Novel multi-purpose bioreduction system for the large-scale production of chiral alcohols

手性醇是许多药物和化学品的有用中间体。酶法不对称还原前手性羰基化合物是制备手性醇的一种很有前途的方法。人们曾多次尝试构建手性醇工业生产的生物还原体系。本研究的重点是利用大肠杆菌转化共表达羰基还原酶和辅因子再生酶基因，建立一种新的生物还原体系。该生物还原系统通过用其他合适的酶基因代替羰基还原酶基因进行羰基还原，适用于生产许多其他有用的手性醇。建立了具有不同底物特异性和立体特异性的羰基还原酶文库。因此，该生物还原体系可作为不对称还原反应的多用途催化剂。除羰基还原酶反应外，该共表达体系可能适用于需要NAD(P)H作为辅助因子的反应，如C=C键还原、还原性胺化反应等。从C. macedoniensis和S. cerevisiae中发现老黄酶催化烯酮化合物C=C键的不对称氢化。定点突变、随机突变、DNA洗牌等直接进化技术是改善或扩展羰基还原酶功能的有效方法。此外，由基因组计划等确定的编码NAD(P) h依赖性羰基还原酶（即氧化还原酶或假设的氧化还原酶）的公共基因数据库也可能是扩展羰基还原酶库的良好来源。这些试验与传统的酶筛选相结合可能更有用和有效。

Chiral alcohols are useful intermediates for many kinds of pharmaceuticals and chemicals. Enzymatic asymmetric reduction of prochiral carbonyl compounds is a promising method for producing chiral alcohols. There have been many attempts to construct bioreduction systems for the industrial production of chiral alcohols. This study focuses on establishment of a novel bioreduction system using an E. coli transformant co-expressing carbonyl reductase and cofactor regeneration enzyme genes. This bioreduction system is applicable to the production of many other useful chiral alcohols, by replacing the carbonyl reductase gene with other appropriate enzyme genes for carbonyl reduction. A good library of carbonyl reduction enzymes with different substrate specificities and stereospecificities has been made. Therefore, this bioreduction system could be useful as a multi-purpose catalyst for the asymmetric reduction reactions.In addition to the carbonyl reductase reactions, this co-expression system might be applicable to the reactions requiring NAD(P)H as a cofactor, such as C=C bond reductions, reductive amination and so on. Old yellow enzyme from C. macedoniensis or S. cerevisiae was found to catalyze the asymmetric hydrogenation of C=C bond of enone compounds.The direct evolution technique, such as site-directed mutagenesis, random mutagenesis, and DNA shuffling, is an effective method for improvement or expansion of carbonyl reductase functions. Furthermore, public databases of genes encoding NAD(P)H-dependent carbonyl reductases (i.e., oxidoreductases or hypothetical oxidoreductases), determined by genome projects and so on, might also be good sources for expansion of the carbonyl reductase library. These trials might be more useful and effective in combination with traditional screening of enzymes.