Enhancing microbial metabolism of C5 organic carbon

• US 10,662,418 B2
• Filed: 03/12/2018
• Issued: 05/26/2020
• 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): Brown, Mindy G
• Application Number:
  US15/918,786
• Publication Number:
  US 20180291363A1
• Time in Patent Office: 806 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: C12P 7/6431 Linoleic acids [18:2[n-6]]
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• 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 method of producing oil comprising

(a) providing recombinant Thraustochytrid microorganisms comprising two or more copies of a nucleic acid sequence encoding xylose isomerase and two or more copies of a nucleic acid sequence encoding a xylulose kinase, wherein the nucleic acid sequences encoding the xylose isomerase and xylulose kinase are is an exogenous nucleic acid sequences;

and

(b) culturing the microorganisms in a culture medium comprising a carbon source under conditions to produce the oil.

2. The method of claim 1, further comprising isolating the oil from the microorganisms and culture medium.

3. The method of claim 1, wherein the carbon source comprises xylose and glucose.

4. The method of claim 1, wherein the culture medium further comprises a nitrogen source.

5. The method of claim 1, recombinant Thraustochytrid microorganisms further comprises at least one nucleic acid sequence encoding a xylose transporter.

6. The method of claim 1, wherein the microorganism comprises more than two copies of the exogenous nucleic acid sequence encoding xylose isomerase.

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

2.

8. The method of claim 1, wherein the microorganism comprises more than two copies of the nucleic acid sequence encoding the xylulose kinase.

9. The method of claim 1, wherein the nucleic acid sequence encoding the xylulose kinase is at least 90% identical to SEQ ID NO:

17.

10. The method of claim 5, wherein the microorganism comprises more than two copies of the nucleic acid sequence encoding the xylose transporter.

11. The method of claim 5, wherein the xylose transporter is GXS1.

12. The method of claim 5, wherein the xylose transporter is AspTx.

13. The method of claim 5, wherein the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:

23.

14. The method of claim 5, wherein the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:

21.

15. The method of claim 1, 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.

16. The method of claim 1, wherein the recombinant Thraustochytrid microorganisms further comprises at least one nucleic acid sequence encoding a xylulose kinase, at least one nucleic acid sequence encoding a xylose transporter, or at least one nucleic acid sequence encoding a xylulose kinase and a xylose transporter.

17. The method of claim 1, wherein the recombinant Thraustochytrid further comprises at least one nucleic acid sequence encoding a xylose kinase and at least one nucleic acid sequence encoding a xylose transporter.

18. The method of claim 17, wherein the nucleic acid sequences encoding the xylose isomerase, the xylulose kinase and the xylose transporter are each operably linked to a promoter.

19. The method of claim 18, wherein the promoter is a tubulin promoter that is at least 80% identical to SEQ ID NO:

25 or SEQ ID NO;

26.

20. The method of claim 17, wherein the nucleic acid sequences encoding the xylose isomerase, the xylulose kinase and the xylose transporter each comprise a terminator.

21. The method of claim 20, 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.