A novel NADH-dependent carbonyl reductase from Kluyveromyces aestuarii and comparison of NADH-regeneration system for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate.
To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.
Yamamoto
Hiroaki
H
Life Science Development Center, CPI Company, Daicel Chemical Industries, Ltd., Tsukuba Research Center, Ibaraki, Japan. h-yamamoto@daicel.co.jp
Mitsuhashi
Kazuya
K
Kimoto
Norihiro
N
Matsuyama
Akinobu
A
Esaki
Nobuyoshi
N
Kobayashi
Yoshinori
Y
D000090:Acetoacetates / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002087:Butyrates / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000235:genetics / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D000090:Acetoacetates; D002087:Butyrates; C121244:ethyl 4-chloro-3-hydroxybutanoate; D009243:NAD; C111982:ethyl 4-chloro-3-oxobutanoate; D000429:Alcohol Oxidoreductases; D042843:Glucose 1-Dehydrogenase; D005560:Formate Dehydrogenases; C020644:Formate dehydrogenase (cytochrome); C036543:short chain trans-2-enoyl-CoA reductase; D009247:NADH, NADPH Oxidoreductases; D064429:Fatty Acid Synthases