Glycerol 3-phosphate dehydrogenase for butanol production

• US 9,944,954 B2
• Filed: 08/04/2016
• Issued: 04/17/2018
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax Advanced Biofuels LLC (International Flavors & Fragrances Incorporated)
• Inventors: Bhalla, Ritu, Chotani, Gopal K., Dauner, Michael, Nelson, Mark J., O'Keefe, Daniel P., Peres, Caroline M., Prasad, Jahnavi Chandra, Tomb, Jean-Francois
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US15/228,770
• Publication Number:
  US 20160376613A1
• Time in Patent Office: 621 Days
• Field of Search: 435160, 435190, 4352523, 43525231, 43525232, 43525233, 43525234, 43525411, 4352542, 43525421, 43525422, 536 231, 536 232, 530350
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12Y 101/01008 Glycerol-3-phosphate dehydrogenase (NAD+) (1.1.1.8)
• CPC Class Codes: C12Y 101/01094 Glycerol-3-phosphate dehydrogenase (NAD(P)+)(1.1.1.94)
• CPC Class Codes: C12Y 101/05003 Glycerol-3-phosphate dehydrogenase (1.1.5.3)
• CPC Class Codes: C12Y 102/01012 Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Provided herein are glycerol-3-phosphate dehydrogenase (GPD) enzymes with increased K_M for NADH and GPD enzymes with substantially the same affinity for NADH and NADPH and/or are feedback inhibited by glycerol-3-phosphate. Also provided herein are recombinant microorganisms comprising a heterologous gene encoding GPD and a deletion or disruption in an endogenous gene encoding GPD. Also provided are recombinant microorganisms comprising a heterologous gene encoding GPD and a butanol biosynthetic pathway. Further provided are methods of producing butanol comprising providing the recombinant microorganisms described herein and contacting the recombinant microorganism with at least one fermentable carbon substrate under conditions wherein butanol is produced.

Claims

1. A method for the production of isobutanol comprising:

(a) providing a recombinant microorganism comprising

i. an engineered isobutanol biosynthetic pathway,

ii. a deletion or disruption in an endogenous gene encoding GPD1, and

iii. an engineered glycerol-3-phosphate dehydrogenase (GPD) enzyme wherein the GPD has at least 90% identity to SEQ ID NO;

195 and comprises at least one substitution at a residue corresponding to position 44, 45, 73, or 129 of SEQ ID NO;

195, and

wherein the engineered GPD has a higher K_M for NADH as compared to the K_M of the endogenous GPD of the microorganism; and

(a) contacting the recombinant microorganism with at least one fermentable carbon substrate under conditions wherein isobutanol is produced.

2. The method of claim 1, wherein the recombinant microorganism is grown under anaerobic conditions.

3. The method of claim 1, wherein the enzyme comprises at least one substitution corresponding to position 73 of SEQ ID NO:

195.

4. The method of claim 1, wherein the enzyme comprises at least one substitution corresponding to position 129 of SEQ ID NO:

195.

5. The method of claim 1, wherein the enzyme comprises a substitution corresponding to position 73 of SEQ ID NO:

195 and a substitution corresponding to position 129 of SEQ ID NO;

195.

6. The method of claim 1, wherein the isobutanol biosynthetic pathway comprises at least one gene encoding a polypeptide that performs at least one of the following substrate to product conversions:

(a) pyruvate to acetolactate, as catalyzed by acetolactate synthase;

(b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by acetohydroxy acid isomeroreductase;

(c) 2,3-dihydroxyisovalerate to α

-ketoisovalerate, as catalyzed by dihydroxyacid dehydratase;

(d) α

-ketoisovalerate to isobutyraldehyde, as catalyzed by a branched chain keto acid decarboxylase; and

(e) isobutyraldehyde to isobutanol, as catalyzed by branched-chain alcohol dehydrogenase.

7. The method of claim 1, wherein the microorganism is from a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Envinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluveromyces, Yarrowia, Pichia, Zygosaccharomyces, Debaryomyces, Candida, Brettanomyces, Pachysolen, Hansenula, Issatchenkia, Trichosporon Yamadazyma, and Saccharomyces.

8. The method of claim 7, wherein the microorganism is from the genus Saccharomyces.

9. The method of claim 1, wherein the microorganism comprises a ketol-acid reductoisomerase (KARI) that utilizes NADH.

10. The method of claim 1, further comprising recovering the isobutanol.

11. The method of claim 10, wherein the isobutanol is recovered by distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, pervaporation, or combinations thereof.