# US 7,745,195 B2 · Method for the preparation of an evolved microorganism for the creation or the modification of metabolic pathways > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,745,195 B2 - Document ID: US7745195B2 - Status: Granted - Kind: B2 - Application number: 10/781,499 - Filed: 2004-02-18 - Published: 2005-03-10 - Granted: 2010-06-29 - Earliest claimed: 2003-02-18 - Assignees: Metabolic Explorer - Inventors: Michel Chateau; Benjamin Gonzalez; Isabelle Meynial-Salles; Philippe Noel Paul Soucaille; Olivier Zink - Web page: https://patentgrep.com/patent/US7745195B2 ## CPC - C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms} - C12N 9/0036: {acting on NADH or NADPH (1.6)} - C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor - C12P 7/18: polyhydric - C12P 7/20: Glycerol - C12P 7/42: Hydroxy-carboxylic acids - C12P 7/52: Propionic acid; Butyric acids - C12P 13/12: Methionine; Cysteine; Cystine - C12P 33/00: Preparation of steroids - 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)} ## Abstract The present invention is directed to a method for the preparation of evolved microorganisms permitting the creation or modification of metabolic pathways. Strains of evolved microorganisms and evolved genes obtained by the method are also provided. The invention is also directed to the use of evolved microorganisms, genes or proteins in a biotransformation process. ## Claims ### Claim 1 A method for producing a microorganism wherein said microorganism is obtained by selection of a modified microorganism, having a modified methionine biosynthesis pathway for the biosynthesis of methionine by metabolism of a simple carbon source and methylmercaptan as a source of sulfur, comprising: a) generating a modified microorganism by inactivating, deleting, or inhibiting expression of metE gene in an initial microorganism, wherein ability of said modified microorganism to grow is impaired when grown on a minimal medium containing no methionine, S-adenosylmethionine, homocysteine or cystathionine; b) culturing said modified microorganism obtained in step (a) on said minimal medium, in the presence of methylmercaptan under selection pressure thereby allowing said modified microorganism to proliferate through a methionine biosynthesis pathway to compensate for impaired growth; and c) selecting said microorganism from step (b) able to grow on said minimal medium in presence of methylmercptan, wherein the methionine biosynthesis pathway modifies such that methionine is produced by the metabolism of a simple carbon source and methylmercaptan as a source of sulfur thereby allowing said microorganism to proliferate on said minimal medium containing no methionine, S-adenosylmethionine, homocysteine or cystathionine. ### Claim 2 (depends on 1) The method as claimed in claim 1, wherein said microorganism possesses at least one modified gene coding for a modified protein-involved in the methionine biosynthesis pathway. ### Claim 3 (depends on 2) The method as claimed in claim 2, comprising a step d) comprising the isolation of said gene coding for a modified protein involved in the methionine biosynthesis pathway. ### Claim 4 (depends on 3) The method as claimed in claim 3, wherein said gene is introduced, into a production microorganism intended for the production of said protein. ### Claim 5 (depends on 1) The method of claim 1, wherein said inactivating, deleting, or inhibiting expression of the metE gene is performed by directed mutation of said metE gene or directed modification of a promoter of said metE gene. ### Claim 6 (depends on 1) The method of claim 1, wherein the deletion of the metE gene includes removal of most of said metE gene. ### Claim 7 (depends on 1) The method of claim 1, wherein said metE gene is replaced with a selection marker gene. ### Claim 8 (depends on 1) The method of claim 1, wherein said microorganism is a bacteria. ### Claim 9 (depends on 1) The method of claim 1, wherein said microorganism is an Escherichia sp. ### Claim 10 (depends on 1) The method of claim 1, wherein the microorganism is E. coli. ### Claim 11 (depends on 1) An evolved microorganism having a modified methionine biosynthesis pathway produced by the method of claim 1. ### Claim 12 (depends on 2) The method of claim 2, wherein said gene coding for said protein involved in the methionine biosynthesis pathway is a mutated metB gene with methionine synthase activity which allows for the direct conversion of O-succinyl-L-homoserine into L-methionine with methylmercaptan as a sulfur source. ### Claim 13 (depends on 3) The method of claim 3, wherein said gene coding for said protein involved in the methionine biosynthesis pathway is a mutated metB gene with methionine synthase activity which allows for the direct conversion of O-succinyl-L-homoserine into L-methionine with methylmercaptan as a sulfur source. ### Claim 14 A method for preparation of modified strains of E. coil having a modified methionine biosynthesis pathway for the biosynthesis of methionine by metabolism of a simple carbon source and methylmercaptan as a source of sulfur, comprising: (a) generating a modified microorganism by inactivating, deleting, or inhibiting expression of metE gene in an initial E. coil strain, wherein ability of said modified microorganism to grow is impaired when grown on a minimal medium containing no methionine, S-adenosylmethionine, homocysteine or cystathionine, b) culturing said modified microorganism obtained in step (a) on said minimal medium in presence of methylmercaptan under selection pressure thereby allowing said modified microorganism to proliferate through the methionine biosynthesis pathway to compensate for the impaired growth; and c) selecting an-evolved said E. coil strain from step (b) able to grow on said minimal medium in the presence of methylmercptan, said microorganism comprising a mutated metB gene with methionine synthase activity and allowing the direct conversion of O-succinyl-L-homoserine into L-methionine with methylmercaptan as a sulfur source. ### Claim 15 (depends on 14) The method of claim 14, wherein said deletion includes the removal of most of said metE gene. ### Claim 16 (depends on 14) The method of claim 14, wherein said metE gene is replaced with a selection marker gene. ### Claim 17 (depends on 14) The method of claim 14, wherein said initial E. coli strain is strain E. coli K12. ### Claim 18 (depends on 14) The method of claim 14, wherein said E. coil strain is strain E. coil 183 deposited at the Collection Nationale de Cultures de Microorganisms (CNCM) and registered under the number I-3005. ### Claim 19 (depends on 1) The method of claim 1, wherein said simple carbon source is selected from the group consisting of glucose, galactose, sucrose, lactose and molasses.