US007556937B2

(12) United States Patent (10) Patent No.: US 7,556,937 B2 Kinkel et al. (45) Date of Patent: Jul. 7, 2009

(54) METHOD FOR PRODUCING Jandrositz, A. et al., “The Gene Encoding Squalene Epoxidase from ERGOSTA-5,7-DIENOLAND/OR Saccharomyces cerevisiae: Cloning and Characterization'. Gene 107 BOSYNTHETIC INTERMEDIATE AND/OR (1991), pp. 155-160. SECONDARY PRODUCTS THEREOF IN Jennings, S. et al., “Molecular Cloning and Characterization of the TRANSGENIC ORGANISMS Yeast Gene for Squalene Synthetase'. Proc. Natl. Acad. Sci. USA 88 (1991), pp. 6038-6042. (75) Inventors: Andreas Kinkel, Neustadt (DE); Tainaka, H. et al., “Effects of Elevated Expression of the CYP51 Markus Veen, Berlin (DE); Christine (P450) Gene on the Sterol Contents of Saccharomyces Lang, Berlin (DE) cerevisiae', Journal of Fermentation and Bioengineering 79 (1995), (73) Assignee: OrganoBalance GmbH, Berlin (DE) pp. 64-66. Polakowski, T., “Molekularbiologische Beeinflussung des (*) Notice: Subject to any disclaimer, the term of this Ergosterolstoffwechsels der Hefe Saccharomyces cerevisiae', patent is extended or adjusted under 35 Shaker Verlag Aachen, Dissertation, Technischen Universität Berlin, U.S.C. 154(b) by 0 days. Germany, 1999, pp. 59-66. Pena-Diaz, J. et al., “A soluble 3-hydroxy-3-methylglutaryl-CoA (21) Appl. No.: 10/549,871 reductase in the protozoan Trypanosoma cruzi'. Biochem. J., (1997), vol. 324, pp. 619-626. (22) PCT Filed: Mar. 12, 2004 Favre, B. et al., “Characterization of Squalene Epoxidase Activity from the Dermatophyte Trichophyton rubrum and its Inhibition by (86). PCT No.: PCT/EP2004/OO2582 Terbinafine and Other Antimycotic Agents'. Antimicrobial Agents S371 (c)(1), and Chemotherapy, Feb. 1996, vol. 40, No. 2, pp. 443-447. (2), (4) Date: Sep. 16, 2005 Robinson, G.W. et al., "Conservation between Human and Fungal Squalene Synthetases: Similarities in Structure, Function, and Regu (87) PCT Pub. No.: WO2004/083407 lation'. Molecular and Cellular Biology, May 1993, vol. 13, No. 5, pp. 2706-2717. PCT Pub. Date: Sep. 30, 2004 Georgopapadakou, N.H. et al., “Effects of Squalene Epoxidase Inhibitors on Candida albicans”. Antimicrobial Agents and Chemo (65) Prior Publication Data therapy, Aug. 1992, vol. 36, No. 8, pp. 1779-1781. US 2006/0269986 A1 Nov.30, 2006 Jennings, S.M. et al., “Molecular cloning and characterization of the yeast gene for squalene synthetase'. Proc. Natl. Acad. Sci. USA, Jul. (30) Foreign Application Priority Data 1991, vol. 88, pp. 6038-6042. Mar. 19, 2003 (DE) ...... 103 12314 Nagumo, A. et al., “Purification and chacterization of recombinant squalene epoxidase'. Journal of Lipid Research, 1995, vol. 36, pp. (51) Int. Cl. 1489-1497. CI2P33/00 (2006.01) Tai, H.H. et al., “Squalene Epoxidase of Rat Liver'. The Journal of CI2N 9/02 (2006.01) Biological Chemistry, Jun. 25, 1972, vol. 247, No. 12 pp. 3767-3773. (52) U.S. Cl...... 435/52:435/189 Basson, M.E. et al., “Structural and Functional Conservation (58) Field of Classification Search ...... 435/52, between Yeast and Human 3-Hydroxy-3-Methylglutaryl Coenzyme 435/189 A Reductases, the Rate-Limiting Enzyme of Sterol Biosynthesis'. See application file for complete search history. Molecular and Cellular Biology, Sep. 1988, vol. 8, No. 9, pp. 3797 3808. (56) References Cited Jandrositz, A. et al., “The gene encoding squalene epoxidase from FOREIGN PATENT DOCUMENTS Saccharomyces cerevisiae: cloning and characterization', 1991, vol. 107, pp. 155-160. CA 2305780 A1 4f1999 DE 19744, 212 4f1999 (Continued) EP O 486 290 A2 5, 1992 WO WO-O2/O61072 A2 8, 2002 Primary Examiner Tekchand Saidha WO WO-03/064650 A1 8, 2003 (74) Attorney, Agent, or Firm Mayer & Williams PC; Ann Wieczorek, Esq.; Keum J. Park, Esq. OTHER PUBLICATIONS (57) ABSTRACT Basson, M. et al., “Structural and Functional Conservation Between Yeast and Human 3-Hydroxy-3-Methylglutaryl Co-enzyme A Reductases, The Rate-Limiting Enzyme of Sterol Biosynthesis'. The present invention relates to a method for the production Molecular and Cellular Biology 8 (1988), pp. 3797-3808. of ergosta-5,7-dienol and/or its biosynthetic intermediates Polakowski, T. et al., “Overexpression of a Cytosolic and/or metabolites by culturing genetically modified organ Hydroxymethylglutaryl-CoA Reductase Leads to Squalene Accu isms, and to the genetically modified organisms, in particular mulation in Yeast”. Appl. Microbiol. Biotechnol. 49 (1998), pp. 66-71 yeasts, themselves. Kalb, V.F. et al., “Isolation of a Cytochrome P-450 Structural Gene from Saccharomyces cerevisiae', Gene 45 (1986), pp. 237-245. 14 Claims, 2 Drawing Sheets US 7,556,937 B2 Page 2

OTHER PUBLICATIONS Expression in Escherichia coli of the himgA Gene, and Purification Bischoff, K.M. et al., “3-Hydroxy-3-Methylglutaryl-Coenzyme A and Kinetic Characterization of the Gene Product’, Journal of Bac Reductase from Haloferax volcanii: Purification, Characterization, teriology, Jun. 1997, vol. 179, No. 11, pp. 3632-3638. and Expression in Escherichia coli, Journal of Bacteriology, Jan. Nakamura, Yuichi et al., “Transcriptional Regulation of Squalene 1996, vol. 178. No. 1, pp. 19-23. Epoxidase by Sterols and Inhibitors in HeLa Cells.” The Journal of Bochar, D.A. et al., “3-Hydroxy-3-Methylglutaryl Coenzyme A Biological Chemistry, vol. 271, No. 14, Issue of Apr. 5, 1996, pp. Reductase of Sulfolobus solfataricus: DNA Sequence, Phylogeny, 8053-8056. U.S. Patent Jul. 7, 2009 Sheet 1 of 2 US 7,556,937 B2

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z?un61-I US 7,556,937 B2 1. 2 METHOD FOR PRODUCING the nucleic acid encoding a squalene epoxidase (ERG1) ERGOSTA-5,7-DIENOLAND/OR (Jandrositz, A., et al (1991) The gene encoding squalene BOSYNTHETIC INTERMEDIATE AND/OR epoxidase from Saccharomyces cerevisiae: cloning and char SECONDARY PRODUCTS THEREOF IN acterization. Gene 107:155-160 and TRANSGENIC ORGANISMS nucleic acids encoding a squalene synthetase (ERG9) (Jen nings, S. M., (1991): Molecular cloning and characterization RELATED APPLICATIONS of the yeast gene for squalene synthetase. Proc Natl Acad Sci USA. July 15: 88(14):6038-42). This application is a national stage application (under 35 There are furthermore known processes which aim at U.S.C. 371) of PCT/EP2004/002582 filed Mar 12, 2004 10 increasing the content in specific intermediates and catabo which claims benefit to German application 103 12 314.8 lites of the sterol metabolism in yeasts and fungi. filed Mar. 19, 2003. It is known from T. Polakowski, Molekularbiologische The present invention relates to a method for the produc Beeinflussung des Ergosterolstoffwechsels der Hefe Saccha tion of ergosta-5,7-dienol and/or its biosynthetic intermedi romyces cerevisiae Molecular-biological effects on the ates and/or metabolites by culturing genetically modified 15 ergosterol metabolism of the yeast Saccharomyces cerevi organisms, and to the genetically modified organisms, in par siae, Shaker Verlag Aachen, 1999, pages 59 to 66, that ticular yeasts, themselves. increasing the expression rate of HMG-CoA reductase leads Ergosta-5,7-dienol and its biosynthetic intermediates of to a slightly increased content in early sterols, such as the sterol metabolism, Such as, for example, farnesol, squalene, while the content in later sterols, such as ergosterol, geraniol, squalene and lanosterol and Zymosterol, and its does not change significantly or even has a tendency to biosynthetic metabolites of the sterol metabolism, for decrease. example in mammals, such as, for example, campesterol, Tainaka et al., J. Ferment. Bioeng. 1995, 79, 64-66 further pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH more describe that the overexpression of ERG 11 (lanosterol progesterone, 11-deoxycortisol, hydrocortisone, deoxycorti C14-demethylase) leads to the accumulation of 4,4-dimeth costerone or corticosterone, are compounds of high economi 25 y1zymosterol, but not ergosterol. In comparison with the wild cal value. type, the Zymosterol content of the transformant is increased Ergosta-5,7-dienol may act as starting compound for the by a factor of 1.1 to 1.47, depending on the fermentation preparation of steroid hormones via biotransformations, conditions. chemical synthesis or biotechnological production. WO 99/16886 describes a method for the production of Hydrocortisone has a weak glucocorticoid effect and is a 30 ergosterol in yeasts which overexpress a combination of the sought-after starting compound for the synthesis of active genes thMG, ERG9, SAT1 and ERG1. ingredients with a highly antiinflammatory, abortive or anti EP 486 290 discloses a method for increasing the squalene, proliferative effect. Zymosterol, ergosta-5,7-24(28)-trienol and ergosta-5,7-di Squalene is used as building block for the synthesis of enol content in yeast by increasing the HMG-CoA reductase terpenes. In its hydrogenated form, it is used as squalane in 35 expression rate and simultaneously interrupting the meta dermatology and cosmetics, and in its various derivatives as bolic pathway of ergosta-5,7,24(28)-trienol-22-dehydroge constituent of skincare and haircare products. nase, hereinbelow also referred to as A22-desaturase (ERG5). Other economically utilizable substances are sterols, such However, the disadvantage of this method is that the as Zymosterol and lanosterol, lanosterol being a pivotal raw ergosta-5,7-dienol yield is still not satisfactory. material and synthetic material for the chemical synthesis of 40 It is an object of the present invention to provide a further saponins and steroid hormones. Owing to its good skin pen method for the production of ergosta-5,7-dienol and/or its etration and spreading properties, lanosterol is used as emul biosynthetic intermediates and/or metabolites with advanta sion auxiliary and active ingredient for skin creams. geous characteristics, such as a higher product yield. An economical method for the production of ergosta-5,7- dienol and/or its biosynthetic intermediates and/or metabo 45 BRIEF DESCRIPTION OF DRAWINGS lites is therefore of great importance. Methods which are particularly economical are biotechno FIG. 1 shows vector puG6 thMG. logical methods exploiting natural organisms or organisms FIG. 2 shows vector puG6 ERG1. optimized by means of genetic modification which produce We have found that this object is achieved by a method for ergosta-5,7-dienol and/or its biosynthetic intermediates and/ 50 producing ergosta-5,7-dienol and/or its biosynthetic interme or metabolites. diates and/or metabolites in which organisms are cultured The genes of the ergosterol metabolism in yeast are largely which have known and cloned, such as, for example, a reduced A22-desaturase activity and nucleic acids encoding an HMG-CoA reductase (HMG) an increased HMG-CoA reductase activity and (Bason M. E. et al. (1988) Structural and functional conser 55 an increased activity of at least one of the activities selected Vation between yeast and human3-hydroxy-3-methylglutaryl from the group consisting of lanosterol C14-demethylase coenzyme A reductases, the rate-limiting enzyme of sterol activity, squalene epoxidase activity and squalene synthetase biosynthesis. Mol Cell Biol 8:3797-3808, activity the nucleic acid encoding a truncated HMG-CoA reductase in comparison with the wild type. (t-HMG)(Polakowski T, Stahl U, Lang C. (1998) Overexpres 60 A reduced activity is understood as meaning not only the sion of a cytosolic hydroxymethylglutaryl-CoA reductase reduction of the activity, but also the complete elimination of leads to squalene accumulation in yeast. Appl Microbiol Bio the activity. Accordingly, a reduction of an activity also technol. January; 49(1):66-71, encompasses a quantitative reduction of the relevant protein the nucleic acid encoding a lanosterol C14-demethylase in the organism through to a complete absence of the relevant (ERG11) (Kalb V F, Loper J C, Dey C R. Woods CW, Sutter 65 protein, which can be assayed, for example, by a lack of T R (1986) Isolation of a cytochrome P-450 structural gene detectability of the relevant enzyme activity or a lack of from Saccharomyces cerevisiae. Gene 45(3):237-45. immunological detectability of the relevant proteins. US 7,556,937 B2 3 4 A22-desaturase activity is understood as meaning the capable of inhibiting the A22-desaturase activity, for example enzyme activity of a A22-desaturase. by inhibiting the expression of endogenous A22-desaturase A A22-desaturase is understood as meaning a protein with activity, the enzymatic activity of converting ergosta-5,7-dienol into b) overexpressing homologous A22-desaturase nucleic ergosta-5,722.24-tetraen-3?-ol. acid sequences, which lead to coSuppression, Accordingly, A22-desaturase activity is understood as c) introducing nonsense mutations into the endogen by meaning the amount of ergosta-5,7-dienol converted, or the introducing RNA/DNA oligonucleotides into the organism, amount of ergosta-5,722.24-tetraen-3?-ol formed, by the d) introducing specific DNA-binding factors, for example protein A22-desaturase within a specific period of time. factors of the Zinc finger transcription factor type, which Thus, in the case of reduced A22-desaturase activity in 10 bring about a reduced gene expression, or comparison with the wild type, the amount of ergosta-5,7- e) generating knock-out mutants, for example with the aid dienol converted, or the amount of ergosta-5.7.22.24-tetraen of T-DNA mutagenesis or homologous recombination. 3f3-ol formed, by the protein A22-desaturase within a specific In a preferred embodiment of the method according to the period of time is reduced in comparison with the wild type. invention, the gene expression of the nucleic acids encoding The A22-desaturase activity is preferably reduced to at 15 a A22-desaturase is reduced by generating knock-out least 90%, more preferably to at least 70%, more preferably to mutants, especially preferably by homologous recombina at least 50%, more preferably to at least 30%, even more tion. preferably by at least 10%, even more preferably by at least Accordingly, it is preferred to use an organism without a 5%, in particular to 0% of the A22-desaturase activity of the functional A22-desaturase gene. wild type. Especially preferred is, accordingly, the elimini In a preferred embodiment, the generation of knock-out nation of the A22-desaturase activity in the organism. mutants, that is to say the deletion of the target locus A22 The A22-desaturase (ERG5) activity can be determined as desaturase gene, is carried out simultaneously with the inte described hereinbelow: gration of an expression cassette comprising at least one of Various concentrations of ergosta-5,7-dienol, isolated the nucleic acids described hereinbelow, encoding a protein from S. cerevisiae Erg5 mutants (Parks et al., 1985. Yeast 25 whose activity is being increased in comparison with the wild sterols.yeast mutants as tools for the study of Sterol metabo type, by homologous recombination. lism. Methods Enzymol. 1 11:333-346) and 50 lug of dilau To this end, it is possible to use nucleic acid constructs roylphosphatidylcholin are mixed and Sonicated until a white which, in addition to the expression cassettes described here Suspension forms. Processed microsomes are added (1 ml)(3 inbelow comprising promoter, coding sequence and, if appro mg/ml protein). NADPH (final concentration 1 mM) is added 30 priate, terminator, and in addition to a selection marker to the test mixture in order to start the enzyme reaction. The described hereinbelow, comprise, at the 3' and 5' end, nucleic mixture is incubated for 20 minutes at 37° C. The reaction is acid sequences which are identical to nucleic acid sequences stopped by addition of 3 ml of methanol, and sterols are at the beginning and at the end of the gene to be deleted. hydrolyzed by addition of 2 ml 60% (wt/vol) KOH in water. Once selection has taken place, it is preferred to remove the The mixture is incubated for 2 hours at 90° C. After cooling, 35 selection marker again by means of recombinase systems, for the mixture is extracted three times with 5 ml of hexane and example by loxP signals at the 3' and 5' end of the selection concentrated by evaporation on a rotary evaporator. The Ste marker, using a Cre recombinase (Cre-LOXP system). rols are subsequently silylated with bis(trimethylsilyl)trifluo In the preferred organism Saccharomyces cerevisiae, the roacetamide (50 ul in 50 ul of toluene) for one hour at 60° C. A22-desaturase gene denotes the gene ERG5 (SEQ. ID. NO. The sterols are analyzed by gas chromatography/mass spec 40 1). SEQ. ID. NO. 2 constitutes the corresponding Saccharo troscopy (GC-MS) (for example Model VG 12-250 gas chro myces cerevisiae A22-desaturase (Skaggs, B. A. et al.: Clon matograph-mass spectrometer, VG Biotech, Manchester, ing and characterization of the Saccharomyces cerevisiae United Kingdom). The resulting A22-desaturated intermedi C-22 sterol desaturase gene, encoding a second cytochrome ate can be identified as a function of the amount of substrate P-450 involved in ergosterol biosynthesis, Gene. 1996 Feb. employed. Microsomes which are not incubated with sub 45 strate act as reference. 22; 169(1):105-9.). This method is a modification of the method described in HMG-CoA reductase activity is understood as meaning the Lamb et al: Purification, reconstitution, and inhibition of enzyme activity of an HMG-CoA reductase (3-hydroxy-3- cytochrome P-450 sterol delta22-desaturase from the patho methylglutaryl-coenzyme A reductase). genic fungus Candida glabrata. Antimicrob Agents 50 An HMG-CoA reductase is understood as meaning a pro Chemother. 1999 July; 43(7): 1725-8. tein with the enzymatic activity of converting 3-hydroxy-3- The A22-desaturase activity can be reduced independently methylglutaryl-coenzyme A into mevalonate. by different cytological mechanisms, for example by inhib Accordingly, HMG-CoA reductase activity is understood iting the corresponding activity at the protein level, for as meaning the amount of 3-hydroxy-3-methylglutaryl-coen example by addition of inhibitors of the enzymes in question, 55 Zyme A converted, or the amount of mevalonate formed, by or by reducing the gene expression of the corresponding the protein HMG-CoA reductase within a specific period of nucleic acids encoding a A22-desaturase in comparison with time. the wild type. Thus, in the case of an increased HMG-CoA reductase In a preferred embodiment of the method according to the activity in comparison with the wild type, the amount of invention, the A22-desaturase activity is reduced in compari 60 3-hydroxy-3-methylglutaryl-coenzyme A converted, or the son with the wild type by reducing the gene expression of the amount of mevalonate formed, by the protein HMG-CoA corresponding nucleic acids encoding a A22-desaturase. reductase within a specific period of time is increased in Reducing the gene expression of the nucleic acids encod comparison with the wild type. ing a A22-desaturase in comparison with the wild type can Preferably, this increase in the HMG-CoA reductase activ likewise be effected in various ways, for example by 65 ity amounts to at least 5%, more preferably to at least 20%, a) introducing nucleic acid sequences which can be tran more preferably to at least 50%, more preferably to at least scribed into an antisense nucleic acid sequence which is 100%, even more preferably to at least 300%, especially US 7,556,937 B2 5 6 preferably to at least 500%, in particular to at least 600% of To carry out the activity determination, a microsome frac the HMG-CoA reductase activity of the wild type. tion (4-10 mg/ml protein in 100 mM potassium phosphate The HMG-CoA reductase activity is determined as buffer) is diluted 1:4 in such a way that the protein concen described in Th. Polakowski, Molekularbiologische Beein tration employed for the assay is 2 mg/ml. The assay is carried flussung des Ergosterolstoffwechsels der Hefe Saccharomy out directly in a cell. ces cerevisiae, Shaker-Verlag, Aachen 1999, ISBN 3-8265 A spatula-tipful of dithionite (SONa) is added to the 6211-9. microsomes. The baseline is recorded with a spectrophotom According to this reference, 10 yeast cells of a 48-hour eter in the 380-500 nm range. old culture are harvested by centrifugation (3500xg, 5 min) Approximately 20-30 CO bubbles are subsequently and washed in 2 ml of buffer I (100 mM potassium phosphate 10 bubbled through the sample. The absorption is now measured buffer, pH 7.0). The cell pellet is taken up in 500 ul of buffer in the same range. The absorption level at 450 nm corre 1 (cytosolic proteins) or 2 (100 mM potassium phosphate sponds to the amount of P450 enzyme in the reaction mixture. buffer pH7.0; 1% Triton X-100) (total proteins), and 1 ul of Squalene epoxidase activity is understood as meaning the 500 mM PMSF in isopropanol is added. 500 ul of glass beads enzyme activity of a squalene epoxidase. (d=0.5 mm) are added to the cells, and the cells are disrupted 15 A squalene epoxidase is understood as meaning a protein by vortexing 5x for one minute. The liquid between the glass with the enzymatic activity of converting squalene into beads is transferred into a fresh Eppendorf tube. Cell debris squalene epoxide. and membrane components are removed by centrifuging for Accordingly, squalene epoxidase activity is understood as 15 minutes (14000xg). The supernatant is transferred into a meaning the amount of squalene converted, or the amount of fresh Eppendorf tube and constitutes the protein fraction. squalene epoxide formed, by the protein squalene epoxidase The HMG-CoA activity is determined by measuring the within a specific period of time. consumption of NADPH-i-H in the reduction of 3-hydroxy Thus, in the case of an increased squalene epoxidase activ 3-methylglutaryl-CoA, which is added as a substrate. ity in comparison with the wild type, the amount of squalene In a reaction volume of 1000 ul there are added 20 ul of converted, or the amount of squalene epoxide formed, by the yeast protein isolate together with 910 ul of buffer I; 50 ul of 25 protein squalene epoxidase within a specific period of time is 0.1 M DTT and 10 ul of 16 mM NADPH-i-H". The reaction increased in comparison with the wild type. mixture is warmed to 30° C. and is measured in a photometer Preferably, this increase in squalene epoxidase activity for 7.5 minutes at 340 nm. The decrease in NADPH which is amounts to at least 5%, more preferably to at least 20%, more measured during this period is the breakdown rate without preferably to at least 50%, more preferably to at least 100%, added substrate and is taken into consideration as back 30 even more preferably to at least 300%, especially preferably ground. to at least 500%, in particular to at least 600% of the squalene Thereafter, substrate is added (10 ul of 30 mM HMG epoxidase activity of the wild type. CoA), and the measurement is continued for 7.5 minutes. The The squalene epoxidase activity is determined as described HMG-CoA reductase activity is calculated by determining in Leber R. Landl K. Zinser E. Ahorn H. Spok A, Kohlwein S the specific NADPH breakdown rate. 35 D, Turnowsky F. Daum G. (1998) Dual localization of Lanosterol C14-demethylase activity is understood as squalene epoxidase, Erg1p, in yeast reflects a relationship meaning the enzyme activity of a lanosterol C14-demethy between the endoplasmic reticulum and lipid particles, Mol. lase. Biol. Cell. 1998, February: 9(2):375-86. Alanosterol C14-demethylase is understood as meaning a This method comprises 0.35 to 0.7 mg of microsomal protein with the enzymatic activity of converting lanosterol 40 protein or 3.5 to 75 ug of lipid particle protein in 100 mM into 4,4-dimethylcholesta-8, 14.24-trienol. Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mM FAD, 3 mM Accordingly, lanosterol C14-demethylase activity is NADPH, 0.1 mM squalene 2,3-epoxidase cyclase inhibitor understood as meaning the amount of lanosterol converted, or U18666A, 32 uMHisqualene dispersed in 0.005% Tween the amount of 4,4-dimethylcholesta-8, 14.24-trienol formed, 80 in a total volume of 500 ul. by the protein lanosterol C14-demethylase within a specific 45 The assay is carried out at 30°C. After pretreatment for 10 period of time. minutes, the reaction is started by addition of squalene and Thus, in the case of an increased lanosterol C14-demethy stopped after 15, 30 or 45 minutes by lipid extraction with 3 lase activity in comparison with the wild type, the amount of ml of chloroform/methanol (2:1 vol/vol) and 750 ul 0.035% lanosterol converted, or the amount of 4.4-dimethylcholesta MgCl2. 8, 14.24-trienol formed, by the protein lanosterol C14-dem 50 The lipids are dried under nitrogen and redissolved in 0.5 ethylase within a specific period of time is increased in com ml of chloroform/methanol (2:1 vol/vol). For a thin-layer parison with the wild type. chromatography, portions are placed on a silica gel 60 plate Preferably, this increase in the lanosterol C14-demethylase (0.2 mm) and separated with chloroform as the eluant. The activity amounts to at least 5%, more preferably to at least positions containing H|2.3-oxidosqualene and H 20%, more preferably to at least 50%, more preferably to at 55 squalene were scraped out and quantified with a scintillation least 100%, even more preferably to at least 300%, especially COunter. preferably to at least 500%, in particular to at least 600%, of Squalene synthetase activity is understood as meaning the the lanosterol C14-demethylase activity of the wild type. enzyme activity of a squalene synthetase. The lanosterol C14-demethylase activity is determined as Squalene synthetase is understood as meaning a protein described in Omura, Tand Sato, R. (1964) The carbon mon 60 with the enzymatic activity of converting farnesyl pyrophos oxide binding pigment in liver microsomes. J. Biol. Chem. phate into squalene. 239, 2370-2378. In this test, the amount of P450 enzyme is Accordingly, squalene synthetase activity is understood as semiquantifiable as the holoenzyme with bound heme. The meaning the amount of farnesyl pyrophosphate converted, or (active) holoenzyme (with heme) can be reduced by CO, and the amount of squalene formed, by the protein squalene Syn only the CO-reduced enzyme has an absorption maximum at 65 thetase within a specific period of time. 450 nm. Thus, the absorption maximum at 450 nm is a mea Thus, in the case of an increased squalene synthetase activ sure for the lanosterol C14-demethylase activity. ity in comparison with the wild type, the amount of farnesyl US 7,556,937 B2 7 8 pyrophosphate converted, or the amount of squalene formed, In accordance with the invention, increasing the gene by the protein squalene synthetase within a specific period of expression of a nucleic acid encoding an HMG-CoA reduc time is increased in comparison with the wild type. tase, lanosterol C14-demethylase, squalene epoxidase or Preferably, this increase in squalene synthetase activity squalene synthetase is also understood as meaning the amounts to at least 5%, more preferably to at least 20%, more manipulation of the expression of the organism’s own, in preferably to at least 50%, more preferably to at least 100%, particular the yeasts own, endogenous HMG-CoA reduc even more preferably to at least 300%, especially preferably tases, lanosterol C14-demethylases, squalene epoxidases or to at least 500%, in particular to at least 600% of the squalene squalene synthetases. synthetase activity of the wild type. This can be achieved for example by modifying the pro The squalene synthetase activity can be determined as 10 moter DNA sequence for genes encoding HMG-CoA reduc described hereinbelow: tase, lanosterol C14-demethylase, squalene epoxidase or The reaction mixtures comprise 50 mM Mops, pH 7.2, 10 squalene synthetase. Such a modification, which results in an mM MgCl2, 1% (v/v) Tween-80, 10% (v/v) 2-propanol. 1 increased expression rate of the gene in question, can be mM DTT, 1 mg/ml BSA, NADPH, FPP (or PSPP) and brought about for example by deletion or insertion of DNA microsomes (protein content 3 mg) in a total Volume of 200ll 15 Sequences. in glass tubes. Reactions with radioactive substrate 1-H As described above, it is possible to modify the expression FPP (15-30 mCi/umol) are incubated for 30 minutes at 30°C., of the endogenous HMG-CoA reductase, lanosterol C14 and the suspension mixture is filled up with 1 volume of 1:1 demethylase, squalene epoxidase or squalene synthetase by (v/v) 40% aqueous KOH:methanol. Liquid NaCl is added applying exogenous stimuli. This can be brought about by until the Solution is Saturated, and 2 ml of ligroin comprising specific physiological conditions, that is to say by the appli 0.5% (v/v) squalene are likewise added. cation of foreign Substances. The suspension is vortexed for 30 seconds. Using a Pasteur Moreover, a modified or increased expression of endog pipette, 1 ml portions of the ligroin layer are applied to a enous HMG-CoA reductase, lanosterol C14-demethylase, packed 0.5x6 cm aluminum column (80-200 mesh, Fisher). squalene epoxidase or squalene synthetase genes can be The columnis preequilibrated with 2 ml of ligroin comprising 25 achieved by a regulator protein which does not occur in the 0.5% (v/v) squalene. The column is subsequently eluted with nontransformed organism interacts with the promoter of 5x1 ml toluene comprising 0.5% (v/v) squalene. The these genes. squalene radioactivity is measured in Cytoscint (ICN) scin Such a regulator may be a chimeric protein consisting of a tillation cocktail using a Scintillation counter (Beckman). DNA binding domain and a transcription activator domain, as This method is a modification of the methods described in 30 described, for example, in WO96/06166. Radisky et al., Biochemistry. 2000 Feb. 22:39(7):1748-60, In a preferred embodiment, the increase inlanosterol C14 Zhang et al. (1993) Arch. Biochem. Biophy's. 304, 133-143 demethylase activity in comparison with the wild type is and Poulter, C. D. et al. (1989).J. Am. Chem. Soc. 111, 3734 effected by increasing the gene expression of a nucleic acid 3739. encoding a lanosterol C14-demethylase. A wild type is understood as meaning the corresponding 35 non-genetically-modified starting organism. Preferably, and In a furthermore preferred embodiment, the increase in the in particular in cases where the organism or the wild type are gene expression of a nucleic acid encoding a lanosterol C14 not unambiguously identifiable, the wild type for the reduc demethylase is effected by introducing, into the organism, tion of the A22-desaturase activity, the increase in the HMG one or more nucleic acids encoding a lanosterol C14-dem CoA reductase activity, the increase in the lanosterol C14 40 ethylase. demethylase activity, the increase in the squalene epoxidase In principle, any lanosterol C14-demethylase gene activity and the increase in the squalene synthetase activity, (ERG11), that is to say any nucleic acid encoding a lanosterol and for the increase in the content in ergosta-5,7-dienol and/ C14-demethylase, may be used for this purpose. In the case of or its biosynthetic intermediates and/or metabolites is under genomic lanosterol C14-demethylase nucleic acid sequences stood as meaning a reference organism. This reference organ 45 from eukaryotic sources, which contain introns, and in the ism is preferably the yeast Strain Saccharomyces cerevisiae event that the host organism is not capable, or cannot be made AH22. capable, of expressing the corresponding lanosterol C14 The HMG-CoA reductase activity, the lanosterol C14 demethylase, it is preferred to use preprocessed nucleic acid demethylase activity, the squalene epoxidase activity or the sequences, such as the corresponding cDNAs. squalene synthetase activity can be increased independently 50 Examples of lanosterol C14-demethylase genes are nucleic in various ways, for example by eliminating inhibiting regu acids encoding a lanosterol C14-demethylase from Saccha latory mechamisms at the expression and protein level, or by romyces cerevisiae (Kalb V F. Loper J C, Dey C R. Woods C increasing the gene expression of the corresponding nucleic W. Sutter T R (1986) Isolation of a cytochrome P-450 struc acids, that is to say nucleic acids encoding an HMG-CoA tural gene from Saccharomyces cerevisiae. Gene 45(3):237 reductase, lanosterol C14-demethylase, squalene epoxidase 55 45), Candida albicans (Lamb DC, Kelly DE, Baldwin B C, or squalene synthetase, in comparison with the wild type. Gozzo F, Boscott P. Richards WG, Kelly S L (1997) Differ Increasing the gene expression of the corresponding ential inhibition of Candida albicans CYP51 with azole anti nucleic acid in comparison with the wild type can likewise be fungal stereoisomers. FEMS Microbiol Lett 149(1):25-30), effected in various ways, for example by inducing the corre Homo sapiens (Stromstedt M. Rozman D. Waterman M. R. sponding genes by activators, that is to say by inducing the 60 (1996) The ubiquitously expressed human CYP51 encodes HMG-CoA reductase gene, the lanosterol C14-demethylase lanosterol 14 alpha-demethylase, a cytochrome P450 whose gene, the squalene epoxidase gene or the squalene synthetase expression is regulated by oxysterols. Arch Biochem Biophys gene by activators or by introducing one or more gene copies 1996 May 1; 329(1): 73-81c) or Rattus norvegicus, AoyamaY. of the corresponding nucleic acids, that is to say by introduc Funae Y, Noshiro M, Horiuchi T, Yoshida Y. (1994) Occur ing, into the organism, one or more nucleic acids encoding an 65 rence of a P450 showing high homology to yeast lanosterol HMG-CoA reductase, lanosterol C14-demethylase, squalene 14-demethylase (P450(14DM)) in the rat liver. Biochem Bio epoxidase or squalene synthetase. phys Res Commun. June 30; 201(3): 1320-6). US 7,556,937 B2 9 10 In the transgenic organisms according to the invention, identity when its sequence is aligned with the sequence SEQ. there thus exists, in this preferred embodiment, at least one ID, NO. 6, in particular inaccordance with the above program furtherlanosterol C14-demethylase gene in comparison with algorithm with the above parameter set. the wild type. In a furthermore preferred embodiment, nucleic acids The number of the lanosterol C14-demethylase genes in encoding proteins comprising the amino acid sequence of the the transgenic organisms according to the invention is at least Saccharomyces cerevisiae lanosterol C14-demethylase two, preferably more than two, especially preferably more (SEQ. ID. NO. 6) are introduced into organisms. than three, very especially preferably more than five. Suitable nucleic acid sequences can be obtained for All of the nucleic acids mentioned in the description may example by backtranslating the polypeptide sequence in be, for example, an RNA, DNA or cDNA sequence. 10 accordance with the genetic code. The above-described method preferably employs nucleic Codons which are preferably used for this purpose are acids encoding proteins comprising the amino acid sequence those which are used frequently in accordance with the organ SEQ. ID. NO. 6 or a sequence derived from this sequence by ism-specific codon usage. The codon usage can be deter Substitution, insertion or deletion of amino acids which has at mined readily with the aid of computer evaluations of other, least 30%, preferably at least 50%, more preferably at least 15 known genes of the organisms in question. 70%, especially preferably at least 90%, most preferably at If, for example, the protein is to be expressed in yeast, it is least 95% identity with the sequence SEQ. ID. NO. 6 at the frequently advantageous to use the yeast codon usage when amino acid level, which proteins have the enzymatic charac backtranslating. teristic of a lanosterol C14-demethylase. In an especially preferred embodiment, a nucleic acid com The sequence SEQ. ID. NO. 6 constitutes the amino acid prising the sequence SEQ. ID. NO. 5 is introduced into the sequence of the Saccharomyces cerevisiae lanosterol C14 organism. demethylase. The sequence SEQ. ID. NO. 5 constitutes the genomic Further examples of lanosterol C14-demethylases and DNA from Saccharomyces cerevisiae (ORF S0001049), lanosterol C14-demethylase genes can be found readily, for which encodes the lanosterol C14-demethylase with the example from various organisms whose genomic sequence is 25 sequence SEQID NO. 6. known, by homology comparisons of the amino acid All of the abovementioned lanosterol C14-demethylase sequences or of the corresponding backtranslated nucleic genes can furthermore be generated from the nucleotide units acid sequences from databases with SEQ. ID. NO. 2. by chemical synthesis in a manner known perse, such as, for Further examples of lanosterol C14-demethylases and example, by fragment condensation of individual overlap lanosterol C14-demethylase genes can be found readily in a 30 ping complementary nucleic acid units of the double helix. manner known per se by hybridization and PCR techniques Oligonucleotides can be synthesized chemically for example from various organisms whose genomic sequence is not in a known manner using the phosphoamidite method (Voet, known, for example starting from the sequence SEQ. ID. NO. Voet, 2nd Edition, Wiley Press New York, pages 896-897). 5. The annealment of synthetic oligonucleotides and the filling In the description, the term “substitution' is understood as 35 in of gaps with the aid of the DNA polymerase Klenow meaning the Substitution of one or more amino acids by one or fragment and ligation reactions are described in Sambrook et more amino acids. It is preferred to perform what are known al. (1989), Molecular cloning: A laboratory manual, Cold as conservative Substitutions, in which the replacement Spring Harbor Laboratory Press, as are general cloning meth amino acid has a similar property to the original amino acid, ods. for example the substitution of Glu by Asp, Gln by ASn, Valby 40 In a preferred embodiment, increasing the HMG-CoA Ile, Leu by Ile, Serby Thr. reductase activity in comparison with the wildtype is effected Deletion is the replacement of an amino acid by a direct by increasing the gene expression of a nucleic acid encoding bond. Preferred positions for deletions are the termini of the an HMG-CoA reductase. polypeptide and the linkages between the individual protein In an especially preferred embodiment of the method domains. 45 according to the invention, increasing the gene expression of Insertions are introductions of amino acids into the a nucleic acid encoding an HMG-CoA reductase is effected polypeptide chain, a direct bond formally being replaced by by introducing, into the organism, a nucleic acid construct one or more amino acids. comprising a nucleic acid encoding an HMG-CoA reductase Identity between two proteins is understood as meaning the whose expression in the organism is Subject to reduced regu identity of the amino acids over in each case the entire protein 50 lation in comparison with the wild type. length, in particular the identity calculated by alignment with Reduced regulation in comparison with the wild type is the aid of the Lasergene software from DNASTAR, inc. understood as meaning a regulation which is reduced in com Madison, Wis. (USA) using the Clustal method (Higgins DG, parison with the above-defined wildtype, preferably no regu Sharp PM. Fast and sensitive multiple sequence alignments lation, at the expression or protein level. on a microcomputer. Comput Appl. Biosci. 1989 April; 5(2): 55 The reduced regulation can preferably be achieved by 151-1), setting the following parameters: means of a promoter which is operably linked to the coding Multiple alignment parameter: sequence in the nucleic acid construct and which, in the Gap penalty 10 organism, is subject to reduced regulation in comparison with Gap length penalty 10 the wild-type promoter. Pairwise alignment parameter: 60 For example, the middle ADH promoter in yeast is only K-tuple 1 Subject to reduced regulation and is therefore particularly Gap penalty 3 preferred as promoter in the above-described nucleic acid Window 5 COnStruct. Diagonals saved 5 This promoter fragment of the ADH12s promoter, herein Accordingly, a protein with an identity of at least 30% with 65 below also referred to as ADH1, shows almost constitutive the sequence SEQ. ID. NO. 6 at the amino acid level is expression (Ruohonen L. Penttila M. Keranen S. (1991) Opti understood as meaning a protein which has at least 30% mization of Bacillus alpha-amylase production by Saccharo US 7,556,937 B2 11 12 myces cerevisiae. Yeast. May-June; 7(4):337-462; Lang C, the organism's own, orthologous nucleic acid and by using a Looman A. C. (1995) Efficient expression and secretion of promoter which is subject to reduced regulation in the organ Aspergillus niger RH5344 polygalacturonase in Saccharo ism in comparison with the wild-type promoter. myces cerevisiae. Appl Microbiol Biotechnol. December; In a preferred embodiment, increasing the squalene epoxi 44(1-2): 147-56.), so that the transcriptional regulation no dase activity in comparison with the wild type is effected by longer proceeds via ergosterol biosynthesis intermediates. increasing the gene expression of a nucleic acid encoding a Further preferred promoters with reduced regulation are squalene epoxidase. constitutive promoters such as, for example, the yeast TEF1 In a furthermore preferred embodiment, increasing the promoter, the yeast GPD promoter or the yeast PGK promoter gene expression of a nucleic acid encoding a squalene epoxi (Mumberg D. Muller R, Funk M. (1995)Yeast vectors for the 10 dase is effected by introducing, into the organism, one or controlled expression of heterologous proteins in different more nucleic acids encoding squalene epoxidase. genetic backgrounds. Gene. 1995 Apr. 14; 156(1): 119-22; In principle, any squalene epoxidase gene (ERG1), that is Chen CY, Oppermann H. Hitzeman R.A. (1984) Homologous to say any nucleic acid which encodes a squalene epoxidase, Versus heterologous gene expression in the yeast, Saccharo may be used for this purpose. In the case of genomic squalene myces cerevisiae. Nucleic Acids Res. December 11; 12(23): 15 epoxidase nucleic acid sequences from eukaryotic sources, 8951-70). which contain introns, and in the event that the host organism In a further preferred embodiment, reduced regulation can is not capable, or cannot be made capable, of expressing the be achieved by using, as the nucleic acid encoding an HMG corresponding squalene epoxidase, it is preferred to use pre CoA reductase, a nucleic acid whose expression in the organ processed nucleic acid sequences, such as the corresponding ism is subject to reduced regulation in comparison with the cDNAS. homologous, orthologous nucleic acid. Examples of nucleic acids encoding a squalene epoxidase It is especially preferred to use a nucleic acid which only are nucleic acids encoding a squalene epoxidase from Sac encodes the catalytic region of the HMG-CoA reductase charomyces cerevisiae (Jandrositz, A., etal (1991) The gene (truncated (t-)HMG-CoA reductase) as the nucleic acid encoding squalene epoxidase from Saccharomyces cerevi encoding an HMG-CoA reductase. This nucleic acid 25 siae: cloning and characterization. Gene 107:155-160, from (t-HMG), which is described in EP 486 290 and WO Mus musculus (Kosuga K, Hata S, OSumi T. Sakakibara J. 99/16886, only encodes the catalytically active portion of the Ono T. (1995) Nucleotide sequence of a cDNA for mouse HMG-CoA reductase while the membrane domain, which is squalene epoxidase, Biochim Biophys Acta, February 21; responsible for the regulation at the protein level, is absent. 1260(3):345-8b), from Rattus norvegicus (Sakakibara J. Thus, this nucleic acid is Subjected to reduced regulation, in 30 Watanabe R. Kanai Y. Ono T. (1995) Molecular cloning and particular in yeast, and leads to an increased gene expression expression of rat squalene epoxidase. J Biol Chem January 6; of the HMG-CoA reductase. 270(1):17-20c) or from Homo sapiens (Nakamura Y. Sakak The above-described nucleic acid construct can be incor ibara J., Izumi T. Shibata A, Ono T. (1996) Transcriptional porated into the host organism either chromosomally using regulation of squalene epoxidase by Sterols and inhibitors in integration vectors or episomally using episomal plasmids, in 35 HeLa cells. J. Biol. Chem. 1996, April 5: 271 (14):8053-6). each case comprising the above-described nucleic acid con In the transgenic organisms according to the invention, Struct. there thus exists, in this preferred embodiment, at least one In an especially preferred embodiment, nucleic acids are further squalene epoxidase gene in comparison with the wild introduced, preferably via the above-described nucleic acid type. construct, which encode proteins comprising the amino acid 40 sequence SEQ. ID. NO. 4 or a sequence derived from this The number of the squalene epoxidase genes in the trans sequence by Substitution, insertion or deletion of amino acids genic organisms according to the invention is at least two, which has at least 30% identity with the sequence SEQ. ID. preferably more than two, especially preferably more than NO. 4 at the amino acid level, which proteins have the enzy three, very especially preferably more than five. matic characteristic of an HMG-CoA reductase. 45 The above-described method preferably employs nucleic The sequence SEQ. ID. NO. 4 constitutes the amino acid acids encoding proteins comprising the amino acid sequence sequence of the truncated HMG-CoA reductase (t-HMG). SEQ. ID. NO. 8 or a sequence derived from this sequence by Further examples of HMG-CoA reductases, and thus also Substitution, insertion or deletion of amino acids which has at of the t-HMG-CoA reductases which are reduced to the cata least 30%, preferably at least 50%, more preferably at least lytic portion, or the coding genes, can be found readily, for 50 70%, especially preferably at least 90%, most preferably at example from various organisms whose genomic sequence is least 95% identity with the sequence SEQ. ID. NO. 8 at the known, by homology comparisons of the amino acid amino acid level, which proteins have the enzymatic charac sequences or of the corresponding back-translated nucleic teristic of a squalene epoxidase. acid sequences from databases with SEQID. NO. 4. The sequence SEQ. ID. NO. 8 constitutes the amino acid Further examples of HMG-CoA reductases, and thus also 55 sequence of the Saccharomyces cerevisiae squalene epoxi of the t-HMG-CoA reductases which are reduced to the cata dase. lytic portion, or the coding genes, can be found readily from Further examples of squalene epoxidases and squalene various organisms whose genomic sequence is not known by epoxidase genes can be found readily, for example from vari hybridization and PCR techniques in a manner known perse, ous organisms whose genomic sequence is known, by homol for example starting from the sequence SEQ. ID. No. 3. 60 ogy comparisons of the amino acid sequences or of the cor It is especially preferred to use a nucleic acid comprising responding backtranslated nucleic acid sequences from the sequence SEQ. ID. NO. 3 as nucleic acid encoding a databases with SEQ. ID. NO. 8. truncated HMG-CoA reductase. Further examples of squalene epoxidase and squalene In an especially preferred embodiment, the reduced regu epoxidase genes can be found readily in a manner known per lation is achieved by using, as nucleic acid encoding an 65 se by hybridization and PCR techniques from various organ HMG-CoA reductase, a nucleic acid whose expression in the isms whose genomic sequence is not known, for example organism is subject to reduced regulation in comparison with starting from the sequence SEQ. ID. NO. 7. US 7,556,937 B2 13 14 In a furthermore preferred embodiment, nucleic acids squalene synthetase from Glycyrrhiza glabra (Hayashi, H. et encoding proteins comprising the amino acid sequence of the al. Molecular cloning and characterization of two cDNAs for Saccharomyces cerevisiae squalene epoxidase (SEQ. ID. Glycyrrhiza glabra squalene synthase, Biol. Pharm. Bull. NO. 8) are introduced into organisms. 1999, September; 22(9):947-50. Suitable nucleic acid sequences can be obtained for 5 In the transgenic organisms according to the invention, example by backtranslating the polypeptide sequence in there thus exists, in this preferred embodiment, at least one accordance with the genetic code. further squalene synthetase gene in comparison with the wild Codons which are preferably used for this purpose are type. those which are used frequently inaccordance with the organ The number of the squalene synthetase genes in the trans ism-specific codon usage. The codon usage can be deter 10 genic organisms according to the invention is at least two, mined readily with the aid of computer evaluations of other, preferably more than two, especially preferably more than known genes of the organisms in question. three, very especially preferably more than five. If, for example, the protein is to be expressed in yeast, it is The above-described method preferably employs nucleic frequently advantageous to use the yeast codon usage when acids encoding proteins comprising the amino acid sequence backtranslating. 15 SEQ. ID. N.O. 10 or a sequence derived from this sequence by In an especially preferred embodiment, a nucleic acid com Substitution, insertion or deletion of amino acids which has at prising the sequence SEQ. ID. NO. 7 is introduced into the least 30%, preferably at least 50%, more preferably at least organism. 70%, especially preferably at least 90%, most preferably at The sequence SEQ. ID. NO. 7 constitutes the genomic least 95% identity with the sequence SEQ. ID. NO. 10 at the DNA from Saccharomyces cerevisiae (ORF S0003407), amino acid level, which proteins have the enzymatic charac which encodes the squalene epoxidase with the sequence teristic of a squalene synthetase. SEQID NO. 8. The sequence SEQ. ID. NO. 10 constitutes the amino acid All of the abovementioned squalene epoxidase genes can sequence of the Saccharomyces cerevisiae squalene Syn furthermore be generated from the nucleotide units by chemi thetase (ERG9). cal synthesis in a manner known perse. Such as, for example, 25 Further examples of squalene synthetases and squalene by fragment condensation of individual overlapping comple synthetase genes can be found readily, for example from mentary nucleic acid units of the double helix. Oligonucle various organisms whose genomic sequence is known, by otides can be synthesized chemically for example in a known homology comparisons of the amino acid sequences or of the manner using the phosphoamidite method (Voet, Voet, 2nd corresponding backtranslated nucleic acid sequences from Edition, Wiley Press New York, pages 896-897). The anneal 30 databases with SEQ. ID. NO. 10. ment of synthetic oligonucleotides and the filling in of gaps Further examples of squalene synthetases and squalene with the aid of the DNA polymerase Klenow fragment and synthetase genes can be found readily in a manner known per ligation reactions are described in Sambrook et al. (1989), se by hybridization and PCR techniques from various organ Molecular cloning: A laboratory manual, Cold Spring Harbor isms whose genomic sequence is not known, for example Laboratory Press, as are general cloning methods. 35 starting from the sequence SEQ. ID. NO. 9. In a preferred embodiment, increasing the squalene syn In a furthermore preferred embodiment, nucleic acids thetase activity in comparison with the wild type is effected encoding proteins comprising the amino acid sequence of the by increasing the gene expression of a nucleic acid encoding Saccharomyces cerevisiae squalene synthetase (SEQ. ID. a squalene synthetase. NO. 10) are introduced into organisms. In a furthermore preferred embodiment, increasing the 40 Suitable nucleic acid sequences can be obtained for gene expression of a nucleic acid encoding a squalene Syn example by backtranslating the polypeptide sequence in thetase is effected by introducing, into the organism, one or accordance with the genetic code. more nucleic acids encoding a squalene synthetase. Codons which are preferably used for this prupose are In principle, any squalene synthetase gene (ERG9), that is those which are used frequently in accordance with the organ to say any nucleic acid which encodes a squalene synthetase, 45 ism-specific codon usage. The codon usage can be deter may be used for this purpose. In the case of genomic squalene mined readily with the aid of computer evaluations of other, synthetase nucleic acid sequences from eukaryotic sources, known genes of the organisms in question. which contain introns, and in the event that the host organism If, for example, the protein is to be expressed in yeast, it is is not capable, or cannot be made capable, of expressing the frequently advantageous to use the codon usage of yeast when corresponding squalene synthetase, it is preferred to use pre 50 backtranslating. processed nucleic acid sequences, such as the corresponding In an especially preferred embodiment, a nucleic acid com cDNAS. prising the sequence SEQ. ID. NO. 9 is introduced into the Examples of nucleic acids encoding a squalene synthetase organism. are nucleic acids encoding a squalene synthetase from Sac The sequence SEQ. ID. NO. 9 constitutes the genomic charomyces cerevisiae (ERG9), (Jennings, S. M., (1991): 55 DNA from Saccharomyces cerevisiae (ORF YHR190W), Molecular cloning and characterization of the yeast gene for which encodes the squalene synthetase of the sequence SEQ. squalene synthetase. Proc Natl Acad Sci USA. July 15; ID, NO. 10. 88(14):6038-42), nucleic acids encoding a squalene Syn All of the abovementioned squalene synthetase genes can thetase from Botryococcus braunii Okada (Devarenne, T. P. et furthermore be generated from the nucleotide units by chemi al. Molecular characterization of squalene synthase from the 60 cal synthesis in a manner known perse, such as, for example, green microalga Botryococcus braunii, raceB, Arch. Bio by fragment condensation of individual overlapping comple chem. Biophys. 2000, Jan. 15,373(2):307-17), nucleic acids mentary nucleic acid units of the double helix. Oligonucle encoding a squalene synthetase from potato tuber (Yoshioka otides can be synthesized chemically for example in a known H. et al.: cDNA cloning of sesquiter penecyclase and manner using the phosphoamidite method (Voet, Voet, 2nd squalene synthase, and expression of the genes in potato tuber 65 Edition, Wiley Press New York, pages 896-897). The anneal infected with Phytophthora infestans, Plant. Cell. Physiol. ment of synthetic oligonucleotides and the filling in of gaps 1999, September, 40(9): 993-8) or nucleic acids encoding a with the aid of the DNA polymerase Klenow fragment and US 7,556,937 B2 15 16 ligation reactions are described in Sambrook et al. (1989), synthetic derivatives of ergosta-5,7-dienol in the organism Molecular cloning: A laboratory manual, Cold Spring Harbor used, that is to say in which ergosta-5,7-dienol occurs as Laboratory Press, as are general cloning methods. intermediate. They may be compounds which the organism The organisms cultured in the method according to the used produces naturally from ergosta-5,7-dienol. invention are organisms which have a reduced A22-desatu However, they are also understood as meaning compounds rase activity and an increased HMG-CoA reductase activity which can only be produced from ergosta-5,7-dienol in the and an increased activity of at least one of the activities organism by introducing genes and enzyme activities from selected from the group consisting of lanosterol C14-dem other organisms to which the starting organism has no ethylase activity, squalene epoxidase activity and squalene orthologous gene. synthetase activity in comparison with the wild type. 10 Owing to the introduction of further plant genes and/or In a preferred embodiment, the organisms cultured are mammalian genes into yeast it is possible, for example, to organisms which have a reduced A22-desaturase activity and produce biosynthetic ergosta-5,7-dienol metabolites which an increased HMG-CoA reductase activity and an increased only occur naturally in plants and/or mammals in this yeast. lanosterol C14-demethylase activity, squalene epoxidase The introduction into yeast of for example, nucleic acids activity or squalene synthetase activity in comparison with 15 encoding a plant A7-reductase (DWF5) or its functional the wild type. equivalents or variants and of nucleic acids encoding mature In an especially preferred embodiment of the method forms of CYP11A1, ADX(FDX1), ADR (FDXR) and according to the invention, the organisms have a reduced 3f3-HSD or their functional equivalents or variants leads to the A22-desaturase activity and an increased HMG-CoA reduc biosynthesis of progesterone in this yeast. A detailed descrip tase activity and an increased activity of at least two of the tion of the procedure and of the methods and materials for the activities selected from the group consisting of lanosterol corresponding genetic modification of yeast is published in C. C14-demethylase activity, squalene epoxidase activity and Duport et al., Nat. Biotechnol. 1998, 16, 186-189 and in the squalene synthetase activity in comparison with the wild references cited therein, which are herewith expressly incor type. porated by reference. Especially preferred combinations are a reduced A22-de 25 The introduction into yeast of for example, nucleic acids saturase activity and an increased HMG-CoA reductase activ encoding a plant A7-reductase (DWF5) or its functional ity and an increased lanosterol C14-demethylase activity and equivalents or variants and of nucleic acids encoding mature squalene epoxidase activity or lanosterol C14-demethylase forms of CYP11A1, ADX(FDX1) and ADR (FDXR) or their activity and squalene synthetase activity or an increased functional equivalents or variants and of nucleic acids encod squalene epoxidase activity and squalene synthetase activity 30 ing mitochondrial forms of ADX and CYP11B1, 3b-HSD, in comparison with the wild type. CYP17A1 and CYP21A1 or their functional equivalents or In a very especially preferred embodiment of the method variants leads to the biosynthesis of hydrocortisone, according to the invention, the organisms have a reduced 11-deoxycortisol, corticosterone and acetalpregnenolone. A22-desaturase activity and an increased HMG-CoA reduc To further increase the content in biosynthetic ergosta-5,7- tase activity and an increased lanosterol C14-demethylase 35 dienol metabolites such as, for example, hydrocortisone, it is activity and an increased squalene epoxidase activity and an additionally advantageous to Suppress wasteful metabolic increased squalene synthetase activity in comparison with the pathways, that is to say biosynthetic pathways which do not wild type. lead to the desired product. For example, the reduction of the Organisms or genetically modified organisms are under activities of the gene products of ATF2, GCY1 and YPR1, stood as meaning, in accordance with the invention, for 40 especially preferably the deletion of these activities, in yeast example bacteria, in particular bacteria of the genus Bacillus, leads to a further increase in the hydrocortisone content. Escherichia coli, Lactobacillus spec. or Streptomyces spec. A detailed description of this procedure and of the methods for example yeasts, in particular yeasts of the genus Sac and materials for the corresponding genetic modification of charomyces cerevisiae, Pichia pastoris or Klyveromyces yeast is published in F. M. Szczebara et al., Nat. Biotechnol. Spec., 45 2003, 21, 143-149 and in the references cited therein, which for example fungi, in particular fungi of the genus are herewith expressly incorporated by reference. Aspergillus spec., Penicillium spec. or Dictyostelium spec. The biosynthetic ergosta-5,7-dienol metabolites are there and, for example, also insect cell lines which are capable of fore understood as meaning in particular campesterol, preg generating ergosta-5,7-dienol and/or its biosynthetic inter nenolone, 17-OH pregnenolone, progesterone, 17-OH mediates and/or metabolites, either as the wild type or owing 50 progesterone, 11-deoxycortisol, hydrocortisone, to preceding genetic modification. deoxycorticosterone and/or corticosterone. Especially preferred organisms or genetically modified Preferred biosynthetic metabolites are progesterone, corti organisms are yeasts, in particular of the species Saccharo costerone and hydrocortisone, especially preferably hydro myces cerevisiae, in particular the yeast strains Saccharomy cortisone. ces cerevisiae AH22, Saccharomyces cerevisiae GRF, Sac 55 Some of the compounds produced in the method according charomyces cerevisiae DBY747 and Saccharomyces to the invention are themselves steroid hormones and can be cerevisiae BY4741. used for therapeutical purposes. The biosynthetic intermediates of ergosta-5,7-dienol are The compounds produced, such as, for example, ergosta understood as meaning all those compounds which occur as 5,7-dienol or hydrocortisone, can furthermore be used for intermediates in the ergosta-5,7-dienol biosynthesis in the 60 preparing steroid hormones or for the synthesis of active organism used, preferably the compounds mevalonate, farne ingredients with a potent antiinflammatory, abortive or anti syl pyrophosphate, geraniol pyrophosphate, squalene proliferative activity viabiotransformation, chemical synthe epoxide, 4-dimethylcholesta-8, 14.24-trienol, 4.4-dimeth sis or biotechnological production. y1zymosterol, squalene, farnesol, geraniol, lanosterol, In the method according to the invention for the production Zymosterone and Zymosterol. 65 of ergosta-5,7-dienol and/or its biosynthetic intermediates The biosynthetic metabolites of ergosta-5,7-dienol are and/or metabolites the step of culturing the genetically modi understood as meaning all those compounds which are bio fied organisms, hereinbelow also referred to as transgenic US 7,556,937 B2 17 18 organisms, is preferably followed by harvesting of the organ Nucleic acid constructs comprising this expression cas isms and isolation of ergosta-5,7-dienol and/or its biosyn sette are, for example, vectors or plasmids. thetic intermediates and/or metabolites from the organisms. The regulatory signals preferably comprise one or more The organisms are harvested in a manner known perse to promoters which ensure the transcription and translation in Suit the organism in question. Microorganisms such as bac organisms, in particular in yeasts. teria, mosses, yeasts and fungi or plant cells which are grown The expression cassettes comprise regulatory signals, that in liquid nutrient media by fermentation can be separated for is to say regulatory nucleic acid sequences which control the example by centrifugation, decanting or filtration. expression of the coding sequence in the host cell. In accor Ergosta-5,7-dienol and/or its biosynthetic intermediates dance with a preferred embodiment, an expression cassette and/or metabolites from the harvested biomass are isolated 10 encompasses a promoter upstream, i.e. at the 5' end of the jointly or separately for each compound in a manner known coding sequence, and a terminator downstream, i.e. at the 3' per se, for example by extraction and, if appropriate, further end, and, if appropriate, further regulatory elements which chemical or physical purification processes Such as, for are linked operably to the interposed coding sequence for at example, precipitation methods, crystallography, thermal least one of the above-described genes. separation methods like rectification methods or physical 15 Operable linkage is understood as meaning the sequential separation methods such as, for example, chromatography. arrangement of promoter, coding sequence, if appropriate The invention furthermore relates to a method for generat terminator and if appropriate further regulatory elements in ing a genetically modified organism in which, starting from a such a way that each of the regulatory elements can fulfill its starting organism, the A22-desaturase activity is reduced and intended function upon expression of the coding sequence. the HMG-CoA reductase activity is increased and at least one By way of example, the preferred nucleic acid constructs, of the activities selected from the group consisting of lanos expression cassettes and plasmids for yeasts and fungi and terol C14-demethylase activity, squalene epoxidase activity methods for generating transgenic yeasts and the transgenic and squalene synthetase activity is increased. yeasts themselves are described in the following text. The methods for deleting the target locus A22-desaturase A suitable promoter for the expression cassette is, in prin gene have already been detailed above. 25 ciple, any promoter which is capable of controlling the The transgenic organisms, in particular yeasts, can prefer expression of foreign genes in organisms, in particular in ably be generated by transforming the starting organisms, in yeasts. particular yeasts, with a nucleic acid construct comprising at A promoter which is preferably used is, in particular, a least one nucleic acid encoding an HMG-CoA reductase and promoter which is subject to reduced regulation in yeast, Such comprising at least one nucleic acid selected from the group 30 as, for example, the middle ADH promoter. consisting of nucleic acids encoding a lanosterol C14-dem This promoter fragment of the ADH12s promoter, herein ethylase, nucleic acids encoding a squalene epoxidase and below also referred to as ADH1, shows approximately con nucleic acids encoding a squalene synthetase, which nucleic stitutive expression (Ruohonen L. Penttila M. Keranen S. acids are linked operably to one or more regulatory signals (1991) Optimization of Bacillus alpha-amylase production which ensure the transcription and translation in the organ 35 by Saccharomyces cerevisiae. Yeast. May-June; 7(4):337 isms. In this embodiment, the transgenic organisms are gen 462: Lang C, Looman A.C. (1995) Efficient expression and erated using a nucleic acid construct. secretion of Aspergillus niger RH5344 polygalacturonase in Nucleic acid constructs which can be used for this purpose Saccharomyces cerevisiae. Appl Microbiol Biotechnol. are those which, in addition to the expression cassettes December; 44(1-2): 147-56.), so that transcriptional regula described hereinbelow and comprising promoter, coding 40 tion is no longer effected by ergosterol biosynthesis interme sequence and, if appropriate, terminator, and in addition to a diates. selection marker described hereinbelow, comprise, at their 3' Further preferred promoters with reduced regulation are and 5' ends, nucleic acid sequences which are identical to constitutive promoters such as, for example, the yeast TEF1 nucleic acid sequences at the beginning and at the end of the 45 promoter, the yeast GPD promoter or the yeast PGK promoter gene to be deleted. (Mumberg D. Muller R, Funk M. (1995)Yeast vectors for the However, the transgenic organisms may also preferably be controlled expression of heterologous proteins in different generated by transforming the starting organisms, in particu genetic backgrounds. Gene. 1995 Apr. 14; 156(1): 119-22; lar yeasts, with a combination of nucleic acid constructs Chen CY, Oppermann H. Hitzeman R.A. (1984) Homologous comprising nucleic acid constructs comprising at least one 50 Versus heterologous gene expression in the yeast, Saccharo nucleic acid encoding an HMG-CoA reductase and compris myces cerevisiae. Nucleic Acids Res. December 11; 12(23): ing nucleic acid constructs or a combination of nucleic acid 8951-70). constructs comprising at least one nucleic acid selected from The expression cassette may also comprise inducible pro the group consisting of nucleic acids encoding a lanosterol moters, in particular chemically inducible promoters, by C14-demethylase, nucleic acids encoding a squalene epoxi 55 means of which the expression, in the organism, of the nucleic dase and nucleic acids encoding a squalene synthetase and acids encoding an HMG-CoA reductase, lanosterol C14 which are in each case linked operably to one or more regu demethylase, squalene epoxidase or squalene synthetase can latory signals which ensure the transcription and translation be controlled at a particular point in time. in organisms. Such promoters such as, for example, the yeast Cupl pro In this embodiment, the transgenic organisms are gener 60 moter, (Etcheverry T. (1990) Induced expression using yeast ated using individual nucleic acid constructs or a combination copper metallothionein promoter. Methods Enzymol. 1990; of nucleic acid constructs. 185:319-29.), the yeast Gall-10 promoter (Ronicke V. Grau Nucleic acid constructs in which the coding nucleic acid lich W. Mumberg D. Muller R, Funk M. (1997) Use of con sequence is linked operably to one or more regulatory signals ditional promoters for expression of heterologous proteins in which ensure the transcription and translation in organisms, 65 Saccharomyces cerevisiae, Methods Enzymol. 283:313-22) in particular in yeasts, are hereinbelow also referred to as or the yeast Pho5 promoter (Bajwa W. Rudolph H, Hinnen A. expression cassettes. (1987) PHO5 upstream sequences confer phosphate control US 7,556,937 B2 19 20 on the constitutive PHO3 gene. Yeast. 1987 March; 3(1):33 These transgenic organisms, in particular yeasts, prefer 42) may be used by way of example. ably have an increased contentinergosta-5,7-dienol and/or its A Suitable terminator for the expression cassette is, in biosynthetic intermediates and/or metabolites in comparison principle, any terminator which is capable of controlling the with the wild type. expression of foreign genes in organisms, in particular in The invention furthermore relates to the use of the above yeasts. described nucleic acids or of the nucleic acid constructs The yeast tryptophan terminator (TRP1 terminator) is pre according to the invention for increasing the content in ferred. ergosta-5,7-dienol and/or its biosynthetic intermediates and/ An expression cassette is preferably generated by fusing a or metabolites in organisms. suitable promoter to the above-described nucleic acids 10 The above-described proteins and nucleic acids can be encoding an HMG-CoA reductase, lanosterol C14-demethy used for producing ergosta-5,7-dienol and/or its biosynthetic lase, squalene epoxidase or squalene synthetase and, if appro intermediates and/or metabolites in transgenic organisms. priate, a terminator using customary recombination and clon The transfer of foreign genes into the genome of an organ ing techniques as are described, for example, in T. Maniatis, ism, in particular of yeast, is referred to as transformation. E. F. Fritsch and J. Sambrook, Molecular Cloning: A Labo 15 Transformation methods which are known per se may be ratory Manual, Cold Spring Harbor Laboratory, Cold Spring used for this purpose, in particular in yeasts. Harbor, N.Y. (1989), in T.J. Silhavy, M. L. Berman and L. W. Suitable methods for transforming yeasts are, for example, Enquist, Experiments with Gene Fusions, Cold Spring Har the LiAC method as described in Schiestl R. H. Gietz, R D. bor Laboratory, Cold Spring Harbor, N.Y. (1984) and in (1989) High efficiency transformation of intact yeast cells Ausubel, F. M. et al., Current Protocols in Molecular Biology, using single stranded nucleic acids as a carrier, Curr Genet. Greene Publishing Assoc. and Wiley-Interscience (1987). December; 16(5-6): 339-46, the electroporation as described in Manivasakam P. Schiestl R. H. (1993) High efficiency The nucleic acids according to the invention can have been transformation of Saccharomyces cerevisiae by electropora synthesized or obtained naturally or comprise a mixture of tion. Nucleic Acids Res. September 11; 21 (18):4414-5, or the synthetic and natural nucleic acid components, or else consist 25 preparation of protoplasts as described in Morgan A.J. (1983) of various heterologous gene segments from various organ Yeast strain improvement by protoplast fusion and transfor 1SS. mation, Experientia Suppl. 46:155-66. Preferred are, as described above, synthetic nucleotide The construct to be expressed is preferably cloned into a sequences with codons which are preferred by yeasts. These vector, in particular into plasmids which are suitable for the codons which are preferred by yeasts can be determined from 30 transformation of yeasts, such as, for example, the vector codons with the highest protein frequency which are systems Yep24 (Naumovski L. Friedberg EC (1982) Molecu expressed in most of the yeast species of interest. lar cloning of eucaryotic genes required for excision repair of When preparing an expression cassette, various DNA frag UV-irradiated DNA: isolation and partial characterization of ments can be manipulated in order to obtain a nucleotide the RAD3 gene of Saccharomyces cerevisiae. J Bacteriol sequence which expediently reads in the correct direction and 35 October; 152(1):323-31), Yep 13 (Broach J R, Strathern J N, is equipped with the correct reading frame. Adapters or link Hicks J. B. (1979) Transformation in yeast: development of a ers may be added to the fragments in order to link the DNA hybrid cloning vector and isolation of the CAN1 gene. Gene. fragments with one another. 1979 December; 8(1):121–33), the pRS vector series (Cen The promoter and terminator regions may expediently be tromer and Episomal) (Sikorski RS, Hieter P. (1989) A sys provided, in the direction of transcription, with a linker or 40 tem of shuttle vectors and yeast host strains designed for polylinker comprising one or more restriction cleavage sites efficient manipulation of DNA in Saccharomyces cerevisiae. for the insertion of this sequence. As a rule, the linker has 1 to Genetics. May: 122(1):19-27) and the vector systems YCp 19 10, in most cases 1 to 8, preferably 2 to 6, restriction cleavage or pYEXBX. sites. In general, the linker within the regulatory regions has a Accordingly, the invention furthermore relates to vectors, size of less than 100 bp, frequently less than 60 bp, but at least 45 in particular plasmids comprising the above-described 5bp. The promoter may be either native, or homologous, or nucleic acids, nucleic acid constructs or expression cassettes. else foreign, or heterologous, with respect to the host organ The invention furthermore relates to a method for the gen ism. The expression cassette preferably comprises, in the 5'-3' eration of genetically modified organisms by functionally direction of transcription, the promoter, a coding nucleic acid inserting, into the starting organism, an above-described sequence or a nucleic acid construct and a region for tran 50 nucleic acid or an above-described nucleic acid construct. Scriptional termination. Various termination regions can be The invention furthermore relates to the genetically modi exchanged for one another as desired. fied organisms, where the genetic modification Manipulations which provide suitable restriction cleavage reduces the A22-desaturase activity and sites or which remove superfluous DNA or restriction cleav 55 increases the HMG-CoA reductase activity and age sites may furthermore be employed. Where insertions, increases at least one of the activities selected from the deletions or Substitutions such as, for example, transitions group consisting of lanosterol C14-demethylase activity, and transversions, are suitable, in vitro mutagenesis, primer squalene epoxidase activity and squalene synthetase activity repair, restriction or ligation may be used. in comparison with the wild type. Suitable manipulations such as, for example, restriction, 60 In a preferred embodiment, the genetically modified organ chewing back or filling in overhangs for blunt ends may isms have a reduced A22-desaturase activity and an increased provide complementary ends of the fragments for the liga HMG-CoA reductase activity and an increased lanosterol tion. C14-demethylase activity in comparison with the wild type. The invention furthermore relates to the use of the above In a further preferred embodiment, the genetically modi described nucleic acids, the above-described nucleic acid 65 fied organisms have a reduced A22-desaturase activity and an constructs or the above-described proteins for the generation increased HMG-CoA reductase activity and an increased of transgenic organisms, in particular yeasts. squalene epoxidase activity in comparison with the wildtype. US 7,556,937 B2 21 22 In a further preferred embodiment, the genetically modi Increasing the content in ergosta-5,7-dienol and/or its bio fied organisms have a reduced A22-desaturase activity and an synthetic intermediates and/or metabolites means, for the increased HMG-CoA reductase activity and an increased purposes of the present invention, preferably the artificially squalene synthetase activity in comparison with the wild acquired ability of an increased biosynthesis rate of at least type. one of these compounds mentioned at the outset in the geneti In an especially preferred embodiment, the genetically cally modified organism in comparison with the non-geneti modified organisms have a reduced A22-desaturase activity cally-modified organism. and an increased HMG-CoA reductase activity and an An increased content in ergosta-5,7-dienol and/or its bio increased lanosterol C14-demethylase activity and an synthetic intermediates and/or metabolites in comparison increased squalene epoxidase activity in comparison with the 10 with the wild type is understood as meaning in particular wild type. increasing the content of at least one of the abovementioned In a further, especially preferred embodiment, the geneti compounds in the organism by at least 50%, by preference cally modified organisms have a reduced A22-desaturase 100%, more preferably 200%, especially preferably 400% in activity and an increased HMG-CoA reductase activity and comparison with the wild type. an increased lanosterol C14-demethylase activity and an 15 The determination of the content in at least one of the increased squalene synthetase activity in comparison with the abovementioned compounds is preferably carried out by ana wild type. lytical methods known perse and preferably relates to those In a further, especially preferred embodiment, the geneti compartments of the organism in which sterols are produced. cally modified organisms have a reduced A22-desaturase The advantage of the present invention in comparison with activity and an increased HMG-CoA reductase activity and the prior art is as follows: an increased squalene epoxidase activity and an increased The method according to the invention makes it possible to squalene synthetase activity in comparison with the wild increase the content in ergosta-5,7-dienol and/or its biosyn type. thetic intermediates and/or metabolites in the production In a very especially preferred embodiment, the genetically organisms. modified organisms have a reduced A22-desaturase activity 25 The invention will now be illustrated by the examples and an increased HMG-CoA reductase activity and an which follow, but is not limited thereto: increased lanosterol C14-demethylase activity and an I. General Experimental Conditions increased squalene epoxidase activity and an increased 1. Restriction squalene synthetase activity in comparison with the wild The plasmids (1 to 10 ug) were restricted in 30 ul reactions. type. 30 To this end, the DNA was taken up in 24 Jul of HO and treated As mentioned above, these activities are preferably with 3 uof the buffer in question, 1 ml of BSA (bovine serum increased by increasing independently, in comparison with albumin) and 2 ul of enzyme. The enzyme concentration was the wild type, the gene expression of nucleic acids encoding 1 unit/ul or 5 units/ul, depending on the DNA quantity. In an HMG-CoA reductase, nucleic acids encoding a lanosterol Some cases, 1 Jul of RNase was also added to the reaction in C14-demethylase, nucleic acids encoding a squalene epoxi 35 order to break down the tRNA. The restriction reaction was dase or nucleic acids encoding a squalene synthetase. incubated for 2 hours at 37° C. The restriction was checked The furthermore preferred embodiments of the preferred with a minigel. genetically modified organisms according to the invention are 2. Gel Electrophoreses described hereinabove in the methods. The gel electrophoreses were carried out in minigel or wide The above-described genetically modified organisms have 40 minigel apparatuses. The minigels (approx. 20 ml, 8 wells) an increased content in ergosta-5,7-dienol and/or its biosyn and the wide minigels (50 ml, 15 or 30 wells) consisted of 1% thetic intermediates and/or metabolites in comparison with agarose in TAE. The running buffer used was 1x TAE. The the wild type. samples (10 ul) were treated with 3 ul of stop solution and Accordingly, the invention relates to an above-described applied. HindIII-cut I-DNA acted as the standard (bands at: genetically modified organism, wherein the genetically 45 23.1 kb; 9.4 kb; 6.6 kb; 4.4 kb; 2.3 kb.: 2.0 kb: 0.6 kb). For the modified organism has an increased content in ergosta-5,7- separation, 80 volts were applied for 45 to 60 minutes. There dienol and/or its biosynthetic intermediates and/or metabo after, the gel was stained in ethidium bromide Solution and, lites in comparison with the wild type. under UV light, recorded with the video documentation sys Organisms or genetically modified organisms are under tem. INTAS or photographed using an orange filter. stood as meaning, in accordance with the invention, for 50 3. Gel Elution example bacteria, in particular bacteria of the genus Bacillus, The desired fragments were isolated by means of gel elu Escherichia coli, Lactobacillus spec. or Streptomyces spec. tion. The restriction reaction was loaded into several wells of for example yeasts, in particular yeasts of the genus Sac a minigel and run. Only u-HindIII and a “sacrificial lane' charomyces cerevisiae, Pichia pastoris or Klyveromyces were stained with ethidium bromide solution and viewed Spec., 55 under UV light, and the desired fragment was marked. Dam for example fungi, in particular fungi of the genus age by the ethidium bromide and the UV light to the DNA in Aspergillus spec., Penicillium spec. or Dictyostelium spec. the remaining wells was thus prevented. By placing the and, for example, also insect cell lines which are capable of stained and the unstained gel slab next to each other, it was generating ergosta-5,7-dienol and/or its biosynthetic inter possible to excise the desired fragment from the unstained gel mediates and/or metabolites, either as the wild type or owing 60 slab with reference to the marker. The agarose section with to preceding genetic modification. the fragment to be isolated was placed into a dialysis tube, Especially preferred organisms or genetically modified sealed with a small amount of TAE buffer without air bubbles organisms are yeasts, in particular of the species Saccharo and placed into the BioRad minigel apparatus. The running myces cerevisiae, in particular the yeast strains Saccharomy buffer consisted of 1x TAE, and the voltage applied was 100 ces cerevisiae AH22, Saccharomyces cerevisiae GRF, Sac 65 V for 40 minutes. Thereafter, the polarity of the current was charomyces cerevisiae DBY747 and Saccharomyces reversed for 2 minutes in order to redissolve the DNA which cerevisiae BY4741. stuck to the dialysis tube. The buffer, of the dialysis tube, US 7,556,937 B2 23 24 which contained the DNA fragments was transferred into ferred into an Eppendorf tube. If the supernatant was not reaction vessels and used for carrying out an ethanol precipi entirely clear, it was recentrifuged. The Supernatant was tation. To this end, /10 volume of 3M sodium acetate, tRNA (1 treated with 360 ul of ice-cold isopropanol and incubated for ul per 50 ul solution) and 2.5 volumes of ice-cold 96% ethanol 30 minutes at -20° C. (DNA precipitation). The DNA was were added to the DNA solution. The reaction was incubated centrifuged off (15 min, 12 000 rpm, 4°C.), the supernatant for 30 minutes at -20°C. and then centrifuged for 30 minutes was discarded, and the pellet was washed in 100 ul of ice-cold at 4°C. at 12 000 rpm. The DNA pellet was dried and taken up 96% ethanol, incubated for 15 minutes at -20° C. and recen in 10 to 50 ul of H2O (depending on the DNA quantity). trifuged (15 min, 12 000 rpm, 4°C.). The pellet was dried in 4. Klenow Treatment a SpeedVac apparatus and then taken up in 100ul of H.O.The The Klenow treatment results in DNA fragment overhangs 10 plasmid DNA was characterized by restriction analysis. To being filled in so that bluntends result. The following mixture this end, 10ul of each reaction were restricted and separated was pipetted together for each ug of DNA: by gel electrophoresis in a wide minigel (see above). DNA pellet--11 ul H20 8. Plasmid Preparation from E. coli (Maxiprep) +1.5 ul 10x Klenow buffer In order to isolate larger amounts of plasmid DNA, the +1 ul 0.1 M DTT 15 maxiprep method was carried out. Two flasks containing 100 +1 ul nucleotide (dNTP 2 mM) ml of LB+ ampicillin medium were inoculated with a colony 25+1 ul Klenow polymerase (1 unit/ul) or with 100 ul of a frozen culture containing the plasmid to be The DNA for this purpose should originate from an ethanol isolated and incubated overnight at 37° C. and 120 rpm. On precipitation in order to prevent contaminants inhibiting the the next day, the culture (200 ml) was transferred into a GSA Klenow polymerase. The mixture was incubated for 30 min beaker and centrifuged for 10 minutes at 4000 rpm (2600xg). utes at 37° C. and the reaction was stopped by a further 5 The cell pellet was taken up in 6 ml of TE buffer. To digest the minutes at 70°C. The DNA was obtained from the mixture by cell wall, 1.2 ml of lysozyme solution (20 mg/ml TE buffer) precipitation of ethanol and taken up in 10 ul of H.O. were added and the mixture was incubated for 10 minutes at 5. Ligation room temperature. The cells were subsequently lysed with 12 The DNA fragments to be ligated were combined. The final 25 ml of 0.2N NaOH, 1% SDS solution and a further 5 minutes volume of 13.1 ul contained approx. 0.5ul of DNA with a incubation at room temperature; The proteins were precipi vectorinsert ratio of 1:5. The sample was incubated for 45 tated by addition of 9 ml of cold 3M sodium acetate solution seconds at 70° C., cooled to room temperature (approx. 3 (pH 4.8) and 15 minutes incubation on ice. After the cen minutes) and then incubated for 10 minutes on ice. Thereaf trifugation (GSA: 13 000 rpm (27500xg), 20 min, 4° C.), the ter, the ligation buffers were added: 2.6 ul 500mMtrishC1 pH 30 supernatant, which contained the DNA, was transferred into a 7.5 and 1.3 ul 100 mM MgCl, and the mixture was incubated fresh GSA beaker and the DNA was precipitated with 15 ml on ice for a further 10 minutes. After addition of 1 Jul 500 mM of ice-cold isopropanol and 30 minutes incubation at -20°C. DTT and 1 Jul 10 mM ATP and another 10 minutes on ice, 1 ul The DNA pellet was washed in 5 ml of ice-cold ethanol and of ligase (1 unit/pl) was added. The whole of the treatment dried in the air (approx. 30-60 min). It was then taken up in 1 should be carried out as free from vibrations as possible in 35 ml of HO. The plasmid was verified by restriction analysis. order not to separate joined-up DNA ends again. The ligation The concentration was determined by applying dilutions to a was carried out overnight at 14°C. minigel. A microdialysis (pore size 0.025um) was carried out 6. E. coli Transformation for 30-60 minutes in order to reduce the salt content. Competent Escherichia coli (E. coli) NM522 cells were 9. Yeast Transformation transformed with the DNA of the ligation reaction. This was 40 A preculture of the strain Saccharomyces cerevisiae AH22 accompanied by a reaction with 50 lug of the pScL3 plasmid was established for the yeast transformation. A flask contain as positive control and a reaction without DNA as Zero con ing 20 ml of YE medium was inoculated with 100 ul of the trol. For each transformation reaction, 100 ul of 8% PEG frozen culture and incubated overnight at 28°C. and 120 rpm. solution, 10ul of DNA and 200 ul of competent cells (E. coli The main culture was carried out under identical conditions in NM522) were pipetted into a tabletop centrifuge tube. The 45 flasks containing 100 ml of YE medium which had been reactions were placed on ice for 30 minutes and shaken occa inoculated with 10 ul, 20 ul or 50 ul of the preculture. sionally. 9.1 Generation of Competent Cells They were then given the thermal shock treatment: 1 On the next day, the flasks were counted using a hemato minute at 42°C. For the regeneration, 1 ml ofLB medium was cytometer and the flask with a cell concentration of 3-5x107 added to the cells and the mixtures were incubated for 90 50 cells/ml was chosen for the following procedure. The cells minutes at 37°C. on a shaker. 100 ul portions of the undiluted were harvested by centrifugation (GSA: 5000 rpm (4000xg) reactions, of a 1:10 dilution and of a 1:100 dilution were 10 min). The cell pellet was taken up in 10 ml of TE buffer and plated onto LB+ amplicillin plates and incubated overnight at divided between two tabletop centrifuge tubes (5 ml each). 370 C. The cells were centrifuged off for 3 minutes at 6000 rpm and 7. Plasmid Isolation from E. coli (Miniprep) 55 washed twice with in each case 5 ml of TE buffer. The cell E. coli colonies were grown overnight in 1.5 ml of LB+ pellet was subsequently taken up in 330 ul of lithium acetate ampicillin medium in tabletop centrifuge tubes at 37° C. and buffer per 10 cells, transferred into a sterile 50 ml Erlenm 120 rpm. On the next day, the cells were centrifuged for 5 eyer flask and shaken for one hour at 28°C. The cells were minutes at 5000 rpm and 4°C. and the pellet was taken up in thus competent for the transformation. 50 ul of TE buffer. Each reaction was treated with 100 ul of 0.2 60 9.2 Transformation N NaOH, 1% SDS solution, mixed and placed on ice for 5 For each transformation reaction, 15 Jul of herring sperm minutes (cell lysis). Thereafter, 400 ul of sodium acetate/ DNA (10 mg/ml), 10ul of DNA to be transformed (approx 0.5 NaCl solution (230 ul of HO, 130 ul of 3 M sodium acetate, ug) and 330 ul of competent cells were pipetted into a tabletop 40 ul of 5M NaCl) were added, and the reaction was mixed centrifuge tube and incubated for 30 minutes at 28°C. (with and placed on ice for a further 15 minutes (protein precipita 65 out shaking). Thereafter, 700 ul 50% PEG 6000 were added tion). After centrifugation for 15 minutes at 11 000 rpm, the and the reactions were incubated for a further hour at 28°C. Supernatant, which contains the plasmid DNA, was trans without shaking. This was followed by 5 minutes heat shock US 7,556,937 B2 25 26 treatment at 42°C. 100 ul of the suspension were plated onto The selection marker used is the resistance to G418. The selection medium (YNB. Difco) in order to select for leucine resulting strain S. cerevisiae GRF-tH1ura3 is Uracil-aux prototrophism. In the case of selection of G418 resistance, the otrophic and contains a copy of the gene thMG under the cells are regenerated following the heat shock treatment (see control of the ADH promoter and tryptophan terminator. 9.3, regeneration phase). In order to subsequently remove the resistance to G418 9.3 Regeneration Phase again, the resulting yeast strain is transformed with the cre Since the selection marker is the resistance to G418, the recombinase vector pSH47 (Guldener U, Heck S. Fielder T, cells required time for expressing the resistance gene. The Beinhauer J, Hegemann J. H. (1996) A new efficient gene transformation reactions were treated with 4 ml of YE disruption cassette for repeated use in budding yeast. Nucleic medium and incubated overnight at 28°C. on a shaker (120 10 Acids Res. July 1:24(13):2519-24.). Owing to this vector, the rpm). On the next day, the cells were centrifuged off (6000 cre recombinase is expressed in the yeast, and, as a conse rpm, 3 min), taken up in 1 ml of YE medium, and 100 ul or 200 quence, the sequence region within the two loXP sequences ul of this were plated onto YE+G418 plates. The plates were recombines out of the gene. The result is that only one of the incubated for several days at 28°C. two loxP sequences and the ADH-thMG-TRP cassette are 15 retained in the URA3 gene locus. As a consequence, the yeast 10. Reaction Conditions for the PCR strain loses the G418 resistance again and is thus Suitable for The reaction conditions for the polymerase chain reaction integrating further genes into the yeast strain by means of this must be optimized for each individual case and are not gen cre-lox system or removing them, respectively. The vector erally valid for each procedure. It is thus possible to vary, inter pSH47 can now be removed again by counterselection on alia, the amount of DNA employed, the salt concentrations YNB agar plates supplemented with Uracil (20 mg/l) and and the melting point. For our approach, it proved Suitable to FOA (5-fluoroorotic acid) (1 g/l). To this end, the cells which combine the following substances in an Eppendorf tube suit bear this plasmid must first be cultured under nonselective able for use in thermocyclers: 5ul of Super Buffer, 8 ul of conditions and Subsequently be grown on FOA-containing dNTPs (0.625 uM each), 5'-primer, 3'-primer and 0.2 ug of selective plates. Only those cells which are not capable of template DNA, dissolved in such an amount of water that a 25 synthesizing Uracil themselves are capable of growing under total volume of 50 ul for the PCR reaction results, were added these conditions. In the present case, these are cells which no to 2 ul (-0.1 U) Super Taq polymerase. The reaction was longer contain plasmid (pSH47). centrifuged briefly and covered with a drop of oil. Between 37 The yeast strain GRFtH 1 ura3 and the original strain GRF and 40 cycles were selected for the amplification. were cultured for 48 hours in WMXIII medium at 28°C. and 30 160 rpm in a culture volume of 20 ml. 500 ul of this preculture II. EXAMPLES were subsequently transferred into a 50 ml main culture of the same medium and cultured for 4 days at 28°C. and 160 rpm Example 1 in a baffle flask. The sterols were extracted after 4 days following the Expression of a Truncated HMG-CoA Reductase in S. cer 35 method as described in Parks L. W. Bottema CD, Rodriguez evisiae GRF RJ, Lewis T.A. (1985)Yeast sterols: yeast mutants as tools for The coding nucleic acid sequence for the expression cas the study of sterol metabolism. Methods Enzymol. 1985; sette consisting of ADH-promoter-thMG-tryptophan-termi 1 11:333-46 and analyzed by gas chromatography. This gives nator was amplified from the vectorYepH2 (Polakowski et al. the data listed in table 1. The percentages are based on the (1998). Overexpression of a cytosolic hydroxymethylglu 40 yeast dry weight. taryl-CoA reductase leads to squalene accumulation in yeast. Appl Microbiol Biotechnol. January: 49(1):66-71) by PCR TABLE 1 using standard methods as detailed above under the general Sterol content peak reaction conditions. area g|DM S. cerevisiae GRFtEH1ura S. cerevisiae GRF 45 The primers used for this purpose are the DNA oligomers Squalene 9.93 O.1 AtEIT-5' (forward: thMGNotF: 5'-CTGCGGCCGCAT Lanosterol O.83 O.31 CATGGACCMTTGGTGAAAACTG-3'; SEQ. ID. NO. 11) Zymosterol 1.18 1.07 and AthT-3' (reverse: thMGXhoR: 5'-MCTCGAGAGACA Fecosterol 1.10 O.64 Episterolfergosta-5,7- 1.04 0.72 CATGGTGCTGTTGTGCTTC-3'; SEQ. ID. No. 12). 50 dienol The resulting DNA fragment was first treated with Klenow Dimethyl- O.34 O.13 and then cloned blunt-ended into the vector, puG6 into the Zymosterol EcoRV cleavage site, giving rise to the vector puG6-tHMG (FIG. 1). Following the isolation of the plasmid, an extended frag 55 Example 2 ment was amplified from the vectorpUG-thMG by means of PCR so that the resulting fragment consists of the following components: loxP-kanMX-ADH-promoter-thMG-tryp Expression of ERG1 in S. cerevisiae GRFtH1ura3 with Simultaneous Deletion of ERG5; Generation of tophan-terminator-loXP. The primers chosen were oligo GRFtH1ura3ERG1erg5 nucleotide sequences which, at the 5' and 3' overhangs, com 60 prise the 5' or the 3' sequence of the URA3 gene, respectively, and in the annealing region the sequences of the loXP regions Example 2.1 5' and 3' of the vectorpUG-thMG. This ensures that firstly the entire fragment including KanR and thMG is amplified and Generation of the Integration Vector puG6-ERG1 secondly that this fragment can Subsequently be transformed 65 The DNA sequence for the cassette consisting of ADH into yeast and the entire fragment integrates into the yeast promoter-ERG1-tryptophan-terminator was isolated from URA3 gene locus by homologous recombination. the vectorpFlat3-ERG1 by restriction with the enzymes Nhel US 7,556,937 B2 27 28 and Bsp681 (Nrul) using standard methods. The resulting RJ, Lewis T.A. (1985)Yeast sterols: yeast mutants as tools for DNA fragment was treated with Klenow and then cloned the study of sterol metabolism. Methods Enzymol. 1985; blunt-ended into the vector puG6 into the EcoRV cleavage 1 11:333-46 and analyzed by gas chromatography. This gives site, giving rise to the vector puG6-ERG1 (FIG. 2). the data listed in table 2. The percentages are based on the yeast dry weight. Example 2.2. TABLE 2 Integrative Transformations Sterol content peak S. cerevisiae Following the isolation of the plasmid, an extended frag area g|DM GRFtH1ura3ERG1erg5 S. cerevisiae GRF ment was amplified from the vector puG6-ERG1 by means of Squalene 8.1 O.1 PCR so that the resulting fragment consists of the following Lanosterol 2.42 O.31 components: loxP-kanMX-loxP-ADH1-Pr-ERG1-Trp Zymosterol 1.35 1.07 Term. The primers used were oligonucleotide sequences Fecosterol 2.01 O.64 Episterolfergosta-5,7- 12.21 0.72 which contain, in the annealing region, the sequences beyond dienol the cassette to be amplified, of the vectorpUG6-ERG1, and at Dimethyl- 1.02 O.13 the 5' and 3' overhangs the 5' or the 3' sequence of the inte Zymosterol gration locus ERG5, respectively. This ensures that firstly the entire fragment including KanR and the target gene ERG1 is amplified and secondly that this fragment can Subsequently Comparative Example 1 be transformed into yeast and integrates into the yeast target gene locus ERG5 by homologous recombination. The follow Deletion of ERG5 in S. cerevisiae GRFtH1ura3; Genera ing primers were used for this purpose: tion of GRFtH1ura3erg5 The deletion of ERG5 in S. cerevisiae GRFt1ura3 was carried out analogously to example 2. In order to delete only ERG5- Crelox-5' (SEQ ID NO : 13) : 25 the ERG5 gene, the same method was used, but the vector 5 - ATGAGTTCTG TCGCAGAAAA. TATAATACAA CATGCCACTC pUG6 was employed instead of the vectorpUG6-ERG1. This CCAGCTGAAGCTTCGTACGC-3' vector pUG6 contains no cassette consisting of ADH-prom and ERG1-Trp-term. By using this vector, it is possible to delete ERG5- Crelox-3" (SEQ ID NO: 14) : one gene, in this case the gene ERG5. 5'-TTATTCGAAG ACTTCTCCAG TAATTGGGTC. TCTCTTTTTG 30 The resulting yeast strain GRFtH1 ura3erg5 was cultured GCATAGGCCA CTAGTGGATC TG-3 for 48 hours in WMVII medium at 28° C. and 160 rpm in a The selection marker used is the resistance to geneticin culture volume of 20 ml. 500 ul of this preculture were sub (G418). The resulting strain contains one copy of the target sequently transferred into a 50 ml main culture of the same gene ERG1 under the control of the ADH1 promoter and the medium and cultured for 3 days at 28°C. and 160 rpm in a tryptophan terminator. By integration of the gene it is simul 35 baffle flask. taneously possible to delete the corresponding gene ERG5 of The sterols were extracted after 4 days following the the target locus. In order to Subsequently remove the resis method as described in Parks L. W. Bottema CD, Rodriguez tance to G418 again, the resulting yeast strain is transformed RJ, Lewis T.A. (1985)Yeast sterols: yeast mutants as tools for with the cre recombinase vectorpSH47. Owing to this vector, the study of sterol metabolism. Methods Enzymol. 1985; the cre recombinase is expressed in the yeast, and, as a con 40 1 11:333-46 and analyzed by gas chromatography. This gives sequence, the sequence region within the two loXP sequences the data listed in table 3. The percentages are based on the recombines out of the gene, the result of which is that only one yeast dry weight. of the two loXP sequences and the cassette consisting of TABLE 3 ADH1-prom.-ERG1-TRP1-term. are retained in the target 45 locus ERG5. As a consequence, the yeast strain loses the GRFtH1ura3erg5 G418 resistance again. The vector pSH47 can now be Sterol content GRFtH1ura3ERG1erg5 (Comparative removed selectively by cultivation on FOA medium. peak areag DMI (Example 2) example) The resulting yeast strain GRFtH1ura3ERG1erg5 was cul Squalene 8.1 13.18 tured for 48 hours in WMVII medium at 28°C. and 160 rpm 50 Lanosterol 2.42 O.78 in a culture volume of 20 ml. 500 ul of this preculture were Zymosterol 1.35 O.10 subsequently transferred into a 50 ml main culture of the same Fecosterol 2.01 1.03 Episterolfergosta-5,7- 12.21 8.98 medium and cultured for 3 days at 28°C. and 160 rpm in a dienol baffle flask. 4,4-Dimethylzymosterol 1.02 O.21 The sterols were extracted after 4 days following the method as described in Parks L. W. Bottema CD, Rodriguez

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS : 14

<210 SEQ ID NO 1 <211 LENGTH: 1617 &212> TYPE: DNA US 7,556,937 B2 29 30

- Continued <213> ORGANISM: Saccharomyces cerevisiae &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1617)

<4 OO SEQUENCE: 1 atgagt tot gtc gca gaa aat ata at a caa cat gcc act cat aat tot 48 Met Ser Ser Wall Ala Glu. ASn Ile Ile Glin His Ala Thir His Asn. Ser 1. 5 1O 15 acg cta cac caa ttg gct aaa gac cag ccc tot gta ggc gtc act act 96 Thr Lieu. His Gln Leu Ala Lys Asp Gln Pro Ser Val Gly Val Thir Thr 2O 25 3O gcc titc agt atc ctd gat aca citt aag tot atgtca tat ttgaaa at a 144 Ala Phe Ser Ile Lieu. Asp Thir Lieu Lys Ser Met Ser Tyr Lieu Lys Ile 35 4 O 45 titt got act tta atc tdt att citt ttg gtt togg gac caa gtt gca tat 192 Phe Ala Thr Lieu. Ile Cys Ile Lieu. Lieu Val Trp Asp Glin Val Ala Tyr SO 55 6 O caa atc aag aaa got toc atc gca ggit coa aag titt aag titc togg ccc 24 O Glin Ile Llys Lys Gly Ser Ile Ala Gly Pro Llys Phe Llys Phe Trp Pro 65 70 7s 8O atc atc ggit coa titt ttg gaa toc tta gat coa aag titt gala gaa tat 288 Ile Ile Gly Pro Phe Lieu. Glu Ser Lieu. Asp Pro Llys Phe Glu Glu Tyr 85 90 95 aag got aag togg gca toc ggit coa citt to a tigt gtt tot att tt c cat 336 Lys Ala Lys Trp Ala Ser Gly Pro Lieu. Ser Cys Val Ser Ile Phe His 1OO 105 11 O aaa ttt gtt gtt atc gca tot act aga gac ttg goa aga aag atc ttg 384 Llys Phe Val Val Ile Ala Ser Thr Arg Asp Lieu Ala Arg Lys Ile Lieu. 115 12 O 125 caa tot to c aaa titc gtc. aaa cct togc gtt gtc gat gtt got gtg aag 432 Glin Ser Ser Llys Phe Val Llys Pro Cys Val Val Asp Wall Ala Wall Lys 13 O 135 14 O atc tta aga cct togc aat tdg gtt ttt ttg gac gigt aaa got cat act 48O Ile Lieu. Arg Pro Cys Asn Trp Val Phe Lieu. Asp Gly Lys Ala His Thr 145 150 155 160 gat tac aga aaa toa tta aac ggit citt titc act aaa caa got ttg got 528 Asp Tyr Arg Llys Ser Lieu. Asn Gly Lieu. Phe Thr Lys Glin Ala Lieu Ala 1.65 17O 17s caa tac tta cct tca ttg gaa caa atc atg gat aag tac atg gat aag 576 Glin Tyr Lieu Pro Ser Lieu. Glu Glin Ile Met Asp Llys Tyr Met Asp Llys 18O 185 19 O titt gtt cqt tta t ct aag gag aat aac tac gag ccc cag gt c titt titc 624 Phe Val Arg Lieu Ser Lys Glu Asn Asn Tyr Glu Pro Glin Val Phe Phe 195 2OO 2O5 cat gala atgaga gaa att citt togc gcc tta toa ttgaac tot titc tigt 672 His Glu Met Arg Glu Ile Lieu. Cys Ala Lieu. Ser Lieu. Asn. Ser Phe Cys 21 O 215 22O ggit aac tat att acc gaa gat caa gtc aga aag att gct gat gat tac 72 O Gly Asn Tyr Ile Thr Glu Asp Glin Val Arg Lys Ile Ala Asp Asp Tyr 225 23 O 235 24 O tat ttg gtt aca gca gca ttg gala tta gtc. aac titc cca att att atc 768 Tyr Lieu Val Thir Ala Ala Lieu. Glu Lieu Val Asn. Phe Pro Ile Ile Ile 245 250 255

Cct tac act aaa aca t t at ggit aag aaa act gca gac atg gCC atg 816 Pro Tyr Thr Lys Thr Trp Tyr Gly Llys Llys Thr Ala Asp Met Ala Met 26 O 265 27 O aag att tt c gaa aac togt gct caa atg got aag gat cat att gct gca 864 Lys Ile Phe Glu Asn. Cys Ala Glin Met Ala Lys Asp His Ile Ala Ala 27s 28O 285

US 7,556,937 B2 33 34

- Continued

35 4 O 45

Phe Ala Thir Luell Ile Ile Luell Luell Wall Trp Asp Glin Wall Ala Tyr SO 55 6 O

Glin Ile Gly Ser Ile Ala Gly Pro Lys Phe Phe Trp Pro 65 70

Ile Ile Pro Phe Lell Glu Ser Luell Asp Pro Phe Glu Glu Tyr 85 90 95

Ala Trp Ala Ser Gly Pro Luell Ser Cys Wall Ser Ile Phe His 1OO 105 11 O

Phe Wall Wall Ile Ala Ser Thir Arg Asp Luell Ala Arg Ile Luell 115 12 O 125

Glin Ser Ser Phe Wall Lys Pro Wall Wall Asp Wall Ala Wall Lys 13 O 135 14 O

Ile Luell Arg Pro Asn Trp Wall Phe Luell Asp Gly Ala His Thir 145 150 155 160

Asp Arg Ser Lell Asn Gly Luell Phe Thir Glin Ala Luell Ala 1.65 17O 17s

Glin Tyr Luell Pro Ser Lell Glu Glin Ile Met Asp Met Asp 18O 185 19 O

Phe Wall Arg Luell Ser Glu Asn Asn Tyr Glu Pro Glin Wall Phe Phe 195

His Glu Met Arg Glu Ile Lell Ala Luell Ser Lell Asn Ser Phe 21 O 215

Gly Asn Tyr Ile Thir Glu Asp Glin Wall Arg Lys Ile Ala Asp Asp Tyr 225 23 O 235 24 O

Luell Wall Thir Ala Ala Lell Glu Luell Wall ASn Phe Pro Ile Ile Ile 245 250 255

Pro Thir Lys Thir Trp Gly Lys Thir Ala Asp Met Ala Met 26 O 265 27 O

Ile Phe Glu Asn Ala Glin Met Ala Lys Asp His Ile Ala Ala 27s 285

Gly Gly Lys Pro Wall Wall Met Asp Ala Trp Cys Luell Met His 29 O 295 3 OO

Asp Ala Asn Ser Asn Asp Asp Asp Ser Arg Ile His Arg Glu 3. OS 310 315

Phe Thir Asn Glu Ile Ser Glu Ala Wall Phe Thir Phe Luell Phe Ala 3.25 330 335

Ser Glin Asp Ala Ser Ser Ser Luell Ala Trp Lell Phe Glin Ile Wall 34 O 345 35. O

Ala Asp Arg Pro Asp Wall Lell Ala Ile Arg Glu Glu Glin Luell Ala 355 360 365

Wall Arg Asn Asn Asp Met Ser Thir Glu Luell ASn Lell Asp Luell Ile Glu 37 O 375

Lys Met Thir Asn Met Wall Ile Glu Thir Lell Arg Arg 385 390 395 4 OO

Pro Pro Wall Luell Met Wall Pro Wall Wall Asn Phe Pro Wall 4 OS 41O 415

Ser Pro Asn Tyr Thir Ala Pro Gly Ala Met Lell Ile Pro Thir Luell 42O 425 43 O

Pro Ala Luell His Asp Pro Glu Wall Glu Asn Pro Asp Glu Phe 435 44 O 445

Ile Pro Glu Arg Trp Wall Glu Gly Ser Ala Ser Glu Ala 450 45.5 460 US 7,556,937 B2 35 36

- Continued

Asn Trp Leu Val Phe Gly Cys Gly Pro His Val Cys Lieu. Gly Glin Thr 465 470 47s 48O Tyr Val Met Ile Thr Phe Ala Ala Lieu. Leu Gly Llys Phe Ala Leu Tyr 485 490 495 Thr Asp Phe His His Thr Val Thr Pro Leu Ser Glu Lys Ile Llys Val SOO 505 51O Phe Ala Thir Ile Phe Pro Lys Asp Asp Lieu. Lieu. Lieu. Thir Phe Llys Llys 515 52O 525 Arg Asp Pro Ile Thr Gly Glu Val Phe Glu 53 O 535

<210 SEQ ID NO 3 <211 LENGTH: 1578 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Description of synthetic sequence: truncated HMG &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1) ... (1578) &223> OTHER INFORMATION: CDS

<4 OO SEQUENCE: 3 atg gac caa ttg gtg aaa act gala gtc acc aag aag tot titt act gct 48 Met Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr Ala 1. 5 1O 15 cct gta caa aag got tot aca cca gtt tta acc aat aaa aca gtc att 96 Pro Val Glin Lys Ala Ser Thr Pro Val Lieu. Thr Asn Lys Thr Val Ile 2O 25 3O tct gga tog aaa gttcaaa agt tta to a tot gog caa tog agc tica to a 144 Ser Gly Ser Llys Val Lys Ser Leu Ser Ser Ala Glin Ser Ser Ser Ser 35 4 O 45 gga cct tca to a tict agt gag gala gat gat tcc cqc gat att gala agc 192 Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu Ser SO 55 6 O ttg gat aag aaa at a cqt cct tta gaa gaa tta gaa goa tta tta agt 24 O Lieu. Asp Llys Lys Ile Arg Pro Lieu. Glu Glu Lieu. Glu Ala Lieu. Lieu. Ser 65 70 7s 8O agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct gcc titg gtt 288 Ser Gly Asn. Thir Lys Glin Lieu Lys Asn Lys Glu Val Ala Ala Lieu Val 85 90 95 att cac ggit aag tita cct ttg tac got ttg gag aaa aaa tta ggit gat 336 Ile His Gly Lys Lieu Pro Lieu. Tyr Ala Lieu. Glu Lys Llys Lieu. Gly Asp 1OO 105 11 O act acg aga gC9 gtt gcg gta Cdt agg aag gCt c tt t ca att ttg gca 384 Thir Thr Arg Ala Val Ala Val Arg Arg Lys Ala Lieu. Ser Ile Lieu Ala 115 12 O 125 gaa got cot gta tta gca tot gat cqt tta cca tat aaa aat tat gac 432 Glu Ala Pro Val Lieu Ala Ser Asp Arg Lieu Pro Tyr Lys Asn Tyr Asp 13 O 135 14 O tac gaC cc gta titt ggc gct tdt tt gala aat gtt at a ggit tac atg 48O Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr Met 145 150 155 160 cct ttg ccc gtt ggt gtt at a ggc ccc titg gtt atc gat ggit aca tot 528 Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly. Thir Ser 1.65 17O 17s tat cat at a cca atg gca act aca gag ggit tdt ttg gta gct tct gcc 576 Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Lieu Val Ala Ser Ala 18O 185 19 O

US 7,556,937 B2 39 40

- Continued aat cqt titg aaa gat gigg toc gtc. acc tigc att aaa toc taa 1578 Asn Arg Lieu Lys Asp Gly Ser Val Thir Cys Ile Llys Ser 515 525

SEQ ID NO 4 LENGTH: 525 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description of synthetic sequence: truncated HMG-CoA reductase (t-HMG)

SEQUENCE: 4.

Met Asp Glin Luell Wall Thir Glu Wall Thir Ser Phe Thir Ala 1. 5 15

Pro Wall Glin Lys Ala Ser Thir Pro Wall Luell Thir Asn Thir Wall Ile 2O 25

Ser Gly Ser Lys Wall Ser Luell Ser Ser Ala Glin Ser Ser Ser Ser 35 4 O 45

Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu Ser SO 55 6 O

Lell Asp Ile Arg Pro Luell Glu Glu Luell Glu Ala Luell Luell Ser 65 70

Ser Gly Asn Thir Lys Glin Lell Asn Lys Glu Wall Ala Ala Luell Wall 85 90 95

Ile His Gly Lys Lell Pro Lell Ala Luell Glu Luell Gly Asp 105 11 O

Thir Thir Arg Ala Wall Ala Wall Arg Arg Ala Lell Ser Ile Luell Ala 115 12 O 125

Glu Ala Pro Wall Lell Ala Ser Asp Arg Luell Pro Tyr Asn Asp 13 O 135 14 O

Tyr Asp Arg Wall Phe Gly Ala Glu ASn Wall Ile Gly Met 145 150 155 160

Pro Luell Pro Wall Gly Wall Ile Gly Pro Luell Wall Ile Asp Gly Thir Ser 1.65 17O 17s

His Ile Pro Met Ala Thir Thir Glu Gly Lell Wall Ala Ser Ala 18O 185 19 O

Met Arg Gly Lys Ala Ile Asn Ala Gly Gly Gly Ala Thir Thir Wall 195

Lell Thir Asp Gly Met Thir Arg Gly Pro Wall Wall Arg Phe Pro Thir 21 O 215 22O

Lell Arg Ser Gly Ala Ile Trp Luell Asp Ser Glu Glu Gly 225 23 O 235 24 O

Glin Asn Ala Ile Lys Ala Phe Asn Ser Thir Ser Arg Phe Ala Arg 245 250 255

Lell Glin His Ile Glin Thir Luell Ala Gly Asp Lell Lell Phe Met Arg 26 O 265 27 O

Phe Arg Thir Thir Thir Gly Asp Ala Met Gly Met Asn Met Ile Ser 27s 285

Gly Wall Glu Ser Lell Lys Glin Met Wall Glu Glu Gly Trp Glu 29 O 295 3 OO

Asp Met Glu Wall Wall Ser Wall Ser Gly Asn Tyr Thir Asp Lys 3. OS 310 315 32O

Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Ser Wall Wall Ala 3.25 330 335 US 7,556,937 B2 41

- Continued Glu Ala Thir Ile Pro Gly Asp Val Val Arg Llys Val Lieu Lys Ser Asp 34 O 345 35. O Val Ser Ala Lieu Val Glu Lieu. Asn. Ile Ala Lys Asn Lieu Val Gly Ser 355 360 365 Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn Lieu 37 O 375 38O Val Thir Ala Val Phe Lieu Ala Lieu. Gly Glin Asp Pro Ala Glin Asn Val 385 390 395 4 OO Glu Ser Ser Asn. Cys Ile Thr Lieu Met Lys Glu Val Asp Gly Asp Lieu. 4 OS 41O 415 Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly 42O 425 43 O Gly Thr Val Lieu. Glu Pro Glin Gly Ala Met Lieu. Asp Lieu. Lieu. Gly Val 435 44 O 445 Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Glin Lieu Ala 450 45.5 460 Arg Ile Val Ala Cys Ala Val Lieu Ala Gly Glu Lieu. Ser Lieu. Cys Ala 465 470 47s 48O Ala Lieu Ala Ala Gly. His Lieu Val Glin Ser His Met Thr His Asn Arg 485 490 495 Llys Pro Ala Glu Pro Thr Llys Pro Asn. Asn Lieu. Asp Ala Thir Asp Ile SOO 505 51O Asn Arg Lieu Lys Asp Gly Ser Val Thir Cys Ile Llys Ser 515 52O 525

<210 SEQ ID NO 5 <211 LENGTH: 1593 &212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1593)

<4 OO SEQUENCE: 5 atgtct gct acc aag tica atc gtt gga gag gCattg gaa tac gta aac 48 Met Ser Ala Thir Lys Ser Ile Val Gly Glu Ala Lieu. Glu Tyr Val Asn 1. 5 1O 15 att ggit tta agt cat titc titg got tta cca ttg gcc caa aga atc. tct 96 Ile Gly Lieu. Ser His Phe Lieu Ala Lieu Pro Lieu Ala Glin Arg Ile Ser 2O 25 3O ttg at cata ata att cot titc att tac aat att gta togg caa tta cta 144 Lieu. Ile Ile Ile Ile Pro Phe Ile Tyr Asn Ile Val Trp Glin Leu Lieu. 35 4 O 45 tat to t titg aga aag gac cqt cca cct ct a gtg ttt tac togg att coa 192 Tyr Ser Lieu. Arg Lys Asp Arg Pro Pro Leu Val Phe Tyr Trp Ile Pro SO 55 6 O tgg gt C ggit agt gct gtt gtg tac ggt atg aag cca tac gag titt tt C 24 O Trp Val Gly Ser Ala Val Val Tyr Gly Met Llys Pro Tyr Glu Phe Phe 65 70 7s 8O gaa gaa tigt caa aag aaa tac ggit gat att ttt to a titc gtt ttgtta 288 Glu Glu. Cys Glin Llys Llys Tyr Gly Asp Ile Phe Ser Phe Val Lieu. Lieu. 85 90 95 gga aga gt C atg act gtg tat tta gga cca aag ggit cac gala titt gt C 336 Gly Arg Val Met Thr Val Tyr Lieu. Gly Pro Lys Gly His Glu Phe Val 1OO 105 11 O titc aac got aag titg gca gat gtt to a goa gaa gct gct tac got cat 384 Phe Asn Ala Lys Lieu Ala Asp Val Ser Ala Glu Ala Ala Tyr Ala His 115 12 O 125 US 7,556,937 B2 43 44

- Continued ttg act act CC a gtt tto ggit a.a.a. ggt gtt att tac gat tgt CC a aat 432 Lell Thir Thir Pro Wall Phe Gly Gly Wall Ile Tyr Asp Cys Pro Asn 13 O 135 14 O tot aga ttg atg gag Cala aag aag titt gtt aag ggit gct Cta acc a.a.a. Ser Arg Luell Met Glu Glin Lys Lys Phe Wall Lys Gly Ala Luell Lys 145 150 155 160 gaa gcc ttic aag agc gtt CC a ttg att gct gaa gaa gtg tac aag 528 Glu Ala Phe Lys Ser Wall Pro Luell Ile Ala Glu Glu Wall Lys 1.65 17O tac ttic aga gac t cc aac ttic cgt ttg aat gaa aga act ggt 576 Phe Arg Asp Ser Asn Phe Arg Luell ASn Glu Arg Thir Gly 18O 185 19 O act att gac gtg atg gtt act Cala cott gala atg act att ttic gct 624 Thir Ile Asp Wall Met Wall Thir Glin Pro Glu Met Thir Ile Phe Ala 195 2OO 2O5 toa aga to a tta ttg ggit aag gala atg aga gca a.a.a. ttg gat gat 672 Ser Arg Ser Luell Lell Gly Lys Glu Met Arg Ala Lys Lell Asp Asp 21 O 215 22O titt gct tac ttg tac agt gat ttg gat aag ggt tto act CC a aac 72 O Phe Ala Luell Ser Asp Luell Asp Lys Gly Phe Thir Pro Asn 225 23 O 235 24 O tto gt C ttic cott aac tta C Ca ttg gala CaC tat aga aag aga gat CaC 768 Phe Wall Phe Pro Asn Lell Pro Luell Glu His Arg Lys Arg Asp His 245 250 255 gct Cala aag gct atc. t cc ggit act tac atg tot ttg att aag gala aga 816 Ala Glin Lys Ala Ile Ser Gly Thir Tyr Met Ser Lell Ile Lys Glu Arg 26 O 265 27 O aga aag aac aac gac att Cala gac aga gat ttg atc. gat to c ttg atg 864 Arg Lys Asn Asn Asp Ile Glin Asp Arg Asp Luell Ile Asp Ser Luell Met 27s 28O 285 aag aac tot acc tac aag gat ggt gtg aag atg act gat Cala gala at C 912 Asn Ser Thir yr Asp Gly Wall Lys Met Thir Asp Glin Glu Ile 29 O 295 3 OO gct aac ttg tta att ggit gtc tta atg ggt ggt Cala Cat act tot gct 96.O Ala Asn Luell Luell le Gly Wall Luell Met Gly Gly Glin His Thir Ser Ala 3. OS 310 315 32O gcc act tot gct att ttg ttg CaC ttg gct gaa aga CC a gat gt C OO8 Ala Thir Ser Ala Ile Lell Luell His Luell Ala Glu Arg Pro Asp Wall 330 335

Cala Cala gala ttg gaa gaa Cala atg cgt gtt ttg gat ggt ggt aag Glin Glin Glu Luell Glu Glu Glin Met Arg Wall Lell Asp Gly Gly Lys 34 O 345 35. O aag gala ttg acc gat tta tta Cala gala atg C Ca ttg aac Cala 104 Glu Luell Thir Asp Lell Luell Glin Glu Met Pro Lell Luell Asn Glin 355 360 365 act att aag gala act Cta aga atg CaC Cat CCa ttg CaC tot ttg ttic 152 Thir Ile Lys Glu Lell Arg Met His His Pro Lell His Ser Luell Phe 37 O 375 38O cgt aag gtt atg a.a.a. gat atg CaC gtt CC a aac act tot tat gt C at C 2OO Arg Lys Wall Met Asp Met His Wall Pro ASn Thir Ser Wall Ile 385 390 395 4 OO

C Ca gca ggt tat CaC gtt ttg gtt tot CC a ggt tac act Cat tta aga 248 Pro Ala Gly His Wall Lell Wall Ser Pro Gly Thir His Luell Arg 4 OS 41O 415 gac gala tac ttic cott aat gct CaC Cala ttic aac att CaC cgt. tgg aac 296 Asp Glu Phe Pro Asn Ala His Glin Phe ASn Ile His Arg Trp Asn 42O 425 43 O a.a.a. gat tot gcc t cc tot tat to c gt C ggt gaa gaa gtc gat tac ggt 344 Asp Ser Ala Ser Ser Ser Wall Gly Glu Glu Wall Asp Gly 435 44 O 445 US 7,556,937 B2 45 46

- Continued titc ggit gcc att tot aag gigt gtc agc tict coa tac tta cct titc ggit 392 Phe Gly Ala Ile Ser Lys Gly Val Ser Ser Pro Tyr Lieu Pro Phe Gly 450 45.5 460 ggt ggit aga cac alga tigt atc ggit gala cac titt gct tac tdt cag ct a 44 O Gly Gly Arg His Arg Cys Ile Gly Glu. His Phe Ala Tyr Cys Glin Lieu. 465 470 47s 48O ggt gtt cta atg tcc att titt atc aga aca tta aaa tdg cat tac cca 488 Gly Val Lieu Met Ser Ile Phe Ile Arg Thr Lieu Lys Trp His Tyr Pro 485 490 495 gag ggit aag acc gtt coa cct colt gac titt aca tot atg gtt act citt 536 Glu Gly Lys Thr Val Pro Pro Pro Asp Phe Thir Ser Met Val Thr Lieu. SOO 505 51O cca acc ggit coa gcc aag atc at C tog gala aag aga aat coa gala caa 584 Pro Thr Gly Pro Ala Lys Ile Ile Trp Glu Lys Arg ASn Pro Glu Glin 515 52O 525 aag atc taa 593

53 O

<210 SEQ ID NO 6 <211 LENGTH: 53 O &212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae

<4 OO SEQUENCE: 6 Met Ser Ala Thir Lys Ser Ile Val Gly Glu Ala Lieu. Glu Tyr Val Asn 1. 5 1O 15 Ile Gly Lieu. Ser His Phe Lieu Ala Lieu Pro Lieu Ala Glin Arg Ile Ser 2O 25 3O Lieu. Ile Ile Ile Ile Pro Phe Ile Tyr Asn Ile Val Trp Glin Leu Lieu. 35 4 O 45 Tyr Ser Lieu. Arg Lys Asp Arg Pro Pro Leu Val Phe Tyr Trp Ile Pro SO 55 6 O Trp Val Gly Ser Ala Val Val Tyr Gly Met Llys Pro Tyr Glu Phe Phe 65 70 7s 8O Glu Glu. Cys Glin Llys Llys Tyr Gly Asp Ile Phe Ser Phe Val Lieu. Lieu. 85 90 95 Gly Arg Val Met Thr Val Tyr Lieu. Gly Pro Lys Gly His Glu Phe Val 1OO 105 11 O Phe Asn Ala Lys Lieu Ala Asp Val Ser Ala Glu Ala Ala Tyr Ala His 115 12 O 125 Lieu. Thir Thr Pro Val Phe Gly Lys Gly Val Ile Tyr Asp Cys Pro Asn 13 O 135 14 O Ser Arg Lieu Met Glu Glin Llys Llys Phe Wall Lys Gly Ala Lieu. Thir Lys 145 150 155 160 Glu Ala Phe Llys Ser Tyr Val Pro Lieu. Ile Ala Glu Glu Val Tyr Lys 1.65 17O 7s Tyr Phe Arg Asp Ser Lys Asn. Phe Arg Lieu. Asn. Glu Arg Thir Thr Gly 18O 185 19 O Thir Ile Asp Val Met Val Thr Glin Pro Glu Met Thr Ile Phe Thr Ala 195 2OO 2O5 Ser Arg Ser Lieu. Lieu. Gly Lys Glu Met Arg Ala Lys Lieu. Asp Thr Asp 21 O 215 22O Phe Ala Tyr Lieu. Tyr Ser Asp Lieu. Asp Llys Gly Phe Thr Pro Ile Asn 225 23 O 235 24 O Phe Val Phe Pro Asn Lieu Pro Lieu. Glu. His Tyr Arg Lys Arg Asp His US 7,556,937 B2 47 48

- Continued

245 250 255

Ala Glin Ala Ile Ser Gly Thr Tyr Met Ser Lieu. Ile Lys Glu Arg 26 O 265 27 O

Arg Asn Asn Asp Ile Glin Asp Arg Asp Lieu. Ile Asp Ser Luell Met 27s 28O 285

Asn Ser Thir yr Lys Asp Gly Val Lys Met Thr Asp Glin Glu Ile 29 O 295 3 OO

Ala Asn Luell Luell le Gly Val Lieu Met Gly Gly Gln His Thir Ser Ala 3. OS 310 315

Ala Thir Ser Ala rp Ile Lieu. Lieu. His Lieu Ala Glu Arg Pro Asp Wall 3.25 330 335

Glin Glin Glu Luell Tyr Glu Glu Gln Met Arg Val Lieu. Asp Gly Gly 34 O 345 35. O

Glu Luell Thir yr Asp Lieu. Lieu. Glin Glu Met Pro Lieu. Luell Asn Glin 355 360 365

Thir Ile Glu Thr Lieu. Arg Met His His Pro Leu. His Ser Luell Phe 37 O 375 38O

Arg Wall Met Lys Asp Met His Val Pro Asn Thr Ser Wall Ile 385 390 395 4 OO

Pro Ala Gly His Val Lieu Val Ser Pro Gly Tyr Thr His Luell Arg 4 OS 41O 415

Asp Glu Phe Pro Asn Ala His Glin Phe ASn Ile His Arg Trp Asn 42O 425 43 O

Asp Ser Ala Ser Ser Tyr Ser Val Gly Glu Glu Val Asp Gly 435 44 O 445

Phe Gly Ala Ile Ser Lys Gly Val Ser Ser Pro Tyr Lieu Pro Phe Gly 450 45.5 460

Gly Gly Arg His Arg Cys Ile Gly Glu. His Phe Ala Tyr Glin Luell 465 470 47s

Gly Wall Luell Met Ser Ile Phe Ile Arg Thr Lieu Lys Trp His Tyr Pro 485 490 495

Glu Gly Thir Val Pro Pro Pro Asp Phe Thir Ser Met Wall Thir Luell SOO 505 51O

Pro Thir Gly Pro Ala Lys Ile Ile Trp Glu Lys Arg Asn Pro Glu Glin 515 52O 525

Ile 53 O

SEO ID NO 7 LENGTH: 1491 TYPE: DNA ORGANISM: Saccharomyces cerevisiae FEATURE: NAME/KEY: CDS LOCATION: (1) ... (1491)

<4 OO SEQUENCE: 7 atg tot gct gtt aac gtt gca cct gala titg att aat gcc gac aac aca 48 Met Ser Ala Wall Asn. Wall Ala Pro Glu Lieu. Ile Asn Ala Asp Asn Thir 1. 15 att acc tac gat gcg att gtc at C ggit gct ggt gtt atc CC a tgt 96 Ile Thir Asp Ala Ile Val Ile Gly Ala Gly Val Ile Pro Cys 2O 25 gtt gct act ggt Cta gca aga aag ggit aag aaa gtt Ctt at C gta gala 144 Wall Ala Thir Gly Lieu Ala Arg Lys Gly Llys Llys Val Lieu. Ile Wall Glu 35 4 O 45 US 7,556,937 B2 49 50

- Continued cgt gac tgg gct atg cott gat aga att gtt ggt gaa ttg atg Cala CC a 192 Arg Asp Trp Ala Met Asp Arg Ile Wall Gly Glu Lell Met Glin Pro SO 55 6 O ggit ggt gtt aga gca ttg aga agt Ctg ggt atg att Cala tot at C aac 24 O Gly Gly Wall Arg Ala Lel Arg Ser Luell Gly Met Ile Glin Ser Ile Asn 65 70 7s 8O aac at C gala gca tat gtt acc ggt tat acc gtc titt ttic aac ggc 288 Asn Ile Glu Ala Tyr Wall Thir Gly Tyr Thir Wall Phe Phe Asn Gly 85 90 95 gaa Cala gtt gat att tac cott tac aag gcc gat atc. cott a.a.a. gtt 336 Glu Glin Wall Asp Ile Pro Tyr Lys Ala Asp Ile Pro Wall 1OO 105 11 O gaa a.a.a. ttg aag gac ttg gtc a.a.a. gat ggt aat gac aag gtC ttg gala 384 Glu Lys Luell Lys Asp Lel Wall Lys Asp Gly ASn Asp Lys Wall Luell Glu 115 12 O 125 gac agc act att CaC atc. aag gat tac gala gat gat gaa aga gala agg 432 Asp Ser Thir Ile His Ile Lys Asp Glu Asp Asp Glu Arg Glu Arg 13 O 135 14 O ggit gtt gct titt gtt Cat ggit aga ttic ttg aac aac ttg aga aac att Gly Wall Ala Phe Wall His Gly Arg Phe Luell ASn Asn Lell Arg Asn Ile 145 150 155 160 act gct Cala gag C Ca aat gtt act aga gtg Cala ggit aac tgt att gag 528 Thir Ala Glin Glu Pro Asn Wall Thir Arg Wall Glin Gly Asn Cys Ile Glu 1.65 17O 17s ata ttg aag gat gaa aag aat gag gtt gtt ggt gcc aag gtt gac att 576 Ile Luell Lys Asp Glu Lys Asn Glu Wall Wall Gly Ala Lys Wall Asp Ile 18O 185 19 O gat ggc cgt ggc aag gtg gaa ttic a.a.a. gcc CaC ttg a Ca titt at C tgt 624 Asp Gly Arg Gly Lys Wall Glu Phe Ala His Lell Thir Phe Ile Cys 195 2OO 2O5 gac ggt at C titt toa cgt tto aga aag gala ttg CaC C Ca gac Cat gtt 672 Asp Gly Ile Phe Ser Arg Phe Arg Lys Glu Luell His Pro Asp His Wall 21 O 215 22O

C Ca act gt C ggt tot tcg titt gt C ggt atg tot ttg tto aat gct aag 72 O Pro Thir Wall Gly Ser Ser Phe Wall Gly Met Ser Lell Phe Asn Ala Lys 225 23 O 235 24 O aat cott gct cott atg CaC ggit CaC gtt att citt ggit gat Cat atg 768 Asn Pro Ala Pro Met His Gly His Wall Ile Luell Gly Ser Asp His Met 245 250 255

C Ca at C ttg gtt tac Cala atc. agt CC a gala gaa a Ca aga at C citt tgt 816 Pro Ile Luell Wall Tyr Glin Ile Ser Pro Glu Glu Thir Arg Ile Luell Cys 26 O 265 27 O gct tac aac tot C Ca aag gtc CC a gct gat atc. aag tgg atg att 864 Ala Asn Ser Pro Wall Pro Ala Asp Ile Ser Trp Met Ile 27s 28O 285 aag gat gt C Cala cott tto att CC a aag agt Cta cgt cott to a titt gat 912 Asp Wall Glin Pro Phe Ile Pro Lys Ser Luell Arg Pro Ser Phe Asp 29 O 295 3 OO gaa gcc gt C agc Cala ggit a.a.a. titt aga gct atg C Ca aac to c tac ttg 96.O Glu Ala Wall Ser Glin Gly Phe Arg Ala Met Pro Asn Ser Luell 3. OS 310 315 32O

C Ca gct aga Cala aac gac gtc act ggt atg gtt atc. ggt gac gct 1008 Pro Ala Arg Glin Asn Asp Wall Thir Gly Met Cys Wall Ile Gly Asp Ala 3.25 330 335

Cta aat atg aga Cat C Ca ttg act ggt ggt ggt atg act gtC ggt ttg 1056 Lell Asn Met Arg His Pro Lell Thir Gly Gly Gly Met Thir Wall Gly Luell 34 O 345 35. O

Cat gat gtt gt C ttg ttg att aag a.a.a. at a ggt gac Cta gac ttic agc 1104 His Asp Wall Wall Lell Lell Ile Lys Ile Gly Asp Lell Asp Phe Ser 355 360 365 US 7,556,937 B2 51

- Continued gac cqt gala aag gtt ttg gat gala tta ct a gac tac Cat ttC gala aga 152 Asp Arg Glu, Llys Val Lieu. Asp Glu Lieu. Lieu. Asp Tyr His Phe Glu Arg 37 O 375 38O aag agt tac gat tcc gtt att aac gtt ttg to a gtg gct ttg tat tot 2OO Llys Ser Tyr Asp Ser Val Ile Asin Val Lieu. Ser Val Ala Lieu. Tyr Ser 385 390 395 4 OO ttgttc gct gct gac agc gat aac titg aag gCa tta caa aaa ggit tt 248 Lieu. Phe Ala Ala Asp Ser Asp Asn Lieu Lys Ala Lieu Gln Lys Gly Cys 4 OS 41O 415 titc aaa tat tt c caa aga got ggc gat tigt gtc. aac aaa ccc gtt gala 296 Phe Llys Tyr Phe Glin Arg Gly Gly Asp Cys Val Asn Llys Pro Val Glu 42O 425 43 O titt ctd tot ggit gtc. ttg cca aag cct ttg caa titg acc agg gtt titc 344 Phe Leu Ser Gly Val Lieu Pro Llys Pro Leu Gln Lieu. Thir Arg Val Phe 435 44 O 445 titc got gtc gct ttt tac acc att tac ttgaac atg gaa gaa cqt ggit 392 Phe Ala Val Ala Phe Tyr Thr Ile Tyr Lieu. Asn Met Glu Glu Arg Gly 450 45.5 460 titc ttg gga tta cca atg gct tta ttg gaa ggit att atg att ttg atc 44 O Phe Lieu. Gly Lieu Pro Met Ala Lieu. Lieu. Glu Gly Ile Met Ile Lieu. Ile 465 470 47s 48O aca got att aga gta t t c acc cca ttt ttg titt ggit gag titg att ggit 488 Thr Ala Ile Arg Val Phe Thr Pro Phe Leu Phe Gly Glu Lieu. Ile Gly 485 490 495 taa 491

<210 SEQ ID NO 8 <211 LENGTH: 496 &212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae

<4 OO SEQUENCE: 8 Met Ser Ala Val Asn. Wall Ala Pro Glu Lieu. Ile Asn Ala Asp Asn. Thir 1. 5 1O 15 Ile Thr Tyr Asp Ala Ile Val Ile Gly Ala Gly Val Ile Gly Pro Cys 2O 25 3O Val Ala Thr Gly Lieu Ala Arg Lys Gly Lys Llys Val Lieu. Ile Val Glu 35 4 O 45 Arg Asp Trp Ala Met Pro Asp Arg Ile Val Gly Glu Lieu Met Glin Pro SO 55 6 O Gly Gly Val Arg Ala Lieu. Arg Ser Lieu. Gly Met Ile Glin Ser Ile Asn 65 70 7s 8O Asn Ile Glu Ala Tyr Pro Val Thr Gly Tyr Thr Val Phe Phe Asin Gly 85 90 95 Glu Glin Val Asp Ile Pro Tyr Pro Tyr Lys Ala Asp Ile Pro Llys Val 1OO 105 11 O Glu Lys Lieu Lys Asp Lieu Val Lys Asp Gly Asn Asp Llys Val Lieu. Glu 115 12 O 125 Asp Ser Thir Ile His Ile Lys Asp Tyr Glu Asp Asp Glu Arg Glu Arg 13 O 135 14 O Gly Val Ala Phe Val His Gly Arg Phe Lieu. Asn. Asn Lieu. Arg Asn. Ile 145 150 155 160 Thr Ala Glin Glu Pro Asn Val Thr Arg Val Glin Gly Asn Cys Ile Glu 1.65 17O 17s Ile Lieu Lys Asp Glu Lys Asn. Glu Val Val Gly Ala Lys Val Asp Ile 18O 185 19 O US 7,556,937 B2 53 54

- Continued

Asp Gly Arg Gly Llys Val Glu Phe Lys Ala His Lell Thir Phe Ile 195 2OO

Asp Gly Ile Phe Ser Arg Phe Arg Lys Glu Lieu. His Pro Asp His Wall 21 O 215 22O

Pro Thr Val Gly Ser Ser Phe Val Gly Met Ser Lell Phe Asn Ala Lys 225 23 O 235 24 O

Asn Pro Ala Pro Met His Gly His Val Ile Leu Gly Ser Asp His Met 245 250 255

Pro Ile Leu Val Tyr Glin Ile Ser Pro Glu Glu Thir Arg Ile Luell 26 O 265 27 O

Ala Tyr Asn. Ser Pro Llys Val Pro Ala Asp Ile Ser Trp Met Ile 27s 28O 285

Asp Val Glin Pro Phe Ile Pro Llys Ser Leu Arg Pro Ser Phe Asp 29 O 295 3 OO

Glu Ala Val Ser Glin Gly Llys Phe Arg Ala Met Pro Asn Ser Luell 3. OS 310 315

Pro Ala Arg Glin Asn Asp Val Thr Gly Met Cys Wall Ile Gly Asp Ala 3.25 330 335

Lell Asn Met Arg His Pro Lieu. Thr Gly Gly Gly Met Thir Wall Gly Luell 34 O 345 35. O

His Asp Val Val Lieu. Lieu. Ile Llys Lys Ile Gly Asp Lell Asp Phe Ser 355 360 365

Asp Arg Glu Lys Val Lieu. Asp Glu Lieu. Lieu. Asp Tyr His Phe Glu Arg 37 O 375

Lys Ser Tyr Asp Ser Val Ile Asin Val Lieu. Ser Wall Ala Luell Ser 385 390 395 4 OO

Lel Phe Ala Ala Asp Ser Asp Asn Lieu Lys Ala Lell Glin Gly 4 OS 41O 415

Phe Llys Tyr Phe Glin Arg Gly Gly Asp Cys Val Asn Pro Wall Glu 42O 425 43 O

Phe Lieu. Ser Gly Val Lieu Pro Llys Pro Lieu. Glin Lell Thir Arg Wall Phe 435 44 O 445

Phe Ala Val Ala Phe Tyr Thr Ile Tyr Lieu. Asn Met Glu Glu Arg Gly 450 45.5 460

Phe Lieu. Gly Lieu Pro Met Ala Lieu. Lieu. Glu Gly Ile Met Ile Luell Ile 465 470 47s

Thir Ala Ile Arg Val Phe Thr Pro Phe Leu Phe Gly Glu Luell Ile Gly 485 490 495

<210 SEQ ID NO 9 <211 LENGTH: 1335 &212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1335)

<4 OO SEQUENCE: 9 atg gga aag ct a tta caa ttg gca ttg cat cog gtc gag atg aag gca 48 Met Gly Lys Lieu. Lieu. Glin Lieu Ala Lieu. His Pro Wall Glu Met Lys Ala 1. 15 gct ttg aag ctgaag titt to aga aca ccg cta tto t cc at C tat gat 96 Ala Lieu Lys Lieu Lys Phe Cys Arg Thr Pro Lieu. Phe Ser Ile Asp 2O 25 3O

Cag to c acg to t c ca tat citc ttg cac togt titc gaa Ctg ttg aac ttg 144 Glin Ser Thr Ser Pro Tyr Lieu. Lieu. His Cys Phe Glu Lell Luell Asn Luell 35 4 O 45 US 7,556,937 B2 55 56

- Continued a CC to c aga tog titt gct gct gtg at C aga gag Ctg Cat CC a gala ttg 192 Thir Ser Arg Ser Phe Ala Ala Wall Ile Arg Glu Lell His Pro Glu Luell SO 55 6 O aga aac tgt gtt act citc. titt tat ttg att tta agg gct ttg gat acc 24 O Arg Asn Cys Wall Thir Lell Phe Luell Ile Luell Arg Ala Luell Asp Thir 65 70 7s 8O atc. gala gac gat atg t cc atc. gala CaC gat a.a.a. att gac ttg ttg 288 Ile Glu Asp Asp Met Ser Ile Glu His Asp Luell Ile Asp Luell Luell 85 90 95 cgt CaC ttic CaC gag ttg ttg tta act a.a.a. tgg ttic gac gga 336 Arg His Phe His Glu Lell Luell Luell Thir Trp Ser Phe Asp Gly 1OO 105 11 O aat gcc cc c gat gtg gac aga gcc gtt ttg a Ca gat ttic gala tog 384 Asn Ala Pro Asp Wall Asp Arg Ala Wall Luell Thir Asp Phe Glu Ser 115 12 O 125 att citt att gala tto CaC a.a.a. ttg a.a.a. CC a gaa tat Cala gala gt C at C 432 Ile Luell Ile Glu Phe His Lys Luell Pro Glu Tyr Glin Glu Wall Ile 13 O 135 14 O aag gag at C acc gag a.a.a. atg ggt aat ggt atg gcc gac tac at C tta Lys Glu Ile Thir Glu Lys Met Gly Asn Gly Met Ala Asp Ile Luell 145 150 155 160 gat gala aat tac aac ttg aat 999 ttg Cala acc gtc CaC gac tac gac 528 Asp Glu Asn Asn Lel Asn Gly Luell Glin Thir Wall His Asp Tyr Asp 1.65 17O 17s gtg tac tgt CaC tac gta gct ggt ttg gt C ggt gat ggit ttg acc cgt 576 Wall Cys His Tyr Wall Ala Gly Luell Wall Gly Asp Gly Luell Thir Arg 18O 185 19 O ttg att gt C att gcc aag titt gcc aac gala tot ttg tat tot aat gag 624 Lell Ile Wall Ile Ala Phe Ala Asn Glu Ser Lell Tyr Ser Asn Glu 195 2OO 2O5

Cala ttg gala agc atg ggit citt ttic Cta Cala a.a.a. a CC aac at C at C 672 Glin Luell Glu Ser Met Gly Luell Phe Luell Glin Lys Thir Asn Ile Ile 21 O 215 22O aga gat aat gaa gat ttg gt C gat ggt aga t cc tto tgg cc c aag 72 O Arg Asp Asn Glu Asp Lell Wall Asp Gly Arg Ser Phe Trp Pro Lys 225 23 O 235 24 O

at C tgg to a Cala tac gct cott cag ttg aag gac tto atg a.a.a. cott 768 Ile Trp Ser Glin Ala Pro Glin Luell Lys Asp Phe Met Lys Pro 245 250 255

aac gala Cala Ctg 999 ttg gac tgt at a aac CaC citc. gtC tta aac 816 Asn Glu Glin Lell Gly Lell Asp Cys Ile ASn His Lell Wall Luell Asn 26 O 265 27 O

ttg agt Cat gtt atc. gat gtg ttg act tat ttg gcc ggt at C CaC 864 Luell Ser His Wall Ile Asp Wall Luell Thir Lell Ala Gly Ile His 27s 28O 285

Cala to c act tto Cala titt gcc att coc Cala gtt atg gcc att 912 Glin Ser Thir Phe Glin Phe Cys Ala Ile Pro Glin Wall Met Ala Ile 29 O 295 3 OO gca acc ttg gct ttg gta tto aac aac cgt gaa gtg Cta Cat ggc aat 96.O Thir Luell Ala Lell Wall Phe Asn Asn Arg Glu Wall Lell His Gly Asn 3. OS 310 315 32O gta aag att cgt aag ggit act acc tgc tat tta att ttg a.a.a. to a agg 1008 Wall Lys Ile Arg Lys Gly Thir Thir Cys Tyr Luell Ile Lell Ser Arg 3.25 330 335 act ttg cgt ggc gtc gag att titt gac tat tac tta cgt. gat at C 1056 Thir Luell Arg Gly Cys Wall Glu Ile Phe Asp Lell Arg Asp Ile 34 O 345 35. O a.a.a. tot a.a.a. ttg gct gtg Cala gat CC a aat titc tta a.a.a. ttg aac att 1104 Ser Luell Ala Wall Glin Asp Pro Asn Phe Lell Luell Asn Ile US 7,556,937 B2 57

- Continued

355 360 365 caa at C to C aag atc gaa cag titt atg gala gala atg tac Cag gat aaa 152 Glin Ile Ser Lys Ile Glu Glin Phe Met Glu Glu Met Tyr Glin Asp Llys 37 O 375 38O tta cct colt aac gitg aag cca aat gala act coa att titc ttgaaa gtt 2OO Lieu Pro Pro Asn Val Llys Pro Asn Glu Thr Pro Ile Phe Leu Lys Val 385 390 395 4 OO aaa gala aga. tcc aga tac gat gat gala ttg gtt cca acc cala caa gala 248 Lys Glu Arg Ser Arg Tyr Asp Asp Glu Lieu Val Pro Thr Glin Glin Glu 4 OS 41O 415 gaa gag tac aag titc aat atg gtt tta tot atc atc titg to c gtt citt 296 Glu Glu Tyr Llys Phe Asn Met Val Lieu. Ser Ile Ile Lieu. Ser Val Lieu. 42O 425 43 O citt ggg ttt tat tat at a tac act tta cac aga gcg toga 335 Lieu. Gly Phe Tyr Tyr Ile Tyr Thr Lieu. His Arg Ala 435 44 O

<210 SEQ ID NO 10 <211 LENGTH: 444 &212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <4 OO SEQUENCE: 10 Met Gly Lys Lieu. Lieu Gln Lieu Ala Lieu. His Pro Val Glu Met Lys Ala 1. 5 1O 15 Ala Lieu Lys Lieu Lys Phe Cys Arg Thr Pro Lieu. Phe Ser Ile Tyr Asp 2O 25 3O Gln Ser Thr Ser Pro Tyr Lieu. Lieu. His Cys Phe Glu Lieu. Lieu. Asn Lieu. 35 4 O 45 Thir Ser Arg Ser Phe Ala Ala Val Ile Arg Glu Lieu. His Pro Glu Lieu. SO 55 6 O Arg Asn. Cys Val Thir Lieu. Phe Tyr Lieu. Ile Lieu. Arg Ala Lieu. Asp Thr 65 70 7s 8O Ile Glu Asp Asp Met Ser Ile Glu. His Asp Lieu Lys Ile Asp Lieu. Lieu. 85 90 95 Arg His Phe His Glu Lys Lieu. Lieu. Lieu. Thir Lys Trp Ser Phe Asp Gly 1OO 105 11 O Asn Ala Pro Asp Wall Lys Asp Arg Ala Val Lieu. Thir Asp Phe Glu Ser 115 12 O 125 Ile Lieu. Ile Glu Phe His Llys Lieu Lys Pro Glu Tyr Glin Glu Val Ile 13 O 135 14 O Lys Glu Ile Thr Glu Lys Met Gly Asn Gly Met Ala Asp Tyr Ile Lieu. 145 150 155 160 Asp Glu Asn Tyr Asn Lieu. Asn Gly Lieu. Glin Thr Val His Asp Tyr Asp 1.65 17O 17s Val Tyr Cys His Tyr Val Ala Gly Lieu Val Gly Asp Gly Lieu. Thir Arg 18O 185 19 O Lieu. Ile Val Ile Ala Lys Phe Ala Asn. Glu Ser Lieu. Tyr Ser Asn. Glu 195 2OO 2O5 Gln Leu Tyr Glu Ser Met Gly Lieu Phe Leu Gln Lys Thr Asn Ile Ile 21 O 215 22O Arg Asp Tyr Asn. Glu Asp Lieu Val Asp Gly Arg Ser Phe Trp Pro Llys 225 23 O 235 24 O Glu Ile Trp Ser Glin Tyr Ala Pro Gln Leu Lys Asp Phe Met Llys Pro 245 250 255 Glu Asn. Glu Glin Lieu. Gly Lieu. Asp Cys Ile Asn His Lieu Val Lieu. Asn US 7,556,937 B2 59

- Continued

26 O 265 27 O Ala Lieu. Ser His Val Ile Asp Val Lieu. Thir Tyr Lieu Ala Gly Ile His 27s 28O 285 Glu Glin Ser Thr Phe Glin Phe Cys Ala Ile Pro Glin Val Met Ala Ile 29 O 295 3 OO Ala Thr Lieu Ala Lieu Val Phe Asn. Asn Arg Glu Val Lieu. His Gly Asn 3. OS 310 315 32O Val Lys Ile Arg Lys Gly. Thir Thr Cys Tyr Lieu. Ile Lieu Lys Ser Arg 3.25 330 335 Thir Lieu. Arg Gly Cys Val Glu Ile Phe Asp Tyr Tyr Lieu. Arg Asp Ile 34 O 345 35. O Llys Ser Lys Lieu Ala Val Glin Asp Pro Asn. Phe Lieu Lys Lieu. Asn. Ile 355 360 365 Glin Ile Ser Lys Ile Glu Glin Phe Met Glu Glu Met Tyr Glin Asp Llys 37 O 375 38O Lieu Pro Pro Asn Val Llys Pro Asn Glu Thr Pro Ile Phe Leu Lys Val 385 390 395 4 OO Lys Glu Arg Ser Arg Tyr Asp Asp Glu Lieu Val Pro Thr Glin Glin Glu 4 OS 41O 415 Glu Glu Tyr Llys Phe Asn Met Val Lieu. Ser Ile Ile Lieu. Ser Val Lieu. 42O 425 43 O Lieu. Gly Phe Tyr Tyr Ile Tyr Thr Lieu. His Arg Ala 435 44 O

<210 SEQ ID NO 11 <211 LENGTH: 35 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Description of synthetic sequence: AtHT-5' &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1) ... (35) &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 11 Ctgcggcc.gc at catggacc aattggtgaa aactg

<210 SEQ ID NO 12 <211 LENGTH: 32 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Description of synthetic sequence: AtHT-3' &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1) ... (32) &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 12 aact cagag acacatggtg Ctgttgttgct tc

<210 SEQ ID NO 13 <211 LENGTH: 60 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Description of synthetic sequence: ERG5 - Crelox-5" &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1) . . (60) &223> OTHER INFORMATION: Primer US 7,556,937 B2 61 62

- Continued

<4 OO SEQUENCE: 13 atgagttctg. tcgcagaaaa tataatacaa catgccactic ccagotgaag citt cqtacgc 6 O

SEQ ID NO 14 LENGTH: 62 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description of synthetic sequence: ERG5 - Crelox-3' FEATURE: NAMEAKEY: misc feature LOCATION: (1) . . (62) OTHER INFORMATION: Primer

SEQUENCE: 14 ttatt cqaag acttct coag taattggg to tct citttittg gcataggcca citagtggat.c 6 O tg 62

We claim: 25 6. The method as claimed in claim 5, wherein the nucleic 1. A method for the production of ergosta-5,7-dienol com acid construct comprises a promoter which, in the organism, prising culturing a genetically modified yeast organism, is subject to reduced regulation in comparison with the wild type promoter. wherein the genetic modification reduces the A22-desaturase 7. The method as claimed in claim 6, wherein the nucleic activity consisting of the enzymatic activity of A22-desatu acid encoding an HMG-CoA reductase is a nucleic acid rase having the amino acid sequence of SEQID.NO: 2 and 30 whose expression in the organism is Subject to reduced regu increases the HMG-CoA reductase activity consisting of the lation in comparison with the homologous, orthologous enzymatic activity of HMG-CoA reductase having the amino nucleic acid. acid sequence of SEQ ID.NO: 4 and increases squalene 8. The method as claimed in claim 7, wherein the nucleic epoxidase activity consisting of the enzymatic activity of acid encoding an HMG-CoA reductase is a nucleic acid squalene epoxidase having the amino acid sequence of SEQ 35 which encodes the catalytic region of HMG-CoA reductase. ID.NO: 8 in comparison with the wild type. 9. The method as claimed in claim 8, wherein the nucleic 2. The method as claimed in claim 1, wherein, in order to acids introduced are nucleic acids encoding proteins compris reduce the A22-desaturase activity, the gene expression of a ing the amino acid sequence SEQ. ID. NO. 4. nucleic acid encoding a A22-desaturase is reduced in com 40 10. The method as claimed in claim 9, wherein a nucleic parison with the wild type organism. acid comprising the sequence SEQ. ID. NO. 3 is introduced. 11. The method as claimed in claim 1, wherein, in order to 3. The method as claimed in claim 2, wherein an organism increase the squalene epoxidase activity, the gene expression without a functional A22-desaturase gene is used. of a nucleic acid encoding a squalene epoxidase is increased 4. The method as claimed in claim 1, wherein, in order to in comparison with the wild type organism. increase the HMG-CoA reductase activity, the gene expres 45 12. The method as claimed in claim 11, wherein, in order to sion of a nucleic acid encoding an HMG-CoA reductase is increase gene expression, one or more nucleic acids encoding increased in comparison with the wild type organism. a squalene epoxidase are introduced into the organism. 5. The method as claimed in claim 4, wherein, in order to 13. The method as claimed inclaim 12, wherein the nucleic increase gene expression, a nucleic acid construct comprising 50 acids introduced are nucleic acids encoding proteins compris a nucleic acid encoding an HMG-CoA reductase is intro ing the amino acid sequence SEQ. ID. NO. 8. duced into the organism and whose expression in the organ 14. The method as claimed in claim 13, wherein a nucleic ism is subject to reduced regulation in comparison with the acid comprising the sequence SEQ. ID. NO. 7 is introduced. wild type organism. k k k k k