# US 9,518,280 B2 · Aldolases, nucleic acids encoding them and methods for making and using them > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,518,280 B2 - Document ID: US9518280B2 - Status: Granted - Kind: B2 - Application number: 14/526,351 - Filed: 2014-10-28 - Published: 2015-04-23 - Granted: 2016-12-13 - Earliest claimed: 2006-03-07 - Assignees: Cargill, Incorporated - Applicants: Cargill, Incorporated - Inventors: Ellen Burke; Steven John Gort; Paula M. Hicks; Peter Luginbuhl; Sara C. McFarlan; Toby Richardson; Christopher Solheid; David P. Weiner; Lishan Zhao - Web page: https://patentgrep.com/patent/US9518280B2 ## CPC - C12P 17/10: Nitrogen as only ring hetero atom - C12N 9/88: Lyases (4.) - C12P 13/001: {Amines; Imines} - Y02P 20/52: using catalysts, e.g. selective catalysts - 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) - Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS ## Abstract This invention relates to polypeptides having aldolase activity, including pyruvate activity such as, without limitation, HMG and/or KHG aldolase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In some embodiments, the invention is directed to polypeptides having aldolase activity, including pyruvate activity such as, without limitation, HMG and/or KHG aldolase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides in accordance with the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts. In some embodiments, the invention provides polypeptides and biosynthetic pathways that are useful in the production of R-2-hydroxy 2-(indol-3ylmethyl)-4-keto glutaric acid (R-MP) and certain stereoisomers of monatin, such as R,R and S,R monatin, and salts thereof, as well as certain stereoisomers of monatin derivatives, such as the R,R and S,R configurations, and salts thereof. ## Claims ### Claim 1 An in vitro method comprising: providing a C3 carbon source selected from pyruvate, oxaloacetate, pyruvate derivatives and salts thereof; providing indole-3-pyruvate; providing a polypeptide capable of facilitating an aldol condensation reaction between said C3 carbon source and said indole-3-pyruvate, resulting in the formation of 2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid; wherein said polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO:27 or a fragment thereof that encodes a polypeptide that retains the capability of facilitating an aldol condensation reaction between said C3 carbon source and said indole-3-pyruvate, resulting in the formation of 2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid. ### Claim 2 (depends on 1) The method of claim 1, wherein the polypeptide is encoded by a polynucleotide sequence that has the sequence of SEQ ID NO:27 or a fragment thereof that encodes a polypeptide capable of facilitating an aldol condensation reaction between said C3 carbon source and said indole-3-pyruvate. ### Claim 3 (depends on 1) The method of claim 1, wherein the reaction preferentially produces R-2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid over S-2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid. ### Claim 4 (depends on 3) The method of claim 3, wherein the method further comprises aminating the R-2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid to produce R,R-monatin. ### Claim 5 (depends on 1) The method of claim 1, wherein the method further comprises aminating the 2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid to produce monatin. ### Claim 6 An in vitro method comprising: providing a C3 carbon source selected from pyruvate, oxaloacetate, pyruvate derivatives and salts thereof; providing indole-3-pyruvate; providing a polypeptide capable of facilitating an aldol condensation reaction between said C3 carbon source and said indole-3-pyruvate, resulting in the formation of 2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid, wherein the polypeptide is encoded by a polynucleotide sequence that has sufficient sequence homology such that the complement would remain hybridized to polynucleotide sequence of SEQ ID NO:27, wherein hybridization wash conditions are no less stringent than washing with 0.1×SSC and 0.5% SDS at 68 degrees C. for 15 minutes. ### Claim 7 (depends on 6) The method of claim 6, wherein the polypeptide is encoded by a polynucleotide sequence that has the sequence of SEQ ID NO:27. ### Claim 8 (depends on 6) The method of claim 6, wherein the reaction preferentially produces R-2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid over S-2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid. ### Claim 9 (depends on 8) The method of claim 8, wherein the method further comprises aminating the R-2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid to produce R,R-monatin. ### Claim 10 (depends on 6) The method of claim 6, wherein the method further comprises aminating the 2-hydroxy-2-(indol-3-yl-methyl)-4-keto-glutaric acid to produce monatin.