DHAD Variants for Butanol Production

Patent metadata

Field	Value
Publication number	US-2017218405-A1
Application number	US-201715485735-A
Country	US
Kind code	A1
Filing date	Apr 12, 2017
Priority date	Dec 28, 2012
Publication date	Aug 3, 2017
Grant date	—

Abstract

Dihydroxy-acid dehydratase (DHAD) variants that display increased DHAD activity are disclosed. Such enzymes can result in increased production of compounds from DHAD requiring biosynthetic pathways. Also disclosed are isolated nucleic acids encoding the DHAD variants, recombinant host cells comprising the isolated nucleic acid molecules, and methods of producing butanol.

First claim

1 - 177 . (canceled)

178 . A method for producing isobutanol comprising:
(a) providing a recombinant host cell comprising an isolated polypeptide or fragment thereof having dihydroxy-acid dehydratase (DHAD) activity, wherein the polypeptide or fragment thereof comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 565, and wherein the amino acid sequence comprises a deletion of about 5 to about 20 amino acids from the C-terminal end of the amino acid sequence; (b) contacting the recombinant host cell with a fermentation medium under conditions whereby isobutanol is produced; and (c) optionally, recovering the isobutanol.

179 . The method of claim 178 , wherein the amino acid sequence further comprises one or more amino acid substitutions selected from P378A, P378G, P378V, P378I, P378L, G383S, G383A, G383V, G383L, G383I, I387V, I387M, I387L, I387G, I387A, L388I, L388V, L388A, and L388M.

180 . The method of claim 178 , wherein the polypeptide or fragment further comprises a polycysteine or polyhistidine tag.

181 . The method of claim 178 , wherein the polypeptide or fragment further comprises the amino acid sequence of SEQ ID NO: 589.

182 . The method of claim 179 , wherein the amino acid comprises the amino acid sequence of SEQ ID NO: 573.

183 . The method of claim 178 , wherein the polypeptide or fragment further comprises a polypeptide sequence selected from SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 728, SEQ ID NO: 729, SEQ ID NO: 730, SEQ ID NO: 731, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 736, SEQ ID NO: 737, SEQ ID NO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 747, and SEQ ID NO: 748.

184 . The method of claim 178 , wherein the amino acid sequence comprises a deletion of about 5 to about 15 amino acids from the C-terminal end of the amino acid sequence.

185 . The method of claim 178 , wherein the amino acid sequence comprises a deletion of 9 C-terminal amino acids.

186 . The method of claim 178 , wherein the recombinant host cell comprises an isobutanol biosynthetic pathway.

187 . The method of claim 186 , wherein the recombinant host cell is genetically modified to disrupt a gene encoding pyruvate decarboxylase (PDC).

188 . The method of claim 186 , wherein the recombinant host cell is genetically modified to disrupt a gene encoding glycerol-3-phosphate dehydrogenase (GPD2).

189 . The method of claim 178 , wherein the recombinant host cell is a yeast cell.

190 . The method of claim 189 , wherein the yeast cell comprises a disruption in one or more endogenous genes affecting iron-sulfur cluster biosynthesis selected from FRA2, GRX3, GRX4, and CCC1.

191 . The method of claim 178 , wherein the isobutanol is recovered by distillation, liquid-liquid extraction, adsorption, decantation, pervaporation, or combinations thereof.

192 . The method of claim 178 , wherein the isobutanol is recovered by contacting the fermentation medium with a water immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase.

193 . The method of claim 192 , wherein the extractant is selected from the group consisting of C 12 to C 22 fatty alcohols, C 12 to C 22 fatty acids, esters of C 12 to C 22 fatty acids, C 12 to C 22 fatty aldehydes, and mixtures thereof.

194 . The method of claim 178 , wherein the isobutanol is recovered by contacting the isobutanol with an organic acid and a catalyst capable of esterifying the isobutanol with the organic acid.

195 . The method of claim 178 , wherein solids are removed from the fermentation medium.

196 . The method of claim 195 , wherein the solids are removed from the fermentation medium by centrifugation, filtration, decantation, or combinations thereof.

197 . The method of claim 195 , wherein the solids are removed before the isobutanol is recovered.

Assignees

• Butamax Advanced Biofuels Llc

Inventors

• Maggio-Hall Lori Ann
• Paul Brian James
• Rothman Steven Cary
• Ye Rick W

Classifications

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12Y402/01009

  Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12P7/16 Primary

  Butanols · CPC title