Methylbutanol as an advanced biofuel
This invention describes genes, metabolic pathways, microbial strains and methods to produce methyl butanol and other compounds of interest from renewable feedstocks.
1. A method for producing a methylbutyl ether product, comprising
culturing a recombinant microorganism in a culture medium containing a carbon source,
wherein said recombinant microorganism comprises at least one exogenous nucleic acid molecule, wherein said at least one exogenous nucleic acid molecule comprises a pyruvate decarboxylase or a pyruvate decarboxylase isoform derived from the genus Pichia and an alcohol dehydrogenase derived from the genus Saccharomyces;
further wherein said recombinant microorganism is a member of a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Pseudomonas, Bacillus, Klebsiella, Corynebacterium, Pichia , and Saccharomyces;
wherein the recombinant microorganism produces spent culture medium from the culture medium by metabolizing the carbon source to 2-methylbutanol;
recovering said 2-methylbutanol from the spent culture medium; and converting said 2-methylbutanol to a methylbutyl ether product comprising one or more of 1-(isopentyloxy)-3-methylbutane, 2-methyl-1-(2-methylbutoxy)butane, 1-(isopentyloxy)-2-methylbutane, 2-methyl-1-(tert-pentyloxy)butane, and 2-methyl-2-(tert-pentyloxy)butane.
2. The method of claim 1 , wherein the pyruvate decarboxylase has at least 90% amino acid sequence identity to a Pichia stipitis pyruvate decarboxylase PDC3-6 gene product (SEQ ID NO:53).
3. The method of claim 2 , wherein the alcohol dehydrogenase has at least 90% amino acid sequence identity to Saccharomyces cerevisiae ADH6 (SEQ ID NO:66) or Saccharomyces cerevisiae SFA1 (SEQ ID NO:76).
4. The method of claim 3 , wherein the alcohol dehydrogenase is Saccharomyces cerevisiae ADH6 (SEQ ID NO:66).
5. The method of claim 1 , wherein said recombinant microorganism is Escherichia coli, Pseudomonas putida, Bacillus subtilis, Corynebacterium glutamicum, Pichia stipitis, Pichia pastoris , or Saccharomyces cerevisiae.
6. The method of claim 5 , wherein said recombinant microorganism is Escherichia coli.
7. The method of claim 5 , wherein said recombinant microorganism is Corynebacterium glutamicum.
8. The method of claim 5 , wherein said recombinant microorganism is Saccharomyces cerevisiae.
9. The method of claim 1 , wherein said 2-methylbutanol is removed from said culture by gas stripping, fractional distillation, chromatography, pervaporation, adsorption, liquid-liquid extraction, or solid-liquid extraction.
10. The method of claim 1 , wherein the recovering step comprises extracting 2-methylbutanol using liquid-liquid extraction, wherein a solvent is used to continuously extract at least 2-methylbutanol from the spent culture medium.
11. The method of claim 10 , wherein the solvent is diisopropyl ether, heptane or isooctane.
12. The method of claim 11 , wherein the solvent is diisopropyl ether; and wherein at least 90% of 2-methylbutanol is extracted from the spent culture medium.
13. The method of claim 1 , wherein the conversion step of converting 2-methylbutanol to a methyl butyl ether product comprises treating the 2-ethylbutanol with an acid resulting in the formation of methyl butyl ether.
14. The method of claim 13 , wherein the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, chromic acid, or a sulfonic acid.
15. The method of claim 14 , wherein the acid is a methane sulfonic acid, an ethane sulfonic acid, a benzene sulfonic acid, a toluene sulfonic acid, trifluoromethyl sulfonic acid, or perfluoroalkane sulfonic acid.
16. The method of claim 14 , wherein the acid is trifluoromethanesulfonic acid.