Intermediate-sensor assisted push–pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli
•An L-Trp biosensor was developed based on native control of the tnaCAB operon.•Deoxyviolacein pathway was divided into two parts, connected by an intermediate.•The senor was used for screening upstream module library based on enhanced output.•Screening downstream enzyme mutant library was based on reduced sensor output•With this push/pull approach, 1.92g/L deoxyviolacein titer was obtained.
Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push–pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an l-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with l-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes. By this means, we increased the deoxyviolacein titer 4.4-fold (1.92 g/L), which represents the greatest deoxyviolacein production reported. This work suggests that a biosynthetic pathway can be enhanced to produce a value-added secondary metabolite(s) without available end-product screening method by using a central metabolic junction molecule biosensor(s).
Journal: Metabolic Engineering - Volume 33, January 2016, Pages 41–51