Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

Rydzak, Thomas; Levin, David B.; Çiçek, Nazim; Sparling, Richard

doi:10.1016/j.jbiotec.2009.01.022

Cited by 66 publications

(77 citation statements)

References 31 publications

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“…Similar levels of NADH-dependent and NADPH-dependent ADH activities have been reported for C. thermocellum strain ATCC 27405 (19), which is quite different from an earlier report that C. thermocellum strains LQRI and AS39 had ≥200-fold higher NADHdependent ADH activities compared with NADPH-dependent ADH activities (20).…”

Section: Nonrandom Distribution Of Mutations Across the Genome And Theircontrasting

confidence: 47%

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Brown

Guss

Karpinets

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

167

145

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Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene ( adhE ), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

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Section: Nonrandom Distribution Of Mutations Across the Genome And Theircontrasting

confidence: 47%

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Brown

Guss

Karpinets

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

167

145

View full text Add to dashboard Cite

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“…Furthermore, given its native ability to produce ethanol and H 2 , C. thermocellum is seen as an attractive microorganism for the production of biofuels via consolidated bioprocessing of lignocellulosic biomass. Unfortunately, current yields and production rates of ethanol and/or H 2 are low due to branched product pathways (3)(4)(5) which redirect carbon and electron flux away from a desired biofuel. These unwanted products include lactate, formate, and/or acetate (6)(7)(8), as well as secreted amino acids (9,10).…”

mentioning

confidence: 99%

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Taillefer

Rydzak

Levin

et al. 2015

Appl Environ Microbiol

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Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a k cat of 45.8 s ؊1 or 14.9 s ؊1 , respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a k cat of 520.8 s ؊1 . The malic enzyme used NADP ؉ as a cofactor along with NH 4 ؉ and Mn 2؉ as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a K i of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH 4 ؉ based on the characterization of the malate dehydrogenase and malic enzyme.C lostridium thermocellum is a Gram-positive, anaerobic thermophile capable of reaching one of the highest growth rates on crystalline cellulose (1, 2). Furthermore, given its native ability to produce ethanol and H 2 , C. thermocellum is seen as an attractive microorganism for the production of biofuels via consolidated bioprocessing of lignocellulosic biomass. Unfortunately, current yields and production rates of ethanol and/or H 2 are low due to branched product pathways (3-5) which redirect carbon and electron flux away from a desired biofuel. These unwanted products include lactate, formate, and/or acetate (6-8), as well as secreted amino acids (9, 10). Thus, redirecting carbon and electron flux away from these secreted products toward either ethanol or H 2 may improve the economic viability of biofuels production using C. thermocellum.A key pathway node involved in the interconversion of phosphoenolpyruvate (PEP) and pyruvate, which may catalyze either substrate level phosphorylation or transhydrogenation reactions between NADH to NADP ϩ , has been revisited with C. thermocellum (10, 11). The interconversion of PEP and pyruvate may be catalyzed using a number of different putative enzymes. In contrast to most clostridia and other fermentative ethanol and/or H 2 -producing organisms (7), C. thermocellum ATCC 27405 (GenBank accession number NC_009012.1) does not encode a pyruvate kinase, which catalyzes PE...

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“…CO 2 , H 2 , and O 2 concentrations were determined using a multiple gas analyzer number 1 gas chromatograph (GC) system (model 8610-0070; SRI Instruments, Torrance, CA) equipped with a thermal conductivity detector (TCD) and argon as the carrier gas. The columns and methods used were those previously described by Rydzak et al (20).…”

Section: Methodsmentioning

confidence: 99%

“…Monoculture purity was determined via 16S rRNA gene PCR amplification and sequencing using primers 8F (5=-AGAGTTTGATCCTGGCTCAG-3=) and 1541R (5=-AAGGAGGTGA TCCAGCCGCA-3=), and the consistency of the end product production phenotype was determined from the literature and previous experiments (16,20). The purity of the cocultures (i.e., confirmation that they contained only C. thermocellum DSM 1237 and C. debilis DSM 16016) was confirmed by PCR using the 16S rRNA interspacer primers ITS-F (5=-GT CGTAACAAGGTAGCCGTA-3=) and ITS-Reub (5=-GCCAAGGCATCC ACC-3=).…”

Section: Methodsmentioning

confidence: 99%

Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum

Wushke

Levin

Cicek

et al. 2015

Appl Environ Microbiol

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bDevelopment of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere. Isolation and characterization of organisms with the ability to degrade and ferment cellulose are essential steps toward enhancing biofuel production via consolidated bioprocessing (CBP) (1). Designing a culture that could achieve both cellulose degradation and ethanol production under nonreduced conditions would reduce the costs and complexity of culturing strictly anaerobic bacteria, as this would eliminate the need for maintaining a reduced environment. Clostridium thermocellum is perhaps the best-studied model organism capable of CBP under anaerobic conditions (2, 3). Coculturing a cellulolytic organism with a noncellulolytic partner capable of producing the desired end products at high rates and yields has been shown to improve the overall biofuel production capability via CBP (4) and increased cellulose hydrolysis rates (4, 5). Many biofuel-producing cellulolytic bacteria, including C. thermocellum, lack aerotolerance and have not been shown to be able to tolerate greater than 2% O 2 in the atmosphere (6, 7). While members of the genus Clostridium are typically described to be strict anaerobes, the genomes of some members, including Clostridium straminisolvens, Clostridium acetobutylicum, and Clostridium intestinalis, encode genes required for aerotolerance, and these organisms have been shown to grow in microaerobic environments (6,8,9). Moreover, genetic modification of C. acetobutylicum resulted in a significant increase in aerotolerance, such that the recombinant strain was able to grow in the presence of atmospheric concentrations of oxygen (10).Previously, it was reported that cocultures containing a Clostridium species paired with facultative aerobes can lead to oxygen removal in a sealed vessel followed by fermentation (11-13) or achieve aerotolerance via respiratory protection under microaerobic conditions (14). The importance of biofilms and the use of mixed cultures to help lignocellulosic degradation h...

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Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

Cited by 66 publications

References 31 publications

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum

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