In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli.
Approaches using metabolic engineering and synthetic biology to overproduce terpenoids, such as the precursors of taxol and artemisinin, in microbial systems have achieved initial success. However, due to the lack of steady-state kinetic information and incomplete understanding of the terpenoid biosynthetic pathway, it has been difficult to build a highly efficient, universal system. Here, we reconstituted the mevalonate pathway to produce farnesene (a precursor of new jet fuel) in vitro using purified protein components. The information from this in vitro reconstituted system guided us to rationally optimize farnesene production in E. coli by quantitatively overexpressing each component. Targeted proteomic assays and intermediate assays were used to determine the metabolic status of each mutant. Through targeted engineering, farnesene production could be increased predictably step by step, up to 1.1 g/L (∼ 2,000 fold) 96 h after induction at the shake-flask scale. The strategy developed to release the potential of the mevalonate pathway for terpenoid overproduction should also work in other multistep synthetic pathways.
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, PR China.
Fayin
F
Zhu
Xiaofang
X
Zhong
Mengzhu
M
Hu
Lei
L
Lu
Zixin
Z
Deng
Tiangang
T
Liu
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D015870:Gene Expression; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008798:Mevalonic Acid / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D005231:Fatty Acids, Unsaturated; D050505:Mutant Proteins; C007702:farnesoic acid; D008798:Mevalonic Acid
biofuel; farnesene; in vitro reconstitution; mevalonate pathway; targeted engineering; terpenoid