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 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 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 Understanding Functional Roles of Native Pentose-Specific Transporters for Activating Dormant Pentose Metabolism in Yarrowia lipolytica

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Seunghyun Ryu ◽  
Cong T. Trinh
Keyword(s):  
Lignocellulosic Biomass ◽  
Yarrowia Lipolytica ◽  
Strain Engineering ◽  
Xylitol Dehydrogenase ◽  
Content Type ◽  
Functional Roles ◽  
Rate Limiting Step ◽  
Pentose Metabolism ◽  
Rate Limiting ◽  
Pentose Sugars

ABSTRACT Pentoses, including xylose and arabinose, are the second most prevalent sugars in lignocellulosic biomass that can be harnessed for biological conversion. Although Yarrowia lipolytica has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that Y. lipolytica (ATCC MYA-2613) has endogenous enzymes for d -xylose assimilation, but inefficient xylitol dehydrogenase causes Y. lipolytica to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in Y. lipolytica . By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parental engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown native l -arabinose assimilation pathway in Y. lipolytica through transcriptomic and metabolic analyses. We discovered that Y. lipolytica can coconsume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose assimilation pathway. Arabinose assimilation is synergistically enhanced in the presence of xylose, while xylose assimilation is competitively inhibited by arabinose. l -Arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Y. lipolytica . Overall, this study sheds light on the cryptic pentose metabolism of Y. lipolytica and, further, helps guide strain engineering of Y. lipolytica for enhanced assimilation of pentose sugars. IMPORTANCE The oleaginous yeast Yarrowia lipolytica is a promising industrial-platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, Y. lipolytica has been known to be incapable of assimilating pentose sugars, xylose and arabinose, that are dominantly present in lignocellulosic biomass. Through bioinformatic, transcriptomic, and enzymatic studies, we have uncovered the dormant pentose metabolism of Y. lipolytica . Remarkably, unlike most yeast strains, which share the same transporters for importing hexose and pentose sugars, we discovered that Y. lipolytica possesses the native pentose-specific transporters. By overexpressing these transporters together with the rate-limiting d -xylitol and l -arabitol dehydrogenases, we activated the dormant pentose metabolism of Y. lipolytica . Overall, this study provides a fundamental understanding of the dormant pentose metabolism of Y. lipolytica and guides future metabolic engineering of Y. lipolytica for enhanced conversion of pentose sugars to high-value chemicals and fuels.

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.

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

scholarly journals Understanding Functional Roles of Native Pentose-Specific Transporters for Activating Dormant Pentose Metabolism in Yarrowia lipolytica

2017 ◽  
Author(s):  
Seunghyun Ryu ◽  
Cong T. Trinh
Keyword(s):  
Lignocellulosic Biomass ◽  
Yarrowia Lipolytica ◽  
Strain Engineering ◽  
Xylitol Dehydrogenase ◽  
Functional Roles ◽  
Rate Limiting Step ◽  
Dehydrogenase Gene ◽  
Pentose Metabolism ◽  
Rate Limiting ◽  
Pentose Sugars

ABSTRACTPentoses including xylose and arabinose are the second-most prevalent sugars of lignocellulosic biomass that can be harnessed for biological conversion. Although Yarrowia lipolytica has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that Y. lipolytica (ATCC MYA-2613) has endogenous enzymes for D-xylose assimilation, but inefficient xylitol dehydrogenase causes Y. lipolytica to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in Y. lipolytica. By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parent engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown, native L-arabinose-assimilating pathway in Y. lipolytica through transcriptomic and metabolic analyses. We discovered that Y. lipolytica can co-consume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose-assimilating pathway. Arabinose assimilation was synergistically enhanced in the presence of xylose while xylose assimilation was competitively inhibited by arabinose. L-arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Y. lipolytica. Overall, this study sheds light on the cryptic pentose metabolism of Y. lipolytica and further helps guide strain engineering of Y. lipolytica for enhanced assimilation of pentose sugars.IMPORTANCEThe oleaginous yeast Yarrowia lipolytica is a promising industrial platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, Y. lipolytica has often been known to be incapable of assimilating pentose sugars, xylose and arabinose, that are dominantly present in lignocellulosic biomass. Through bioinformatic, transcriptomic and enzymatic studies, we have uncovered the dormant pentose metabolism of Y. lipolytica. Remarkably, unlike most yeast strains that share the same transporters for importing hexose and pentose sugars, we discovered that Y. lipolytica possess the native pentose-specific transporters. By overexpressing these transporters together with the rate-limiting D-xylitol and L-arabitol dehydrogenases, we activated the dormant pentose metabolism of Y. lipolytica. Overall, this study provides a fundamental understanding of the dormant pentose metabolism of Y. lipolytica and guides future metabolic engineering of Y. lipolytica for enhanced conversion of pentose sugars to high-value chemicals and fuels.

scholarly journals Two-dimensional isobutyl acetate production pathways to improve carbon yield

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Yohei Tashiro ◽  
Shuchi H. Desai ◽  
Shota Atsumi
Keyword(s):  
Two Dimensional ◽  
Carbon Yield ◽  
Acetate Production ◽  
Isobutyl Acetate
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