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YEAST STRAINS ELIMINATED OF ACETYL-CoA PREGNENOLONE ACETYL-TRANSFERASE ACTIVITY AND METHODS IN-VIVO OXIDATION OF STEROLS AND STEROIDS AND USES
YEAST STRAINS ELIMINATED OF ACETYL-CoA PREGNENOLONE ACETYL-TRANSFERASE ACTIVITY AND METHODS IN-VIVO OXIDATION OF STEROLS AND STEROIDS AND USES
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机译:消除了乙酰辅酶A孕烯醇酮乙酰转移酶活性的酵母菌株以及体内甾醇和甾体氧化的方法及用途
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摘要
1. A modified yeast strain in which the acetyl-CoA pregnenolone acetyltransferase (APAT) activity is eliminated by obtaining the gene ATF2 gene or YGR 177c, or a homologue thereof, resulting in stabilization of 3beta-hydroxy steroids. 2. A strain according to claim 1, which is S. cerevisiae. 3. A strain according to claim 2 in which the ATF2 gene is altered by inserting a DNA sequence that has at least one nucleotide. 4. A strain according to claim 3 in which the ATF2 gene is altered by inserting URA3 selection gene. 5. A strain according to claim 3 in which the ATF2 gene is altered by inserting expression block TEF1prom/PGKterm. 6. A strain according to claim 4, which is S. Cerevisiae TGY156. 7. A strain according to claim 4, which is S. Cerevisiae TGY158. 8. A strain according to claim 4, which is S. Cerevisiae TGY186. 9. A strain according to any one of claims 1-5, further expressing at least one of the enzymes of the route for biosynthesis of hydrocortisone from cholesterol selected from among: - the side chain cleavage ensyme of cholesterol (P450SCC), -3beta-hydroxy-Delta5-steroid dehydrogenase /Delta5-Delta4 -steroid isomerase (3beta-HSD); and 17alpha-steroid-hydroxylase (P45017alpha). 10. A strain according to claim 9, further expressing 3beta-HSD. 11. A strain S. Cerevisiae transformed according to claim 4 and 10 designated TGY158/pTG10862. 12. A strain according to claim 9, further expressing P45017alpha. 13. A strain of S. cerevisiae transformed according to claim 5 and 12, designated TGY158/pTG10862. 14. A strain according to claim 9, further co-expressing 3beta-HSD and P45017alpha. 15. A strain of S. cerevisiae transformed according to claim 5 and 14, designated TGY186/pTG10417. 16. A method of in-vivo oxidation of sterol or steroid in which a transformed yeast strain according to one of the claims 9 to 15 is cultivated and oxidized endogenous sterol is isolated. 17. A method of in-vivo oxidation of sterol or steroid in which a transformed yeast strain according to one of the claims 9 to 15 is incubated with the exogenous sterol or steroid and the oxidized compound obtained is isolated. 18. A method according to claim 17 in which the substrate is a 3beta-hydroxysteroid and in which a transformed yeast strain according to claim 10 is used and the 3-oxo-Delta4-steroid obtained is isolated. 19. A method according to claim 18 in which the 3beta-hydroxysteroid is chosen between pregnenolone or 17alpha-hydroxypregnenolone. 20. A method according to claim 17 in which the substrate is a steroid and in which a transformed yeast strain according to claim 12 is used and the 17alpha hydroxyl steroid obtained is isolated. 21. A method according to claim 20 in which the steroid substrate is pregnenolone or progesterone. 22. A method according to claim 17 in which the substrate is 3beta hydroxyl steroid and in which a transformed yeast strain according to claim 12 is used and the 3-oxo-Delta4-steroid obtained is isolated. 23. A method according to claim 22 in which the steroid substrate is pregnenolone.
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