Establishment Of Gibson Assembly And Combinatorial Optimization Of MEP Pathway

One goal of metabolic engineering is improving yields with robust and simply executed methods.Nowadays it is not difficult to import a metabolic pathway into engineering strain.But the microbial biosynthesis efficiency is often limited by metabolic pathway flux unbalanced utilization and inappropriate enzymes activity.Previous research used to focus on single element optimization,such as overexpression of one rate-limiting enzyme.Due to the complexity of metabolic network and organism,this kind of optimization may not work well.With the rapid development of synthetic biology,two pivotal techniques: DNA cloning and assembly techniques;reliable and quantitative biological function elements,which used to limit the application of synthetic biology,have made great progress.Thanks to these new techniques,we can adopt semi-rationalizing design,combinatorially optimize the metabolic pathway to improve the yield of target products.Based on these new technologies and bioinformatics,it is a method to solve the current problem by semi-rationalizing the metabolic pathway and improving the yield of target products by means of combinatorial optimization.This work is Gibson Assembly and bicistronic design standard initiation elements.We chose MEP pathway as an example,combinatorially optimized 2 rate-limiting enzymes coding genes: dxs,idi.We constructed a combinatorial optimization library in limited steps and adopted a ?-carotene high-thro?ghput screening method to obtain a recombinant strain that improved 10.2 folds for the production of ?-carotene.This result demonstrated the effectiveness of this combinatorial optimization approach and the potential of pathway optimization engineering.We believe that this approach could be applicable for a range of secondary metabolic pathways.