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1. WO2020112497 - VOIES DE BIOSYNTHÈSE MODIFIÉES POUR LA PRODUCTION DE 1,5-DIAMINOPENTANE PAR FERMENTATION

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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CLAIMS

What is claimed is:

1. An engineered microbial cell that expresses a non-native lysine decarboxylase, wherein the engineered microbial cell produces 1,5-diaminopentane.

2. The engineered microbial cell of claim 1, wherein the engineered microbial cell also expresses a non-native 1,5-diaminopentane transporter.

3. The engineered microbial cell of claim 1 or claim 2, wherein the engineered microbial cell expresses one or more additional enzyme(s) selected from an additional non-native lysine decarboxylase and/or an additional non-native 1,5-diaminopentane transporter.

4. The engineered microbial cell of claim 3, wherein the additional enzyme(s) are from a different organism than the corresponding enzyme in claim 1 or claim 2.

5. The engineered microbial cell of claim 3 or claim 4, wherein the additional enzyme(s) comprise(s) one or more additional copies of the corresponding enzyme in claim 1 or claim 2.

6. The engineered microbial cell of any of claims 1-5, wherein the engineered microbial cell comprises increased activity of one or more upstream lysine pathway enzyme(s), said increased activity being increased relative to a control cell.

7. The engineered microbial cell of any of claims 1-6, wherein the engineered microbial cell comprises increased activity of one or more enzyme(s) that increase the supply of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), said increased activity being increased relative to a control cell.

8. The engineered microbial cell of claim 7, wherein the one or more enzyme(s) that increase the supply of the reduced form of NADPH is selected from the group consisting of pentose phosphate pathway enzymes, NADP+-dependent

glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and NADP+-dependent glutamate dehydrogenase.

9. The engineered microbial cell of any one of claims 1-8, wherein the engineered microbial cell comprises reduced activity of one or more enzyme(s) that consume one or more lysine pathway precursors, said reduced activity being reduced relative to a control cell.

10. The engineered microbial cell of any one of claims 1-9, wherein the engineered microbial cell comprises reduced activity of a native lysine exporter, said reduced activity being reduced relative to a control cell.

11. The engineered microbial cell of claim 10, wherein the native lysine exporter is Corynebacterium glutamicum lysE or an ortholog thereof.

12. The engineered microbial cell of any one of claims 1-11, wherein the engineered microbial cell comprises reduced expression of the C. glutamicum NCgl0561 gene or an ortholog thereof, said reduced expression being reduced relative to a control cell.

13. The engineered microbial cell of any one of claims 1-12, wherein the engineered microbial cell comprises reduced expression of the C. glutamicum trpB gene or an ortholog thereof, said reduced expression being reduced relative to a control cell.

14. The engineered microbial cell of any one of claims 9-13, wherein the reduced activity is achieved by one or more means selected from the group consisting of gene deletion, gene disruption, altering regulation of a gene, and replacing a native promoter with a less active promoter.

15. An engineered microbial cell, wherein the engineered microbial cell comprises means for expressing a non-native lysine decarboxylase, and wherein the engineered microbial cell produces 1,5-diaminopentane.

16. The engineered microbial cell of claim 15, wherein the engineered microbial cell also comprises means for expressing a non-native 1,5-diaminopentane transporter.

17. The engineered microbial cell of claim 15 or claim 16, wherein the engineered microbial cell means for expressing one or more additional enzyme(s) selected from an additional non-native lysine decarboxylase and/or an additional non- native 1,5-diaminopentane transporter.

18. The engineered microbial cell of claim 17, wherein the additional enzyme(s) are from a different organism than the corresponding enzyme in claim 15 or claim 16.

19. The engineered microbial cell of any of claims 15-18 wherein the engineered microbial cell comprises means for increasing activity of one or more upstream lysine pathway enzyme(s), said activity being increased relative to a control cell.

20. The engineered microbial cell of any of claims 15-19, wherein the engineered microbial cell comprises means for increasing activity of one or more enzyme(s) that increase the NADPH supply, said activity being increased relative to a control cell.

21. The engineered microbial cell of claim 20, wherein the one or more enzyme(s) that increase the supply of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) is selected from the group consisting of pentose phosphate pathway enzymes, NADP+-dependent glyceraldehyde 3-phosphate

dehydrogenase (GAPDH), and NADP+-dependent glutamate dehydrogenase.

22. The engineered microbial cell of any one of claims 15-21, wherein the engineered microbial cell comprises means for reducing activity of one or more enzyme(s) that consume one or more lysine pathway precursors, said activity being reduced relative to a control cell.

23. The engineered microbial cell of any one of claims 15-22, wherein the engineered microbial cell comprises means for reducing activity of a native lysine exporter, said activity being reduced relative to a control cell.

24. The engineered microbial cell of claim 23, wherein the native lysine exporter is Corynebacterium glutamicum lysE or an ortholog thereof.

25. The engineered microbial cell of any one of claims 15-24, wherein the engineered microbial cell comprises means for reducing expression of the C.

glutamicum NCgl0561 gene or an ortholog thereof, said expression being reduced relative to a control cell.

26. The engineered microbial cell of any one of claims 15-25, wherein the engineered microbial cell comprises means for reducing expression of the C.

glutamicum trpB gene or an ortholog thereof, said expression being reduced relative to a control cell.

27. The engineered microbial cell of any one of claims 1-26, wherein the engineered microbial cell is a bacterial cell.

28. The engineered microbial cell of claim 27, wherein the bacterial cell is a cell of the genus Corynebacteria.

29. The engineered microbial cell of claim 28, wherein the bacterial cell is a cell of the species glutamicum.

30. The engineered microbial cell of claim 29, wherein the non-native lysine decarboxylase comprises a lysine decarboxylase having at least 70% amino acid sequence identity with a lysine decarboxylase selected from the group consisting of Escherichia coli, Vibrio cholerae, Candidatus Burkholderia crenata, butyrate-producing bacterium, and any combination thereof.

31. The engineered microbial cell of claim 30, wherein the cell comprises at least three different lysine decarboxylases.

32. The engineered microbial cell of claim 31, wherein the engineered microbial cell comprises three non-native lysine decarboxylases having at least 70% amino acid sequence identity with each of the lysine decarboxylases from Escherichia coli, Candidatus Burkholderia crenata, and butyrate-producing bacterium.

33. The engineered microbial cell of claim 32, wherein the engineered microbial cell additionally comprises a non-native lysine decarboxylase having at least 70% amino acid sequence identity with a lysine decarboxylase from a mine drainage

metagenome.

34. The engineered microbial cell of claim 33, wherein the lysine decarboxylases from Escherichia coli, Candidatus Burkholderia crenata, butyrate-producing bacterium, and the mine drainage metagenome comprise SEQ ID NOs:87, 97, 30, and 93.

35. The engineered microbial cell of claim 27, wherein the bacterial cell is a cell of the genus Bacillus.

36. The engineered microbial cell of claim 35, wherein the bacterial cell is a cell of the species subtilis.

37. The engineered microbial cell of claim 36, wherein the non-native lysine decarboxylase comprises a lysine decarboxylase having at least 70% amino acid sequence identity with a lysine decarboxylase selected from the group consisting of a Clostridium species, Staphylococcus aureus, and any combination thereof.

38. The engineered microbial cell of claim 37, wherein the cell comprises at least three different lysine decarboxylases.

39. The engineered microbial cell of claim 38, wherein the engineered microbial cell comprises three non-native lysine decarboxylases having at least 70% amino acid sequence identity with each of the lysine decarboxylases from Clostridium CAG:221, Clostridium CAG:288, and Staphylococcus aureus.

40. The engineered microbial cell of any one of claims 1-26, wherein the engineered microbial cell comprises a fungal cell.

41. The engineered microbial cell of claim 40, wherein the engineered microbial cell comprises a yeast cell.

42. The engineered microbial cell of claim 41, wherein the yeast cell is a cell of the genus Saccharomyces.

43. The engineered microbial cell of claim 42, wherein the yeast cell is a cell of the species cerevisiae.

44. The engineered microbial cell of any one of claims 1-43, wherein the non-native lysine decarboxylase comprises a lysine decarboxylase having at least 70% amino acid sequence identity with a lysine decarboxylase selected from the group consisting of Yersinia enterocolitica, Castellaniella detragans, Prochorococcus marinus, and any combination thereof.

45. The engineered microbial cell of claim 44, wherein the cell comprises at least three different lysine decarboxylases.

46. The engineered microbial cell of claim 45, wherein the engineered microbial cell comprises three non-native lysine decarboxylases having at least 70% amino acid sequence identity with each of the lysine decarboxylases from Yersinia enterocolitica, Castellaniella detragans, and Prochorococcus marinus.

47. The engineered microbial cell of any one of claims 1-46, wherein, when cultured, the engineered microbial cell produces 1,5-diaminopentane at a level at least 5 mg/L of culture medium.

48. The engineered microbial cell of claim 47, wherein, when cultured, the engineered microbial cell produces 1,5-diaminopentane at a level at least 5 gm/L of culture medium.

49. The engineered microbial cell of claim 48, wherein, when cultured, the engineered microbial cell produces 1,5-diaminopentane at a level at least 25 gm/L of culture medium.

50. A method of culturing engineered microbial cells according to any one of claims 1-49, the method comprising culturing the cells under conditions suitable for producing 1,5-diaminopentane.

51. The method of claim 50, wherein the method comprises fed-batch culture, with an initial glucose level in the range of 1-100 g/L, followed controlled sugar feeding.

52. The method of claim 50 or claim 51, wherein the fermentation substrate comprises glucose and a nitrogen source selected from the group consisting of urea, an ammonium salt, ammonia, and any combination thereof.

53. The method of any one of claims 50-52, wherein the culture is pH-controlled during culturing.

54. The method of any one of claims 50-53, wherein the culture is aerated during culturing.

55. The method of any one of claims 50-54, wherein the engineered microbial cells produce 1,5-diaminopentane at a level at least 5 mg/L of culture medium.

56. The method of any one of claims 50-55, wherein the method additionally comprises recovering 1,5-diaminopentane from the culture.

57. A method for preparing 1,5-diaminopentane using microbial cells engineered to produce 1,5-diaminopentane, the method comprising:

(a) expressing a non-native lysine decarboxylase in microbial cells; (b) cultivating the microbial cells in a suitable culture medium under conditions that permit the microbial cells to produce 1,5- diaminopentane, wherein the 1,5-diaminopentane is released into the culture medium; and

(c) isolating 1,5-diaminopentane from the culture medium.