DECARBOXYLASE PROTEINS WITH HIGH KETO-ISOVALERATE DECARBOXYLASE ACTIVITY
The present invention relates to recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise at least one nucleic acid molecule encoding a polypeptide with keto-isovalerate decarboxylase (KIVD) activity, wherein said polypeptide is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a polypeptide selected from SEQ ID NOs: 1-214. Also provided are modified decarboxylases exhibiting an improved ability to utilize α-ketoisovalerate as a substrate in various beneficial enzymatic conversions.
1 .- 101 . (canceled)
102 . A recombinant microorganism comprising at least one nucleic acid molecule encoding a modified decarboxylase enzyme, wherein the modified decarboxylase enzyme has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) serine 286 of the L. lactis KIVD (SEQ ID NO: 197); (b) glutamine 377 of the L. lactis KIVD (SEQ ID NO: 197); (c) phenylalanine 381 of the L. lactis KIVD (SEQ ID NO: 197); (d) valine 461 of the L. lactis KIVD (SEQ ID NO: 197); (e) isoleucine 465 of the L. lactis KIVD (SEQ ID NO: 197); (f) methionine 538 of the L. lactis KIVD (SEQ ID NO: 197); and (g) phenylalanine 542 of the L. lactis KIVD (SEQ ID NO: 197).
103 . The recombinant microorganism of claim 102 , wherein the residue corresponding to position 286 of the L. lactis KIVD (SEQ ID NO: 197) is replaced with a residue selected from serine, threonine, asparagine, glycine, alanine, proline, glutamine, and aspartic acid.
104 . The recombinant microorganism of claim 102 , wherein the residue corresponding to position 377 of the L. lactis KIVD (SEQ ID NO: 197) is replaced with a residue selected from glutamine, serine, threonine, and asparagine.
105 . The recombinant microorganism of claim 102 , wherein the residue corresponding to position 381 of the L. lactis KIVD (SEQ ID NO: 197) is replaced with a residue selected from phenylalanine, alanine, isoleucine, leucine, methionine, tryptophan, tyrosine, and valine.
106 . The recombinant microorganism of claim 102 , wherein the residue corresponding to position 461 of the L. lactis KIVD (SEQ ID NO: 197) is replaced with a residue selected from valine, phenylalanine, alanine, isoleucine, leucine, methionine, tryptophan, and tyrosine.
107 . The recombinant microorganism of claim 102 , wherein the residue corresponding to position 465 of the L. lactis KIVD (SEQ ID NO: 197) is replaced with a residue selected from isoleucine, valine, phenylalanine, alanine, leucine, methionine, tryptophan, and tyrosine.
108 . The recombinant microorganism of claim 102 , wherein the residue corresponding to position 538 of the L. lactis KIVD (SEQ ID NO: 197) is replaced with a residue selected from methionine, isoleucine, leucine, valine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, and tyrosine.
109 . The recombinant microorganism of claim 102 , wherein the residue corresponding to position 542 of the L. lactis KIVD (SEQ ID NO: 197) is replaced with a residue selected from phenylalanine, isoleucine, leucine, methionine, valine, alanine, cysteine, glycine, proline, tryptophan, and tyrosine.
110 .- 122 . (canceled)
123 . The recombinant microorganism of claim 102 , wherein the modified decarboxylase enzyme is derived from a corresponding unmodified decarboxylase enzyme selected from SEQ ID NOs 1-214.
124 .- 137 . (canceled)
138 . The recombinant microorganism of claim 102 , wherein the modified decarboxylase enzyme is derived from a corresponding unmodified decarboxylase enzyme selected from the group consisting of PDC1 (SEQ ID NO: 241), PDC5 (SEQ ID NO: 242), and PDC6 (SEQ ID NO: 243) of Saccharomyces cerevisiae.
139 . (canceled)
140 . The recombinant microorganism of claim 102 , wherein the recombinant microorganism comprises a deletion or disruption of one or more endogenous pyruvate decarboxylase genes.
141 . The recombinant microorganism of claim 102 , wherein the recombinant microorganism comprises an isobutanol producing metabolic pathway comprising one or more isobutanol metabolic pathway enzymes selected from acetolactate synthase, ketol-acid reductoisomerase, dihydroxy acid dehydratase, and alcohol dehydrogenase.
142 . The recombinant microorganism of claim 141 , wherein the recombinant microorganism comprises a ketol-acid reductoisomerase and the ketol-acid reductoisomerase is an NADH-dependent ketol-acid reductoisomerase (NKR).
143 . The recombinant microorganism of claim 141 , wherein the recombinant microorganism comprises an alcohol dehydrogenase and the alcohol dehydrogenase is an NADH-dependent alcohol dehydrogenase.
144 . The recombinant microorganism of claim 102 , wherein the recombinant microorganism comprises a metabolic pathway for the production of a metabolite selected from 1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol.
145 . The recombinant microorganism of claim 102 , wherein the recombinant microorganism is a yeast microorganism.
146 . The recombinant microorganism of claim 102 , wherein the recombinant microorganism is a prokaryotic microorganism.
147 . A method of producing isobutanol, comprising:
(a) providing a recombinant microorganism of claim 141 ; and
(b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the isobutanol is produced.
148 .- 150 . (canceled)
151 . An isolated nucleic acid molecule encoding a modified decarboxylase enzyme, wherein the modified decarboxylase enzyme has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) serine 286 of the L. lactis KIVD (SEQ ID NO: 197); (b) glutamine 377 of the L. lactis KIVD (SEQ ID NO: 197); (c) phenylalanine 381 of the L. lactis KIVD (SEQ ID NO: 197); (d) valine 461 of the L. lactis KIVD (SEQ ID NO: 197); (e) isoleucine 465 of the L. lactis KIVD (SEQ ID NO: 197); (f) methionine 538 of the L. lactis KIVD (SEQ ID NO: 197); and (g) phenylalanine 542 of the L. lactis KIVD (SEQ ID NO: 197).
152 .- 154 . (canceled)