构建多酶复合体强化异戊二烯生物合成

In view of the problems of insufficient precursors, accumulation of toxic intermediate metabolites and product loss in the synthesis of isoprene in Escherichia coli cell factories, we used synthetic biology and metabolic engineering methods to express isoprene synthase from Populus alba in Escherichia coli and overexpressed single or multiple genes of Dxs, Dxr and IspD in the form of polycistronic to strengthen the endogenous MEP pathway of Escherichia coli. In order to shorten the transport distance and time of intermediate metabolites between enzyme molecules and enhance the substrate channeling effect of the pathway, the protein scaffold strategy was applied to Dxs, Dxr and IspD enzyme colocalization in the MEP pathway to construct a multi-enzyme complex for isoprene synthesis, and obtained the engineered strain BL21(DE3) -ScaS produced isoprene yield of 24 mg/L, which was 35.7% higher than the engineered strain BL21(DE3)-FreeS in free enzyme form. And through the stability study of the engineered strain BL21(DE3) -ScaS plasmid, it was found that the supplementation of antibiotics during the fermentation process could significantly improve the stability of the incompatible recombinant plasmids pET28aDxsDxrIspD and pET21b-GSP-IspS. At the same time, flux balance analysis (FBA) was used to explore the metabolic flux distribution of key enzymes in the synthetic pathway, the effectiveness of the above strategies is proved theoretically, which provides a reference for the development and application of isoprene microbial cell factories.