Consolidated Bioprocessing of Lignocellulosic Biomass Poplar to Produce Short-Chain Esters by Clostridium thermocellum

Consolidated bioprocessing (CBP) of lignocellulosic biomass using cellulolytic microorganisms presents a promising sustainable and economical biomanufacturing platform where enzyme production, biomass hydrolysis, and fermentation to produce biofuels, biochemicals, and biomaterials occur in a single step. However, understanding and redirecting metabolism of microorganisms to be compatible with CBP to produce non-native metabolites are limited. In this study, we metabolically engineered a cellulolytic thermophile Clostridium thermocellum and demonstrated its compatibility with CBP integrated with a mild Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment for conversion of hardwood poplar into short-chain esters (i.e., ethyl acetate, ethyl isobutyrate, isobutyl acetate, isobutyl isobutyrate) with broad use as solvents, flavors, fragrances, and biofuels. A recombinant C. thermocellum engineered with deletion of carbohydrate esterases and stable overexpression of a thermostable alcohol acetyltransferase improved the target esters production without compromised deacetylation activities. We discovered these esterases exhibited promiscuous thioesterase activities and their deletion improved ester production by increasing isobutanol flux and rerouting the native electron and carbon fermentative metabolism besides their known major function of ester degradation. The total ester production could be further enhanced up to 80-fold and the composition of short-chain esters could be modified by deleting lactate biosynthesis and/or CELF-pretreated poplar under different pretreatment conditions.