Targeted Engineering of Biochemicals and Drug Precursors Guided By in Vitro Reconstitution

Metabolic Engineering has become a major research field in the past several years. Several milestone and representative successes have been achieved for bioproduction in bacteria. However, due to the lack of steady-state kinetic data as well as a dearth of information regarding the factors controlling the overall flux through many multistep biosynthetic pathways, even the successful cases of amorphadiene production provide little guidance as to how much room for improvement has been left, or directions for further engineering.

Our strategy is that we reconstitute the biosynthetic pathway in vitro, and we can test the contributions of cofactors, substrates and each components. After we know much better about this system, we transfered this information back in vivo and guided us to engineer this pathway in bacteria. So we call this strategy "Targeted Engineering". In our first case, we successfully in vitro reconstituted E. coli fatty acid biosynthesis system and these kinetic information are ont only help us to understand fatty acid biosynthesis better, but also guide us to improve fatty acids production (Yu X, et.al. PNAS 2011). Furthermore, we reconstituted fatty alcohol synthetic pathway, and we used this strategy to identify the rate-limited step and select the suitable condidates. We prove that the propose candidates can directly take fatty acyl-ACPs as the substrates, and this pathway save more ATPs than current available pathways (Liu R, et.al. Metabolic Engineering 2013).

To prove our concept, we transfered this targeted engineering strategy to terpenoids overproduction. 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 hrs 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. In the future, this  targeted engineering strategy will be applied into many pathways.