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(12) United States Patent (10) Patent No.: US 7,556,937 B2 Kinkel Et Al 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 61,TH09GI ?,aun61-) U.S. Patent US 7,556,937 B2 Yhduy 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.
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