Microbial production process for stereospecific synthesis of nitrogen-containing chiral compounds

手性化合物是生产药物等的重要中间体。由于具有较高的立体专一性和环境因素，生物催化合成手性化合物引起了人们的极大关注。另一方面，含氮的手性化合物也是合成医药、农用化学品等许多有用化合物的重要原料。(1)从根癌农杆菌中分离得到3-喹诺酮还原酶，并对其进行了详细的鉴定。该酶属于SDR超家族。根据该酶的氨基酸序列，克隆了编码3-喹诺酮还原酶的基因，并在大肠杆菌转化细胞中高效表达。在此基础上，构建了共表达3-喹诺酮还原酶和葡萄糖脱氢酶基因的大肠杆菌，并建立了利用转化细胞不对称还原生产(R)-3-喹诺酮醇的体系。(2)红球红球菌氨基醇脱氢酶是一种很有前途的生产d-伪麻黄碱的酶。但d-伪麻黄碱对该酶有抑制作用。为了提高氨基醇脱氢酶的效率，采用容易出错的聚合酶链式反应进行了随机突变。最后，对几种突变酶在高浓度d-伪麻黄碱存在下的活性进行了测定。氨基醇脱氢酶是由几种氨基醇诱导的。通过对氨基醇脱氢酶基因的遗传和转录分析，发现一个与GntR家族转录调控基因同源的ORF可能是红腹鱼氨基醇脱氢酶基因的负转录调控因子。

Chiral compounds are useful intermediates for the production of pharmaceuticals and so on. Because of high stereospecificity and environmental aspects, biocatalytic production of chiral compounds has been attracting considerable attention. On the other hand, nitrogen-containing chiral compounds are also important for the synthesis of many useful compounds, such as pharmaceuticals and agrochemicals.(1) 3-Quinuclidinone reductase was isolated from Agrobacterium tumefaciens, and characterized in some details. The enzyme belongs to SDR superfamily. Based on amino acid sequence of the enzyme, the gene encoding 3-quinuclidinone reductase was cloned and overexpressed in E. coli transformant cells. Furthermore, E. coli coexpressing 3-quinuclidinone reductase and glucose dehydrogenase genes was constructed, and the production system for (R)-3-quinuclidinol via asymmetric reduction with E. coil transformant cells was established.(2) Aminoalcohol dehydrogenase from Rhodococcus erythropolis is a promising enzyme for the production of d-pseudoephedrine. However, the enzyme was inhibited by d-pseudoephedrine. To improve aminoalcohol dehydrogenase, a random mutagenesis was performed by error-prone PCR. Finally, several mutant enzymes showing the activity in the presence of high concentration of d-pseudoephedrine. Aminoalcohol dehydrogenase was induced by several aminoalcohols. Through the genetic and transcriptional analysis of aminoalcohol dehydrogenase gene, a homologous orf of the GntR family transcriptional regulators was suggested to be a negative transcriptional regulator of aminoalcohol dehydrogenase gene in R. erythropolis.