Fermentive production of four carbon alcohols
View Patent ↗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.
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.