Yeast strain and microbial method for production of pentacyclic triterpenes and/or triterpenoids

1. A modified yeast strain for production of pentacyclic triterpenoids, comprising:

i. at least one copy of a gene for encoding an oxidosqualene cyclase,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

ii. at least one copy of a gene for encoding a NADPH-cytochrome P450 reductase,

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 66, 68 or 69; and

iii. at least one copy of a gene for encoding a cytochrome P450 monooxygenase,

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 83, 84 or 88.

2. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 66; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 83.

3. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 66; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 83.

4. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 66; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 84.

5. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 66; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 84.

6. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 66; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 88.

7. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 66; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 88.

8. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 68; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 83.

9. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 68; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 83.

10. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 68; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 84.

11. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 68; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 84.

12. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 68; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 88.

13. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 68; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 88.

14. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 69; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 83.

15. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 69; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 83.

16. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 69; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 84.

17. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 69; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 84.

18. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 69; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 88.

19. The modified yeast strain according to claim 1 ,

wherein the oxidosqualene cyclase has an amino acid sequence of SEQ ID NO: 55;

wherein the NADPH-cytochrome P450 reductase has an amino acid sequence of SEQ ID NO: 69; and

wherein the cytochrome P450 monooxygenase has an amino acid sequence of SEQ ID NO: 88.

20. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulin with an intracellular concentration of more than 1 mg per gram of dry biomass.

21. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulin with an intracellular concentration of more than 3 mg per gram of dry biomass.

22. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulin with an intracellular concentration of more than 10 mg per gram of dry biomass.

23. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulin with a concentration of more than 50 mg per liter of culture medium.

24. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulin aldehyde with an intracellular concentration of more than 1 mg per gram of dry biomass.

25. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulin aldehyde with an intracellular concentration of more than 2 mg per gram of dry biomass.

26. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulin aldehyde with an intracellular concentration of more than 3 mg per gram of dry biomass.

27. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulin aldehyde with a concentration of more than 25 mg per liter of culture medium.

28. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulinic acid with an intracellular concentration of more than 1 mg per gram of dry biomass.

29. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulinic acid with an intracellular concentration of more than 2 mg per gram of dry biomass.

30. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulinic acid with an intracellular concentration of more than 5 mg per gram of dry biomass.

31. The modified yeast strain according to claim 1 , wherein the strain is capable of producing betulinic acid with a concentration of more than 25 mg per liter of culture medium.

32. The modified yeast strain according to claim 1 , wherein the yeast strain comprises a tHMG1 expression cassette.

33. The modified yeast strain according to claim 1 , wherein this strain is Saccharomyces cerevisiae.

34. The modified yeast strain according to claim 1 , wherein the strain is a Saccharomyces cerevisiae GEN.PK, CEN.PK111-61A or AH22tH3ura8 strain.

35. A method for producing the modified yeast strain according to claim 1 comprising:

a) providing a Saccharomyces cerevisiae strain,

b) transforming the strain with a vector comprising a gene for encoding an oxidosqualene cyclase, wherein the oxidosqualene cyclase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 55;

c) transforming the strain with a vector comprising a gene for encoding an NADPH-cytochrome P450 reductase, wherein the NADPH-cytochrome P450 reductase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 66, 68 or 69; and

d) transforming the strain with a vector comprising a gene for encoding a cytochrome P450 monooxygenase, wherein the cytochrome P450 monooxygenase has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 83, 84 or 88.

36. A method for producing a triterpene and/or triterpenoid, comprising cultivating the modified yeast strain according to claim 1 in a medium.