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

2020 ◽  
Vol 117 (7) ◽  
pp. 2223-2236 ◽  
Author(s):  
Hyeongmin Seo ◽  
Preston N. Nicely ◽  
Cong T. Trinh
Keyword(s):  
Clostridium Thermocellum ◽  
Acetate Production ◽  
Isobutyl Acetate ◽  
Carbohydrate Esterases

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

2019 ◽  
Author(s):  
Hyeongmin Seo ◽  
Preston N. Nicely ◽  
Cong T. Trinh
Keyword(s):  
Lignocellulosic Biomass ◽  
Clostridium Thermocellum ◽  
Ester Hydrolysis ◽  
Acetate Production ◽  
Functional Roles ◽  
Medium Chain ◽  
Isobutyl Acetate ◽  
Acetate Degradation ◽  
Dependent Pathway ◽  
Carbohydrate Esterases

ABSTRACTMedium chain esters are potential drop-in biofuels and versatile chemicals. 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 esterases/lipases or alcohol acyltransferase (AAT), but the AAT-dependent pathway is more thermodynamically favorable in aqueous fermentation environment. Even though cellulolytic thermophiles such as Clostridium thermocellum harboring the engineered AAT-dependent pathway can directly convert lignocellulosic biomass into esters, the production is currently not efficient and requires optimization. One potential bottleneck is the ester degradation caused by the endogenous carbohydrate esterases (CEs) whose functional roles are poorly understood. In this study, we developed a simple, high-throughput colorimetric assay to screen the endogenous esterases of C. thermocellum responsible for ester hydrolysis. We identified, characterized, and disrupted two critical endogenous esterases that significantly contributes to isobutyl acetate degradation in C. thermocellum. We demonstrated that not only did the engineered esterase-deficient strain alleviate ester hydrolysis but also helped improve isobutyl acetate production while not affecting its robust metabolism for effective cellulose assimilation.IMPORTANCECarbohydrate esterases (CEs) are important enzymes in the deconstruction of lignocellulosic biomass by the cellulolytic thermophile C. thermocellum, yet some are potential ester degraders in a microbial ester production system. Currently, the functional roles of CEs for hydrolyzing medium chain esters and negatively affecting the ester microbial biosynthesis are not well understood. This study discovered novel CEs responsible for isobutyl acetate degradation in C. thermocellum and hence identified one of the critical bottlenecks for direct conversion of lignocellulosic biomass into esters.

scholarly journals Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables thermophilic isobutyl acetate production directly from cellulose by Clostridium thermocellum

2019 ◽  
Author(s):  
Hyeongmin Seo ◽  
Jong-Won Lee ◽  
Sergio Garcia ◽  
Cong T. Trinh
Keyword(s):  
Lignocellulosic Biomass ◽  
Clostridium Thermocellum ◽  
Consolidated Bioprocessing ◽  
Potential Drop ◽  
Chloramphenicol Acetyltransferase ◽  
Single Mutation ◽  
Acetate Production ◽  
Isobutyl Acetate ◽  
Ester Biosynthesis ◽  
Conserved Region

ABSTRACTBackgroundEsters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows thermophilic production with advantageous downstream product separation. However, due to the limited thermal stability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for thermophilic ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum.ResultsIn this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperature. We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, we demonstrated the engineered C. thermocellum could produce isobutyl acetate directly from cellulose.ConclusionsThis study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.

scholarly journals Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Hyeongmin Seo ◽  
Jong-Won Lee ◽  
Sergio Garcia ◽  
Cong T. Trinh
Keyword(s):  
Lignocellulosic Biomass ◽  
Clostridium Thermocellum ◽  
Elevated Temperatures ◽  
Consolidated Bioprocessing ◽  
Chloramphenicol Acetyltransferase ◽  
Single Mutation ◽  
Acetate Production ◽  
Isobutyl Acetate ◽  
Ester Biosynthesis ◽  
Conserved Region

Abstract Background Esters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows high-temperature fermentation with advantageous downstream product separation. However, due to the limited thermostability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum. Results In this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperatures. We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, we demonstrated direct conversion of cellulose into isobutyl acetate by an engineered C. thermocellum at elevated temperatures. Conclusions This study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.

A study of acetate production from cellulose using Clostridium thermocellum

Biomass ◽  
1984 ◽  
Vol 4 (4) ◽  
pp. 295-303 ◽  
Author(s):  
G. Florenzano ◽  
M. Poulain ◽  
G. Goma
Keyword(s):  
Clostridium Thermocellum ◽  
Acetate Production

Exemplar Abstract for Clostridium thermocellum Viljoen et al. 1926 (Approved Lists 1980).

2003 ◽  
Author(s):  
Charles Thomas Parker ◽  
Dorothea Taylor ◽  
George M Garrity
Keyword(s):  
Clostridium Thermocellum

Revalorizing Lignocellulose for the Production of Natural Pharmaceuticals and Other High Value Bioproducts

2019 ◽  
Vol 26 (14) ◽  
pp. 2475-2484 ◽  
Author(s):  
Congqiang Zhang ◽  
Heng-Phon Too
Keyword(s):  
Clostridium Thermocellum ◽  
White Rot Fungi ◽  
Brown Rot ◽  
Cost Effective ◽  
White Rot ◽  
Chemical Pretreatment ◽  
Significant Challenge ◽  
Drug Molecules ◽  
Natural Medicine ◽  
Renewable Natural Resource

Lignocellulose is the most abundant renewable natural resource on earth and has been successfully used for the production of biofuels. A significant challenge is to develop cost-effective, environmentally friendly and efficient processes for the conversion of lignocellulose materials into suitable substrates for biotransformation. A number of approaches have been explored to convert lignocellulose into sugars, e.g. combining chemical pretreatment and enzymatic hydrolysis. In nature, there are organisms that can transform the complex lignocellulose efficiently, such as wood-degrading fungi (brown rot and white rot fungi), bacteria (e.g. Clostridium thermocellum), arthropods (e.g. termite) and certain animals (e.g. ruminant). Here, we highlight recent case studies of the natural degraders and the mechanisms involved, providing new utilities in biotechnology. The sugars produced from such biotransformations can be used in metabolic engineering and synthetic biology for the complete biosynthesis of natural medicine. The unique opportunities in using lignocellulose directly to produce natural drug molecules with either using mushroom and/or ‘industrial workhorse’ organisms (Escherichia coli and Saccharomyces cerevisiae) will be discussed.

scholarly journals Glu280 is the nucleophile in the active site of Clostridium thermocellum CelC, a family A endo-beta-1,4-glucanase

1993 ◽  
Vol 268 (19) ◽  
pp. 14096-14102
Author(s):  
Q. Wang ◽  
D. Tull ◽  
A. Meinke ◽  
N.R. Gilkes ◽  
R.A. Warren ◽  
...  
Keyword(s):  
Active Site ◽  
Clostridium Thermocellum
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