Biofuel production by recombinant microorganisms

Patent metadata

Field	Value
Publication number	US-9416378-B2
Application number	US-201514637336-A
Country	US
Kind code	B2
Filing date	Mar 3, 2015
Priority date	Feb 9, 2007
Publication date	Aug 16, 2016
Grant date	Aug 16, 2016

Abstract

Provided herein are metabolically-modified microorganisms useful for producing biofuels. More specifically, provided herein are methods of producing high alcohols including isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol from a suitable substrate.

First claim

What is claimed is:

1. A recombinant prokaryote comprising:
(a) an α-isopropylmalate synthase polypeptide having at least 90% identity to SEQ ID NO:50 and having α-isopropylmalate synthase activity;
(b) a β-isopropylmalate dehydrogenase polypeptide having at least 90% identity to SEQ ID NO:52 and having β-isopropylmalate dehydrogenase activity;
(c) an α-isopropylmalate isomerase polypeptide having at least 90% identity to SEQ ID NO:54;
(d) an α-isopropylmalate isomerase polypeptide having at least 90% identity to SEQ ID NO:56 and having α-isopropylmalate isomerase activity in combination with the α-isopropylmalate isomerase polypeptide of (c);
(e) a 2-keto-acid decarboxylase polypeptide having at least 90% identity to SEQ ID NO:28 and having 2-keto acid decarboxylase activity;
(f) a NADH-dependent alcohol dehydrogenase having at least 90% identity to SEQ ID NO:38;
(g) an acetohydroxy acid synthase polypeptide having at least 90% identity to SEQ ID NO:42;
(h) an acetohydroxy acid synthase polypeptide having at least 90% identity to SEQ ID NO:40 and having acetohydroxy acid synthase activity in combination with the acetohydroxy acid synthase polypeptide of (g);
(i) an acetohydroxy acid isomeroreductase polypeptide having at least 90% identity to SEQ ID NO:44 and having acetohydroxy acid isomeroreductase activity; and
(j) a dihydroxy-acid dehydratase polypeptide having at least 90% identity to SEQ ID NO:46 and having dihydroxy-acid dehydratase activity;
and wherein the recombinant prokaryote produces 3-methyl-1-butanol.

2. The recombinant prokaryote of claim 1 , wherein the recombinant prokaryote is E. coli.

3. The recombinant prokaryote of claim 2 , wherein the recombinant prokaryote has a disruption of an endogenous adhE gene, or the endogenous adhE gene and an endogenous IdhA gene.

4. The recombinant prokaryote of claim 3 , wherein the recombinant prokaryote produces less than about 240 mg/L of ethanol at about 112 h of culture.

5. The recombinant prokaryote of claim 1 , wherein said 2-keto-acid decarboxylase polypeptide is KivD of Lactococcus lactis or a homolog, or variant of KivD having at least 98% identity to SEQ ID NO:28 and having 2-keto-acid decarboxylase activity.

6. The recombinant prokaryote of claim 1 , wherein said NADH-dependent alcohol dehydrogenase has at least 98% identity to SEQ ID NO:38 and converts 3 methylbutyraldehyde to 3-methyl-1-butanol.

Assignees

• Univ California

Inventors

• Liao James C
• Atsumi Shota
• Smith Kevin M
• Shen Roa Pu Claire
• Cann Anthony F
• Connor Michael R

Classifications

• C12N9/1022

  transferring aldehyde or ketonic groups (2.2) · CPC title

• C12N9/1025

  Acyltransferases (2.3) · CPC title

• C12P7/16 Primary

  Butanols · CPC title

• C12Y101/01086

  Ketol-acid reductoisomerase (1.1.1.86) · CPC title

• C12Y202/01006

  Acetolactate synthase (2.2.1.6) · CPC title