Fermentive production of four carbon alcohols

Patent metadata

Field	Value
Publication number	US-9297029-B2
Application number	US-201414585261-A
Country	US
Kind code	B2
Filing date	Dec 30, 2014
Priority date	Oct 26, 2005
Publication date	Mar 29, 2016
Grant date	Mar 29, 2016

Abstract

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway.

First claim

What is claimed is:

1. A method comprising:
a) providing a recombinant yeast host cell that expresses an engineered biosynthetic isobutanol pathway, wherein the engineered biosynthetic isobutanol pathway comprises an acetolactate synthase (ALS) enzyme, a ketol-acid reductoisomerase (KARI) enzyme, a dihydroxy-acid dehydratase (DHAD) enzyme, a branched chain keto acid decarboxylase enzyme (DC), and an alcohol dehydrogenase (ADH) enzyme, each of which is encoded by a heterologous gene that lacks a mitochondrial targeting sequence, and wherein the yeast host cell is provided in a growth phase;
b) growing the yeast host cells in fermentation medium, whereby isobutanol is bioproduced; and
c) recovering the bioproduced isobutanol;
wherein the yeast host cells are capable of producing 7- to 8-fold more isobutanol when grown on glucose under aerobic conditions compared to a recombinant yeast host cell lacking said engineered biosynthetic isobutanol pathway.

2. The method of claim 1 , further comprising blending the bioproduced isobutanol with a fossil fuel to make a fuel or fuel additive.

3. The method of claim 1 , wherein the bioproduced isobutanol is a chemical feedstock.

4. The method of claim 1 , wherein the ALS enzyme has an increased affinity for pyruvate over ketobuytrate.

5. The method of claim 1 , wherein the ALS enzyme is from Lactococcus lactis.

6. The method of claim 1 , wherein the ALS enzyme is from Klebsiella pneumonia.

7. The method of claim 1 , wherein the ALS enzyme is from Bacillus subtilis.

8. The method of claim 1 , wherein the ALS enzyme is capable of producing an activity of 8 units/mg as measured in a cell free extract when expressed on a pTrc99A plasmid in E. coli TOP10 cells grown at 37° C. for three hours following induction with 0.4 mM isopropyl β-D-1-thiogalactopyran (IPTG).

9. The method of claim 1 , wherein the DC enzyme is capable of producing an activity of 3.7 units/mg as measured in a cell free extract when expressed on a pTrc99A plasmid in E. coli TOP10 cells grown at 37° C. for three hours following induction with 0.4 mM isopropyl β-D-1-thiogalactopyran (IPTG).

10. The method of claim 1 , wherein the ALS enzyme has an amino acid sequence selected from SEQ ID NOs: 2, 178, or 180.

11. The method of claim 1 , wherein the KARL enzyme has an amino acid sequence selected from SEQ ID NOs: 4, 181, 183, or 185.

12. The method of claim 1 , wherein the DHAD enzyme has an amino acid sequence selected from SEQ ID NOs: 6, 186, 188, or 190.

13. The method of claim 1 , wherein the branched chain keto acid decarboxylase enzyme has an amino acid sequence selected from SEQ ID NOs: 8, 193, 195, or 197.

14. The method of claim 1 , wherein the ADH enzyme has an amino acid sequence selected from SEQ ID NOs: 10, 199, 201, 203, or 204.

15. The method of claim 1 , further comprising removing solids from the fermentation medium.

16. The method of claim 1 , wherein the recovering is by distillation, liquid-liquid extraction, adsorption, decantation, pervaporation, or combinations thereof.

17. The method of claim 15 , wherein the removing is by centrifugation, filtration, or decantation.

18. The method of claim 15 , wherein the removing occurs before the recovering.

Assignees

• Butamax Advanced Biofuels Llc

Inventors

• Donaldson Gail K
• Eliot Andrew C
• Flint Dennis
• Maggio-Hall Lori Ann
• Nagarajan Vasantha

Classifications

• C12N9/0016

  with NAD or NADP as acceptor (1.4.1) · CPC title

• C12Y102/04004

  3-Methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring) (1.2.4.4), i.e. branched-chain-alpha-ketoacid dehydrogenase · CPC title

• C12N15/746

  for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12N9/1022

  transferring aldehyde or ketonic groups (2.2) · CPC title