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

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, rhlAB) 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rhlAB基因和rhaBDAC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，12 h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。

Abstract:

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 (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-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.