Construction and co-expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl (S)-4-chloro-3-hydroxybutanoate.
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.
Ye
Qi
Q
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing 210009, PR China.
Cao
Hou
H
Yan
Ming
M
Cao
Fei
F
Zhang
Yueyuan
Y
Li
Ximu
X
Xu
Lin
L
Chen
Yong
Y
Xiong
Jian
J
Ouyang
Pingkai
P
Ying
Hanjie
H
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
D002087:Butyrates; D017931:DNA Primers; C121244:ethyl 4-chloro-3-hydroxybutanoate; D000429:Alcohol Oxidoreductases; D042843:Glucose 1-Dehydrogenase