Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid

Qi-Fan Sun; Yu-Cong Zheng; Qi Chen; Jian-He Xu; Jiang Pan

doi:10.1016/j.bbrc.2020.12.039

Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid

Biochem Biophys Res Commun. 2021 Jan 22:537:64-70. doi: 10.1016/j.bbrc.2020.12.039. Epub 2020 Dec 30.

Authors

Qi-Fan Sun¹, Yu-Cong Zheng¹, Qi Chen², Jian-He Xu³, Jiang Pan⁴

Affiliations

¹ State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China.
² State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China; State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing and Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237, China.
³ State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China; State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing and Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237, China. Electronic address: jianhexu@ecust.edu.cn.
⁴ State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China; State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing and Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237, China. Electronic address: panjiang@ecust.edu.cn.

PMID: 33387884
DOI: 10.1016/j.bbrc.2020.12.039

Abstract

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C₁₀-functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (PaOH) with significantly improved catalytic efficiency (from 33 s^-1 mM^-1 to 119 s^-1 mM^-1), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered PaOH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L^-1 day^-1 and 160 g L^-1 day^-1, respectively.

Keywords: Biocatalysis; Directed evolution; Fatty acid functionalization; Oleate hydratase; Oleic acid.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biocatalysis
Carbon / metabolism*
Genetic Engineering*
High-Throughput Screening Assays
Hydro-Lyases / metabolism*
Kinetics
Molecular Dynamics Simulation
Mutagenesis / genetics
Oleic Acid / metabolism*
Paracoccus / enzymology
Stearic Acids / metabolism

Substances

Stearic Acids
Oleic Acid
10-hydroxystearic acid
Carbon
Hydro-Lyases

Supplementary concepts

Paracoccus aminophilus