DHAD variants and methods of screening
First Claim
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1. A method for the production of isobutanol comprising(a) providing a recombinant host cell that comprises(i) an isolated polypeptide having dihydroxy-acid dehydratase (DHAD) activity, wherein the polypeptide is at least 95% identical to a Streptococcus mutans DHAD of SEQ ID NO:
- 168 and comprises amino acid substitutions at position 33 and position 563, and(ii) an isobutanol biosynthetic pathway, wherein the isobutanol biosynthetic pathway comprises the following substrate to product conversions;
(1) pyruvate to acetolactate catalyzed by an acetolactate synthase,(2) acetolactate to 2,3-dihydroxyisovalerate catalyzed by a ketol-acid reductoisomerase,(3) 2,3-dihydroxyisovalerate to α
-ketoisovalerate catalyzed by DHAD,(4) α
-ketoisovalerate to isobutyraldehyde catalyzed by an α
-keto acid decarboxylase, and(5) isobutyraldehyde to isobutanol catalyzed by an alcohol dehydrogenase;
(b) culturing the recombinant host cell in a fermentation medium under suitable conditions to produce isobutanol; and
(c) recovering the isobutanol.
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Abstract
Methods of screening for dihydroxy-acid dehydratase (DHAD) variants that display increased DHAD activity are disclosed, along with DHAD variants identified by these methods. Such enzymes can result in increased production of compounds from DHAD requiring biosynthetic pathways. Also disclosed are isolated nucleic acids encoding the DHAD variants, recombinant host cells comprising the isolated nucleic acid molecules, and methods of producing butanol.
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14 Claims
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1. A method for the production of isobutanol comprising
(a) providing a recombinant host cell that comprises (i) an isolated polypeptide having dihydroxy-acid dehydratase (DHAD) activity, wherein the polypeptide is at least 95% identical to a Streptococcus mutans DHAD of SEQ ID NO: - 168 and comprises amino acid substitutions at position 33 and position 563, and
(ii) an isobutanol biosynthetic pathway, wherein the isobutanol biosynthetic pathway comprises the following substrate to product conversions; (1) pyruvate to acetolactate catalyzed by an acetolactate synthase, (2) acetolactate to 2,3-dihydroxyisovalerate catalyzed by a ketol-acid reductoisomerase, (3) 2,3-dihydroxyisovalerate to α
-ketoisovalerate catalyzed by DHAD,(4) α
-ketoisovalerate to isobutyraldehyde catalyzed by an α
-keto acid decarboxylase, and(5) isobutyraldehyde to isobutanol catalyzed by an alcohol dehydrogenase; (b) culturing the recombinant host cell in a fermentation medium under suitable conditions to produce isobutanol; and (c) recovering the isobutanol. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
- 168 and comprises amino acid substitutions at position 33 and position 563, and
Specification