# US 10,246,728 B2 · Variant thioesterases and methods of use > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,728 B2 - Document ID: US10246728B2 - Status: Granted - Kind: B2 - Application number: 15/684,941 - Filed: 2017-08-24 - Published: 2018-05-31 - Granted: 2019-04-02 - Earliest claimed: 2014-07-24 - Assignees: Corbion Biotech, Inc. - Applicants: Corbion Biotech, Inc. - Inventors: David Davis; Scott Franklin; Jeffrey L. Moseley; Riyaz Bhat - Web page: https://patentgrep.com/patent/US10246728B2 ## CPC - C12P 7/6463: obtained from glyceride producing microorganisms, e.g. single cell oil - C12N 9/16: acting on ester bonds (3.1) - C12N 15/8247: {involving modified lipid metabolism, e.g. seed oil composition} - C12Y 301/02014: Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract The present invention relates to variant thioesterases and their use in plants, e.g., to increase enzymatic activity and to promote increased production of mid-chain length fatty acids (e.g., 8 to 14 carbons) and at desired ratios. Further disclosed herein are methods of manufacturing renewable chemicals through the manufacture of novel triglyceride oils followed by chemical modification of the oils. Oils containing fatty acid chain lengths of C8, C10, C12 or C14 are also disclosed and are useful as feedstocks in the methods described herein. ## Claims ### Claim 1 A non-natural fatty acyl-ACP thioesterase wherein the non-natural thioesterase has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 and comprises: i) Tyrosine (Y) or phenylalanine (F) at the position corresponding to position 163 of SEQ ID NO: 1 and lysine (K), or alanine (A) at the position corresponding to position 186 of SEQ ID NO: 1; or ii) Phenylalanine (F) at the position corresponding to position 163 of SEQ ID NO: 1 and/or proline (P), lysine (K), or alanine (A) at the position corresponding to position 186 of SEQ ID NO: 1, wherein the non-natural thioesterase catalyzes the production of increased levels of C8:0 and C10:0 fatty acids in comparison to a wild-type thioesterase. ### Claim 2 (depends on 1) A method for producing a triglyceride oil, the method comprising expressing, in a host cell, the non-natural thioesterase of claim 1; cultivating the host cell; and isolating the oil. ### Claim 3 (depends on 1) A method for increasing the C8 and/or C10 fatty acids in a fatty acid profile of an oil produced by an optionally oleaginous host cell, the method comprising, providing a parent gene encoding a fatty acyl-ACP thioesterase enzyme, mutating the gene to so as to encode a non-natural thioesterase of claim 1; expressing the mutated gene in the host cell; and producing the oil, whereby the C8 and/or C10 fatty acids in the fatty acid profile of the oil are increased. ### Claim 4 (depends on 1) The non-natural fatty acyl-ACP thioesterase of claim 1, further comprising a lysine (K) at the position corresponding to position 228 of SEQ ID NO: 1. ### Claim 5 (depends on 4) A method for producing a triglyceride oil, the method comprising expressing, in a host cell, the non-natural thioesterase of claim 4. ### Claim 6 (depends on 3) The method of claim 3, comprising further mutating the gene so as to encode a non-natural fatty acyl-ACP thioesterase further comprising a lysine (K) at the position corresponding to position 228 of SEQ ID NO: 1. ### Claim 7 An isolated polynucleotide encoding a non-natural fatty acyl-ACP thioesterase wherein the non-natural thioesterase has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 and comprises: i) Tyrosine (Y) or phenylalanine (F) at the position corresponding to position 163 of SEQ ID NO: 1 and lysine (K), or alanine (A) at the position corresponding to position 186 of SEQ ID NO: 1; or ii) Phenylalanine (F) at the position corresponding to position 163 of SEQ ID NO: 1 and/or proline (P), lysine (K), or alanine (A) at the position corresponding to position 186 of SEQ ID NO: 1, wherein the non-natural thioesterase catalyzes the production of increased levels of C8:0 and C10:0 fatty acids in comparison to a wild-type thioesterase. ### Claim 8 (depends on 7) The isolated polynucleotide of claim 7, wherein the polynucleotide encodes a non-natural thioesterae further comprising a lysine (K) at the position corresponding to position 228 of SEQ ID NO: 1. ### Claim 9 (depends on 7) An expression cassette comprising the isolated polynucleotide of claim 7. ### Claim 10 (depends on 9) A host cell comprising the expression cassette of claim 9. ### Claim 11 (depends on 10) The host cell of claim 10, wherein the cell is an oleaginous microalga cell. ### Claim 12 (depends on 11) The host cell of claim 11, wherein the microalga cell is classified as Chlorophyta, Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. ### Claim 13 (depends on 11) The host cell of claim 11, wherein the microalga cell is of a genus selected from the group consisting of Chlorella, Dunaliella, and Prototheca. ### Claim 14 (depends on 9) The host cell of claim 9, wherein the microalga cell is of a species selected from the group consisting of Prototheca moriformis and Chlorella protothecoides.