大肠杆菌四氢嘧啶合成途径的构建与优化

Construction and Optimization of Ectoine Synthesis Pathway in Escherichia coli

摘要:

为了构建重组工程菌株发酵合成四氢嘧啶，解决野生型菌株对高盐环境的依赖，作者克隆了来自伸长盐单胞菌（Halomonas elongata ATCC 33173）的四氢嘧啶合成相关基因簇ectABC并在大肠杆菌（Escherichia coli，E. coli）中重构了四氢嘧啶的合成途径。通过宿主比较发现，与E. coli W3110、E. coli DH5α和高产天冬氨酸的大肠杆菌（命名为E. coli（asp））相比，E. coli BL21（DE3）更适用于四氢嘧啶的合成（185.23 mg/L）。进一步分析了不同拷贝数的表达系统对四氢嘧啶合成的影响，发现高拷贝的pRSFDuet-1为载体时产四氢嘧啶量最高，达267.3 mg/L。在此基础上，采用核糖体结合位点（RBS）优化策略，对四氢嘧啶合成途径3个酶EctA、EctB与EctC进行了组合优化表达，四氢嘧啶产量提高至521.24 mg/L。为强化前体天冬氨酸和天冬氨酸β-半醛的供给，对天冬氨酸激酶、天冬氨酸半醛脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸转氨酶和天冬氨酸裂解酶进行了过量表达。研究表明，单独强化天冬氨酸激酶的表达更有利于四氢嘧啶的合成（551.24 mg/L），为构建高产四氢嘧啶的工程菌种提供了新的策略。

Abstract:

Ectoine is widely used in medicine, cosmetics and other fields. This study aimed to construct recombinant Escherichia coli strain to synthesize ectoine to solve the dependence of wild-type strain on high salt environment. Ectoine synthesis encoding gene cluster ectABC was cloned from Halomonas elongata ATCC 33173 and reconstructed in Escherichia coli. In order to confirm a suitable host, four commonly used E. coli strains, i.e., E. coli W3110, E. coli DH5α and E. coli (asp) and E. coli BL21(DE3) were compared, and E. coli BL21(DE3) was found to be the best host for ectoine synthesis(185.23 mg/L). Further analysis of the effect of expression systems with different copy numbers on ectoine synthesis was carried out, and the results showed that pRSFDuet-1 with high copy number generated the highest titer of ectoine(267.3 mg/L). On this basis, the ribosomal binding site optimization strategy was introduced to optimize the expression of three enzymes including EctA, EctB and EctC, producing ectoine to 521.24 mg/L. To increase the supply of precursors aspartate and aspartate semialdehyde, aspartate kinase, aspartate hemialdehyde dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate lyase were overexpressed. The results demonstrated that the single overexpression of aspartate kinase was beneficial to ectoine biosynthesis and the production was eventually enhanced to 551.24 mg/L. The present study provides a new strategy to construct ectoine-producing strain.