DHAD variants and methods of screening

• US 9,580,705 B2
• Filed: 03/13/2014
• Issued: 02/28/2017
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax Advanced Biofuels LLC (International Flavors & Fragrances Incorporated)
• Inventors: Kelly, Kristen J., Ye, Rick W.
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US14/207,823
• Publication Number:
  US 20140273116A1
• Time in Patent Office: 1,083 Days
• US Class Current: 1/1
• CPC Class Codes: B01D 11/04 of solutions which are liquid
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12Y 101/01001 Alcohol dehydrogenase (1.1.1.1)
• CPC Class Codes: C12Y 101/01086 Ketol-acid reductoisomerase (1.1.1.86)
• CPC Class Codes: C12Y 202/01006 Acetolactate synthase (2.2.1.6)
• CPC Class Codes: C12Y 402/01009 Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

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.

Claims

1. An isolated polypeptide having dihydroxy-acid dehydratase (DHAD) activity, wherein the polypeptide is a Streptococcus mutans DHAD and comprises one or more amino acid substitutions selected from:

(a) aspartic acid at a position corresponding to position 33 of SEQ ID NO;

168;

(b) glutamic acid at a position corresponding to position 62 of SEQ ID NO;

168;

(c) valine at a position corresponding to position 115 of SEQ ID NO;

168;

(d) glutamic acid at a position corresponding to position 116 of SEQ ID NO;

168;

(e) serine at a position corresponding to position 119 of SEQ ID NO;

168;

(f) arginine at a position corresponding to position 158 of SEQ ID NO;

168;

(g) glutamine at a position corresponding to position 176 of SEQ ID NO;

168;

(h) leucine at a position corresponding to position 179 of SEQ ID NO;

168;

(i) arginine at a position corresponding to position 322 of SEQ ID NO;

168;

(j) serine at a position corresponding to position 425 of SEQ ID NO;

168;

(k) glycine at a position corresponding to position 524 of SEQ ID NO;

168;

(l) valine or leucine at a position corresponding to position 562 of SEQ ID NO;

168;

(m) arginine, cysteine, or glycine at a position corresponding to position 563 of SEQ ID NO;

168;

(n) glutamic acid at a position corresponding to position 564 of SEQ ID NO;

168; and

(o) aspartic acid at a position corresponding to position 567 of SEQ ID NO;

168.

2. The isolated polypeptide of claim 1, wherein the polypeptide comprises one or more amino acid substitutions selected from:

(a) glycine to aspartic acid at a position corresponding to position 33 of SEQ ID NO;

168;

(b) aspartic acid to glutamic acid at a position corresponding to position 62 of SEQ ID NO;

168;

(c) methionine to valine at a position corresponding to position 115 of SEQ ID NO;

168;

(d) glycine to glutamic acid at a position corresponding to position 116 of SEQ ID NO;

168;

(e) asparagine to serine at a position corresponding to position 119 of SEQ ID NO;

168;

(f) glycine to arginine at a position corresponding to position 158 of SEQ ID NO;

168;

(g) histidine to glutamine at a position corresponding to position 176 of SEQ ID NO;

168;

(h) histidine to leucine at a position corresponding to position 179 of SEQ ID NO;

168;

(i) glutamine to arginine at a position corresponding to position 322 of SEQ ID NO;

168;

(j) alanine to serine at a position corresponding to position 425 of SEQ ID NO;

168;

(k) glutamic acid to glycine at a position corresponding to position 524 of SEQ ID NO;

168;

(l) phenylalanine to valine or leucine at a position corresponding to position 562 of SEQ ID NO;

168;

(m) tryptophan to arginine, cysteine, or glycine at a position corresponding to position 563 of SEQ ID NO;

168;

(n) lysine to glutamic acid at a position corresponding to position 564 of SEQ ID NO;

168; and

(o) glutamic acid to aspartic acid at a position corresponding to position 567 of SEQ ID NO;

168.

3. The isolated polypeptide of claim 1, wherein the polypeptide having DHAD activity comprises three conserved cysteines corresponding to positions 56, 129, and 201 of SEQ ID NO:

168.

4. A method of converting 2,3-dihydroxyisovalerate to α

-ketoisovalerate or 2,3-dihydroxymethylvalerate to α

-ketomethylvalerate, comprising providing the isolated polypeptide of claim 1;

wherein the isolated polypeptide catalyzes the conversion of 2,3-dihydroxyisovalerate to α

-ketoisovalerate or 2,3-dihydroxymethylvalerate to α

-ketomethylvalerate.

5. The method of claim 4, wherein the isolated polypeptide is comprised within a recombinant host cell.

6. The isolated polypeptide of claim 1, wherein the polypeptide is selected from SEQ ID NO:

528, 532, 534, 537, 540, 545, 548, 552, 555, 557, 561, 563, 566, 569, and 572.