科研之友
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摘要
Medium-chain fatty acids (MCFAs) are key intermediates in the synthesis of medium-chain chemicals including alpha-olefins and dicarboxylic acids. In bacteria, microbial production of MCFAs is limited by the activity and product profile of fatty acyl-ACP thioesterases. Here, we engineer a heterologous bacterial medium-chain fatty acyl-ACP thioesterase for improved MCFA production in Escherichia coli. Electrostatically matching the interface between the heterologous medium-chain Acinetobacter baylyi fatty acyl-ACP thioesterase (AbTE) and the endogenous E. coli fatty acid ACP (E. coli AcpP) by replacing small nonpolar amino acids on the AbTE surface for positively charged ones increased secreted MCFA titers more than 3-fold. Nuclear magnetic resonance titration of E. coli N-15-octanoyl-AcpP with a single AbTE point mutant and the best double mutant showed a progressive and significant increase in the number of interactions when compared to AbTE wildtype. The best AbTE mutant produced 131 mg/L of MCFAs, with MCFAs being 80% of all secreted fatty acid chain lengths after 72 h. To enable the future screening of larger numbers of AbTE variants to further improve MCFA titers, we show that a previously developed G-protein coupled receptor (GPCR)-based MCFA sensor differentially detects MCFAs secreted by E. coli expressing different AbTE variants. This work demonstrates that engineering the interface of heterologous enzymes to better couple with endogenous host proteins is a useful strategy to increase the titers of microbially produced chemicals. Further, this work shows that GPCR-based sensors are producer microbe agnostic and can detect chemicals directly in the producer microbe supernatant, setting the stage for the sensor-guided engineering of MCFA producing microbes.
- 出版日期2018-5
