Patent EA-004629-B1 (EA)
Title: YEAST STRAINS ELIMINATED OF ACETYL-CoA PREGNENOLONE ACETYL-TRANSFERASE ACTIVITY AND METHODS IN-VIVO OXIDATION OF STEROLS AND STEROIDS AND USES
Inventor: ACHSTETTER TILMAN
IPC: C12P33/00
Filed: 1999-02-04
Abstract: 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-Delta<5>-steroid dehydrogenase /Delta<5>-Delta<4> -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-Delta<4>-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-Delta<4>-steroid obtained is isolated. 23. A method according to claim 22 in which the steroid substrate is pregnenolone.