Biosynthesis of Ethyl (S)-4-Chloro-3-Hydroxybutanoate by NADH-Dependent Reductase from E. coli CCZU-Y10 Discovered by Genome Data Mining Using Mannitol as Cosubstrate

• Published in: Applied biochemistry and biotechnology Vol. 173; no. 8; pp. 2042 - 2053
• Main Authors: He, Yu-Cai, Yang, Zhen-Xing, Zhang, Dan-Ping, Tao, Zhi-Cheng, Chen, Chao, Chen, Yi-Tong, Guo, Fei, Xu, Jian-He, Huang, Lei, Chen, Rui-Jie, Ma, Xiao-Feng
• Format: Journal Article
• Language: English
• Published: Boston Springer-Verlag 01.08.2014; Springer US; Springer; Springer Nature B.V
• Subjects: Acetoacetates - chemistry; Acetoacetates - metabolism; Biochemistry; Biological and medical sciences; Biosynthesis; Biotechnology; Biotransformation; Chemistry; Chemistry and Materials Science; Data Mining; E coli; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; genome; Genome, Bacterial; Genomes; Industrial production; Kinetics; mannitol; Mannitol - metabolism; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - genetics; NADH, NADPH Oxidoreductases - metabolism; Stereoisomerism; Substrate Specificity; temperature; Asymmetric reduction; CCZU-Y10; Biphasic system; Biosynthesis; Ethyl 4-chloro-3-oxobutanoate; Ethyl; Recombinant; 4-chloro-3-hydroxybutanoate; Enzyme; Recombinant E. coli CCZU-Y10; Data mining; Ethyl (S)-4-chloro-3-hydroxybutanoate; Reduction; NADH; Mannitol; Oxidoreductases; Genome; Reductase
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-014-1001-4

The reductase (PgCR) from recombinant Escherichia coli CCZU-Y10 displayed high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). To efficiently synthesize (S)-CHBE (>99 % enantiomeric excess (ee)), the highly stereoselective bioreduction of COBE into (S)-CHBE with the whole cells of E. coli CCZU-Y10 was successfully demonstrated in a dibutyl phthalate-water biphasic system. The appropriate ratio of the organic phase to water phase was 1:1 (v/v). The optimum reaction temperature, reaction pH, cosubstrate, NAD⁺, and cell dosage of the biotransformation of 100 mM COBE in this biphasic system were 30 °C, 7.0, mannitol (2.5 mmol/mmol COBE), 0.1 μmol/(mmol COBE), and 0.1 g (wet weight)/mL, respectively. Moreover, COBE at a high concentration of (1,000 mM) could be asymmetrically reduced to (S)-CHBE in a high yield (99.0 %) and high enantiometric excess value (>99 % ee). Significantly, E. coli CCZU-Y10 shows high potential in the industrial production of (S)-CHBE (>99 % ee).