Fermentive Production of Isobutanol Using Highly Effective Ketol-Acid Reductoisomerase Enzymes
Ketol-acid reductoisomerase enzymes have been identified that provide high effectiveness in vivo as a step in an isobutanol biosynthetic pathway in bacteria and in yeast. These KARIs are members of a clade identified through molecular phylogenetic analysis called the SLSL Clade.
1 - 18 . (canceled)
19 . A recombinant yeast cell comprising at least one nucleic acid molecule encoding a polypeptide having ketol-acid reductoisomerase (KARI) activity wherein the polypeptide having KARI activity has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28, 36, 38, 40, 56, 60, or 68 and wherein the yeast cell comprises:
(i) at least one inactivated pyruvate decarboxylase (PDC) gene,
(ii) a modification to reduce glycerol-3-phosphate dehydrogenase activity, and
(iii) a heterologous polynucleotide encoding a polypeptide with phosphoketolase activity.
20 . The recombinant yeast cell of claim 19 , wherein the inactivated pyruvate decarboxylase (PDC) gene is PDC1, PDC5, or PDC6.
21 . The recombinant yeast cell of claim 19 , wherein the recombinant yeast cell further comprises at least one deletion, mutation, or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis.
22 . The recombinant yeast cell of claim 19 , wherein the yeast cell expresses an engineered isobutanol biosynthetic pathway.
23 . The recombinant yeast cell of claim 22 , wherein the engineered isobutanol biosynthetic pathway comprises the following substrate to product conversions:
(i) pyruvate to acetolactate catalyzed by acetolactate synthase,
(ii) 2,3-dihydroxyisovalerate to a-ketoisovalerate catalyzed by dihydroxy-acid dehydratase, and
(iii) α-ketoisovalerate to isobutyraldehyde catalyzed by branched-chain a-keto acid decarboxylase.
24 . The recombinant yeast cell of claim 23 , wherein the acetolactate synthase has an EC number 1.1.1.86.
25 . The recombinant yeast cell of claim 23 , wherein the dihydroxy-acid dehydratase has an EC number 4.2.1.9.
26 . The recombinant yeast cell of claim 23 , wherein the branched-chain a-keto acid decarboxylase has an EC number 4.1.1.72.
27 . The recombinant yeast cell of claim 23 , wherein the acetolactate synthase has an amino acid sequence selected from the group consisting of SEQ ID NO: 70, 73, 75, 77, 79, 81, 83, 85, and 87.
28 . The recombinant yeast cell of claim 23 , wherein the dihydroxy-acid dehydratase has an amino acid sequence selected from the group consisting of SEQ ID NO: 95, 97, and 99.
29 . The recombinant yeast cell of claim 23 , wherein the branched-chain a-keto acid decarboxylase has an amino acid sequence selected from the group consisting of SEQ ID NO: 101 and 102.
30 . The recombinant yeast cell of claim 19 , the yeast cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia , and Pichia.
31 . A method for the production of isobutanol comprising:
a) providing a recombinant yeast cell comprising an isobutanol biosynthetic pathway comprising at least one nucleic acid molecule encoding a polypeptide having ketol-acid reductoisomerase activity, wherein the polypeptide having KARI activity has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28, 36, 38, 40, 56, 60, or 68;
b) growing the recombinant yeast cell of step (a) under conditions wherein isobutanol is produced.
32 . The method of claim 31 , wherein the isobutanol is recovered.
33 . The method of claim 32 , wherein the isobutanol is recovered by distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.