Increased ethanol production by thermophilic microorganisms with deletion of individual hfs hydrogenase subunits

Patent metadata

Field	Value
Publication number	US-10619172-B2
Application number	US-201715656665-A
Country	US
Kind code	B2
Filing date	Jul 21, 2017
Priority date	Jul 21, 2016
Publication date	Apr 14, 2020
Grant date	Apr 14, 2020

Abstract

Disclosed are methods for engineering bacteria, for example, Thermoanaerobacterium saccharolyticum, that convert biomass to ethanol at high yield by deleting a single gene. Deletion of subunit A or subunit B of the hfs hydrogenase, but not deletion of subunit C or subunit D, results in an increase in ethanol yield.

First claim

What is claimed is:

1. A genetically engineered microorganism comprising a functional hfsC protein subunit and a functional hfsD protein subunit of hfs hydrogenase, wherein expression of hfsA and hfsB protein subunits of the hfs hydrogenase is disrupted,
wherein said microorganism is selected from the group consisting of Thermoanaerobacterium sp., Thermoanaerobacter mathranii and Clostridium thermocellum.

2. The microorganism of claim 1 , wherein said microorganism is selected from the group consisting of Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum , and Thermoanaerobacterium xylanolyticum.

3. The microorganism of claim 1 , wherein said disruption of the protein subunits hfsA and hfsB of the hfs hydrogenase in said microorganism is by targeted mutation.

4. The microorganism of claim 1 , wherein said microorganism has an ethanol yield greater than 80% of theoretical yield.

5. The microorganism of claim 1 , wherein the pta and ack genes in said microorganism are identical to the pta and ack genes in a wildtype strain of the same species.

6. The microorganism of claim 1 , wherein ldh the gene in the microorganism is identical to the ldh gene in a wildtype strain of the same species.

7. The microorganism of claim 1 , wherein amino acid sequence of said hfsA protein subunit is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

8. The microorganism of claim 1 , wherein amino acid sequence of said hfsB protein subunit is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

9. The microorganism of claim 1 , wherein said functional hfsC protein subunit has an amino acid sequence identical to that of wild-type hfsC protein subunit in said microorganism, and wherein said functional hfsD protein subunit has an amino acid sequence identical to that of wild-type hfsD protein subunit in said microorganism.

10. A method of producing ethanol from biomass, comprising contacting the biomass with the genetically engineered microorganism of claim 1 to produce ethanol.

11. The method of claim 10 , wherein the microorganism is co-cultured with Clostridium thermocellum to produce ethanol from the biomass, wherein the Clostridium thermocellum is genetically engineered.

12. A method of generating the genetically engineered microorganism of claim 1 , comprising disrupting expression of at least the hfsA and hfsB protein subunits of the hfs hydrogenase, said hfsB protein subunit having an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, and said hfsA protein subunit having an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

Assignees

• Dartmouth College

Inventors

• Eminoglu Aysenur
• Olson Daniel G
• Lynd Lee R

Classifications

• C12N9/0067

  acting on hydrogen as donor (1.12) · CPC title

• C12Y112/07002

  Ferredoxin hydrogenase (1.12.7.2) · CPC title

• C12P7/065 Primary

  with microorganisms other than yeasts · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title