Endogenous esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose

Abstract

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.

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

“…2A). Due to optimal growth of C. thermocellum at elevated temperatures (>50℃) and its unique capability to make cellulolytic enzymes (e.g., esterases) to degrade lignocellulosic biomass, a combined heterologous expression of a thermostable AAT and deletion of the select endogenous esterdegrading carbohydrate esterases (CEs) are required to activate the short-chain ester biosynthesis pathways in C. thermocellum 29,38 . Since the biosynthesis of the target alcohols and acyl-CoAs is important for the anabolic and redox metabolisms of C. thermocellum, which is responsive to both genetic and environmental perturbations [39][40][41][42][43] , we first investigated how these two genetic manipulations activated the biosynthesis of short-chain esters and affected cell growth, biomass degradation, and redox metabolism using the model cellulose substrate Avicel, which is a commercial, pure crystalline cellulose derived from wood pulp.…”

“…The esterase-deficient strain HSCT2105 produced 0.29 g/L isobutanol, about 3.0-fold higher than the parent strain HSCT0102 (0.11 g/L) without two CE deletions 38 . Likewise, the esterasedeficient strain HSCT2108 produced 0.81 g/L of isobutanol, about 2.5-fold higher than the parent strain HSCT0103 (0.33 g/L).…”

“…While the effects of deleting these two CEs on lignocellulose degradation and short-chain ester hydrolysis were previously reported 38,44 , it was still unknown of how their deletion improved both the target alcohol and ester production besides their main function. Since many esterases also function as phospholipases, thioesterases, and/or proteases 45,46 , we hypothesized that the two CEs might have exhibited promiscuous activities affecting the endogenous isobutyryl-CoA metabolism.…”

Follow your nose ‐ Traveling the world of odorants in new cars

“…The group of the identified esters is well‐known odorants in food, leather, and plastics. The fruity‐smelling substances are used as solvents for the production of materials of the vehicle interior or can be generated during processing by esterification of alcohols with acids 33 . Butyl acrylate is a monomer in the production of acrylic resins and acrylic coatings and can potentially be released from these materials into the vehicle cabin 34 …”

“…The fruity-smelling substances are used as solvents for the production of materials of the vehicle interior or can be generated during processing by esterification of alcohols with acids. 33 Butyl acrylate is a monomer in the production of acrylic resins and acrylic coatings and can potentially be released from these materials into the vehicle cabin. 34 The coconut-smelling γ-nonalactone, on the other side, can be generated by oxidation in plastics.…”