# US 2021/0340506 A1 · Directed evolution of cyp52a12 gene and its use in dicarboxylic acid production > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0340506 A1 - Document ID: US20210340506A1 - Status: Published application - Kind: A1 - Application number: 17/373,341 - Filed: 2021-07-12 - Published: 2021-11-04 - Earliest claimed: 2018-04-04 - Assignees: Cathay Biotech Inc.; Cibt America Inc. - Applicants: Cathay Biotech Inc.; Cibt America Inc. - Inventors: Wenbo LIU; Min XU; Howard CHOU; Xiucai LIU - Web page: https://patentgrep.com/patent/US20210340506A1 ## CPC - C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)} - C12N 15/52: Genes encoding for enzymes or proenzymes - C12P 7/44: Polycarboxylic acids - C12N 9/001: {acting on the CH-CH group of donors (1.3)} - C12P 7/6409: Fatty acids - C12Y 114/14: with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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)} - C12Y: ENZYMES ## Abstract The invention is directed to a method of preparing a long chain dicarboxylic acid producing strain by using directed evolution and homologous recombination, a strain obtained by this method that is capable of producing a long chain dicarboxylic acid under an acidic condition and the use of the strain. In particular, the invention is directed to a method of preparing a long chain dicarboxylic acid producing strain by using directed evolution of CYP52A12 gene and homologous recombination, a strain obtained by this method that is capable of producing a long chain dicarboxylic acid under an acidic condition and the use of the strain. ## Claims ### Claim 1 A vector, comprising an isolated nucleic acid molecule which encoding an isolated modified CYP52A12 protein, wherein the vector is a prokaryotic vector, a virus vector or a eukaryotic vector; wherein, with reference to the amino acid numbering of SEQ ID NO: 20, the modified CYP52A12 protein comprises Asp at a position corresponding to the position 76 of SEQ ID NO: 20 and Ile at a position corresponding to the position 470 of SEQ ID NO: 20, compared to an unmodified CYP52A12 protein, and the modified CYP52A12 protein comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO: 20 and has the capability of producing a long chain dicarboxylic acid under an acidic culture condition that is at least about 95% or more of that of the modified CYP52A12 protein comprising the amino acid sequence set forth in SEQ ID NO: 21. ### Claim 2 (depends on 1) The vector according to claim 1, wherein the modified CYP52A12 protein comprises the amino acid sequence set forth in SEQ ID NO: 21. ### Claim 3 (depends on 1) The vector according to claim 1, wherein the long chain dicarboxylic acid is at least one selected from C12 to C16 dicarboxylic acids or at least one selected from normal C12 to C16 dicarboxylic acids. ### Claim 4 (depends on 1) A microorganism containing an isolated CYP52A12 protein or the vector of claim 1, wherein said microorganism is selected from the group consisting of Corynebacterium, Geotrichum candidum, Candida, Pichia, Rhodotroula, Saccharomyces and Yarrowia; wherein, with reference to the amino acid numbering of SEQ ID NO: 20, the modified CYP52A12 protein comprises Asp at a position corresponding to the position 76 of SEQ ID NO: 20 and Ile at a position corresponding to the position 470 of SEQ ID NO: 20, compared to an unmodified CYP52A12 protein, and the modified CYP52A12 protein comprises an amino acid sequence having at least about 99% sequence identity with SEQ ID NO: 20 and has the capability of producing a long chain dicarboxylic acid under an acidic culture condition that is at least about 95% or more of that of the modified CYP52A12 protein comprising the amino acid sequence set forth in SEQ ID NO: 21. ### Claim 5 (depends on 4) The microorganism according to claim 4, wherein the modified CYP52A12 protein comprises the amino acid sequence set forth in SEQ ID NO: 21. ### Claim 6 (depends on 4) The microorganism according to claim 4, wherein the long chain dicarboxylic acid is selected from the group consisting of C9-C18 long chain dicarboxylic acids. ### Claim 7 (depends on 4) The microorganism according to claim 4, wherein the long chain dicarboxylic acid is selected from one or more of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid. ### Claim 8 (depends on 4) The microorganism according to claim 4, wherein the acidic culture condition is controlling the pH of the fermentation system to be 7.0 or below. ### Claim 9 (depends on 4) The microorganism according to claim 4, wherein the acidic culture condition is controlling the pH of the fermentation system to 4.0 to 6.8. ### Claim 10 (depends on 4) The microorganism according to claim 4, wherein controlling the pH of the fermentation system is controlling the pH of the fermentation system during conversion phase. ### Claim 11 (depends on 4) The microorganism according to claim 4, wherein said microorganism is yeast. ### Claim 12 (depends on 4) The microorganism according to claim 4, wherein said microorganism is Candida tropicalis or Candida sake. ### Claim 13 (depends on 4) A method for producing a long chain dicarboxylic acid using the microorganism according to claim 4, comprising a step of culturing the microorganism according to claim 4, optionally further comprising the step of isolating and purifying the long chain dicarboxylic acid from cultured products. ### Claim 14 (depends on 13) The method of claim 13, wherein the long chain dicarboxylic acid is selected from the group consisting of C9-C18 long chain dicarboxylic acids. ### Claim 15 (depends on 13) The method of claim 13, wherein the long chain dicarboxylic acid is selected from one or more of sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid. ### Claim 16 (depends on 13) The method of claim 13, wherein the long chain dicarboxylic acid is at least one selected from C12 to C16 dicarboxylic acids or at least one selected from normal C12 to C16 dicarboxylic acids. ### Claim 17 (depends on 13) The method of claim 13, wherein the modified long chain dicarboxylic acid producing strain is able to produce the long chain dicarboxylic acid at pH7.0 or below, compared to the strain before modified. ### Claim 18 (depends on 13) The method of claim 13, for producing the long chain dicarboxylic acid under an acidic culture condition. ### Claim 19 (depends on 13) The method of claim 13, wherein the modified long chain dicarboxylic acid producing strain is able to produce the long chain dicarboxylic acid at 4.0 to 6.8, compared to the strain before modified. ### Claim 20 (depends on 13) The method of claim 13, wherein the modified long chain dicarboxylic acid producing strain is able to produce the long chain dicarboxylic acid at 5.0 to 6.5, compared to the strain before modified.