Endogenous carbohydrate esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose
- 25 April 2020
- journal article
- research article
- Published by Wiley in Biotechnology & Bioengineering
- Vol. 117 (7), 2223-2236
- https://doi.org/10.1002/bit.27360
Abstract
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.This publication has 50 references indexed in Scilit:
- Acetylation of woody lignocellulose: significance and regulationFrontiers in Plant Science, 2013
- Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellumBiotechnology for Biofuels, 2012
- High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic MicrobesApplied and Environmental Microbiology, 2011
- eQuilibrator--the biochemical thermodynamics calculatorNucleic Acids Research, 2011
- Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factorsProceedings of the National Academy of Sciences, 2010
- Characterization of a New Rhamnogalacturonan Acetyl Esterase from Bacillus halodurans C-125 with a New Putative Carbohydrate Binding DomainJournal of Bacteriology, 2008
- CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choiceNucleic Acids Research, 1994
- Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13Microbiology, 1991
- Basic local alignment search toolJournal of Molecular Biology, 1990
- Studies on the mechanism of fatty acid synthesisBiochimica et Biophysica Acta, 1959