Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst.
A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.
Ye
Qi
Q
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing 210009, People's Republic of China.
Cao
Hou
H
Zang
Guanglou
G
Mi
Lan
L
Yan
Ming
M
Wang
Yan
Y
Zhang
Yueyuan
Y
Li
Ximu
X
Li
Jian
J
Xu
Lin
L
Xiong
Jian
J
Ouyang
Pingkai
P
Ying
Hanjie
H
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D002087:Butyrates / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D005948:Glucose Dehydrogenases / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D002087:Butyrates; D011994:Recombinant Proteins; C121244:ethyl 4-chloro-3-hydroxybutanoate; D000429:Alcohol Oxidoreductases; D005948:Glucose Dehydrogenases