利用RBS序列策略在大肠杆菌中高效合成异戊二烯

Improvement of isoprene production in Escherichia coli by rational optimization of RBSs

[目的]提高现有产异戊二烯大肠杆菌工程菌株合成异戊二烯的产量。[方法]首先对异戊二烯合成酶这一关键酶进行RBS序列优化,提高了异戊二烯的产量;然后通过构建异戊二烯焦磷酸异构酶和异戊二烯合酶的融合蛋白,研究这两个蛋白间的交互作用,将异戊二烯的产量提高了40%;最后通过RBS策略,对甲羟戊酸下游途径的非关键酶进行表达调控,继续提高了异戊二烯的产量。[结果]利用优化RBS序列和融合蛋白策略,异戊二烯的产量最终提高到60 mg/L,比原始工程菌株提高了10倍。[结论]RBS序列优化可能成为菌株代谢工程改造的有力手段。此外,为了提高工程菌株的产量,不仅要关注关键酶,还要关注非关键酶。

[Objective]To improve the production of isoprene in the engineered E.coli strain.[Method]Firstly,we optimized the RBS sequence of isoprene synthase,a key enzyme in isoprene productive system,and increased the yield of isoprene.Then we focused on the protein interaction between isopentenyl diphosphate isomerase and isoprene synthase,and increased isoprene production by 40%by using the fusion protein strategy.Finally,RBS strategy was adopted to regulate the expression of non-key enzymes in the downstream of mevalonate pathway,which further increased the yield of isoprene.[Result]By utilization of RBS optimization and the fusion protein strategy,the yield of isoprene was finally increased to 60 mg/L,which was 10 times higher than that of the original strain.[Conclusion]RBS optimization could be a powerful strategy for metabolic engineering of strain.Moreover,to increase the production of engineered strain,our attention should not only be focused on the key enzymes,but also on the non-key enzymes.