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.

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 The Role of Yeast-Surface-Display Techniques in Creating Biocatalysts for Consolidated BioProcessing

Catalysts ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 94 ◽  
Author(s):  
Ian Dominic Flormata Tabañag ◽  
I-Ming Chu ◽  
Yu-Hong Wei ◽  
Shen-Long Tsai
Keyword(s):  
Climate Change ◽  
Lignocellulosic Biomass ◽  
Surface Engineering ◽  
Consolidated Bioprocessing ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Qualitative Assessment ◽  
The Status ◽  
Yeast Surface

Climate change is directly linked to the rapid depletion of our non-renewable fossil resources and has posed concerns on sustainability. Thus, imploring the need for us to shift from our fossil based economy to a sustainable bioeconomy centered on biomass utilization. The efficient bioconversion of lignocellulosic biomass (an ideal feedstock) to a platform chemical, such as bioethanol, can be achieved via the consolidated bioprocessing technology, termed yeast surface engineering, to produce yeasts that are capable of this feat. This approach has various strategies that involve the display of enzymes on the surface of yeast to degrade the lignocellulosic biomass, then metabolically convert the degraded sugars directly into ethanol, thus elevating the status of yeast from an immobilization material to a whole-cell biocatalyst. The performance of the engineered strains developed from these strategies are presented, visualized, and compared in this article to highlight the role of this technology in moving forward to our quest against climate change. Furthermore, the qualitative assessment synthesized in this work can serve as a reference material on addressing the areas of improvement of the field and on assessing the capability and potential of the different yeast surface display strategies on the efficient degradation, utilization, and ethanol production from lignocellulosic biomass.

Consolidated bioprocessing performance of a two‐species microbial consortium for butanol production from lignocellulosic biomass

2020 ◽  
Vol 117 (10) ◽  
pp. 2985-2995 ◽  
Author(s):  
Yujia Jiang ◽  
Yang Lv ◽  
Ruofan Wu ◽  
Jiasheng Lu ◽  
Weiliang Dong ◽  
...  
Keyword(s):  
Lignocellulosic Biomass ◽  
Microbial Consortium ◽  
Consolidated Bioprocessing ◽  
Butanol Production

scholarly journals Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum

2015 ◽  
Vol 31 ◽  
pp. 44-52 ◽  
Author(s):  
Paul P. Lin ◽  
Luo Mi ◽  
Amy H. Morioka ◽  
Kouki M. Yoshino ◽  
Sawako Konishi ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Consolidated Bioprocessing

scholarly journals Development of a genome-scale metabolic model of Clostridium thermocellum and its applications for integration of multi-omics datasets and strain design

2020 ◽  
Author(s):  
Sergio Garcia ◽  
R. Adam Thompson ◽  
Richard J. Giannone ◽  
Satyakam Dash ◽  
Costas D. Maranas ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Lignocellulosic Biomass ◽  
Clostridium Thermocellum ◽  
System Analysis ◽  
Plant Biomass ◽  
Metabolic Model ◽  
Efficient Production ◽  
Branch Points ◽  
A Genome ◽  
Genome Scale

AbstractSolving environmental and social challenges such as climate change requires a shift from our current non-renewable manufacturing model to a sustainable bioeconomy. To lower carbon emissions in the production of fuels and chemicals, plant biomass feedstocks can replace petroleum using microorganisms as catalysts. The anaerobic thermophile Clostridium thermocellum is a promising bacterium for bioconversion due to its capability to efficiently degrade untreated lignocellulosic biomass. However, the complex metabolism of C. thermocellum is not fully understood, hindering metabolic engineering to achieve high titers, rates, and yields of targeted molecules. In this study, we developed an updated genome-scale metabolic model of C. thermocellum that accounts for recent metabolic findings, has improved prediction accuracy, and is standard-conformant to ensure easy reproducibility. We illustrated two applications of the developed model. We first formulated a multi-omics integration protocol and used it to understand redox metabolism and potential bottlenecks in biofuel (e.g., ethanol) production in C. thermocellum. Second, we used the metabolic model to design modular cells for efficient production of alcohols and esters with broad applications as flavors, fragrances, solvents, and fuels. The proposed designs not only feature intuitive push-and-pull metabolic engineering strategies, but also novel manipulations around important central metabolic branch-points. We anticipate the developed genome-scale metabolic model will provide a useful tool for system analysis of C. thermocellum metabolism to fundamentally understand its physiology and guide metabolic engineering strategies to rapidly generate modular production strains for effective biosynthesis of biofuels and biochemicals from lignocellulosic biomass.

scholarly journals Clostridium thermocellum as a Promising Source of Genetic Material for Designer Cellulosomes: An Overview

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 996
Author(s):  
Dung Minh Ha-Tran ◽  
Trinh Thi My Nguyen ◽  
Chieh-Chen Huang
Keyword(s):  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Genetic Material ◽  
Economic Feasibility ◽  
Biofuel Production ◽  
Cellulolytic Bacterium ◽  
Microbial Conversion ◽  
Promising Source

Plant biomass-based biofuels have gradually substituted for conventional energy sources thanks to their obvious advantages, such as renewability, huge quantity, wide availability, economic feasibility, and sustainability. However, to make use of the large amount of carbon sources stored in the plant cell wall, robust cellulolytic microorganisms are highly demanded to efficiently disintegrate the recalcitrant intertwined cellulose fibers to release fermentable sugars for microbial conversion. The Gram-positive, thermophilic, cellulolytic bacterium Clostridium thermocellum possesses a cellulolytic multienzyme complex termed the cellulosome, which has been widely considered to be nature’s finest cellulolytic machinery, fascinating scientists as an auspicious source of saccharolytic enzymes for biomass-based biofuel production. Owing to the supra-modular characteristics of the C. thermocellum cellulosome architecture, the cellulosomal components, including cohesin, dockerin, scaffoldin protein, and the plentiful cellulolytic and hemicellulolytic enzymes have been widely used for constructing artificial cellulosomes for basic studies and industrial applications. In addition, as the well-known microbial workhorses are naïve to biomass deconstruction, several research groups have sought to transform them from non-cellulolytic microbes into consolidated bioprocessing-enabling microbes. This review aims to update and discuss the current progress in these mentioned issues, point out their limitations, and suggest some future directions.

Sign in / Sign up

Export Citation Format

Share Document