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

含氮手性化合物立体定向合成的微生物生产工艺

基本信息

批准号：
18380057
负责人：
KATAOKA Michihiko
金额：
$ 10.5万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

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.

手性化合物是生产药物等的有用中间体。由于具有较高的立体特异性和环境因素，手性化合物的生物催化生产一直受到人们的广泛关注。另一方面，含氮手性化合物对许多有用化合物的合成也很重要，如药物和农用化学品。(1)从农杆菌中分离得到3-醌二酮还原酶，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3-喹啉酮还原酶基因，并在大肠杆菌转化细胞中过表达。构建了共表达3-喹啉酮还原酶和葡萄糖脱氢酶基因的大肠杆菌，建立了与E. coil转化细胞不对称还原生产(R)-3-喹啉醇的体系。(2)红红球菌的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。为了改善氨基醇脱氢酶，采用易出错PCR进行随机诱变。最后，几种突变酶在高浓度d-伪麻黄碱存在下表现出活性。几种氨基醇均可诱导氨基醇脱氢酶。通过对氨基脱氢酶基因的遗传和转录分析，认为GntR家族的一个同源转录调控因子可能是红藓属氨基脱氢酶基因的负转录调控因子。