Medium‐chain esters are versatile chemicals with broad applications as flavors, fragrances, solvents, and potential drop‐in biofuels. Currently, these esters are largely produced by the conventional chemical process that uses harsh operating conditions and requires high energy input. Alternatively, the microbial conversion route has recently emerged as a promising platform for sustainable and renewable ester production. The ester biosynthesis pathways can utilize either lipases or alcohol acyltransferase (AAT), but the AAT‐dependent pathway is more thermodynamically favorable in an aqueous fermentation environment. Even though a cellulolytic thermophile Clostridium thermocellum harboring an AAT‐dependent pathway has recently been engineered for direct conversion of lignocellulosic biomass into esters, the production is not efficient. One potential bottleneck is the ester degradation caused by the endogenous carbohydrate esterases (CEs) whose functional roles are poorly understood. The challenge is to identify and disrupt CEs that can alleviate ester degradation while not negatively affecting the efficient and robust capability of C. thermocellum for lignocellulosic biomass deconstruction. In this study, by using bioinformatics, comparative genomics, and enzymatic analysis to screen a library of CEs, we identified and disrupted the two most critical CEs, Clo1313_0613 and Clo1313_0693, that significantly contribute to isobutyl acetate degradation in C. thermocellum. We demonstrated that an engineered esterase‐deficient C. thermocellum strain not only reduced ester hydrolysis but also improved isobutyl acetate production while maintaining effective cellulose assimilation.
The cover image is based on the Original Article Endogenous carbohydrate esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose by Cong Trinh, Hyeongmin Seo, and Preseton Nicely, https://doi.org/10.1002/bit.27360.
13Medium chain esters are potential drop-in biofuels and versatile chemicals. Currently, these 14 esters are largely produced by the conventional chemical process that uses harsh operating 15 conditions and requires high energy input. Alternatively, the microbial conversion route has 16 recently emerged as a promising platform for sustainable and renewable ester production. The 17 ester biosynthesis pathways can utilize either esterases/lipases or alcohol acyltransferase (AAT), 18 but the AAT-dependent pathway is more thermodynamically favorable in aqueous fermentation 19 environment. Even though cellulolytic thermophiles such as Clostridium thermocellum harboring 20 the engineered AAT-dependent pathway can directly convert lignocellulosic biomass into esters, 21 the production is currently not efficient and requires optimization. One potential bottleneck is the 22 31 lignocellulosic biomass by the cellulolytic thermophile C. thermocellum, yet some are potential 32 ester degraders in a microbial ester production system. Currently, the functional roles of CEs for 33 hydrolyzing medium chain esters and negatively affecting the ester microbial biosynthesis are not 34 well understood. This study discovered novel CEs responsible for isobutyl acetate degradation in 35 3 C. thermocellum and hence identified one of the critical bottlenecks for direct conversion of 36 lignocellulosic biomass into esters. 37 38 Keywords: Carbohydrate esterase, bioester, consolidated bioprocessing, Clostridium 39 thermocellum, ethyl isobutyrate, isobutyl acetate, isobutyl isobutyrate, ethyl acetate. 40 A developed high-throughput screening enabled identification of two esterases hydrolyzing 102 isobutyl acetate 103Even though the AAT-dependent pathway is shown to be feasible in C. thermocellum (16), 104 the production is inefficient. One of the potential bottlenecks might be due to relatively high 105 endogenous activity of esterases in C. thermocellum that limit the ester biosynthesis. Even though 106 C. thermocellum harnesses a large number of CEs to deacetylate O-acetyl groups of hemicelluloses 107 responsible for lignocellulosic biomass deconstruction (22-24), their functional roles for ester 108 degradation are largely unknown. Therefore, it is critical to identify CEs hydrolyzing isobutyl 109 control, the reaction with Novozyme 435, an immobilized CalB lipase, resulted in a dramatic pH 129 change, validating that the hydrolysis of isobutyl acetate lowers pH. We found that only 130Clo1313_0613 and Clo1313_0693 out of the seven CEs characterized could hydrolyze isobutyl 131 acetate with pH changes of -0.9 and -0.4, respectively. The hydrolysis was further confirmed with 132 detection of isobutanol and acetate by HPLC analysis. For the negative control, we observed that 133 157 chain esters: Enzymes, pathways, and applications.
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