title: Biosynthesis of (S)-4-chloro-3-hydroxybutanoate ethyl using Escherichia coli co-expressing a novel NADH-dependent carbonyl reductase and a glucose dehydrogenase.
abstract: A novel NADH-dependent carbonyl reductase (PsCR II) gene with an open reading frame of 855bp encoding 285 amino acids was cloned from Pichia stipitis. Analysis of the amino acid sequence of PsCR II revealed less than 55% identity to known reductases that produce (S)-4-chloro-3-hydroxybutanoates ethyl [(S)-CHBE]. When NADH was provided as an electron donor, Escherichia coli with pET-22b-PsCRII exhibited an activity of 15U/mg protein using 4-chloro-3-oxobutanoate ethyl (COBE) as a substrate. This activity was the highest ever reported for reductases, with the exception of PsCR I, which in our previous analysis required NADPH for catalysis. Biocatalysis of COBE to (S)-CHBE was investigated using E. coli with a polycistronic plasmid pET-BP II co-expressing PsCR II and a glucose dehydrogenase in a water/butyl acetate system for 24h. The transformants gave a molar yield of 91%, and an optical purity of the (S)-isomer of higher than 99% enantiomeric excess.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, PR China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Ye
authors.forename: Hou
authors.initials: H
authors.lastname: Cao
authors.forename: Lan
authors.initials: L
authors.lastname: Mi
authors.forename: Ming
authors.initials: M
authors.lastname: Yan
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Qinting
authors.initials: Q
authors.lastname: He
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Lin
authors.initials: L
authors.lastname: Xu
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.forename: Jian
authors.initials: J
authors.lastname: Xiong
authors.forename: Pingkai
authors.initials: P
authors.lastname: Ouyang
authors.forename: Hanjie
authors.initials: H
authors.lastname: Ying
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics*; D002087:Butyrates / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000502:physiology*; D042843:Glucose 1-Dehydrogenase / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002087:Butyrates; D011994:Recombinant Proteins; C121244:ethyl 4-chloro-3-hydroxybutanoate; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+); D042843:Glucose 1-Dehydrogenase