Enhancing microbial metabolism of C5 organic carbon

• US 9,951,326 B2
• Filed: 07/13/2016
• Issued: 04/24/2018
• Est. Priority Date: 07/13/2015
• Status: Active Grant

• Current Assignee: Mara Renewables Corporation
• Original Assignee: Mara Renewables Corporation
• Inventors: Merkx-Jacques, Alexandra, Woodhall, David, Scaife, Mark, Armenta, Roberto E., Muise, Denise, Rasmussen, Holly, Benjamin, Jeremy
• Primary Examiner(s): Ketter, James S
• Application Number:
  US15/208,849
• Publication Number:
  US 20170015988A1
• Time in Patent Office: 650 Days
• Field of Search: None
• CPC Class Codes: C07K 14/38 from Aspergillus
• CPC Class Codes: C07K 14/40 from Candida
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C12N 1/10 Protozoa; Culture media therefor
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/92 Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12Y 207/01017 Xylulokinase (2.7.1.17)
• CPC Class Codes: C12Y 503/01005 Xylose isomerase (5.3.1.5)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Provided herein are recombinant microorganisms having two or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is an exogenous nucleic acid. Optionally, the recombinant microorganisms include at least one nucleic acid sequence encoding a xylulose kinase and/or at least one nucleic acid sequence encoding a xylose transporter. The provided recombinant microorganisms are capable of growing on xylose as a carbon source.

Claims

1. A recombinant Thraustochytrid microorganism comprising two or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the nucleic acid encoding xylose isomerase is an exogenous nucleic acid.

2. The recombinant Thraustochytrid microorganism of claim 1, further comprising at least one nucleic acid sequence encoding a xylulose kinase.

3. The recombinant Thraustochytrid microorganism of claim 2, further comprising at least one nucleic acid sequence encoding a xylose transporter.

4. The recombinant Thraustochytrid microorganism of claim 1, wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the exogenous nucleic acid sequence encoding xylose isomerase.

5. The recombinant Thraustochytrid microorganism of claim 1, wherein the nucleic acid sequence encoding the xylose isomerase is at least 90% identical to SEQ ID NO:

2.

6. The recombinant Thraustochytrid microorganism of claim 2, wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylulose kinase.

7. The recombinant Thraustochytrid microorganism of claim 2, wherein the nucleic acid sequence encoding the xylulose kinase is at least 90% identical to SEQ ID NO:

17.

8. The recombinant Thraustochytrid microorganism of claim 3, wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylose transporter.

9. The recombinant Thraustochytrid microorganism of claim 8, wherein the xylose transporter is GXS1 from Candida intermedia.

10. The recombinant Thraustochytrid microorganism of claim 8, wherein the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:

23.

11. The recombinant Thraustochytrid microorganism of claim 3, wherein the recombinant microorganism has increased xylose transport activity as compared to a non-recombinant control microorganism, increased xylose isomerase activity as compared to a non-recombinant control microorganism, increased xylulose kinase activity as compared to a non-recombinant control microorganism, or any combination thereof.

12. The recombinant Thraustochytrid microorganism of claim 3, wherein the recombinant microorganism grows with xylose as the sole carbon source.

13. The recombinant Thraustochytrid microorganism of claim 3, wherein the nucleic acid sequence encoding the xylose isomerase, the xylulose kinase and/or the xylose transporter is operably linked to a promoter.

14. The recombinant Thraustochytrid microorganism of claim 13, wherein the promoter is a tubulin promoter that is at least 80% identical to SEQ ID NO:

25 or SEQ ID NO;

26.

15. The recombinant Thraustochytrid microorganism of claim 3, wherein the nucleic acid sequence encoding the xylose isomerase, the xylulose kinase and/or the xylose transporter comprises a terminator.

16. The recombinant Thraustochytrid microorganism of claim 15, wherein the terminator is a tubulin terminator that is at least 80% identical to SEQ ID NO:

27, SEQ ID NO;

28, or SEQ ID NO;

30.

17. The recombinant Thraustochytrid microorganism of claim 1, wherein the microorganism is ONC-T18.

18. A method of making a recombinant xylose-metabolizing Thraustochytrid microorganism comprising:

providing one or more nucleic acid constructs comprising a nucleic acid sequence encoding a xylose isomerase, a nucleic acid sequence encoding a xylulose kinase and a nucleic acid sequence encoding a xylose transporter;

transforming the Thraustochytrid microorganism with the one or more nucleic acid constructs; and

isolating recombinant Thraustochytrid microorganisms comprising at least two or more copies of the nucleic acid sequences encoding the xylose isomerase.

19. The method of claim 18, further comprising isolating recombinant Thraustochytrid microorganisms comprising at least one copy of the nucleic acid sequence encoding the xylulose kinase.

20. The method of claim 19, isolating recombinant Thraustochytrid microorganisms comprising at least one copy of the xylose transporter.

21. The method of claim 18, wherein the providing comprises providing a first nucleic acid construct comprising a nucleic acid sequence encoding a xylose isomerase, a second nucleic acid construct comprising a nucleic acid sequence encoding a xylulose kinase and a third nucleic acid construct comprising a nucleic acid sequence encoding a xylose transporter.

22. The method of claim 21, wherein the first, second, and/or third nucleic acid construct comprises a promoter, a selectable marker, a nucleic acid sequence encoding a 2A peptide, the nucleic acid sequence encoding the xylose isomerase, and a terminator.

23. The method of claim 22, wherein the promoter is a tubulin promoter that is at least 80% identical to SEQ ID NO:

25 or SEQ ID NO;

26.

24. The method of claim 22, wherein the terminator is a tubulin terminator that is at least 80% identical to SEQ ID NO:

27, SEQ ID NO;

28, or SEQ ID NO;

30.

25. The method of claim 18, wherein the isolated recombinant Thraustochytrid microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the exogenous nucleic acid sequence encoding xylose isomerase.

26. The method of claim 18, wherein the nucleic acid sequence encoding the xylose isomerase is at least 90% identical to SEQ ID NO:

2.

27. The method of claim 18, wherein the isolated recombinant Thraustochytrid microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylulose kinase.

28. The method of claim 18, wherein the nucleic acid sequence encoding the xylulose kinase is at least 90% identical to SEQ ID NO:

17.

29. The method of claim 18, wherein the isolated recombinant Thraustochytrid microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylose transporter.

30. The method of claim 29, wherein the xylose transporter is GXS1 from Candida intermedia.

31. The method of claim 29, wherein the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:

23.

32. The method of claim 18, wherein the isolated recombinant Thraustochytrid microorganism has increased xylose transport activity as compared to a control non-recombinant microorganism, increased xylose isomerase activity as compared to a control non-recombinant microorganism, increased xylulose kinase activity as compared to a control non-recombinant microorganism, or a combination thereof.

33. The method of claim 18, wherein the isolated recombinant Thraustochytrid microorganism grows with xylose as the sole carbon source.

34. The method of claim 18, wherein the Thraustochytrid microorganism is ONC-T18.