# US 10,308,972 B2 · Whole-cell system for cytochrome P450 monooxygenases biocatalysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,972 B2 - Document ID: US10308972B2 - Status: Granted - Kind: B2 - Application number: 14/898,655 - Filed: 2014-06-17 - Published: 2016-12-29 - Granted: 2019-06-04 - Earliest claimed: 2013-06-17 - Assignees: Sanofi Chimie - Applicants: Sanofi - Inventors: Adrian Gerber; Frank Hannemann; Sabrina Bleif; Michael Kleser; Rita Bernhardt - Web page: https://patentgrep.com/patent/US10308972B2 ## CPC - C12P 33/16: Acting at 17 position - C12N 9/0081: {Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)} - C12N 9/0095: {acting on iron-sulfur proteins as donor (1.18)} - C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)} - C12P 7/625: Polyesters of hydroxy carboxylic acids - C12P 33/00: Preparation of steroids - C12Y 114/15006: Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc - C12Y 203/01: transferring groups other than amino-acyl groups (2.3.1) - 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 subject of the present invention is a whole-cell catalysis process for converting substrates of cytochrome P450 monooxygenases of eukaryotic origin into valuable biotechnological products. The subject of the present invention is also microorganisms genetically engineered to achieve those biotransformations with high rates and processes to prepare these microorganism strains. ## Claims ### Claim 1 A genetically engineered microorganism capable of converting cholesterol, cholesterol analogs, and derivatives thereof into steroid hormones precursors, wherein said microorganism comprises at least one DNA sequence encoding a cytochrome P450 of eukaryotic origin, an exogenous DNA sequence encoding Adx, and an exogenous DNA sequence encoding AdR, wherein said microorganism is Bacillus megaterium, wherein said cytochrome P450 of eukaryotic origin is selected from the group consisting of CYP11A1, CYP17A1, CYP11B1, CYP11B2, CYP3A4, CYP46A1, CYP27A1, CYP21A1, and CYP21A2, and wherein the gene PhaC is overexpressed by introducing into said microorganism an exogenous DNA sequence encoding PhaC. ### Claim 2 (depends on 1) The genetically engineered microorganism according to claim 1, wherein said exogenous DNA sequences have been introduced into said microorganism by means of genetic engineering techniques. ### Claim 3 (depends on 2) The genetically engineered microorganism according to claim 2, wherein said microorganism has been transformed with at least one plasmid comprising said exogenous DNA sequences. ### Claim 4 (depends on 1) The genetically engineered microorganism according to claim 1, wherein said exogenous DNA sequences are integrated into the genome of said microorganism. ### Claim 5 (depends on 1) The genetically engineered microorganism according to claim 1, wherein said microorganism further comprises a functional endogenous polymerase system capable of building polyhydroxyalkanoate bodies. ### Claim 6 (depends on 1) The genetically engineered microorganism according to claim 1, wherein said microorganism is Bacillus megaterium MS941 strain. ### Claim 7 (depends on 1) A method for producing steroid hormones precursors, comprising the steps of: a. Providing a microorganism according to claim 1, b. Culturing said microorganism under conditions allowing the expression of said exogenous DNA sequences, c. Contacting said microorganism culture with a substrate selected from the group consisting of cholesterol, cholesterol analogs, and derivatives thereof, and d. Recovering steroid hormones precursors. ### Claim 8 (depends on 7) The method according to claim 7, wherein: a. said substrate is selected from the group consisting of cholesterol, campesterol, ergostadienol, desmosterol, beta-sitosterol, generol and a mixture of oxysterols, and b. said steroid hormones precursor is pregnenolone. ### Claim 9 (depends on 7) The method according to claim 7, wherein said substrate is solubilized into β-cyclodextrin and saponin. ### Claim 10 (depends on 1) A method of preparing a genetically engineered microorganism according to claim 1 capable of converting cholesterol, cholesterol analogs, and derivatives thereof into steroid hormones precursors, comprising the steps of: a. Providing a microorganism, wherein said microorganism is Bacillus megaterium; and b. Introducing by means of genetic engineering techniques into said microorganism at least one DNA sequence encoding a cytochrome P450 of eukaryotic origin, an exogenous DNA sequence encoding Adx, an exogenous DNA sequence encoding AdR, and an exogenous DNA sequence encoding PhaC, to overexpress gene PhaC, wherein said cytochrome P450 of eukaryotic origin is selected from the group consisting of CYP11A1, CYP17A1, CYP11B1, CYP11B2, CYP3A4, CYP46A1, CYP27A1, CYP21A1, and CYP21A2. ### Claim 11 (depends on 10) The method according to claim 10, wherein said microorganism comprises a functional endogenous polymerase system capable of building polyhydroxyalkanoate bodies. ### Claim 12 (depends on 10) The method according to claim 10, wherein said microorganism is Bacillus megaterium MS941 strain.