title: Construction and co-expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl (S)-4-chloro-3-hydroxybutanoate.
abstract: Biocatalysis of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] was carried out using Escherichia coli co-expressing a carbonyl reductase gene from Pichia stipitis and a glucose dehydrogenase gene from Bacillus megaterium. An efficient polycistronic plasmid with a high-level of enzyme co-expression was constructed by changing the order of the genes, altering the Shine-Dalgarno (SD) regions, and aligned spacing (AS) between the SD sequence and the translation initiation codon. The optimal SD sequence was 5-TAAGGAGG-3, and the optimal AS distance was eight nucleotides. Asymmetric reduction of COBE to (S)-CHBE with more than 99% enantiomeric excess was demonstrated by transformants, using a water/ethyl caprylate system. The recombinant cells produced 1260 mM product in the organic phase, and the total turnover number, defined as moles (S)-CHBE formed per mole NADP(+), was 12,600, which was more than 10-fold higher than in aqueous systems.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing 210009, PR China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Ye
authors.forename: Hou
authors.initials: H
authors.lastname: Cao
authors.forename: Ming
authors.initials: M
authors.lastname: Yan
authors.forename: Fei
authors.initials: F
authors.lastname: Cao
authors.forename: Yueyuan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Ximu
authors.initials: X
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 / Q000378:metabolism*; D001407:Bacillus / Q000235:genetics; D001483:Base Sequence; D055162:Biocatalysis; D002087:Butyrates / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D010957:Plasmids
chemical_list: D002087:Butyrates; D017931:DNA Primers; C121244:ethyl 4-chloro-3-hydroxybutanoate; D000429:Alcohol Oxidoreductases; D042843:Glucose 1-Dehydrogenase