Structure-guided reshaping of the acyl binding pocket of 'TesA thioesterase enhances octanoic acid production in E. coli
Structure-guided reshaping of the acyl binding pocket of 'TesA thioesterase enhances octanoic acid production in E. coli
复制标题
'TesA 硫酯酶酰基结合口袋的结构引导重塑可增强大肠杆菌中的辛酸产量
DOI：
10.1016/j.ymben.2020.04.010
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发表时间：
2020-09-01
影响因子：
8.4
通讯作者：
Yang, Guang-Yu
中科院分区：
文献类型：
--
作者：
Deng, Xi;Chen, Liuqing;Yang, Guang-Yu
中链脂肪酸（C6 - C10）由于与长链脂肪酸相比具有独特的性质，包括低熔点和相对较高的碳转化率，最近受到了广泛关注。硫酯酶能够催化酰基 - ACP（酰基载体蛋白）水解以释放游离脂肪酸（FAs），它调节细菌和酵母发酵培养物中总的脂肪酸产量以及酰基链长度分布。这些酶通常更倾向于较长链的底物。在此，为了提高细菌对中链脂肪酸的产量，我们对大肠杆菌硫酯酶‘TesA的底物结合口袋中的多个残基进行了基于结构的突变筛选。我们的假设是增强对中链酰基底物的底物选择性将促进中链脂肪酸的总体产量，通过实验我们证实了这一假设，即发现用更疏水的残基替换口袋底部的残基可极大地提高‘TesA对C8底物的选择性。具体而言，两轮饱和诱变导致鉴定出‘TesARD - 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，在β - 氧化途径受阻的大肠杆菌菌株中重组表达该变体，使得体内辛酸（C8）的生产效价提高了1030%。当该菌株在5升补料分批式生物反应器中发酵时，它产生了2.7克/升的游离C8（摩尔分数为45%）和7.9克/升的总游离脂肪酸，这是迄今为止使用大肠杆菌II型脂肪酸合成途径所报道的最高游离C8效价。因此，通过改变多个残基的疏水性来重塑细菌硫酯酶的底物结合口袋，改变了底物选择性，进而改变了细胞内脂肪酸产物的分布。我们的研究证明了这种策略对于提高作为发酵产物的具有工业吸引力的中链脂肪酸的效价的相关性。
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 `TesARD-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 beta-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.