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. coli¹⁵N-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.

中文翻译：

---

匹配的蛋白质界面可改善中链脂肪酸的生产

中链脂肪酸（MCFA）是合成中链化学物质（包括α-烯烃和二羧酸）的关键中间体。在细菌中，MCFA的微生物产生受到脂肪酰基ACP硫酯酶的活性和产物特性的限制。在这里，我们设计了一种异源细菌中链脂肪酰基-ACP硫酯酶，用于改善大肠杆菌中的MCFA生产。静电匹配异源中链巴氏不动杆菌脂肪酰基-ACP硫酯酶（AbTE）与内源性大肠杆菌脂肪酸ACP（E. coli）之间的界面通过将AbTE表面上的小的非极性氨基酸替换为带正电荷的氨基酸，可以使分泌的MCFA滴度提高3倍以上。与AbTE野生型相比，具有单个AbTE点突变体和最佳双突变体的大肠杆菌¹⁵ N-辛酰基-AcpP的核磁共振滴定显示出相互作用数量的逐步增加和显着增加。最好的AbTE突变体产生131 mg / L的MCFA，其中72小时后MCFA占所有分泌的脂肪酸链长度的80％。为了能够在将来筛选出更多的AbTE变异体，以进一步提高MCFA效价，我们显示了以前开发的基于G蛋白偶联受体（GPCR）的MCFA传感器可差异检测大肠杆菌分泌的MCFA 。表达不同的AbTE变体。这项工作表明，工程化异源酶的界面以更好地与内源宿主蛋白偶联是提高微生物产生的化学品滴度的有用策略。此外，这项工作表明，基于GPCR的传感器与生产者微生物无关，可以直接在生产者微生物上清液中检测化学物质，为MCFA生产微生物的传感器指导工程奠定了基础。