Methods and compositions related to thioesterase enzymes

• US 9,175,234 B2
• Filed: 12/23/2009
• Issued: 11/03/2015
• Est. Priority Date: 12/23/2008
• Status: Active Grant

• Current Assignee: Genomatica Incorporated, LS9, Inc.
• Original Assignee: REG Life Sciences, LLC (Genomatica Incorporated)
• Inventors: Alibhai, Murtaza, Hu, Zhihao, Arlagadda, Vikranth, Hom, Louis, Trinh, Na, Groban, Eli, Clarke, Elizabeth
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US12/645,497
• Publication Number:
  US 20100154293A1
• Time in Patent Office: 2,141 Days
• US Class Current: 1/1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C10L 1/02 essentially based on components consisting of carbon, hydrogen, and oxygen only
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention relates to novel mutant thioesterase enzymes and naturally-occurring equivalents thereof, compositions made from such enzymes and uses of thioesterase enzymes. In particular, the present invention provides mutant thioesterase enzymes that have altered properties, for example, altered substrate specificity, altered activity, altered selectivity, and/or altered proportional yields in the product mixtures. The present invention also provides polynucleotides encoding such mutant thioesterase enzymes, and vectors and host cells comprising such polynucleotides. The invention further provides for novel uses of thioesterases in the production of various fatty acid derivatives, which are useful as, or as components of, industrial chemicals and fuels.

Claims

1. An engineered thioesterase enzyme which converts a C10, C12, or C14 acyl-ACP substrate to a fatty acid derivative with a greater activity as compared to a wild-type thioesterase enzyme, wherein the engineered thioesterase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

73 and has a substitution at an amino acid position selected from the group consisting of position 78, 80, 101, 108, 111, 117, 118, 122, 145, 152, and 178.

2. The engineered thioesterase enzyme of claim 1, wherein the amino acid sequence has a feature selected from the group consisting of:

(a) the amino acid residue at position 78 is methionine;

(b) the amino acid residue at position 80 is tryptophan;

(c) the amino acid residue at position 101 is leucine, alanine, or valine;

(d) the amino acid residue at position 108 is tryptophan;

(e) the amino acid residue at position 111 is tyrosine or tryptophan;

(f) the amino acid residue at position 117 is arginine;

(g) the amino acid residue at position 122 is arginine;

(h) the amino acid residue at position 145 is alanine, cysteine, aspartic acid, glutamic acid, isoleucine, leucine, methionine, glutamine, arginine, serine, or threonine;

(i) the amino acid residue at position 152 is arginine; and

(j) the amino acid residue at position 178 is threonine or tryptophan.

3. The engineered thioesterase enzyme of claim 1, wherein the amino acid residue at position 101 is leucine, the amino acid residue at position 111 is tyrosine, the amino acid residue at position 145 is cysteine, or the amino acid residue at position 178 is threonine.

4. The engineered thioesterase enzyme of claim 1, wherein the amino acid residue at position 111 is tyrosine or the amino acid residue at position 145 is cysteine.

5. The engineered thioesterase enzyme of claim 1, wherein the amino acid residue at 78 is methionine, the amino acid residue at position 108 is tryptophan, or the amino acid residue at position 145 is cysteine.

6. The engineered thioesterase enzyme of claim 1, wherein the amino acid residue at 78 is methionine, the amino acid residue at position 108 is tryptophan, or the amino acid residue at position 145 is leucine.

7. An engineered thioesterase enzyme which converts an acyl-ACP substrate to a fatty acid derivative with a greater yield or an increased percentage of short-chain fatty acid derivatives as compared to a wild-type thioesterase enzyme, wherein the engineered thioesterase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

73 and has a substitution at an amino acid position selected from the group consisting of position 78, 80, 101, 108, 111, 117, 118, 122, 145, 152 and 178.

8. The engineered thioesterase enzyme of claim 7, wherein the amino acid sequence has a feature selected from the group consisting of:

(a) the amino acid residue at position 78 is methionine;

(b) the amino acid residue at position 80 is tryptophan;

(c) the amino acid residue at position 101 is leucine, alanine or valine;

(d) the amino acid residue at position 108 is tryptophan;

(e) the amino acid residue at position 111 is tyrosine or tryptophan;

(f) the amino acid residue at position 117 is arginine;

(g) the amino acid residue at position 122 is arginine;

(h) the amino acid residue at position 145 is alanine, cysteine, aspartic acid, glutamic acid, isoleucine, leucine, methionine, glutamine, arginine, serine or threonine;

(i) the amino acid residue at position 152 is arginine; and

(j) the amino acid residue at position 178 is threonine or tryptophan.

9. The engineered thioesterase enzyme of claim 7, wherein the amino acid residue at position 145 is cysteine.