Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid Via 7β-Hydroxylation of Lithocholic Acid

Overview

Journal Angew Chem Int Ed Engl

Specialty Chemistry

Date 2020 Oct 21

PMID 33085147

Citations 13

Authors

Sascha Grobe

Christoffel P S Badenhorst

Thomas Bayer

Emil Hamnevik

Shuke Wu

Christoph W Grathwol

Andreas Link

Sven Koban

Henrike Brundiek

Beatrice Grossjohann

Uwe T Bornscheuer

Affiliations

Soon will be listed here.

Abstract

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7β-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β-position but LCA is exclusively hydroxylated at the 6β-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity-determining residues. Alanine scanning identified S240A as a UDCA-producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio- and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10-fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA.

Citing Articles

Microbial transformations of bile acids and their receptors in the regulation of metabolic dysfunction-associated steatotic liver disease.

Gao Y, Lin J, Ye C, Guo S, Jiang C Liver Res. 2025; 7(3):165-176.

PMID: 39958385 PMC: 11792070. DOI: 10.1016/j.livres.2023.09.002.

Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling.

Wang X, Xu K, Fu H, Chen Q, Zhao B, Zhao X Synth Syst Biotechnol. 2024; 10(1):185-193.

PMID: 39552758 PMC: 11564792. DOI: 10.1016/j.synbio.2024.10.003.

Optimization and Impurity Control Strategy for Lithocholic Acid Production Using Commercially Plant-Sourced Bisnoralcohol.

Zeng C, Xu S, Yin Z, Cui Y, Xu X, Li N ACS Omega. 2023; 8(25):23130-23141.

PMID: 37396276 PMC: 10308411. DOI: 10.1021/acsomega.3c02548.

Development of HPLC-CAD method for simultaneous quantification of nine related substances in ursodeoxycholic acid and identification of two unknown impurities by HPLC-Q-TOF-MS.

Huang Y, Lu H, Li Z, Zeng Y, Xu Q, Wu Y J Pharm Biomed Anal. 2023; 229:115357.

PMID: 36966621 PMC: 10033147. DOI: 10.1016/j.jpba.2023.115357.

Biological synthesis of ursodeoxycholic acid.

Song P, Zhang X, Feng W, Xu W, Wu C, Xie S Front Microbiol. 2023; 14:1140662.

PMID: 36910199 PMC: 9998936. DOI: 10.3389/fmicb.2023.1140662.

References

Kuipers R, Joosten H, van Berkel W, Leferink N, Rooijen E, Ittmann E . 3DM: systematic analysis of heterogeneous superfamily data to discover protein functionalities. Proteins. 2010; 78(9):2101-13. DOI: 10.1002/prot.22725. View

Li A, Acevedo-Rocha C, DAmore L, Chen J, Peng Y, Garcia-Borras M . Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants. Angew Chem Int Ed Engl. 2020; 59(30):12499-12505. PMC: 7384163. DOI: 10.1002/anie.202003139. View

Gotoh O . Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem. 1992; 267(1):83-90. View

Goossens J, Bailly C . Ursodeoxycholic acid and cancer: From chemoprevention to chemotherapy. Pharmacol Ther. 2019; 203:107396. DOI: 10.1016/j.pharmthera.2019.107396. View

Park H, Park G, Jeon W, Ahn J, Yang Y, Choi K . Whole-cell biocatalysis using cytochrome P450 monooxygenases for biotransformation of sustainable bioresources (fatty acids, fatty alkanes, and aromatic amino acids). Biotechnol Adv. 2020; 40:107504. DOI: 10.1016/j.biotechadv.2020.107504. View

Grobe S, Badenhorst C, Bayer T, Hamnevik E, Wu S, Grathwol C . Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β-Hydroxylation of Lithocholic Acid. Angew Chem Int Ed Engl. 2020; 60(2):753-757. PMC: 7839452. DOI: 10.1002/anie.202012675. View

Wei Y, Lui Ang E, Zhao H . Recent developments in the application of P450 based biocatalysts. Curr Opin Chem Biol. 2017; 43:1-7. DOI: 10.1016/j.cbpa.2017.08.006. View

Liu L, Aigner A, Schmid R . Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Appl Microbiol Biotechnol. 2010; 90(1):127-35. DOI: 10.1007/s00253-010-3052-y. View

Qu G, Li A, Acevedo-Rocha C, Sun Z, Reetz M . The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes. Angew Chem Int Ed Engl. 2019; 59(32):13204-13231. DOI: 10.1002/anie.201901491. View

10.

Li A, Acevedo-Rocha C, Sun Z, Cox T, Xu J, Reetz M . Beating Bias in the Directed Evolution of Proteins: Combining High-Fidelity on-Chip Solid-Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction. Chembiochem. 2017; 19(3):221-228. DOI: 10.1002/cbic.201700540. View

11.

Appel D, Schmid R, Dragan C, Bureik M, Urlacher V . A fluorimetric assay for cortisol. Anal Bioanal Chem. 2005; 383(2):182-6. DOI: 10.1007/s00216-005-0022-9. View

12.

Kollerov V, Monti D, Deshcherevskaya N, Lobastova T, E Ferrandi E, Larovere A . Hydroxylation of lithocholic acid by selected actinobacteria and filamentous fungi. Steroids. 2013; 78(3):370-8. DOI: 10.1016/j.steroids.2012.12.010. View

13.

Goel A, Kim W . Natural History of Primary Biliary Cholangitis in the Ursodeoxycholic Acid Era: Role of Scoring Systems. Clin Liver Dis. 2018; 22(3):563-578. DOI: 10.1016/j.cld.2018.03.007. View

14.

Gaisser S, Lill R, Staunton J, Mendez C, Salas J, Leadlay P . Parallel pathways for oxidation of 14-membered polyketide macrolactones in Saccharopolyspora erythraea. Mol Microbiol. 2002; 44(3):771-81. DOI: 10.1046/j.1365-2958.2002.02910.x. View

15.

Gulamhusein A, Hirschfield G . Primary biliary cholangitis: pathogenesis and therapeutic opportunities. Nat Rev Gastroenterol Hepatol. 2019; 17(2):93-110. DOI: 10.1038/s41575-019-0226-7. View

16.

Park R . The major neutral products of the aerobic catabolism of cattle bile by Pseudomonas sp. ATCC 31752. Steroids. 1981; 38(4):383-95. DOI: 10.1016/0039-128x(81)90073-8. View

17.

Chascsa D, Lindor K . Emerging therapies for PBC. J Gastroenterol. 2020; 55(3):261-272. PMC: 7026299. DOI: 10.1007/s00535-020-01664-0. View

18.

Agematu H, Matsumoto N, Fujii Y, Kabumoto H, Doi S, Machida K . Hydroxylation of testosterone by bacterial cytochromes P450 using the Escherichia coli expression system. Biosci Biotechnol Biochem. 2006; 70(1):307-11. DOI: 10.1271/bbb.70.307. View

19.

Montemiglio L, Parisi G, Scaglione A, Sciara G, Savino C, Vallone B . Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis. Biochim Biophys Acta. 2015; 1860(3):465-75. DOI: 10.1016/j.bbagen.2015.10.009. View

20.

Urlacher V, Girhard M . Cytochrome P450 Monooxygenases in Biotechnology and Synthetic Biology. Trends Biotechnol. 2019; 37(8):882-897. DOI: 10.1016/j.tibtech.2019.01.001. View