaac aaatccaaag 480agacgcagcg gaataattgg catcaacgaa cgatttggga tactctggcg agaatgccga 540aatatttcgc ttgtcttgtt gtttctcttg agtgagttgt ttgtgaagtc gtttggaaga 600aggttcccaa tgtcacaaac cataccaact cgttacagcc agcttgtaat cccccacctc 660ttcaatacat actaacgcag acccgatcct acgccacttc cgtggcctct ttcaccggcc 720agaagaactc caacggcaag tacactgtgt ctctgattga gggagacggt atcggaaccg 780agatctccaa ggctgtcaag gacatctacc atgccgccaa ggtccccatc gactgggagg 840ttgtcgacgt cacccccact ctggtcaacg gcaagaccac catccccgac agcgccattg 900agtccatcaa ccgaaacaag gttgccctca agggtcccct cgccaccccc atcggtaagg 960gccacgtttc catgaacctg actctgcgac gaaccttcaa cctgttcgcc aacgtccgac 1020cttgcaagtc cgtcgtgggc tacaagaccc cttacgagaa cgtcgacacc ctgctcatcc 1080gagagaacac tgagggtgag tactccggta tcgagcacac cgtcgtcccc ggtgtcgttc 1140agtccatcaa gctgatcacc cgagaggctt ccgagcgagt catccggtac gcttacgagt 1200acgccctgtc ccgaggcatg aagaaggtcc ttgttgtcca caaggcctct attatgaagg 1260tctccgatgg tcttttcctt gaggttgctc gagagctcgc caaggagtac ccctccattg 1320acctttccgt cgagctgatc gacaacacct gtctgcgaat ggtccaggac cccgctctct 1380accgagatgt cgtcatggtc atgcccaacc tttacggtga cattctgtcc gatcttgcct 1440ccggtcttat cggtggtctt ggtctgaccc cctccggtaa catgggtgac gaggtctcca 1500tcttcgaggc cgtccacgga tccgctcccg acattgctgg caagggtctt gctaacccca 1560ctgctctgct gctctcctcc gtgatgatgc tgcgacacat gggtctcaac gacaacgcca 1620ccaacatcga gcaggccgtc tttggcacca ttgcttccgg ccccgagaac cgaaccaagg 1680atcttaaggg taccgccacc acttctcact ttgctgagca gattatcaag cgactcaagt 1740ag 174292369PRTYarrowia lipolytica 92Met Leu Asn Leu Arg Thr Ala Leu Arg Ala Val Arg Pro Val Thr Leu 1 5 10 15 Thr Arg Ser Tyr Ala Thr Ser Val Ala Ser Phe Thr Gly Gln Lys Asn 20 25 30 Ser Asn Gly Lys Tyr Thr Val Ser Leu Ile Glu Gly Asp Gly Ile Gly 35 40 45 Thr Glu Ile Ser Lys Ala Val Lys Asp Ile Tyr His Ala Ala Lys Val 50 55 60 Pro Ile Asp Trp Glu Val Val Asp Val Thr Pro Thr Leu Val Asn Gly 65 70 75 80 Lys Thr Thr Ile Pro Asp Ser Ala Ile Glu Ser Ile Asn Arg Asn Lys 85 90 95 Val Ala Leu Lys Gly Pro Leu Ala Thr Pro Ile Gly Lys Gly His Val 100 105 110 Ser Met Asn Leu Thr Leu Arg Arg Thr Phe Asn Leu Phe Ala Asn Val 115 120 125 Arg Pro Cys Lys Ser Val Val Gly Tyr Lys Thr Pro Tyr Glu Asn Val 130 135 140 Asp Thr Leu Leu Ile Arg Glu Asn Thr Glu Gly Glu Tyr Ser Gly Ile 145 150 155 160 Glu His Thr Val Val Pro Gly Val Val Gln Ser Ile Lys Leu Ile Thr 165 170 175 Arg Glu Ala Ser Glu Arg Val Ile Arg Tyr Ala Tyr Glu Tyr Ala Leu 180 185 190 Ser Arg Gly Met Lys Lys Val Leu Val Val His Lys Ala Ser Ile Met 195 200 205 Lys Val Ser Asp Gly Leu Phe Leu Glu Val Ala Arg Glu Leu Ala Lys 210 215 220 Glu Tyr Pro Ser Ile Asp Leu Ser Val Glu Leu Ile Asp Asn Thr Cys 225 230 235 240 Leu Arg Met Val Gln Asp Pro Ala Leu Tyr Arg Asp Val Val Met Val 245 250 255 Met Pro Asn Leu Tyr Gly Asp Ile Leu Ser Asp Leu Ala Ser Gly Leu 260 265 270 Ile Gly Gly Leu Gly Leu Thr Pro Ser Gly Asn Met Gly Asp Glu Val 275 280 285 Ser Ile Phe Glu Ala Val His Gly Ser Ala Pro Asp Ile Ala Gly Lys 290 295 300 Gly Leu Ala Asn Pro Thr Ala Leu Leu Leu Ser Ser Val Met Met Leu 305 310 315 320 Arg His Met Gly Leu Asn Asp Asn Ala Thr Asn Ile Glu Gln Ala Val 325 330 335 Phe Gly Thr Ile Ala Ser Gly Pro Glu Asn Arg Thr Lys Asp Leu Lys 340 345 350 Gly Thr Ala Thr Thr Ser His Phe Ala Glu Gln Ile Ile Lys Arg Leu 355 360 365 Lys 93960DNAYarrowia lipolytica 93cctctcactt tgtgaatcgt gaaacatgaa tcttcaagcc aagaatgtta ggcaggggaa 60gctttctttc agactttttg gaattggtcc tcttttggac attattgacg atattattat 120tttttccccg tccaatgttg acccttgtaa gccattccgg ttctggagcg catctcgtct 180gaaggagtct tcgtgtggct ataactacaa gcgttgtatg gtggatccta tgaccgtcta 240tatagggcaa cttttgctct tgttcttccc cctccttgag ggacgtatgg caatggctat 300gacaactatc gtagtgagcc tctataaccc attgaagtac aagtcctcca ccttgctgcc 360aaactcgcga gaaaaaaagt ccaccaactc cgccgggaaa tactggagaa cacctctaag 420acgtgggctt ctgcacctgt gtggcttggg tctgggtttt gcgagctctg agccacaacc 480taaggacggt gtgattggga gataagtagt cgttggtttt ctaatcgcac gtgatatgca 540agccacactt ataacacaat gaagacaggc cgatgaactg catgtcattg tacaggtgcg 600gagagcaaga aactctgggg cggaggtgaa agatgagaca aaaagcctca ggtgcaaggt 660agggagttga tcaacgtcaa acacaaataa tctaggttgt taggcagcta aacatgtata 720taactgggct gccaccgagt gttacttgtc attaacgtcg cattttcgcc tacacaaaat 780ttgggttact cgccactaca ctgctcaaat ctttcagctg tgcaacaagc tttcaggtca 840cacatagact cgcataagga cccgggtcat ctgttattct ccactggtaa accaatagtc 900ctagctgatt tgggtacaga agctcacttt cacatctttt catcttcttc tacaaccatc 96094855DNAYarrowia lipolytica 94atctgtgagg agcccctggc gtcactgtcg actgtgccgg catttctgat ggtatttcca 60gccccgcagt tctcgagacc cccgaacaaa tgtgccacac ccttgccaaa atgacgaata 120cacggcgtcg cggccgggaa tcgaactctt ggcaccgcca caggagtgaa atttgaaatt 180tgaaatttga aaaataattc acattttgag tttcaataat atatcgatga ccctcccaaa 240agacccaagt cgagacgcaa aaaaacaccc agacgacatg gatgcggtca cgtgaccgca 300aaaaccgccc cggaaatccg tttgtgacgt gttcaattcc atctctatgt ttttctgcgg 360tttctacgat gccgcaatgg tggccaatgt gcgtttcact gccgtagtgg ctggaacaag 420ccacaggggg tcgtcgggcc aatcagacgg tccctgacat ggttctgcgc cctaacccgg 480gaactctaac ccccgtggtg gcgcaatcgc tgtcttcatg tgctttatct cacgtgacgg 540ctggaatctg gcagaagacg gagtatgtac attttgtcgt tggtcacgtt atccctaaaa 600cgtggtgttt aaactggtcg aatgcttggc ccagaacaca agaagaaaaa aacgagacaa 660cttgatcagt ttcaacgcca cagcaagctt gtcttcactg tggttggtct tctccacgcc 720acaagcaaca cgtacatgtc aattacgtca gggtctttta agttctgtgg cttttgaacc 780agttataaag aaccaaccac ccttttttca aagctaatca agacggggaa attttttttt 840tgatattttt cgaca 85595897DNAYarrowia lipolytica 95gatactgcag acggtgcatt acttacccgt gtcgactgag agtctacttg gtacttggcc 60ctgtggctaa gcagtatttg agcaacaatg caatgcagtt gctgactcgg ttccagatcc 120ccttgccccg atgtgtggaa gcgttgtttt tggggcaagg gcatgtgggg gctgcatcat 180actgtggctg gggccgttgg aagagccgtc ggcagcgagc ctgagtcgct tctcggggcc 240ttattccccc cgcctctagg tcagcggcgg ccgaagtgtc gtactcagct cgcctgtaca 300gtatgacgtg accgaatagc ctctggaagg ttggagaagt acagtgcaaa aaaaagttgc 360aaaatttcat tttagcgttc gatccgacgt ggcagttgga caatgaatcg atggagacat 420gatcatgggc agaaatagaa ggtctccatg ttcaatggca gtaccaattg agcaacagac 480gggtcgacag gcggcgggca caccatccgc cctccacatg gcgcaatcgt cagtgcagcg 540attcgtactc ggattgcatc atgttgcacc gaaagttggg gcccgcacgt tggagaggcg 600aggagccagg gttagctttg gtggggtcct ttgttgtcac gtggcatcag cgaatggcgt 660cctccaatca gggccgtcag cgaagtcggc gtgtgatagt gcgtggggag cgaatagagt 720ttctgggggg gggcggccca aaacgtgaaa tccgagtacg catgtagagt gtaaattggg 780tgtatagtga cattgtttga ctctgaccct gagagtaata tataatgtgt acgtgtcccc 840ctccgttggt cttctttttt tctcctttct cctaaccaac acccaaacta atcaatc 89796972DNAYarrowia lipolytica 96aagtcgtatt aacataactt tccttacatt tttttaaagc acgtcactat ccacgtgacc 60tagccacgcg ataccaagta ttcatccata atgacacact catgacgtcc ggaggacgtc 120atcatcgtcc agtcacgtgc caaggcacat gactaatcat aacaccttat gactagcttc 180tgaatcgcta cacagttcca attcgcaaat aaactcgaaa tgacgaaatg ccataataaa 240aatgacgaaa ctcgagattg agagcagcac atgcactgaa gtggtggaca accagcgtat 300ccggagacac gacggatcca gcaccatgga agctggccga aaaagagatc cccagcacat 360tgagcaacca agtcagctca attgagtaac atcacacact cagatcgagt ctgatggtgg 420tccccttttg ttccttcact tgaaaaataa ttgaaaataa caataacaat aaaaataaaa 480acaaaataaa aataaaaata aaaataaaaa taaaaaaata aaaaaacctt gccgcattta 540gcgtcagcca ccccccgcat tgacctgagt acgttggatt gaccccgatc ctgcacgtcg 600agcgtggtcg gccaaaaagc gcccgtggct ggtgagtcag aaatagcagg gttgcaagag 660agagctgcgc aacgagcaat aaacggtgtt tttttcgctt ctgtgctgct tagagtggag 720agccgaccct cgccatgctc acgtgaccat tcacgtggtt gcaaactcca ccttagtata 780gccgtgtccc tctcgctacc cattatcgca tcgtactcca gccacatttt tttgttcccc 840gctaaatccg gaaccttatc tgggtcacgt gaaattgcaa tctcgacagg aggttatact 900tatagagtga gacactccac gcaaggtgtt gcaagtcaat tgacaccacc tcacctcaga 960ctaacatcca ca 972971425DNAYarrowia lipolytica 97gaatctgccc ccacatttta tctccgcttt tgactgtttt tctcccccct ttcacactct 60gcttttggct acataaaccc cgcaccgttt ggaactctgt tggtccgggg aagccgccgt 120taggtgtgtc agatggagag cgccagacga gcagaaccga gggacagcgg atcgggggag 180ggctgtcacg tgacgaaggg cactgttgac gtggtgaatg tcgcccgttc tcacgtgacc 240cgtctcctct atatgtgtat ccgcctcttt gtttggtttt ttttctgctt cccccccccc 300ccccccaccc caatcacatg ctcagaaagt agacatctgc atcgtcctgc atgccatccc 360acaagacgaa caagtgatag gccgagagcc gaggacgagg tggagtgcac aaggggtagg 420cgaatggtac gattccgcca agtgagactg gcgatcggga gaagggttgg tggtcatggg 480ggatagaatt tgtacaagtg gaaaaaccac tacgagtagc ggatttgata ccacaagtag 540cagagatata cagcaatggt gggagtgcaa gtatcggaat gtactgtacc tcctgtactc 600gtactcgtac ggcactcgta gaaacggggc aatacggggg agaagcgatc gcccgtctgt 660tcaatcgcca caagtccgag taatgctcga gtatcgaagt cttgtacctc cctgtcaatc 720atggcaccac tggtcttgac ttgtctattc atactggaca agcgccagag ttaagcttgt 780agcgaatttc gccctcggac atcaccccat acgacggaca cacatgcccg acaaacagcc 840tctcttattg tagctgaaag tatattgaat gtgaacgtgt acaatatcag gtaccagcgg 900gaggttacgg ccaaggtgat accggaataa ccctggcttg gagatggtcg gtccattgta 960ctgaagtgtc cgtgtcgttt ccgtcactgc cccaattgga catgtttgtt tttccgatct 1020ttcgggcgcc ctctccttgt ctccttgtct gtctcctgga ctgttgctac cccatttctt 1080tggcctccat tggttcctcc ccgtctttca cgtcgtctat ggttgcatgg tttcccttat 1140acttttcccc acagtcacat gttatggagg ggtctagatg gaggcctaat tttgacgtgc 1200aaggggcgaa ttggggcgag aaacacgtcg tggacatggt gcaaggcccg cagggttgat 1260tcgacgcttt tccgcgaaaa aaacaagtcc aaataccccc gtttattctc cctcggctct 1320cggtatttca catgaaaact ataacctaga ctacacgggc aaccttaacc ccagagtata 1380cttatatacc aaagggatgg gtcctcaaaa atcacacaag caacg 142598500DNAYarrowia lipolytica 98cgccattcgg ttccttccag accattccag atcaatccac ctcttcttat ctcaggtggg 60tgtgctgaca tcagaccccg tagcccttct cccagtggcg aacagcaggc ataaaacagg 120gccattgagc agagcaaaca aggtcggtga aatcgtcgaa aaagtcggaa aacggttgca 180agaaattgga gcgtcacctg ccaccctcca ggctctatat aaagcattgc cccaattgct 240aacgcttcat atttacacct ttggcacccc agtccatccc tccaataaaa tgtactacat 300gggacacaac aagagaggat gcgcgcccaa accctaacct agcacatgca cgatgattct 360ctttgtctgt gaaaaaattt ttccaccaaa atttccccat tgggatgaaa ccctaaccgc 420aaccaaaagt ttttaactat catcttgtac gtcacggttt ccgattcttc tcttctcttt 480catcatcatc acttgtgacc 50099494DNAYarrowia lipolytica 99aactaccata aagtaccgag aaatataggc aattgtacaa attgtccacc tccttcactt 60acattaccga accatggcca tatcaccaaa ataccccgag tgctaaaaca cctccctcca 120aatgttctct taccttccac cgaaaaccga tcttattatc ccaacgcttg ttgtggcttg 180acgcgccgca cccgctgggc ttgccatttc gataccaatc caagaggaaa agctcatgag 240aaacaatcgg aatatcacga gaacggcctg gcgaaccaac aggatatttt tgaatataat 300tacccctcga atctagtcat atctatgtct actgtagact tgggcggcat catgatgtac 360attattttag cgtctggaac cctaaagttc acgtacaatc atgtgacaaa cgaggctaaa 420aaatgtcaat ttcgtatatt agtgttatta cgtggctcac atttccgaat catctaccac 480cccccaccta aaaa 494100440DNAYarrowia lipolytica 100tttttttaat tttcatattt attttcatat ttattttcat atttattttc atttatttat 60tcatgtattt atttattact ttttaagtat tttaaactcc tcactaaacc gtcgattgca 120caatattaac cttcattaca cctgcagcgt ggtttttgtg gtcgttagcc gaagtcttcc 180aacgtgggta taagtaggaa caattgggcc gattttttga gccgtctaaa tctctcgact 240caattgatct gctgtcgaaa atccggctct ctagctcctt ttccccgtcc gctggagctc 300ctcttcattg tgccgttttt ccaacattta actttgccac ccaccaccac ccccactacc 360atcacccact cgatctctgt tcgtgtcacc acgactttgt cttctcacac atactctgtt 420tgtgcaccac acattgcgaa 440101434DNAYarrowia lipolytica 101gctacaatag ctttattggc cctattgagc acgctacaat tcggtccagt atgtacaacg 60tctatgcgca ctaacggcca tacagtgagt tacagcacac ccaaaagtaa ccctgcctga 120cctgtctgcc tgagacagga agattaactc ttgtagtgac cgagctcgat aagactcaag 180ccacacaatt tttttatagc cttgcttcaa gagtcgccaa aatgacatta cacaactcca 240cggaccgtcg gttccatgtc cacacccttg gatgggtaag cgctccacgc acgtaccacg 300tgcattgagt ttaaccacaa acataggtct gtgtcccaga gttaccctgc tgcatcagcc 360aagtcttgaa agcaaaattt cttgcacaat ttttcctctt cttttcttca ctgatcgcag 420tccaaacaca aaca 434102752DNAYarrowia lipolytica 102gcgctctgat ccacttgtat ggctccaagt tcagtgtacc aagtagttgg tgatgcaggg 60agggatgtct ctatccacca ataatgaact catgggcgaa attgtttctg ttaaacactc 120caactgtcgt tttaaatctc attctctttg catttggact ccattcgctt ccgttgggcc 180aatataatcc atcgtaacgt actttagatg gaaatttagt tacctgctac ttgtctcaac 240accccaacag gggctgttcg acagaggtaa tagagcgtca atgggttaat aaaaacacac 300tgtcgatttt cactcattgt ctttatgata ttacctgttt tccgctgtta tcaatgccga 360gcatcgtgtt atatcttcca ccccaactac ttgcatttac ttaactatta cctcaactat 420ttacaccccg aattgttacc tcccaataag taactttatt tcaaccaatg ggacgagagc 480atctctgaga acatcgatct atctctgtca atattgccca gaatcgttcg aaaaaaaaca 540ccaaaaggtt tacagcgcca ttataaatat aaattcgttg tcaattcccc cgcaatgtct 600gttgaaatct cattttgaga ccttccaaca ttaccctctc tcccgtctgg tcacatgacg 660tgactgcttc ttcccaaaac gaacactccc aactcttccc ccccgtcagt gaaaagtata 720catccgacct ccaaatcttt tcttcactca ac 752103402DNAYarrowia lipolytica 103agagacgggt tggcggcgta tttgtgtccc aaaaaacagc cccaattgcc ccaattgacc 60ccaaattgac ccagtagcgg gcccaacccc ggcgagagcc cccttcaccc cacatatcaa 120acctcccccg gttcccacac ttgccgttaa gggcgtaggg tactgcagtc tggaatctac 180gcttgttcag actttgtact agtttctttg tctggccatc cgggtaaccc atgccggacg 240caaaatagac tactgaaaat ttttttgctt tgtggttggg actttagcca agggtataaa 300agaccaccgt ccccgaatta cctttcctct tcttttctct ctctccttgt caactcacac 360ccgaaatcgt taagcatttc cttctgagta taagaatcat tc 402104491DNAYarrowia lipolytica 104gctgcgctga tctggacacc acagaggttc cgagcacttt aggttgcacc aaatgtccca 60ccaggtgcag gcagaaaacg ctggaacagc gtgtacagtt tgtcttagca aaaagtgaag 120gcgctgaggt cgagcagggt ggtgtgactt gttatagcct ttagagctgc gaaagcgcgt 180atggatttgg ctcatcaggc cagattgagg gtctgtggac acatgtcatg ttagtgtact 240tcaatcgccc cctggatata gccccgacaa taggccgtgg cctcattttt ttgccttccg 300cacatttcca ttgctcggta cccacacctt gcttctcctg cacttgccaa ccttaatact 360ggtttacatt gaccaacatc ttacaagcgg ggggcttgtc tagggtatat ataaacagtg 420gctctcccaa tcggttgcca gtctcttttt tcctttcttt ccccacagat tcgaaatcta 480aactacacat c 4911051213DNAYarrowia lipolytica 105gatgaggaat agacaagcgg gtatttattg tatgaataaa gattatgtat tgattgcaaa 60aaagtgcatt tgtagatgtg gtttattgta gagagtacgg tatgtactgt acgaacatta 120ggagctactt ctacaagtag attttcttaa caagggtgaa atttactagg aagtacatgc 180atatttcgtt agtagaatca caaaagaaat gtacaagcac gtactacttg tactccacaa 240tgtggagtgg gagcaaaaaa attggacgac accggaatcg aaccggggac ctcgcgcatg 300ctaagcgcat gtgataacca actacaccag acgcccaaga actttcttgg tgattatgga 360atacgtggtc tgctatatct caattttgct gtaatgaatc attagaatta aaaaaaaaac 420cccatttttg tgtgattgtc ggccaagaga tggaacagga agaatacgtg aacaagcgag 480cacgaatgcc atatgctctt ctgaacaacc gagtccgaat ccgatttgtg ggtatcacat 540gtctcaagta gctgaaatgt atttcgctag aataaaataa atgagattaa gaattaaaaa 600tattggaata tattttccta gaatagaaac tttggatttt ttttcggcta ttacagtctg 660aactggacaa acggctgact atatataaat attattgggt ctgttttctt gtttatgtcg 720aaattatctg ggttttacta ctgtgtcgtc gagtatagag tggcctgact ggagaaaatg 780cagtagtatg gacagtaggt actgccagcc agagaagttt ttggaattga tacttgagtc 840atttttccat tccccattcc ccattccaac acaatcaact gtttctgaac attttccaaa 900acgcggagat gtatgtcact tggcactgca agtctcgatt caaaatgcat ctctttcaga 960ccaaagtgtc atcagctttg tttggcccca aattaccgca aatacttgtc gaaattgaag 1020tgcaatacgg cctcgtctgc catgaaacct gcctattctc ttcaaattgg cgtcaggttt 1080cacgtccagc attcctcgcc cagacagagt tgctatggtt gaatcgtgta

ctgttaatat 1140atgtatgtat tatactcgta ctacgatata ctgttcaata gagtctctta taatcgtacg 1200acgattctgg gca 121310634PRTYarrowia lipolytica 106Met Leu Ser Arg Asn Leu Ser Lys Phe Ala Arg Ala Gly Leu Ile Arg 1 5 10 15 Pro Ala Thr Thr Ser Thr His Thr Arg Leu Phe Ser Val Ser Ala Arg 20 25 30 Arg Leu 10732PRTYarrowia lipolytica 107Met Leu Arg Leu Ile Arg Pro Arg Leu Ala Ala Leu Ala Arg Pro Thr 1 5 10 15 Thr Arg Ala Pro Gln Ala Leu Asn Ala Arg Thr His Ile Val Ser Val 20 25 30 10853PRTSaccharomyces cerevisiae 108Met Phe Gln Arg Ser Gly Ala Ala His His Ile Lys Leu Ile Ser Ser 1 5 10 15 Arg Arg Cys Arg Phe Lys Ser Ser Phe Ala Val Ala Leu Asn Ala Ala 20 25 30 Ser Lys Leu Val Thr Pro Lys Ile Leu Trp Asn Asn Pro Ile Ser Leu 35 40 45 Val Ser Lys Glu Met 50 10977PRTYarrowia lipolytica 109Met Leu Arg Val Gly Arg Ile Gly Thr Lys Thr Leu Ala Ser Ser Ser 1 5 10 15 Leu Arg Phe Val Ala Gly Ala Arg Pro Lys Ser Thr Leu Thr Glu Ala 20 25 30 Val Leu Glu Thr Thr Gly Leu Leu Lys Thr Thr Pro Gln Asn Pro Glu 35 40 45 Trp Ser Gly Ala Val Lys Gln Ala Ser Arg Leu Val Glu Thr Asp Thr 50 55 60 Pro Ile Arg Asp Pro Phe Ser Ile Val Ser Gln Glu Met 65 70 75 1101125PRTAspergillus nidulans 110Met Ala Ser Val Leu Ile Arg Arg Lys Phe Gly Thr Glu Gly Gly Ser 1 5 10 15 Asp Ala Glu Pro Ser Trp Leu Lys Arg Gln Val Thr Gly Cys Leu Gln 20 25 30 Ser Ile Ser Arg Arg Ala Cys Ile His Pro Ile His Thr Ile Val Val 35 40 45 Ile Ala Leu Leu Ala Ser Thr Thr Tyr Val Gly Leu Leu Glu Gly Ser 50 55 60 Leu Phe Asp Ser Phe Arg Asn Ser Asn Asn Val Ala Gly His Val Asp 65 70 75 80 Val Asp Ser Leu Leu Leu Gly Asn Arg Ser Leu Arg Leu Gly Glu Gly 85 90 95 Thr Ser Trp Lys Trp Gln Val Glu Asp Ser Leu Asn Gln Asp Asp Gln 100 105 110 Lys Val Gly Asn Pro Glu Leu Lys Arg Glu Val Asp Gln His Leu Ala 115 120 125 Leu Thr Thr Leu Ile Phe Pro Asp Ser Ile Ser Lys Ser Ala Ser Thr 130 135 140 Ala Pro Ala Ala Asp Ala Leu Pro Val Pro Ala Asn Ala Ser Ala Gln 145 150 155 160 Leu Leu Pro His Thr Pro Asn Leu Phe Ser Pro Phe Ser His Asp Ser 165 170 175 Ser Leu Val Phe Thr Leu Pro Phe Asp Gln Val Pro Gln Phe Leu Arg 180 185 190 Ala Val Gln Glu Leu Pro Asp Pro Thr Leu Glu Asp Asp Glu Gly Glu 195 200 205 Gln Lys Arg Trp Ile Met Arg Ala Thr Arg Gly Pro Val Ser Gly Pro 210 215 220 Asn Gly Thr Ile Ser Ser Trp Leu Ser Asp Ala Trp Ser Ser Phe Val 225 230 235 240 Asp Leu Ile Lys His Ala Glu Thr Ile Asp Ile Ile Ile Met Thr Leu 245 250 255 Gly Tyr Leu Ala Met Tyr Leu Ser Phe Ala Ser Leu Glu Phe Ser Met 260 265 270 Lys Gln Leu Gly Ser Lys Phe Trp Leu Ala Thr Thr Val Leu Phe Ser 275 280 285 Gly Met Phe Ala Phe Leu Phe Gly Leu Leu Val Thr Thr Lys Phe Gly 290 295 300 Val Pro Leu Asn Leu Leu Leu Leu Ser Glu Gly Leu Pro Phe Leu Val 305 310 315 320 Thr Thr Ile Gly Phe Glu Lys Pro Ile Ile Leu Thr Arg Ala Val Leu 325 330 335 Ser Ala Ser Ile Asp Lys Lys Arg Gln Gly Ser Ala Thr Ser Thr Pro 340 345 350 Ser Ser Ile Gln Asp Ser Ile Gln Thr Ala Ile Arg Glu Gln Gly Phe 355 360 365 Glu Ile Ile Arg Asp Tyr Cys Ile Glu Ile Ser Ile Leu Ile Ala Gly 370 375 380 Ala Ala Ser Gly Val Gln Gly Gly Leu Gln Gln Phe Cys Phe Leu Ala 385 390 395 400 Ala Trp Ile Leu Phe Phe Asp Cys Ile Leu Leu Phe Thr Phe Tyr Thr 405 410 415 Thr Ile Leu Cys Ile Lys Leu Glu Ile Thr Arg Ile Arg Arg His Val 420 425 430 Thr Leu Arg Lys Ala Leu Glu Glu Asp Gly Thr Thr Gln Ser Val Ala 435 440 445 Glu Lys Val Ala Ser Ser Asn Asp Trp Phe Gly Ala Gly Ser Asp Asn 450 455 460 Ser Asp Ala Asp Asp Ala Ser Val Phe Gly Arg Lys Ile Lys Ser Asn 465 470 475 480 Asn Val Arg Arg Phe Lys Phe Leu Met Val Gly Gly Phe Val Leu Val 485 490 495 Asn Val Val Asn Met Thr Ala Ile Pro Phe Arg Asn Ser Ser Leu Ser 500 505 510 Pro Leu Cys Asn Val Phe Ser Pro Thr Pro Ile Asp Pro Phe Lys Val 515 520 525 Ala Glu Asn Gly Leu Asp Ala Thr Tyr Val Ser Ala Lys Ser Gln Lys 530 535 540 Leu Glu Thr Leu Val Thr Val Val Pro Pro Ile Lys Val Lys Leu Glu 545 550 555 560 Tyr Pro Ser Val His Tyr Ala Lys Leu Gly Glu Ser Gln Ser Ile Glu 565 570 575 Ile Glu Tyr Thr Asp Gln Leu Leu Asp Ala Val Gly Gly His Val Leu 580 585 590 Asn Gly Val Leu Lys Ser Ile Glu Asp Pro Val Ile Ser Lys Trp Ile 595 600 605 Thr Ala Val Leu Thr Ile Ser Ile Val Leu Asn Gly Tyr Leu Phe Asn 610 615 620 Ala Ala Arg Trp Ser Ile Lys Glu Pro Gln Ala Ala Pro Ala Pro Lys 625 630 635 640 Glu Pro Ala Lys Pro Lys Val Tyr Pro Lys Thr Asp Leu Asn Ala Gly 645 650 655 Pro Lys Arg Ser Met Glu Glu Cys Glu Ala Met Leu Lys Ala Lys Lys 660 665 670 Ala Ala Tyr Leu Ser Asp Glu Leu Leu Ile Glu Leu Ser Leu Ser Gly 675 680 685 Lys Leu Pro Gly Tyr Ala Leu Leu Lys Ser Leu Glu Asn Glu Glu Leu 690 695 700 Met Ser Arg Val Asp Ala Phe Leu Arg Ala Val Lys Leu Arg Arg Ala 705 710 715 720 Val Val Ser Arg Thr Pro Ala Thr Ser Ala Val Thr Ser Ser Leu Glu 725 730 735 Thr Ser Lys Leu Pro Tyr Lys Asp Tyr Asn Tyr Ala Leu Val His Gly 740 745 750 Ala Cys Cys Glu Asn Val Ile Gly Thr Leu Pro Leu Pro Leu Gly Val 755 760 765 Ala Gly Pro Leu Val Thr Asp Gly Gln Ser Tyr Phe Ile Pro Met Ala 770 775 780 Thr Ile Glu Gly Val Leu Val Ala Ser Ala Ser Arg Gly Ala Lys Ala 785 790 795 800 Ile Asn Ala Gly Gly Gly Ala Val Ile Val Leu Thr Gly Asp Gly Met 805 810 815 Thr Arg Gly Pro Cys Val Gly Phe Pro Thr Leu Ala Arg Ala Ala Ala 820 825 830 Ala Lys Val Trp Leu Asp Ser Glu Glu Gly Lys Ser Val Met Thr Ala 835 840 845 Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln His Leu Lys Thr 850 855 860 Ala Leu Ala Gly Thr Tyr Leu Tyr Ile Arg Phe Lys Thr Thr Thr Gly 865 870 875 880 Asp Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu Lys Ala Leu 885 890 895 His Val Met Ala Thr Glu Cys Gly Phe Asp Asp Met Ala Thr Ile Ser 900 905 910 Val Ser Gly Asn Phe Cys Thr Asp Lys Lys Ala Ala Ala Leu Asn Trp 915 920 925 Ile Asp Gly Arg Gly Lys Ser Val Val Ala Glu Ala Ile Ile Pro Gly 930 935 940 Asp Val Val Arg Asn Val Leu Lys Ser Asp Val Asp Ala Leu Val Glu 945 950 955 960 Leu Asn Thr Ser Lys Asn Leu Ile Gly Ser Ala Met Ala Gly Ser Leu 965 970 975 Gly Gly Phe Asn Ala His Ala Ser Asn Ile Val Thr Ala Ile Phe Leu 980 985 990 Ala Thr Gly Gln Asp Pro Ala Gln Asn Val Glu Ser Ser Ser Cys Ile 995 1000 1005 Thr Thr Met Lys Asn Thr Asn Gly Asn Leu Gln Thr Ala Val Ser 1010 1015 1020 Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly Gly Thr Ile Leu 1025 1030 1035 Glu Ala Gln Gly Ala Met Leu Asp Leu Leu Gly Val Arg Gly Ser 1040 1045 1050 His Pro Thr Asn Pro Gly Asp Asn Ala Arg Gln Leu Ala Arg Ile 1055 1060 1065 Val Ala Ala Ala Val Leu Ala Gly Phe Leu Ser Leu Cys Ser Ala 1070 1075 1080 Leu Ala Ala Gly His Leu Val Arg Ala His Met Ala His Asn Arg 1085 1090 1095 Ser Ala Ala Pro Thr Arg Ser Ala Thr Pro Val Ser Ala Ala Val 1100 1105 1110 Gly Ala Thr Arg Gly Leu Ser Met Thr Ser Ser Arg 1115 1120 1125 1111210PRTGibberella zeae 111Met Ala Ser Ile Leu Leu Pro Lys Lys Phe Arg Gly Glu Thr Ala Pro 1 5 10 15 Ala Glu Lys Thr Thr Pro Ser Trp Ala Ser Lys Arg Leu Thr Pro Ile 20 25 30 Ala Gln Phe Ile Ser Arg Leu Ala Cys Ser His Pro Ile His Thr Val 35 40 45 Val Leu Val Ala Val Leu Ala Ser Thr Ser Tyr Val Gly Leu Leu Gln 50 55 60 Glu Ser Phe Phe Ser Thr Asp Leu Pro Thr Val Gly Lys Ala Asp Trp 65 70 75 80 Ser Ser Leu Val Glu Gly Ser Arg Val Leu Arg Ala Gly Pro Glu Thr 85 90 95 Ala Trp Asn Trp Lys Ala Ile Glu Gln Asp Ser Ile Gln His Ala Gly 100 105 110 Ala Asp Ala Asp His Leu Ala Leu Leu Thr Leu Val Phe Pro Asp Thr 115 120 125 His Ser Ala Glu Ser Ser Ser Thr Ala Pro Arg Ser Ser His Val Pro 130 135 140 Val Pro Gln Asn Leu Ser Ile Thr Pro Leu Pro Ser Thr Lys Asn Ser 145 150 155 160 Phe Thr Ala Tyr Ser Gln Asp Ser Ile Leu Ala Tyr Ser Leu Pro Tyr 165 170 175 Ala Glu Gly Pro Asp Val Val Gln Trp Ala Asn Asn Ala Trp Thr Glu 180 185 190 Phe Leu Asp Leu Leu Lys Asn Ala Glu Thr Leu Asp Ile Val Ile Met 195 200 205 Phe Leu Gly Tyr Thr Ala Met His Leu Thr Phe Val Ser Leu Phe Leu 210 215 220 Ser Met Arg Lys Ile Gly Ser Lys Phe Trp Leu Gly Ile Cys Thr Leu 225 230 235 240 Phe Ser Ser Val Phe Ala Phe Leu Phe Gly Leu Ile Val Thr Thr Lys 245 250 255 Leu Gly Val Pro Ile Ser Val Ile Leu Leu Ser Glu Gly Leu Pro Phe 260 265 270 Leu Val Val Thr Ile Gly Phe Glu Lys Asn Ile Val Leu Thr Arg Ala 275 280 285 Val Met Ser His Ala Ile Glu His Arg Arg Gln Ile Gln Asn Ser Lys 290 295 300 Ser Gly Lys Gly Ser Pro Glu Arg Ser Met Gln Asn Val Ile Gln Tyr 305 310 315 320 Ala Val Gln Ser Ala Ile Lys Glu Lys Gly Phe Glu Ile Met Arg Asp 325 330 335 Tyr Ala Ile Glu Ile Val Ile Leu Ala Leu Gly Ala Ala Ser Gly Val 340 345 350 Gln Gly Gly Leu Gln His Phe Cys Phe Leu Ala Ala Trp Thr Leu Phe 355 360 365 Phe Asp Phe Ile Leu Leu Phe Thr Phe Tyr Thr Ala Ile Leu Ser Ile 370 375 380 Lys Leu Glu Ile Asn Arg Ile Lys Arg His Val Asp Met Arg Met Ala 385 390 395 400 Leu Glu Asp Asp Gly Val Ser Arg Arg Val Ala Glu Asn Val Ala Lys 405 410 415 Ser Asp Gly Asp Trp Thr Arg Val Lys Gly Asp Ser Ser Leu Phe Gly 420 425 430 Arg Lys Ser Ser Ser Val Pro Thr Phe Lys Val Leu Met Ile Leu Gly 435 440 445 Phe Ile Phe Val Asn Ile Val Asn Ile Cys Ser Ile Pro Phe Arg Asn 450 455 460 Pro Arg Ser Leu Ser Thr Ile Arg Thr Trp Ala Ser Ser Leu Gly Gly 465 470 475 480 Val Val Ala Pro Leu Ser Val Asp Pro Phe Lys Val Ala Ser Asn Gly 485 490 495 Leu Asp Ala Ile Leu Ala Ala Ala Lys Ser Asn Asn Arg Pro Thr Leu 500 505 510 Val Thr Val Leu Thr Pro Ile Lys Tyr Glu Leu Glu Tyr Pro Ser Ile 515 520 525 His Tyr Ala Leu Gly Ser Ala Ile Asn Gly Asn Asn Ala Glu Tyr Thr 530 535 540 Asp Ala Phe His His His Phe Gln Gly Tyr Gly Val Gly Gly Arg Met 545 550 555 560 Val Gly Gly Ile Leu Lys Ser Leu Glu Asp Pro Val Leu Ser Lys Trp 565 570 575 Ile Val Ile Ala Leu Ala Leu Ser Val Ala Leu Asn Gly Tyr Leu Phe 580 585 590 Asn Val Ala Arg Trp Gly Ile Lys Asp Pro Asn Val Pro Glu His Asn 595 600 605 Ile Asp Arg Asn Glu Leu Ala Arg Ala Gln Gln Phe Asn Asp Thr Gly 610 615 620 Ser Ala Thr Leu Pro Leu Gly Glu Tyr Val Pro Pro Thr Pro Met Arg 625 630 635 640 Thr Glu Pro Ser Thr Pro Ala Ile Thr Asp Asp Glu Ala Glu Gly Leu 645 650 655 Gln Met Thr Lys Ala Arg Ser Asp Lys Leu Pro Asn Arg Pro Asn Glu 660 665 670 Glu Leu Glu Lys Leu Leu Ala Glu Lys Arg Val Lys Glu Met Ser Asp 675 680 685 Glu Glu Leu Val Ser Leu Ser Met Arg Cys Lys Ile Pro Gly Tyr Ala 690 695 700 Leu Leu Lys Thr Leu Gly Asp Phe Thr Arg Ala Val Lys Ile Arg Arg 705 710 715 720 Ser Ile Ile Ala Arg Asn Arg Ala Thr Ser Asp Leu Thr His Ser Leu 725 730 735 Glu Arg Ser Lys Leu Pro Phe Glu Lys Tyr Asn Trp Glu Arg Val Phe 740 745 750 Cys Ala Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Leu Pro Val Gly 755 760 765 Val Ala Gly Arg Leu Val Thr Asp Gly Gln Ser Tyr Phe Ile Pro Met 770 775 780 Ala Thr Thr Glu Gly Val Leu Val Ala Ser Ala Ser Arg Gly Cys Lys 785 790 795 800 Ala Ile Asn Ala Gly Gly Gly Ala Val Thr Val Leu Thr Ala Asp Gly 805 810 815 Met Thr Arg Gly Pro Cys Val Ala Phe Glu Thr Leu Glu Arg Ala Gly 820 825 830 Ala Ala Lys Leu Trp Ile Asp Ser Glu Ala Gly Ser Asp Ile Met Lys 835 840 845 Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln Ser Met Lys 850 855 860 Thr Ala Leu Ala Gly Thr Asn Leu Tyr Ile Arg Phe Lys Thr Thr Thr 865 870 875 880 Gly Asp Ala Met Gly Met Asn Ile Ile Ser Lys Gly Val Glu His Ala 885 890 895 Leu Ser Val Met Ser Asn Glu Ala Gly Phe Asp Asp Met Gln Ile Val 900 905 910 Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys Ala Ala Ala Leu Asn 915 920 925 Trp Ile Asp Gly Arg Gly Lys Gly

Val Val Ala Glu Ala Ile Ile Pro 930 935 940 Gly Asp Val Val Arg Ser Val Leu Lys Ser Asp Val Asp Ala Leu Val 945 950 955 960 Glu Leu Asn Ile Ser Lys Asn Ile Ile Gly Ser Ala Met Ala Gly Ser 965 970 975 Val Gly Gly Phe Asn Ala His Ala Ala Asn Ile Val Ala Ala Ile Phe 980 985 990 Leu Ala Thr Gly Gln Asp Pro Ala Gln Val Val Glu Ser Ala Asn Cys 995 1000 1005 Ile Thr Leu Met Lys Asn Leu Arg Gly Ala Leu Gln Thr Ser Val 1010 1015 1020 Ser Met Pro Ser Leu Glu Val Gly Thr Leu Gly Gly Gly Thr Ile 1025 1030 1035 Leu Glu Pro Gln Ser Ala Met Leu Asp Leu Leu Gly Val Arg Gly 1040 1045 1050 Ser His Pro Thr Asn Pro Gly Asp Asn Ser Arg Arg Leu Ala Arg 1055 1060 1065 Ile Ile Gly Ala Ser Val Leu Ala Gly Glu Leu Ser Leu Cys Ser 1070 1075 1080 Ala Leu Ala Ala Gly His Leu Val Arg Ala His Met Gln His Asn 1085 1090 1095 Arg Ser Ala Ala Pro Ser Arg Ser Thr Thr Pro Ala Pro Met Thr 1100 1105 1110 Pro Val Arg Ser Phe Asp Thr Lys Val Arg Cys Gln Pro Asn Asn 1115 1120 1125 Lys Asp Ile Arg Asn Ile Leu Leu Thr Gln His Pro Ser Lys Pro 1130 1135 1140 Thr Ile Thr Tyr Ser Lys Arg Val Ile Lys Ser Thr Ile His Leu 1145 1150 1155 Asn Pro Leu Ile Leu Ala Leu Phe Asp Asn Ser Val Gln Thr Arg 1160 1165 1170 Asp Val Gln Leu Gly Asp Gln Val Ser Thr Arg Gly Thr Leu Asp 1175 1180 1185 Ala Val Gly Gly Pro Gln Gly Gly Gly Val Ala Ala Gly Gly Val 1190 1195 1200 Ala Arg Arg Val Val Gly Ser 1205 1210 1121173PRTNeurospora crassa 112Met Ile Ala Ser Ser Leu Leu Pro Ser Lys Phe Arg Gly Glu Gln Pro 1 5 10 15 Ala Thr Gln Ala Ala Thr Pro Ser Trp Ile Asn Lys Lys Val Thr Pro 20 25 30 Pro Leu Gln Lys Leu Ser Lys Ile Thr Ser Ser Asn Pro Ile His Thr 35 40 45 Ile Val Ile Val Ala Leu Leu Ala Ser Ser Ser Tyr Ile Gly Leu Leu 50 55 60 Gln Asn Ser Leu Phe Asn Val Thr Arg Ser Val Arg Lys Ala Glu Trp 65 70 75 80 Glu Ser Leu Gln Ala Gly Ser Arg Met Leu Arg Ala Gly Ala Asn Thr 85 90 95 Glu Trp Asn Trp Gln Asn His Asp Pro Glu Ala Pro Val Pro Ala Asn 100 105 110 Ala Asn His Leu Ala Leu Leu Thr Leu Val Phe Pro Asp Thr Ala Glu 115 120 125 Ser Gly Pro Val Val Ala Gln Thr Asn Thr Val Pro Leu Pro Ser Asn 130 135 140 Leu Ser Thr Thr Pro Leu Pro Ser Thr Ala Ile Ser Phe Thr Tyr Ser 145 150 155 160 Gln Asp Ser Ala Leu Ala Phe Ser Leu Pro Tyr Ser Gln Ala Pro Glu 165 170 175 Phe Leu Ala Asn Ala Gln Glu Ile Pro Asn Ala Val Ser Ser Gln Glu 180 185 190 Thr Ile Glu Thr Glu Arg Gly His Glu Lys Lys Met Trp Ile Met Lys 195 200 205 Ala Ala Arg Val Gln Thr Arg Ser Ser Thr Val Lys Trp Val Gln Asn 210 215 220 Ala Trp Val Glu Phe Thr Asp Leu Leu Arg Asn Ala Glu Thr Leu Asp 225 230 235 240 Ile Ile Ile Met Ala Leu Gly Tyr Ile Ser Met His Leu Thr Phe Val 245 250 255 Ser Leu Phe Leu Ser Met Arg Arg Met Gly Ser Asn Phe Trp Leu Ala 260 265 270 Thr Ser Val Ile Phe Ser Ser Ile Phe Ala Phe Leu Phe Gly Leu Leu 275 280 285 Val Thr Thr Lys Leu Gly Val Pro Met Asn Met Val Leu Leu Ser Glu 290 295 300 Gly Leu Pro Phe Leu Val Val Thr Ile Gly Phe Glu Lys Asn Ile Val 305 310 315 320 Leu Thr Arg Ala Val Leu Ser His Ala Ile Asp His Arg Arg Pro Thr 325 330 335 Glu Lys Ser Gly Lys Pro Ser Lys Gln Ala Asp Ser Ala His Ser Ile 340 345 350 Gln Ser Ala Ile Gln Leu Ala Ile Lys Glu Lys Gly Phe Asp Ile Val 355 360 365 Lys Asp Tyr Ala Ile Glu Ala Gly Ile Leu Val Leu Gly Ala Ala Ser 370 375 380 Gly Val Gln Gly Gly Leu Gln Gln Phe Cys Phe Leu Ala Ala Trp Ile 385 390 395 400 Leu Phe Phe Asp Cys Ile Leu Leu Phe Ser Phe Tyr Thr Ala Ile Leu 405 410 415 Cys Ile Lys Leu Phe Ile Asn Arg Ile Lys Arg His Val Gln Met Arg 420 425 430 Lys Ala Leu Glu Glu Asp Gly Val Ser Arg Arg Val Ala Glu Lys Val 435 440 445 Ala Gln Ser Asn Asp Trp Pro Arg Ala Asp Gly Lys Asp Gln Pro Gly 450 455 460 Thr Thr Ile Phe Gly Arg Gln Leu Lys Ser Thr His Ile Pro Lys Phe 465 470 475 480 Lys Val Met Met Val Thr Gly Phe Val Leu Ile Asn Val Leu Asn Leu 485 490 495 Cys Thr Ile Pro Phe Arg Ser Ala Asn Ser Ile Ser Ser Ile Ser Ser 500 505 510 Trp Ala Arg Gly Leu Gly Gly Val Val Thr Pro Pro Pro Val Asp Pro 515 520 525 Phe Lys Val Ala Ser Asn Gly Leu Asp Ile Ile Leu Glu Ala Ala Arg 530 535 540 Ala Asp Gly Arg Glu Thr Thr Val Thr Val Leu Thr Pro Ile Arg Tyr 545 550 555 560 Glu Leu Glu Tyr Pro Ser Thr His Tyr Asp Leu Pro Gln Lys Ser Ala 565 570 575 Glu Val Glu Gly Gly Asp Tyr Ala Asn Leu Gly Gly Tyr Gly Gly Arg 580 585 590 Met Val Gly Ser Ile Leu Lys Ser Leu Glu Asp Pro Thr Leu Ser Lys 595 600 605 Trp Ile Val Val Ala Leu Ala Leu Ser Val Ala Leu Asn Gly Tyr Leu 610 615 620 Phe Asn Ala Ala Arg Trp Gly Ile Lys Asp Pro Asn Val Pro Asp His 625 630 635 640 Pro Ile Asn Pro Lys Glu Leu Asp Glu Ala Gln Lys Phe Asn Asp Thr 645 650 655 Ala Ser Ala Thr Leu Pro Leu Gly Glu Tyr Met Lys Pro Thr Ala Pro 660 665 670 Ser Ser Pro Val Ala Pro Leu Thr Pro Ser Ser Thr Asp Asp Glu Asn 675 680 685 Asp Ala Gln Ala Lys Glu Asn Arg Ala Val Thr Leu Ala Ala Gln Arg 690 695 700 Ala Thr Thr Ile Arg Ser Gln Gly Glu Leu Asp Lys Met Thr Ala Glu 705 710 715 720 Lys Arg Thr His Glu Leu Asn Asp Glu Glu Thr Val His Leu Ser Leu 725 730 735 Lys Gly Lys Ile Pro Gly Tyr Ala Leu Glu Lys Thr Leu Lys Asp Phe 740 745 750 Thr Arg Ala Val Lys Val Arg Arg Ser Ile Ile Ser Arg Thr Lys Ala 755 760 765 Thr Thr Glu Leu Thr Asn Ile Leu Asp Arg Ser Lys Leu Pro Tyr Gln 770 775 780 Asn Val Asn Trp Ala Gln Val His Gly Ala Cys Cys Glu Asn Val Ile 785 790 795 800 Gly Tyr Met Pro Leu Pro Val Gly Val Ala Gly Pro Leu Val Thr Asp 805 810 815 Gly Gln Ser Phe Phe Val Pro Met Ala Thr Thr Glu Gly Val Leu Val 820 825 830 Ala Ser Thr Ser Arg Gly Cys Lys Ala Ile Asn Ser Gly Gly Gly Ala 835 840 845 Val Thr Val Leu Thr Ala Asp Gly Met Thr Arg Gly Pro Cys Val Gln 850 855 860 Phe Glu Thr Leu Glu Arg Ala Gly Ala Ala Lys Leu Trp Leu Asp Ser 865 870 875 880 Glu Lys Gly Gln Ser Ile Met Lys Lys Ala Phe Asn Ser Thr Ser Arg 885 890 895 Phe Ala Arg Leu Glu Thr Met Lys Thr Ala Met Ala Gly Thr Asn Leu 900 905 910 Tyr Ile Arg Phe Lys Ile Thr Thr Gly Asp Ala Met Gly Met Asn Met 915 920 925 Ile Ser Lys Gly Val Glu His Ala Leu Ser Val Met Tyr Asn Glu Gly 930 935 940 Phe Glu Asp Met Asn Ile Val Ser Leu Ser Gly Asn Tyr Cys Thr Asp 945 950 955 960 Lys Lys Ala Ala Ala Ile Asn Val Ile Asp Gly Arg Gly Lys Ser Val 965 970 975 Val Ala Glu Ala Ile Ile Pro Ala Asp Val Val Lys Asn Val Leu Lys 980 985 990 Thr Asp Val Asp Thr Leu Val Glu Leu Asn Val Asn Lys Asn Thr Ile 995 1000 1005 Gly Ser Ala Met Ala Gly Ser Met Gly Gly Phe Asn Ala His Ala 1010 1015 1020 Ala Asn Ile Val Ala Ala Ile Phe Leu Ala Thr Gly Gln Asp Pro 1025 1030 1035 Ala Gln Val Val Glu Ser Ala Asn Cys Ile Thr Leu Met Arg Asn 1040 1045 1050 Leu Arg Gly Asn Leu Gln Ile Ser Val Ser Met Pro Ser Ile Glu 1055 1060 1065 Val Gly Thr Leu Gly Gly Gly Thr Ile Leu Glu Pro Gln Ser Ala 1070 1075 1080 Met Leu Asp Met Leu Gly Val Arg Gly Pro His Pro Thr Asn Pro 1085 1090 1095 Gly Glu Asn Ala Arg Arg Leu Ala Arg Ile Val Ala Ala Ala Val 1100 1105 1110 Leu Ala Gly Glu Leu Ser Leu Cys Ser Ala Leu Ala Ala Gly His 1115 1120 1125 Leu Val Lys Ala His Met Ala His Asn Arg Ser Ala Pro Pro Thr 1130 1135 1140 Arg Thr Ser Thr Pro Ala Pro Ala Ala Ala Ala Gly Leu Thr Met 1145 1150 1155 Thr Ser Ser Asn Pro Asn Ala Ala Ala Val Glu Arg Ser Arg Arg 1160 1165 1170 1131045PRTSaccharomyces cerevisiae 113Met Ser Leu Pro Leu Lys Thr Ile Val His Leu Val Lys Pro Phe Ala 1 5 10 15 Cys Thr Ala Arg Phe Ser Ala Arg Tyr Pro Ile His Val Ile Val Val 20 25 30 Ala Val Leu Leu Ser Ala Ala Ala Tyr Leu Ser Val Thr Gln Ser Tyr 35 40 45 Leu Asn Glu Trp Lys Leu Asp Ser Asn Gln Tyr Ser Thr Tyr Leu Ser 50 55 60 Ile Lys Pro Asp Glu Leu Phe Glu Lys Cys Thr His Tyr Tyr Arg Ser 65 70 75 80 Pro Val Ser Asp Thr Trp Lys Leu Leu Ser Ser Lys Glu Ala Ala Asp 85 90 95 Ile Tyr Thr Pro Phe His Tyr Tyr Leu Ser Thr Ile Ser Phe Gln Ser 100 105 110 Lys Asp Asn Ser Thr Thr Leu Pro Ser Leu Asp Asp Val Ile Tyr Ser 115 120 125 Val Asp His Thr Arg Tyr Leu Leu Ser Glu Glu Pro Lys Ile Pro Thr 130 135 140 Glu Leu Val Ser Glu Asn Gly Thr Lys Trp Arg Leu Arg Asn Asn Ser 145 150 155 160 Asn Phe Ile Leu Asp Leu His Asn Ile Tyr Arg Asn Met Val Lys Gln 165 170 175 Phe Ser Asn Lys Thr Ser Glu Phe Asp Gln Phe Asp Leu Phe Ile Ile 180 185 190 Leu Ala Ala Tyr Leu Thr Leu Phe Tyr Thr Leu Cys Cys Leu Phe Asn 195 200 205 Asp Met Arg Lys Ile Gly Ser Lys Phe Trp Leu Ser Phe Ser Ala Leu 210 215 220 Ser Asn Ser Ala Cys Ala Leu Tyr Leu Ser Leu Tyr Thr Thr His Ser 225 230 235 240 Leu Leu Lys Lys Pro Ala Ser Leu Leu Ser Leu Val Ile Gly Leu Pro 245 250 255 Phe Thr Val Val Ile Ile Gly Glu Lys His Lys Val Arg Leu Ala Ala 260 265 270 Phe Ser Leu Gln Lys Phe His Arg Ile Ser Ile Asp Lys Lys Ile Thr 275 280 285 Val Ser Asn Ile Ile Tyr Glu Ala Met Phe Gln Glu Gly Ala Tyr Leu 290 295 300 Ile Arg Asp Tyr Leu Phe Tyr Ile Ser Ser Phe Ile Gly Cys Ala Ile 305 310 315 320 Tyr Ala Arg His Leu Pro Gly Leu Val Asn Phe Cys Ile Leu Ser Thr 325 330 335 Phe Met Leu Val Phe Asp Leu Leu Leu Ser Ala Thr Phe Tyr Ser Ala 340 345 350 Ile Leu Ser Met Lys Leu Phe Ile Asn Ile Ile His Arg Ser Thr Val 355 360 365 Ile Arg Gln Thr Leu Glu Glu Asp Gly Val Val Pro Thr Thr Ala Asp 370 375 380 Ile Ile Tyr Lys Asp Glu Thr Ala Ser Glu Pro His Phe Leu Arg Ser 385 390 395 400 Asn Val Ala Ile Ile Leu Gly Lys Ala Ser Val Ile Gly Leu Leu Leu 405 410 415 Leu Ile Asn Leu Tyr Val Phe Thr Asp Lys Leu Asn Ala Thr Ile Leu 420 425 430 Asn Thr Val Tyr Phe Asp Ser Thr Ile Tyr Ser Leu Pro Asn Phe Ile 435 440 445 Asn Tyr Lys Asp Ile Gly Asn Leu Ser Asn Gln Val Ile Ile Ser Val 450 455 460 Leu Pro Lys Gln Tyr Tyr Thr Pro Leu Lys Lys Tyr His Gln Ile Glu 465 470 475 480 Asp Ser Val Leu Leu Ile Ile Asp Ser Val Ser Asn Ala Ile Arg Asp 485 490 495 Gln Phe Ile Ser Lys Leu Leu Phe Phe Ala Phe Ala Val Ser Ile Ser 500 505 510 Ile Asn Val Tyr Leu Leu Asn Ala Ala Lys Ile His Thr Gly Tyr Met 515 520 525 Asn Phe Gln Pro Gln Ser Asn Lys Ile Asp Asp Leu Val Val Gln Gln 530 535 540 Lys Ser Ala Thr Ile Glu Phe Ser Glu Thr Arg Ser Met Pro Leu Ala 545 550 555 560 Ser Gly Leu Glu Thr Pro Val Thr Ala Lys Asp Ile Ile Ile Ser Glu 565 570 575 Glu Ile Gln Asn Asn Glu Cys Val Tyr Ala Leu Ser Ser Gln Asp Glu 580 585 590 Pro Ile Arg Pro Leu Ser Asn Leu Val Glu Leu Met Glu Lys Glu Gln 595 600 605 Leu Lys Asn Met Asn Asn Thr Glu Val Ser Asn Leu Val Val Asn Gly 610 615 620 Lys Leu Pro Leu Tyr Ser Leu Glu Lys Lys Leu Glu Asp Thr Leu Arg 625 630 635 640 Ala Val Leu Val Arg Arg Lys Ala Leu Ser Thr Leu Ala Glu Ser Pro 645 650 655 Ile Leu Val Ser Glu Lys Leu Pro Phe Arg Asn Tyr Asp Tyr Asp Arg 660 665 670 Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Ile Pro 675 680 685 Val Gly Val Ile Gly Pro Leu Ile Thr Asp Gly Thr Ser Tyr His Ile 690 695 700 Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser Ala Met Pro Gly 705 710 715 720 Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr Val Leu Thr Lys 725 730 735 Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro Thr Leu Ile Arg 740 745 750 Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu Gly Gln Asn Ser 755 760 765 Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln His 770 775 780 Thr Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met Arg Phe Arg Thr 785 790 795 800 Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu 805 810 815 Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp Glu Asp Met Glu

820 825 830 Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys Pro Ala Ala 835 840 845 Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val Ala Glu Ala Thr 850 855 860 Ile Pro Gly Asp Val Val Lys Ser Val Leu Lys Ser Asp Val Ser Ala 865 870 875 880 Leu Val Glu Leu Asn Ile Ser Lys Asn Ile Val Gly Ser Ala Met Ala 885 890 895 Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn Leu Val Thr Ala 900 905 910 Leu Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn Val Glu Ser Ser 915 920 925 Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp Leu Arg Ile Ser 930 935 940 Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly Gly Thr Val 945 950 955 960 Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly Val Arg Gly Pro 965 970 975 His Pro Thr Glu Pro Gly Ala Asn Ala Arg Gln Leu Ala Arg Ile Ile 980 985 990 Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys Ser Ala Leu Ala 995 1000 1005 Ala Gly His Leu Val Gln Ser His Met Thr His Asn Arg Lys Thr 1010 1015 1020 Asn Lys Ala Asn Glu Leu Pro Gln Pro Ser Asn Lys Gly Pro Pro 1025 1030 1035 Cys Lys Thr Ser Ala Leu Leu 1040 1045 1141053PRTSaccharomyces cerevisiae 114Met Pro Pro Leu Phe Lys Gly Leu Lys Gln Met Ala Lys Pro Leu Ala 1 5 10 15 Tyr Val Ser Arg Phe Ser Ala Lys Pro Pro Ile His Ile Ile Leu Phe 20 25 30 Ser Leu Ile Ile Ser Ala Phe Ala Tyr Leu Ser Val Ile Gln Tyr Tyr 35 40 45 Phe Asn Gly Trp Gln Leu Asp Ser Asn Ser Val Phe Glu Thr Ala Pro 50 55 60 Asn Lys Asp Ser Asn Thr Leu Phe Gln Glu Cys Ser His Tyr Tyr Arg 65 70 75 80 Asp Ser Ser Leu Asp Gly Trp Val Ser Ile Thr Ala His Glu Ala Ser 85 90 95 Glu Leu Pro Ala Pro His His Tyr Tyr Leu Leu Asn Leu Asn Phe Ser 100 105 110 Pro Asn Glu Thr Asp Ser Ile Pro Glu Leu Ala Asn Thr Val Phe Glu 115 120 125 Lys Asp Asn Thr Lys Tyr Leu Leu Gln Glu Asp Leu Ser Val Ser Lys 130 135 140 Glu Ile Ser Ser Thr Asp Gly Thr Lys Trp Arg Leu Arg Ser Asp Arg 145 150 155 160 Lys Ser Leu Phe Asp Val Lys Thr Leu Ala Tyr Ser Leu Tyr Asp Val 165 170 175 Phe Ser Glu Asn Val Thr Gln Ala Asp Pro Phe Asp Val Leu Ile Met 180 185 190 Val Thr Ala Tyr Leu Met Met Phe Tyr Thr Ile Phe Gly Leu Phe Asn 195 200 205 Asp Met Arg Lys Thr Gly Ser Asn Phe Trp Leu Ser Ala Ser Thr Val 210 215 220 Val Asn Ser Ala Ser Ser Leu Phe Leu Ala Leu Tyr Val Thr Gln Cys 225 230 235 240 Ile Leu Gly Lys Glu Val Ser Ala Leu Thr Leu Phe Glu Gly Leu Pro 245 250 255 Phe Leu Val Val Val Val Gly Phe Lys His Lys Ile Lys Ile Ala Gln 260 265 270 Tyr Ala Leu Glu Lys Phe Glu Arg Val Gly Leu Ser Lys Arg Ile Thr 275 280 285 Thr Asp Glu Ile Val Phe Glu Ser Val Ser Glu Glu Gly Gly Arg Leu 290 295 300 Ile Gln Asp His Leu Leu Cys Ile Phe Ala Phe Ile Gly Cys Ser Met 305 310 315 320 Tyr Ala His Gln Leu Lys Thr Leu Thr Asn Phe Gly Ile Leu Ser Ala 325 330 335 Phe Ile Leu Ile Phe Glu Leu Ile Leu Thr Pro Thr Phe Tyr Ser Ala 340 345 350 Ile Leu Ala Leu Arg Leu Phe Met Asn Val Ile His Arg Ser Thr Ile 355 360 365 Ile Lys Gln Thr Leu Glu Glu Asp Gly Val Val Pro Ser Thr Ala Arg 370 375 380 Ile Ile Ser Lys Ala Glu Lys Lys Ser Val Ser Ser Phe Leu Asn Leu 385 390 395 400 Ser Val Val Val Ile Thr Met Lys Leu Ser Val Ile Leu Leu Phe Val 405 410 415 Phe Ile Asn Phe Tyr Asn Phe Gly Ala Asn Trp Val Asn Asp Ala Phe 420 425 430 Asn Ser Leu Tyr Phe Asp Lys Glu Arg Val Ser Leu Pro Asp Phe Ile 435 440 445 Thr Ser Asn Ala Ser Glu Asn Phe Lys Glu Gln Ala Ile Val Ser Val 450 455 460 Thr Pro Leu Leu Tyr Tyr Lys Pro Ile Lys Ser Tyr Gln Arg Ile Glu 465 470 475 480 Asp Met Val Leu Leu Leu Leu Arg Asn Val Ser Val Ala Ile Arg Asp 485 490 495 Arg Phe Val Ser Lys Leu Val Leu Ser Ala Leu Val Cys Ser Ala Val 500 505 510 Ile Asn Val Tyr Leu Leu Asn Ala Ala Arg Ile His Thr Ser Tyr Thr 515 520 525 Ala Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr Ala 530 535 540 Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val Ile 545 550 555 560 Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser Ser 565 570 575 Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu Ser 580 585 590 Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu Ser 595 600 605 Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu Val 610 615 620 Thr His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly Asp 625 630 635 640 Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu Ala 645 650 655 Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr Asp 660 665 670 Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr Met 675 680 685 Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Thr Asp Gly Thr Ser 690 695 700 Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser Ala 705 710 715 720 Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr Val 725 730 735 Leu Thr Lys Asp Gly Met Ile Arg Gly Pro Val Val Arg Phe Pro Thr 740 745 750 Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu Gly 755 760 765 Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg 770 775 780 Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met Arg 785 790 795 800 Phe Arg Thr Thr Thr Ser Asp Ala Met Gly Met Asn Met Ile Ser Lys 805 810 815 Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp Glu 820 825 830 Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys 835 840 845 Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val Ala 850 855 860 Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser Asp 865 870 875 880 Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly Ser 885 890 895 Ala Met Ala Gly Ser Val Gly Gly Glu Asn Ala His Ala Ala Asn Leu 900 905 910 Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn Val 915 920 925 Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp Leu 930 935 940 Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly 945 950 955 960 Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Ser Val 965 970 975 Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu Ala 980 985 990 Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys Ala 995 1000 1005 Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn 1010 1015 1020 Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr 1025 1030 1035 Asp Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser 1040 1045 1050 115999PRTYarrowia lipolytica 115Met Leu Gln Ala Ala Ile Gly Lys Ile Val Gly Phe Ala Val Asn Arg 1 5 10 15 Pro Ile His Thr Val Val Leu Thr Ser Ile Val Ala Ser Thr Ala Tyr 20 25 30 Leu Ala Leu Leu Asp Ile Ala Ile Pro Gly Glu Glu Gly Thr Gln Pro 35 40 45 Ile Ser Tyr Tyr His Pro Ala Ala Lys Ser Tyr Asp Asn Pro Ala Asp 50 55 60 Trp Thr His Ile Ala Glu Ala Asp Ile Pro Ser Asp Ala Tyr Arg Leu 65 70 75 80 Ala Phe Ala Gln Ile Arg Val Ser Asp Val Gln Gly Gly Glu Ala Pro 85 90 95 Thr Ile Pro Gly Ala Val Ala Val Ser Asp Leu Asp His Arg Ile Val 100 105 110 Met Asp Tyr Lys Gln Trp Ala Pro Trp Thr Ala Ser Asn Glu Gln Ile 115 120 125 Ala Ser Glu Asn His Ile Trp Lys His Ser Phe Lys Asp His Val Ala 130 135 140 Phe Ser Trp Ile Lys Trp Phe Arg Trp Ala Tyr Leu Arg Leu Ser Thr 145 150 155 160 Leu Ile Gln Gly Ala Asp Asn Phe Asp Ile Ala Val Val Ala Leu Gly 165 170 175 Tyr Leu Ala Met His Tyr Thr Phe Phe Ser Leu Phe Arg Ser Lys Arg 180 185 190 Lys Val Gly Ser His Phe Trp Leu Ala Ser Met Ala Leu Val Ser Ser 195 200 205 Phe Phe Ala Phe Leu Leu Ala Val Val Ala Ser Ser Ser Leu Gly Tyr 210 215 220 Arg Pro Ser Met Ile Thr Met Ser Glu Gly Leu Pro Phe Leu Val Val 225 230 235 240 Ala Ile Gly Phe Asp Arg Lys Val Asn Leu Ala Ser Glu Val Leu Thr 245 250 255 Ser Lys Ser Ser Gln Leu Ala Pro Met Val Gln Val Ile Thr Lys Ile 260 265 270 Ala Ser Lys Ala Leu Phe Glu Tyr Ser Leu Glu Val Ala Ala Leu Phe 275 280 285 Ala Gly Ala Tyr Thr Gly Val Pro Arg Leu Ser Gln Phe Cys Phe Leu 290 295 300 Ser Ala Trp Ile Leu Ile Phe Asp Tyr Met Phe Leu Leu Thr Phe Tyr 305 310 315 320 Ser Ala Val Ile Ala Ile Lys Phe Leu Ile Asn His Ile Lys Phe Asn 325 330 335 Arg Met Ile Gln Asp Ala Leu Lys Glu Asp Gly Val Ser Ala Ala Val 340 345 350 Ala Glu Lys Val Ala Asp Ser Ser Pro Asp Ala Lys Leu Asp Arg Lys 355 360 365 Ser Asp Val Ser Leu Phe Gly Ala Ser Gly Ala Ile Ala Val Phe Lys 370 375 380 Ile Phe Met Val Leu Gly Phe Leu Gly Leu Asn Leu Ile Asn Leu Thr 385 390 395 400 Ala Ile Pro His Leu Gly Lys Ala Ala Ala Ala Ala Gln Ser Val Thr 405 410 415 Pro Ile Thr Leu Ser Pro Glu Leu Leu His Ala Ile Pro Ala Ser Val 420 425 430 Pro Val Val Val Thr Phe Val Pro Ser Val Val Tyr Glu His Ser Gln 435 440 445 Leu Ile Leu Gln Leu Glu Asp Ala Leu Thr Phe Phe Leu Ala Ala Cys 450 455 460 Ser Lys Thr Ile Gly Asp Pro Val Ile Ser Lys Tyr Ile Phe Leu Cys 465 470 475 480 Leu Met Val Ser Thr Ala Leu Asn Val Tyr Leu Phe Gly Ala Thr Arg 485 490 495 Glu Val Val Arg Thr Gln Ser Val Lys Val Val Glu Lys His Val Pro 500 505 510 Ile Val Ile Glu Lys Pro Ser Glu Lys Glu Glu Asp Thr Ser Ser Glu 515 520 525 Asp Ser Ile Glu Leu Thr Val Gly Lys Gln Pro Lys Pro Val Thr Glu 530 535 540 Thr Arg Ser Leu Asp Asp Leu Glu Ala Thr Met Lys Ala Gly Lys Thr 545 550 555 560 Lys Leu Leu Glu Asp His Glu Val Val Lys Leu Ser Leu Glu Gly Lys 565 570 575 Leu Pro Leu Tyr Ala Leu Phe Lys Gln Leu Gly Asp Asn Thr Arg Ala 580 585 590 Val Gly Ile Arg Arg Ser Ile Ile Ser Gln Gln Ser Asn Thr Lys Thr 595 600 605 Leu Glu Thr Ser Lys Leu Pro Tyr Leu His Tyr Asp Tyr Asp Arg Val 610 615 620 Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Leu Pro Val 625 630 635 640 Gly Val Ala Gly Pro Met Asn Thr Asp Gly Lys Asn Tyr His Ile Pro 645 650 655 Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser Thr Met Arg Gly Cys 660 665 670 Lys Ala Ile Asn Ala Gly Gly Gly Val Thr Thr Val Leu Thr Gln Asp 675 680 685 Gly Met Thr Arg Gly Pro Cys Val Ser Phe Pro Ser Leu Lys Arg Ala 690 695 700 Gly Ala Ala Lys Ile Trp Leu Asp Glu Ser Glu Gly Leu Lys Ser Met 705 710 715 720 Arg Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln Ser Leu 725 730 735 His Ser Thr Leu Ala Gly Asn Leu Leu Phe Ile Arg Phe Arg Thr Thr 740 745 750 Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu His 755 760 765 Ser Leu Ala Val Met Val Lys Glu Tyr Gly Phe Pro Leu Met Asp Ile 770 775 780 Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys Pro Ala Ala Ile 785 790 795 800 Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val Ala Glu Ala Thr Ile 805 810 815 Pro Ala His Ile Val Lys Ser Val Leu Lys Ser Glu Val Asp Ala Leu 820 825 830 Val Glu Leu Asn Ile Ser Lys Asn Leu Ile Gly Ser Ala Met Ala Gly 835 840 845 Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn Leu Val Thr Ala Ile 850 855 860 Tyr Leu Ala Thr Gly Gln Asp Pro Ala Gln Asn Val Glu Ser Ser Asn 865 870 875 880 Cys Ile Thr Leu Met Ser Asn Val Asp Gly Asn Leu Leu Ile Ser Val 885 890 895 Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly Gly Thr Ile Leu 900 905 910 Glu Pro Gln Gly Ala Met Leu Glu Met Leu Gly Val Arg Gly Pro His 915 920 925 Ile Glu Thr Pro Gly Ala Asn Ala Gln Gln Leu Ala Arg Ile Ile Ala 930 935 940 Ser Gly Val Leu Ala Ala Glu Leu Ser Leu Cys Ser Ala Leu Ala Ala 945 950 955 960 Gly His Leu Val Gln Ser His Met Thr His Asn Arg Ser Gln Ala Pro 965 970 975 Thr Pro Ala Lys Gln Ser Gln Ala Asp Leu Gln Arg Leu Gln Asn Gly 980 985 990 Ser Asn Ile Cys Ile Arg Ser 995 1161289PRTArtificial SequenceDescription of Artificial Sequence Synthetic consensus sequence 116Xaa Met Ala Ser Xaa Leu Leu Xaa Xaa Arg Phe Xaa Xaa Glu Xaa

Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Ala Xaa Pro Ser Trp Xaa Xaa Lys Xaa Leu Thr Xaa 20 25 30 Pro Ile Gln Xaa Ile Ser Arg Phe Ala Ala Xaa His Pro Ile His Thr 35 40 45 Ile Val Leu Val Ala Leu Leu Ala Ser Thr Ala Tyr Leu Gly Leu Leu 50 55 60 Gln Xaa Ser Leu Phe Xaa Trp Xaa Leu Xaa Ser Asn Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Asp Xaa Thr Ser Leu Xaa Xaa Gly Ser Arg Xaa Leu Arg Xaa 85 90 95 Gly Xaa Xaa Thr Xaa Trp Arg Trp Xaa Xaa Ile Asp Xaa Xaa Xaa Ile 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Ala Asp Ala Xaa 115 120 125 His Leu Ala Leu Xaa Thr Leu Val Phe Pro Asp Thr Gln Ser Xaa Glu 130 135 140 Xaa Ala Ser Thr Ile Pro Xaa Ala Xaa Xaa Val Pro Val Pro Xaa Asn 145 150 155 160 Xaa Ser Ile Xaa Leu Leu Pro Xaa Thr Xaa Xaa Ile Phe Thr Xaa Tyr 165 170 175 Ser Gln Asp Ser Ser Leu Xaa Phe Ser Leu Pro Tyr Ser Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 220 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Ile Val Xaa Trp Xaa Xaa 225 230 235 240 Asn Ala Trp Xaa Xaa Phe Ser Asp Leu Ile Lys Asn Ala Asp Thr Phe 245 250 255 Asp Ile Ile Ile Met Xaa Leu Gly Tyr Leu Ala Met His Tyr Thr Phe 260 265 270 Xaa Ser Leu Phe Xaa Ser Met Arg Lys Leu Gly Ser Lys Phe Trp Leu 275 280 285 Ala Thr Ser Xaa Leu Phe Ser Ser Ile Phe Ala Phe Leu Leu Gly Leu 290 295 300 Leu Val Thr Thr Lys Leu Gly Xaa Val Pro Ile Ser Met Leu Leu Leu 305 310 315 320 Ser Glu Gly Leu Pro Phe Leu Val Val Thr Ile Gly Phe Glu Lys Lys 325 330 335 Ile Val Leu Thr Arg Ala Val Leu Ser Xaa Ala Ile Asp Xaa Arg Arg 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa 355 360 365 Xaa Xaa Ser Ile Gln Xaa Ala Ile Gln Xaa Ala Ile Lys Glu Xaa Gly 370 375 380 Phe Glu Ile Ile Arg Asp Tyr Ala Ile Glu Ile Ser Ile Leu Ile Ala 385 390 395 400 Gly Ala Ala Ser Gly Val Gln Gly Gly Xaa Leu Xaa Gln Phe Cys Phe 405 410 415 Leu Ala Ala Trp Ile Leu Phe Phe Asp Xaa Ile Leu Leu Phe Thr Phe 420 425 430 Tyr Ser Ala Ile Leu Ala Ile Lys Leu Glu Ile Asn Arg Ile Lys Arg 435 440 445 His Val Ile Ile Arg Xaa Ala Leu Glu Glu Asp Gly Val Ser Xaa Ser 450 455 460 Val Ala Glu Lys Val Ala Lys Ser Glu Xaa Asp Trp Xaa Xaa Xaa Lys 465 470 475 480 Gly Ser Asp Ser Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 485 490 495 Lys Ser Xaa Ala Ile Xaa Ile Phe Lys Val Leu Met Ile Leu Gly Phe 500 505 510 Val Leu Ile Asn Leu Val Asn Leu Thr Ala Ile Pro Phe Arg Xaa Ala 515 520 525 Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Gly Val 530 535 540 Xaa Ser Pro Xaa Xaa Val Asp Pro Phe Lys Val Ala Xaa Asn Leu Leu 545 550 555 560 Asp Ala Ile Xaa Ala Ala Ala Lys Ser Asn Xaa Arg Glu Thr Leu Val 565 570 575 Thr Val Val Thr Pro Ile Lys Tyr Glu Leu Glu Tyr Pro Ser Ile His 580 585 590 Tyr Xaa Glu Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 595 600 605 Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Gly Gly Xaa Met Leu 610 615 620 Gly Ser Val Ser Lys Ser Ile Glu Asp Pro Val Ile Ser Lys Trp Ile 625 630 635 640 Val Ile Ala Leu Ala Leu Ser Ile Ala Leu Asn Val Tyr Leu Phe Asn 645 650 655 Ala Ala Arg Trp Xaa Ile Lys Asp Pro Asn Val Xaa Xaa Xaa Xaa Xaa 660 665 670 Glu Val Xaa Glu Leu Xaa Xaa Xaa Gln Xaa Xaa Asn Xaa Xaa Xaa Ser 675 680 685 Ala Xaa Leu Xaa Xaa Xaa Xaa Xaa Ile Xaa Xaa Thr Xaa Xaa Xaa Xaa 690 695 700 Xaa Xaa Xaa Xaa Xaa Thr Pro Ala Xaa Thr Asp Asp Glu Xaa Xaa Ser 705 710 715 720 Xaa Xaa Ser Xaa Xaa Xaa Xaa Val Xaa Lys Ile Xaa Xaa Xaa Xaa Xaa 725 730 735 Xaa Ile Arg Ser Leu Glu Glu Leu Glu Ala Leu Leu Ala Ala Lys Lys 740 745 750 Thr Lys Xaa Leu Xaa Asp Glu Glu Val Val Xaa Leu Ser Leu Xaa Gly 755 760 765 Lys Leu Pro Leu Tyr Ala Leu Glu Lys Thr Leu Gly Xaa Xaa Xaa Xaa 770 775 780 Xaa Xaa Xaa Xaa Xaa Asp Phe Thr Arg Ala Val Lys Ile Arg Arg Ser 785 790 795 800 Ile Ile Ser Arg Xaa Xaa Ala Thr Ser Ala Leu Thr Xaa Ser Leu Glu 805 810 815 Ser Ser Lys Leu Pro Tyr Lys Asn Tyr Asn Tyr Asp Arg Val Phe Gly 820 825 830 Ala Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Leu Pro Val Gly Val 835 840 845 Ala Gly Pro Leu Val Ile Asp Gly Gln Ser Tyr His Ile Pro Met Ala 850 855 860 Thr Thr Glu Gly Val Leu Val Ala Ser Ala Ser Arg Gly Cys Lys Ala 865 870 875 880 Ile Asn Ala Gly Gly Gly Ala Val Thr Val Leu Thr Ala Asp Gly Met 885 890 895 Thr Arg Gly Pro Cys Val Xaa Phe Pro Thr Leu Xaa Arg Ala Gly Ala 900 905 910 Ala Lys Ile Trp Leu Asp Ser Glu Glu Gly Gln Xaa Ser Met Lys Lys 915 920 925 Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln His Ile Lys Thr 930 935 940 Ala Leu Ala Gly Thr Leu Leu Phe Ile Arg Phe Lys Thr Thr Thr Gly 945 950 955 960 Asp Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu His Ala Leu 965 970 975 Ser Val Met Val Xaa Glu Tyr Gly Phe Glu Asp Met Glu Ile Val Ser 980 985 990 Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys Pro Ala Ala Ile Asn Trp 995 1000 1005 Ile Asp Gly Arg Gly Lys Ser Val Val Ala Glu Ala Thr Ile Pro 1010 1015 1020 Gly Asp Val Val Lys Ser Val Leu Lys Ser Asp Val Asp Ala Leu 1025 1030 1035 Val Glu Leu Asn Ile Ser Lys Asn Leu Ile Gly Ser Ala Met Ala 1040 1045 1050 Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn Ile Val Thr 1055 1060 1065 Ala Ile Phe Leu Ala Thr Gly Gln Asp Pro Ala Gln Asn Val Glu 1070 1075 1080 Ser Ser Asn Cys Ile Thr Leu Met Lys Asn Val Asp Gly Asn Leu 1085 1090 1095 Gln Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly 1100 1105 1110 Gly Gly Thr Ile Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu 1115 1120 1125 Gly Val Arg Gly Pro His Pro Thr Asn Pro Gly Asp Asn Ala Arg 1130 1135 1140 Gln Leu Ala Arg Ile Ile Ala Ala Ala Val Leu Ala Gly Glu Leu 1145 1150 1155 Ser Leu Cys Ser Ala Leu Ala Ala Gly His Leu Val Gln Ala His 1160 1165 1170 Met Thr His Asn Arg Ser Ala Ala Pro Thr Arg Ser Xaa Thr Pro 1175 1180 1185 Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Ile Xaa Ser 1190 1195 1200 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1205 1210 1215 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1220 1225 1230 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1235 1240 1245 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1250 1255 1260 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1265 1270 1275 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1280 1285 11734DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 117ctctagacac aaaaatgtcg caaccccaga acgt 3411827DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 118cacgcgtcta ctgcttgatc tcgtact 2711936DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 119cgctagccac aaaaatggac tacatcattt cggcgc 3612027DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 120cacgcgtcta atgggtccag ggaccga 2712135DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 121ctctagacac aaaaatgacc acctattcgg ctccg 3512229DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 122cggcgcgccc tacttgaacc ccttctcga 2912336DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 123ctctagacac aaaaatgatc caccaggcct ccacca 3612427DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 124cacgcgtcta cttgctgttc ttcagag 2712534DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 125ctctagacac aaaaatgacg acgtcttaca gcga 3412627DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 126cacgcgtcta cttgatccac cgccgaa 2712735DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 127ctctagacac aaaaatgtcc aaggcgaaat tcgaa 3512827DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 128cacgcgtcta cttctgtcgc ttgtaaa 2712934DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 129ctctagacac aaaaatgtta cgactacgaa ccat 3413026DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 130cacgcgtcta gtcgtaaccc gcacat 2613134DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 131cgctagccac aaaaatgccg cagcaagcaa tgga 3413227DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 132cacgcgttta accatgcagc cgctcaa 2713334DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 133ctctagacac aaaaatgttc cgaacccgag ttac 3413427DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 134cacgcgttta agggttctgc ttgacaa 2713534DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 135ctctagacac aaaaatgaca caaacgcaca atct 3413627DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 136cacgcgttta catcttgtac gcagggt 2713734DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 137ctctagacac aaaaatggaa gccaaccccg aagt 3413827DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 138cacgcgttca tttcagaagg tacttct 2713932DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 139ctctagacac aaaaatgcga ctcactctgc cc 3214029DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 140cacgcgtcta ctcgacagaa gagaccttc 2914137DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 141ttctagaccc aaaaatgtct gccaacgaga acatctc 3714229DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 142aacgcgtcta tgatcgagtc ttggccttg 2914337DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 143ttctagacac aaaaatgtca gcgaaatcca ttcacga 3714426DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 144cacgcgttaa actccgagag gagtgg 2614534DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 145ctctagacac aaaagtggtt aaagctgtcg ttgc 3414627DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 146cacgcgttta cttggcagga ggagggt 2714734DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 147ctctagacac aaaaatgact ggcaccttac ccaa 3414827DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 148cacgcgttca cgaggagccc ttggtga 2714934DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 149ctctagacac aaaaatgact gacacttcaa acat 3415027DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 150cacgcgttta agcatcgtaa gtggaag 2715134DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 151ctctagacac aaaaatgctc aaccttagaa ccgc 3415227DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 152cacgcgtcta cttgagtcgc ttgataa 27

Claims

1. A recombinant fungus characterized by: a. the recombinant fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and b. the recombinant fungus produces at least one quinone derived compound, and can accumulate the produced quinone derived compound to at least about 1% of its dry cell weight; wherein the recombinant fungus comprises at least one modification as compared with a parental fungus, which parental fungus both is not oleaginous and does not accumulate the quinone derived compound to at least about 1% of its dry cell weight, the at least one modification being selected from the group consisting of quinonogenic modifications, oleaginic modifications, and combinations thereof, and wherein the at least one modification alters oleaginicity of the recombinant fungus, confers to the recombinant fungus oleaginy, confers to the recombinant fungus the ability to produce the at least one quinone derived compound to a level at least about 1% of its dry cell weight, or confers to the recombinant fungus the ability to produce at least one quinone derived compound which the parental fungus does not produce.

2. A recombinant fungus characterized by: a. the recombinant fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and b. the recombinant fungus produces at least one quinone derived compound selected from the group consisting of: a ubiquinone, a vitamin K compound, and a vitamin E compound, and combinations thereof, and can accumulate the produced quinone derived compound to at least about 1% of its dry cell weight; wherein the recombinant fungus comprises at least one modification as compared with a parental fungus, the at least one modification being selected from the group consisting of quinonogenic modifications, oleaginic modifications, and combinations thereof, and wherein the at least one modification alters oleaginicity of the recombinant fungus, confers to the recombinant fungus oleaginy, confers to the recombinant fungus the ability to produce the at least one quinone derived compound to a level at least about 1% of its dry cell weight, or confers to the recombinant fungus the ability to produce at least one quinone derived compound which the parental fungus does not naturally produce.

3-4. (canceled)

5. The recombinant fungus of claim 1 wherein the recombinant fungus is a member of a genus selected from the group consisting of: Saccharomyces, Xanthophyllomyces (Phaffia), and Yarrowia.

6-12. (canceled)

13. The recombinant fungus of claim 5 wherein the recombinant fungus is of the species Yarrowia lipolytica.

14-21. (canceled)

22. The recombinant fungus of claim 1 wherein the quinone derived compound is a ubiquinone.

23-25. (canceled)

26. The recombinant fungus of claim 1 wherein the quinone derived compound is a vitamin K compound.

27-28. (canceled)

29. The recombinant fungus of claim 1 wherein the quinone derived compound is a vitamin E compound.

30-53. (canceled)

54. The recombinant fungus of claim 1 wherein the fungus contains at least one quinonogenic modification.

55-59. (canceled)

60. The recombinant fungus of claim 54, wherein the at least one quinonogenic modification increases expression or activity of a quinonogenic polypeptide.

61-72. (canceled)

73. A recombinant fungus according to claim 1 wherein the fungus accumulates the produced at least one quinone derived compound to a level selected from the group consisting of: above about 1%, above about 2%, above about 3%, above about 5%, and above about 10% of the fungus' dry cell weight.

74-75. (canceled)

76. A strain of Yarrowia lipolytica comprising one or more modifications selected from the group consisting of an oleaginic modification, a quinonogenic modification, and combinations thereof, such that the strain accumulates from 1% to 15% of its dry cell weight as at least one quinone derived compound.

77-329. (canceled)