Development of production process for chiral building blocks using microbial carbonyl reductases

具有额外官能团的手性醇是用于合成对映体纯药物和其它化学品的有用结构单元。我们构建了一种新型的微生物羰基还原酶催化合成手性醇（4-氯-3-羟基丁酸乙酯，CHBE）的生物还原体系。在该系统中，大肠杆菌E.coli将共表达羰基还原酶的细胞（来自Sporobolomyces salmonicolor或Candida magnoliae）和葡萄糖脱氢酶（GDH）基因，因为GDH与大肠杆菌细胞中的羰基还原酶共同产生，通过将葡萄糖氧化成α-内酯，再生还原反应所需的NADPH。的共表达系统分别用于（R）-或（S）-CHBE的生产。这些酶催化NADPH依赖性立体特异性还原4-氯乙酰乙酸乙酯（CAAE）为（R）-或（S）-CHBE。还原反应在含有CAAE、葡萄糖、NADP^+和共表达AR 1（或S1）和GDH基因的大肠杆菌细胞的混合物中进行。使用表达AR 1和GDH基因的大肠杆菌细胞，将300 μ g/l的CAAE化学计量转化为（R）-CHBE（92% e.e.）。表达S1和GDH基因的大肠杆菌细胞产生约350 g/l的（S）-CHBE，光学纯度为96% e.e.。通过用其它合适的还原酶基因代替羰基还原酶基因，认为使用共表达羰基还原酶和辅因子再生酶（GDH）基因的大肠杆菌细胞的这种生物还原系统适用于各种其它手性醇的生产。

Chiral alcohols with additional functional groups are useful building blocks for the synthesis of enantiomeric pure pharmaceuticals and other chemicals. We constructed a novel bioreduction system for the production of chiral alcohols (ethyl 4-chloro-3-hydroxybutanoate, CHBE) using microbial carbonyl reductases as catalysts. In this system, E.coli transformant cells co-expressing a carbonyl reductase (from Sporobolomyces salmonicolor or Candida magnoliae) and glucose dehydrogenase (GDH) genes were used, because GDH co-produced with carbonyl reductase in E.coli cells regenerated NADPH required for the reduction reaction through oxidation of glucose to gluconolactone.Aldehyde reductase (ART) of S.salmonicolor or carbonyl reductase (S1) of C.magnoliae were applied to the co-expression system for (R)- or (S)-CHBE production, respectively. These enzymes catalyze NADPH-dependent stereospecific reduction of ethyl 4-chloroacetoacetate (CAAE) to (R)- or (S)-CHBE.The reduction reaction is carried out in a mixture containing CAAE, glucose, NADP^+ and E.coli cells co-expressing AR1 (or S1) and GDH genes. Using E.coli cells expressing AR1 and GDH genes, 300 g/l of CAAE was stoichiometrically converted to (R)-CHBE (92% e.e.). E.coli cells expressing S1 and GDH genes produced about 350 g/l of (S)-CHBE with optical purity of 96% e.e.This bioreduction system using E.coli cells co-expressing carbonyl reductase and cofactor-regeneration enzyme (GDH) genes is thought to be applicable to the production of various kinds of other chiral alcohols, by replacing the carbonyl reductase gene for other appropriate reductase genes.