6LFC

E. coli Thioesterase I mutant DG

6LFC の概要

• エントリーDOI: 10.2210/pdb6lfc/pdb
• 分子名称: Acyl-CoA thioesterase I also functions as protease I (2 entities in total)
• 機能のキーワード: thioesterase, mutant, hydrolase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 122473.15

構造登録者

Deng, X.,Chen, L.,Yang, G. (登録日: 2019-12-01, 公開日: 2020-07-15, 最終更新日: 2023-11-22)

主引用文献

Deng, X.,Chen, L.,Hei, M.,Liu, T.,Feng, Y.,Yang, G.Y.
Structure-guided reshaping of the acyl binding pocket of 'TesA thioesterase enhances octanoic acid production in E. coli.
Metab. Eng., 61:24-32, 2020

PubMed Abstract: 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 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.

PubMed: 32339761
DOI: 10.1016/j.ymben.2020.04.010

実験手法

X-RAY DIFFRACTION (2.7 Å)