Production of isobutanol in yeast mitochondria
View Patent ↗Yeast cells with modified expression of certain enzyme activities in the mitochondria are described for isobutanol production. Modifications described provide an isobutanol biosynthesis pathway in the yeast mitochondria.
1. A recombinant yeast cell comprising a heterologous gene encoding a mitochondrial localized polypeptide having α-keto acid decarboxylase activity and wherein the mitochondria is substantially devoid of a functional polypeptide having threonine deaminase activity and a functional polypeptide having isopropylmalate synthase activity.
2. The yeast cell of claim 1 wherein the cell produces isobutanol in the mitochondria.
3. The yeast cell of claim 2 wherein the cell further comprises genes encoding mitochondrial localized polypeptides having ketol-acid reductoisomerase activity and dihydroxy-acid dehydratase activity which are overexpressed.
4. The yeast cell of claim 3 wherein the mitochondria is further substantially devoid of a functional polypeptide having an enzyme activity selected from branched chain amino acid transaminase activity, pyruvate dehydrogenase activity, or a combination thereof.
5. The yeast cell of claim 1 selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.
6. The yeast cell of claim 1 wherein the α-keto acid decarboxylase activity is defined by the enzyme classification number EC 4.1.1.72.
7. The yeast cell of claim 4 wherein:
a) the threonine deaminase activity is defined by the enzyme classification number EC 4.3.1.19;
b) the isopropylmalate synthase activity is defined by the enzyme classification number EC 2.3.3.13;
c) the branched chain amino acid transaminase activity is defined by the enzyme classification number EC 2.6.1.42; and
d) the pyruvate dehydrogenase activity is defined by the enzyme classification number EC 1.2.4.1.
8. The yeast cell of claim 4 wherein the polypeptide having the pyruvate dehydrogenase activity is a multienzyme complex comprising proteins selected from the group consisting of: PDA1, PDA1, PDB1, LAT1, LPD1, and PDX1.
9. A method for making isobutanol comprising growing the yeast host cell of any of claim 3 , and 4 under conditions whereby isobutanol is produced.