产鼠李糖脂生物表面活性剂大肠杆菌的构建与优化  

Construction and optimization of Escherichia coli for producing rhamnolipid biosurfactant

作　　者：巩志金 彭彦峰[2] 张煜婷 宋国田 陈五九[2] 贾士儒[1] 王钦宏[2] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所中国科学院系统微生物工程重点实验室,天津300308

出　　处：《生物工程学报》2015年第7期1050-1062,共13页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2014AA022104);天津市科学技术委员会工业生物技术专项(No.12ZCZDSY13000)资助~~

摘　　要：目前鼠李糖脂生物表面活性剂主要由条件致病的铜绿假单胞菌生产获得,从而影响工业应用。为了开发一种相对安全的鼠李糖脂生产菌,将带有不同强度组成型合成启动子的鼠李糖基转移酶基因(Rhamnosyltransferase gene,rhl AB)以单、中、高3种拷贝数分别在大肠杆菌ATCC 8739中异源表达,实现了不同产量的鼠李糖脂异源合成。对rhl AB基因和rha BDAC基因簇(TDP-L-鼠李糖合成的基因簇)进一步利用合成启动子进行组合调控,筛选获得了最优生产鼠李糖脂工程菌——大肠杆菌TIB-RAB226。对大肠杆菌TIB-RAB226进行发酵温度优化,鼠李糖脂产量达到124.3 mg/L,是优化前的1.17倍。通过分批补料发酵,12h时鼠李糖脂产量达到209.2 mg/L。对发酵产物进行高效液相色谱-质谱联用技术分析,共检出相对含量变化的5类质核比不同的鼠李糖脂同系物。本研究可为异源合成产鼠李糖脂提供重要参考。Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase（rhl AB） gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhl AB gene and rha BDAC gene cluster for d TDP-l-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain—E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry（LC-MS） analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.

关 键 词：鼠李糖脂 鼠李糖基转移酶 rhaBDAC基因簇 组成型合成启动子 组合调控 大肠杆菌 

分 类 号：Q939.9[生物学—微生物学]