초록
<P>Medium-chain fatty acids (MCFAs) are key intermediates in the synthesis of medium-chain chemicals including α-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 <I>Escherichia coli</I>. Electrostatically matching the interface between the heterologous medium-chain <I>Acinetobacter baylyi</I> fatty acyl-ACP thioesterase (AbTE) and the endogenous <I>E. coli</I> fatty acid ACP (<I>E. coli</I> 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 <I>E. coli</I><SUP>15</SUP>N-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 <I>E. coli</I> 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.</P><BR>[FIG OMISSION]</BR>