title: Engineering 7β-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution.
abstract: Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7β-HSDH. The best variant (V3-1) showed a specific activity 5.5-fold higher than and a half-life 3-fold longer than those of the wild type. In addition, the pH optimum of the variant was shifted to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with V3-1increased to 942 g L-1day-1, in contrast to 141 g L-1day-1with the wild type. Therefore, this study provides a useful strategy for improving the catalytic efficiency of a key enzyme that significantly facilitated the bioproduction of UDCA.
authors.affiliation: State Key Laboratory of Bioreactor Engineering and ‡Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology , Shanghai 200237, P. R. China.
authors.forename: Ming-Min
authors.identifier: http://orcid.org/0000-0001-7313-1886
authors.initials: MM
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Bioreactor Engineering and ‡Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology , Shanghai 200237, P. R. China.
authors.forename: Ke-Cai
authors.initials: KC
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering and ‡Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology , Shanghai 200237, P. R. China.
authors.forename: Ru-Feng
authors.initials: RF
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering and ‡Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology , Shanghai 200237, P. R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioreactor Engineering and ‡Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology , Shanghai 200237, P. R. China.
authors.forename: Chun-Xiu
authors.initials: CX
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioreactor Engineering and ‡Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology , Shanghai 200237, P. R. China.
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008095:Lithocholic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014580:Ursodeoxycholic Acid / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C023020:7-ketolithocholic acid; D008095:Lithocholic Acid; D014580:Ursodeoxycholic Acid; D006913:Hydroxysteroid Dehydrogenases; C031932:7 beta-hydroxysteroid dehydrogenase
keywords: 7β-hydroxysteroid dehydrogenase; biocatalysis; cascade reaction; multiobjective directed evolution; ursodeoxycholic acid