Biofuel production by recombinant microorganisms
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.
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.