他克莫司工程菌的构建及初步发酵工艺优化  

Construction of Recombinant Engineering Strains Over-producing Tacrolimus and Preliminary Optimization of Fermentation Process

作　　者：王亚[1] 黄鹤[1] 边佳昕 周斌[1] 胡海峰[1] 

机构地区：[1]中国医药工业研究总院上海医药工业研究院,创新药物与制药工艺国家重点实验室,上海201203

出　　处：《中国医药工业杂志》2016年第2期152-157,共6页Chinese Journal of Pharmaceuticals

基　　金：十二五大平台项目(Z-2-3)

摘　　要：基于他克莫司的生物合成途径和机制,根据生物合成基因簇序列设计引物,通过PCR扩增天蓝色链霉菌(Streptomyces coelicolor)乙酰CoA羧化酶和游动放线菌(Actinoplanes sp.)N902-109赖氨酸环化脱氨酶/非核糖体肽合成酶(rapL/rapP)基因,将其整合到筑波链霉菌(Streptomyces tsukubaensis)No.9993基因组上,成功构建了分别过表达乙酰CoA羧化酶基因和rapL/rapP基因的重组工程菌,分别命名为SIPI-F002和SIPI-F003。试验结果显示,添加0.1%丙酸钠和0.01%油酸甲酯均可以促进工程菌SIPI-F002合成他克莫司,相应摇瓶产量为392和383mg/ml。调整发酵培养基复合碳源为4%糊精和2%可溶性淀粉,可以促进SIPI-F003发酵生产他克莫司,摇瓶产量达到694mg/ml。Based on the synthetic pathway and mechanism of tacrolimus, the genes encoding Streptomyces coelicolor acetyl-CoA carboxylase and Actinoplanes sp. N902-109 lysine cyclodeaminase/non-ribosomal peptide synthetase（rapL/rapP） were obtained by PCR amplification using the primers designed according to the biosynthetic gene clusters. Two recombinant engineering strains, named as SIPI-F002 and SIPI-F003, were constructed by integrating acetyl CoA carboxylase gene, or rapL/rapP gene into Streptomyces tsukubaensis No. 9993 genome, respectively. The results showed that the production of tacrolimus by recombinant strain SIPI-F002 was greatly improved through the addition of 0.1% sodium propionate and 0.01% oleic acid methyl ester into the fermentation medium, with a yield of 392 and 383 mg/ml in the shake flask, respectively. An optimized composite carbon source consisting of 4% dextrin and 2% soluble starch was verified to be effective for the promotion of the yield of tacrolimus by SIPI-F003, which could reach 694 mg/ml in the shake flask.

关 键 词：他克莫司 乙酰CoA羧化酶基因 rapL/rapP基因 基因工程菌 PCR扩增 

分 类 号：TQ465[化学工程—制药化工]