文献类型：期刊文献

中文题名：棘白霉素脱酰酶基因工程菌的构建及应用研究

英文题名：The Construction and Application of EchDA Genetic Engineering Bacteria

作者：赵国玲[1];陶欣艺[1];王风清[1];魏东芝[1]

机构：[1]华东理工大学生物反应器国家重点实验室,上海200237

年份：2015

卷号：35

期号：1

起止页码：67

中文期刊名：中国生物工程杂志

外文期刊名：China Biotechnology

收录：CSTPCD;;北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；

基金：国家"863"科技重大专项资助项目(2012ZX09304009)

语种：中文

中文关键词：变铅青链霉菌;棘白霉素脱酰酶;基因工程;犹他游动放线菌

外文关键词：Streptomyces lividans TK24;Echinocandindeacylase;Genetic engineering;Actinoplanesutahensis

摘要：目的:以Streptomyces lividans TK24为表达宿主,构建高效表达棘白霉素脱酰酶(Ech DA)的基因工程菌。方法:从Actinoplanesutahensis NRRL 12052中获取Ech DA的基因片段,连接到链霉菌表达载体p NW-S1,采用诱导型启动子Plac与组成型启动子Perm E*和PSau3A,依次构建基因工程菌株ZNW-S11、ZNW-S12和ZNW-S13。通过分析启动子对Ech DA分泌表达的影响,确定工程菌的表达能力,随后完善发酵放大的工艺过程,将工程菌株发酵放大到300 L发酵罐水平。结果:成功实现了Ech DA在S.lividans TK24中的诱导分泌表达;在摇瓶水平,Ech DA的酶活可达到125U/L,在24h内可实现对15g/L米卡芬净前体FR901379的完全转化;在300L发酵罐水平,Ech DA的酶活可达到80U/L,可用于10g/LFR901379的完全转化。当前,国内在工业生产中仍使用原始菌A.utahensis来表达Ech DA,而开发的Ech DA基因工程菌,与之相比在生产效率上有了大幅度提升,有助于改良当前棘白霉素类抗生素的生产工艺。
The EchDA was systematically overexpressed by genetic engineering in the heterologous hosts Streptomyces lividans TK24. Mature peptide coding fragement of Ech DA was amplified from Actinoplanesutahensis NRRL 12052. The expression plasmids p NW-S11,p NW-S12 and p NW-S13 are constructed by inserting Ech DA,Perm E＊promoter and PSau3 Apromoter into vector p NW-S1. As a result,The Ech DA was secretion expressed after induction with IPTGand the enzyme activity can reach 125 U / L under the best conditions for industrialized production. With optimization of the reaction conditions with 15 g / Liter FR901379,the molar conversion can achieve 100% within 24 hours. The highest enzyme activity by 300 L fermentation tank can reach 80 U / L,and the molar conversion can achieve100% with 10 g / L FR901379. Ech DA in industry have been relying on fermentationof original producer,Actinoplanesutahensis,which have low yield and activity. The construction of recombinant Ech DA producing strain will be must to improve the current production process of echinocandin antifungal drugs.