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 for the production of isobutanol comprising:
providing a recombinant yeast host cell comprising genes encoding an engineered isobutanol biosynthetic pathway comprising the following substrate to product conversions:
i) pyruvate to acetolactate catalyzed by acetolactate synthase,
ii) acetolactate to 2,3-dihydroxyisovalerate catalyzed by acetohydroxy acid isomeroreductase,
iii) 2,3-dihydroxyisovalerate to a-ketoisovalerate catalyzed by acetohydroxy acid dehydratase,
iv) α-ketoisovalerate to isobutyryl-CoA catalyzed by branched-chain keto acid dehydrogenase,
v) isobutyryl-CoA to isobutyraldehyde catalyzed by acylating aldehyde dehydrogenase,
vi) isobutyraldehyde to isobutanol catalyzed by branched-chain alcohol dehydrogenase;
wherein the substrate to produce conversions of i)-v) are encoded by genes that are heterologous to the recombinant yeast host cell;
contacting the recombinant yeast host cell with a fermentable carbon in a fermentation medium substrate under conditions whereby isobutanol is produced;
recovering the isobutanol; and
removing solids from the fermentation medium.
2. The method of claim 1 , wherein recovering is by distillation, liquid-liquid extraction, membrane-based separation, adsorption, decantation, pervaporation, or combinations thereof.
3. The method of claim 1 , wherein the recombinant yeast host cell further comprises an inactivated gene thereby reducing yield loss from competing pathways for carbon flow.
4. The method of claim 1 , wherein the acetolactate synthase is from Bacillus subtilis, Klebsiella pneumoniae , or Lactococcus lactis.
5. The method of claim 1 , wherein the acetohydroxy acid isomeroreductase is from Escherichia coli, Saccharomyces cerevisiae, Methanococcus maripaludis , or Bacillus subtilis.
6. The method of claim 1 , wherein the acetohydroxy acid dehydratase is from Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae , or Methanococcus maripaludis.
7. The method of claim 1 , wherein the branched-chain alcohol dehydrogenase is from Saccharomyces cerevisiae, Escherichia coli , or Clostridium acetobutylicum.
8. The method of claim 1 , wherein the branched-chain keto acid dehydrogenase is from Bacillus subtilis or Pseudomonas putida.
9. The method of claim 1 , wherein the acylating aldehyde dehydrogenase is from Clostridium beijerinckii, Clostridium acetobutylicum, Pseudomonas putida , or Thermus thermophilus.
10. The method of claim 1 , further comprising blending the isobutanol with a fossil fuel to make a fuel or fuel additive.
11. The method of claim 1 , wherein the isobutanol is a chemical feedstock.
12. The method of claim 1 , wherein the removing is by centrifugation, filtration, or decantation.
13. The method of claim 1 , wherein the removing occurs before the recovering.