Engineering microorganisms to increase ethanol production by metabolic redirection

Patent metadata

Field	Value
Publication number	US-9803221-B2
Application number	US-201214348231-A
Country	US
Kind code	B2
Filing date	Sep 28, 2012
Priority date	Sep 30, 2011
Publication date	Oct 31, 2017
Grant date	Oct 31, 2017

Abstract

The present invention provides for the manipulation of carbon flux in a recombinant host cell to increase the formation of desirable products. The invention relates to cellulose-digesting organisms that have been genetically modified to allow the production of ethanol at a high yield by redirecting carbon flux at key steps of central metabolism.

First claim

What is claimed is:

1. A recombinant prokaryotic microorganism comprising:
(a) a heterologous nucleic acid encoding a pyruvate kinase (EC 2.7.1.40);
(b) a genetic modification that leads to the inactivation of malic enzyme (EC 1.1.1.38, EC 1.1.1.39, or EC 1.1.1.40) wherein the inactivation results from a deletion of one or more nucleic acids of an endogenous gene encoding malic enzyme or an insertion of one or more nucleic acids into an endogenous gene encoding malic enzyme; and
(c) one or more genetic modifications that leads to the inactivation of one or more enzymes selected from the group consisting of: lactate dehydrogenase (LDH) (EC 1.1.1.27 or EC 1.1.1.28); phosphotransacetylase (PTA) (EC 2.3.1.8); and acetate kinase (ACK) (EC 2.7.2.1), wherein the inactivation results from a deletion of one or more nucleic acids of an endogenous gene encoding the one or more enzymes or an insertion of one or more nucleic acids into the endogenous gene encoding the one or more enzymes.

2. The recombinant prokaryotic microorganism of claim 1 , wherein the pyruvate kinase is from a microorganism of the genus Thermoanaerobacterium.

3. The recombinant prokaryotic microorganism of claim 1 , wherein the pyruvate kinase is from the bacterium Thermoanaerobacteriuin saccharolyticum.

4. The recombinant prokaryotic microorganism of claim 1 , further comprising:
(d) a heterologous nucleic acid encoding a pyruvate formate lyase (PFL) (EC 2.3.1.54); and
(e) a heterologous nucleic acid encoding a PFL-activating enzyme (EC 1.97.1.4).

5. The recombinant prokaryotic microorganism of claim 4 , further comprising:
(f) a genetic modification that leads to the inactivation of the enzymes pyruvate oxidoreductase (EC 1.2.7.1) or NADH-dependent reduced ferredoxin:NADP+oxidoreductase, wherein the inactivation results from the deletion of one or more nucleic acids of an endogenous gene encoding the enzyme or the insertion of one or more nucleic acids into the endogenous gene encoding the enzyme.

6. The recombinant prokaryotic microorganism of claim 4 , further comprising:
(g) a heterologous nucleic acid encoding formate dehydrogenase (EC 1.2.1.43 (NAD+-specific) or EC 1.2.1.2 (NADP+-specific).

7. The recombinant prokaryotic microorganism of claim 1 , wherein the microorganism is a thermophilic bacterium.

8. The recombinant prokaryotic microorganism of claim 7 , wherein the microorganism is a cellulose-digesting bacterium.

9. The recombinant prokaryotic microorganism of claim 8 , wherein the microorganism is in the genus Clostridium.

10. The recombinant prokaryotic microorganism of claim 9 , wherein the microorganism is the bacterium Clostridium thermocellum.

11. The recombinant prokaryotic microorganism of claim 1 , wherein the microorganism comprises an ethanol producing pathway comprising the following substrate to product conversions: a) phosphoenolpyruvate to pyruvate; b) pyruvate to acetyl-CoA; c) acetyl-CoA to acetaldehyde; and, d) acetaldehyde to ethanol.

12. The recombinant prokaryotic microorganism of claim 1 , wherein the microorganism produces ethanol at a higher yield than a microorganism lacking the genetic modifications.

13. The recombinant prokaryotic microorganism of claim 1 , wherein the microorganism further comprises a bifunctional acetaldehyde-alcohol dehydrogenase (EC 1.2.1.4, EC 1.2.1.10, EC 1.1.1.2or EC 1.1.1.1).

14. The recombinant prokaryotic microorganism of claim 13 , wherein the bifunctional acetaldehyde-alcohol dehydrogenase is AdhB or AdhE.

15. A composition comprising the recombinant prokaryotic microorganism of claim 1 having faster growth by culturing it repeatedly on a growth medium or in a continuous culture device.

16. A composition comprising the recombinant prokaryotic microorganism of claim 1 and a carbon containing feed stock.

17. The composition of claim 16 , wherein the feed stock is selected from the group consisting of woody biomass, grasses, sugar-processing residues, agricultural wastes, and any combination thereof.

18. The composition of claim 17 , wherein the feed stock comprises recycled wood pulp fiber, sawdust, hardwood, softwood, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, succulents, agave, cane bagasse, switchgrass, miscanthus, paper sludge, municipal waste or any combination thereof.

19. A co-culture comprising at least two microorganisms wherein (a) one of the microorganisms comprises the recombinant prokaryotic microorganism of claim 1 ; and, (b) one of the microorganisms is genetically distinct from (a).

20. The co-culture of claim 19 , wherein the genetically distinct microorganism is a yeast or bacterium.

21. The co-culture of claim 20 , wherein the genetically distinct microorganism is any organism from the genus Issatchenkia, Pichia, Clavispora, Candida, Hansenula, Kluyveromyces, Trichoderma, Thermoascus, Escherichia, Clostridium, Caldicellulosiruptor, Zymomonas, Thermoanaerobacter or Thermoanaerobacterium.

22. A method of producing ethanol comprising: (a) providing the recombinant prokaryotic microorganism of claim 1 ; (b) culturing the recombinant prokaryotic microorganism of claim 1 in the presence of a carbon containing feedstock for sufficient time to produce ethanol; and, optionally (c) extracting the ethanol.

Assignees

• Enchi Corp
• Dartmouth College

Inventors

• Deng Yu
• Olson Daniel G
• Van Dijken Johannes Pieter
• Shaw Iv Arthur J
• Argyros Aaron
• Barrett Trisha
• Caiazza Nicky
• Herring Christopher D
• Rogers Stephen R
• Agbogbo Frank

Classifications

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12P7/065 Primary

  with microorganisms other than yeasts · CPC title

• C12N9/1217

  Phosphotransferases with a carboxyl group as acceptor (2.7.2) · CPC title

• C12N9/1205

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title