A substrate-coupled biocatalytic process was developed based on the reactions catalyzed by an NADPH-dependent sorbose reductase (SOU1) from Candida albicans in which ethyl 4-chloro-3-oxobutanoate (COBE) was reduced to (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], while NADPH was regenerated by the same enzyme via oxidation of sugar alcohols. (S)-CHBE yields of 1,140, 1,150, and 780 mM were obtained from 1,220 mM COBE when sorbitol, mannitol, and xylitol were used as co-substrates, respectively. Optimization of COBE and sorbitol proportions resulted in a maximum yield of (S)-CHBE (2,340 mM) from 2,500 mM COBE, and the enantiomeric excess was 99.6 %. The substrate-coupled system driven by SOU1 maintained a stable pH and a robust intracellular NADPH circulation; thus, pH adjustment and addition of extra coenzymes were unnecessary.
A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP+/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.
(2015) Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candidaalbicans with two co-existing recombinant Escherichiacoli strains, Bioscience, Biotechnology, and Biochemistry, 79:7, 1090-1093,
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