Ketol-Acid Reductoisomerases With Improved Performance Properties
The present invention relates to recombinant microorganisms comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI) or modified NADH-dependent variant thereof, wherein said KARI is at least about 60% identical to SEQ ID NO: 2. In various aspects of the invention, the recombinant microorganisms may comprise an isobutanol producing metabolic pathway and can be used in methods of making isobutanol.
1 . A recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 60% identical to SEQ ID NO: 2.
2 . The recombinant microorganism of claim 1 , wherein said KARL is derived from the genus Slackia.
3 . The recombinant microorganism of claim 2 , wherein said KARI is derived from Slackia exigua.
4 . The recombinant microorganism of claim 1 , wherein said KARI is encoded by SEQ ID NO: 1.
5 - 10 . (canceled)
11 . The recombinant microorganism of claim 1 , wherein said KARI is modified to be an NADH-dependent ketol acid reductoisomerase.
12 - 23 . (canceled)
24 . A mutant ketol-acid reductoisomerase (KARI) comprising one or more mutations or modifications at positions corresponding to amino acids selected from the group consisting of: (a) tyrosine 35 of the S. exigua KARI (SEQ ID NO: 2); (b) leucine 57 of the S. exigua KARI (SEQ ID NO: 2); (c) arginine 58 of the S. exigua KARI (SEQ ID NO: 2); (d) glycine 60 of the S. exigua KARI (SEQ ID NO: 2); (e) serine 61 of the S. exigua KARI (SEQ ID NO: 2); (f) serine 62 of the S. exigua KARI (SEQ ID NO: 2); (g) serine 63 of the S. exigua KARI (SEQ ID NO: 2); (h) isoleucine 95 of the S. exigua KARI (SEQ ID NO: 2); and (i) valine 99 of the S. exigua KARI (SEQ ID NO: 2).
25 . The mutant KARI of claim 24 , wherein said tyrosine 35 is replaced with a histidine residue.
26 . The mutant KARI of claim 24 , wherein said leucine 57 is replaced with a residue selected from serine and arginine.
27 . The mutant KARI of claim 24 , wherein said arginine 58 is replaced with a residue selected from proline, alanine, or arginine.
28 . The mutant KARI of claim 24 , wherein said glycine 60 is replaced with a valine residue.
29 . The mutant KARI of claim 24 , wherein said serine 61 is replaced with a residue selected from aspartic acid, glutamic acid, or cysteine.
30 . The mutant KARI of claim 24 , wherein said serine 62 is replaced with a residue selected from proline, alanine, or glutamic acid.
31 . The mutant KARI of claim 24 , wherein said serine 63 is replaced with a residue selected from aspartic acid, glutamic acid, histidine, isoleucine, methionine, arginine, or glutamine.
32 . The mutant KARI of claim 24 , wherein said isoleucine 95 is replaced with a residue selected from alanine, threonine, valine, and asparagine.
33 . The mutant KARI of claim 24 , wherein said valine 99 is replaced with a leucine residue.
34 . A recombinant microorganism comprising at least one nucleic acid molecule encoding a mutant KARI of claim 24 .
35 - 36 . (canceled)
37 . The recombinant microorganism of claim 1 , wherein said recombinant microorganism further expresses exogenous genes encoding an acetolactate synthase, a dihydroxy acid dehydratase, a keto-isovalerate decarboxylase, and an alcohol dehydrogenase, and wherein said recombinant microorganism produces isobutanol.
38 . (canceled)
39 . The recombinant microorganism of claim 37 , wherein said recombinant microorganism is a yeast microorganism.
40 . (canceled)
41 . A method of producing isobutanol, comprising:
(a) providing a recombinant microorganism of claim 37 ; and
(b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source, until the isobutanol is produced.
42 . A method of producing isobutanol, comprising:
(a) providing a recombinant microorganism of claim 39 ; and
(b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source, until the isobutanol is produced.