Medium-chain fatty acids (C6–C10) have attracted much attention recently for their unique properties compared to their long-chain counterparts, including low melting points and relatively higher carbon conversion yield. Thioesterase enzymes, which can catalyze the hydrolysis of acyl-ACP (acyl carrier protein) to release free fatty acids (FAs), regulate both overall FA yields and acyl chain length distributions in bacterial and yeast fermentation cultures. These enzymes typically prefer longer chain substrates. Herein, seeking to increase bacterial production of MCFAs, we conducted structure-guided mutational screening of multiple residues in the substrate-binding pocket of the E. coli thioesterase enzyme ‘TesA. Confirming our hypothesis that enhancing substrate selectivity for medium-chain acyl substrates would promote overall MCFA production, we found that replacement of residues lining the bottom of the pocket with more hydrophobic residues strongly promoted the C8 substrate selectivity of ‘TesA. Specifically, two rounds of saturation mutagenesis led to the identification of the ‘TesA^RD−2 variant that exhibited a 133-fold increase in selectivity for the C8-ACP substrate as compared to C16-ACP substrate. Moreover, the recombinant expression of this variant in an E. coli strain with a blocked β-oxidation pathway led to a 1030% increase in the in vivo octanoic acid (C8) production titer. When this strain was fermented in a 5-L fed-batch bioreactor, it produced 2.7 g/L of free C8 (45%, molar fraction) and 7.9 g/L of total free FAs, which is the highest-to-date free C8 titer to date reported using the E. coli type II fatty acid synthetic pathway. Thus, reshaping the substrate binding pocket of a bacterial thioesterase enzyme by manipulating the hydrophobicity of multiple residues altered the substrate selectivity and therefore fatty acid product distributions in cells. Our study demonstrates the relevance of this strategy for increasing titers of industrially attractive MCFAs as fermentation products.

中链脂肪酸（C6-C10）最近因其与长链脂肪酸相比的独特性质而备受关注，包括低熔点和相对较高的碳转化率。硫酯酶可催化酰基-ACP（酰基载体蛋白）水解以释放游离脂肪酸 (FA)，调节细菌和酵母发酵培养物中的总 FA 产量和酰基链长度分布。这些酶通常更喜欢较长链的底物。在此，为了增加 MCFA 的细菌产量，我们对大肠杆菌的底物结合口袋中的多个残基进行了结构引导的突变筛选硫酯酶'TesA。证实了我们的假设，即提高对中链酰基底物的底物选择性将促进整体 MCFA 的产生，我们发现用更疏水的残基替换口袋底部的残基强烈促进了 'TesA 的 C8 底物选择性。具体而言，两轮饱和诱变导致鉴定出“TesA ^RD-2”变体，与 C16-ACP 底物相比，该变体对 C8-ACP 底物的选择性提高了 133 倍。此外，该变体在β-氧化途径受阻的大肠杆菌菌株中的重组表达导致体内β-氧化途径增加 1030% 。辛酸 (C8) 生产效价。当该菌株在 5-L 分批补料生物反应器中发酵时，它产生 2.7 g/L 的游离 C8（45%，摩尔分数）和 7.9 g/L 的总游离 FA，这是迄今为止最高的游离 FA迄今为止，使用大肠杆菌II 型脂肪酸合成途径报道的 C8 滴度。因此，通过操纵多个残基的疏水性来重塑细菌硫酯酶的底物结合口袋会改变底物选择性，从而改变细胞中的脂肪酸产物分布。我们的研究证明了该策略对于提高具有工业吸引力的 MCFA 作为发酵产品的效价的相关性。