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 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-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.

Across a wide range of biomarker detection schemes, carboxylterminated thiol self-assembled monolayers (COOH-SAMs) on Au are utilized to functionalize the sensing surface with a bioreceptor via amine coupling. However, commonly used COOH-SAM preparation methods result in large defect densities due to cooperative hydrogen bonding between carboxylic acid end groups, which in turn leads to large nonselective adsorption (NSA) of proteins to hydrophobic surfaces exposed by these defects. In this work, X-ray photoelectron spectroscopy is used to characterize the quality of COOH-SAMs by differentiating properly and improperly bound S groups. NSA of model small (lysozyme), medium (bovine serum albumin, BSA), and large (fibrinogen) proteins on COOH-SAMs is presented. Due to large NSA to COOH-SAMs, blocking is necessary for sensor reliability. However, conventional blocking techniques occur after functionalization (postblocking) and fail to prevent receptor NSA to the sensor surface during functionalization, which can cause receptor denaturation and allow the receptor to wash off the surface during later sensing. Here, a procedure is developed where the surface of COOH-SAMs is pretreated by blocking agents before functionalization. Preblocking can shield the COOH-SAM from oxidation, improve baseline stability, and prevent receptor denaturation. In this method, a preblocking protein orthogonal to the immunological system of interest is used to cover hydrophobic, nonselective sites on the sensor surface while still leaving carboxylic acid head groups available for covalent functionalization. Amine functionalization of BSA, antibody BSA, and antibody haptoglobin (aHp) is successfully completed after gelatin preblocking. Haptoglobin detection via surface plasmon resonance with a preblocked aHp sensor is shown to perform similarly to conventional postblocking, while demonstrating improved baseline stability and percentage of active receptors.

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