Nitrogen and sulfur requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on cellulosic substrates in minimal nutrient media

Kridelbaugh, Donna M.; Nelson, Joshua; Engle, Nancy L.; Tschaplinski, Timothy J.; Graham, David E.

doi:10.1016/j.biortech.2012.12.006

Cited by 31 publications

(30 citation statements)

References 31 publications

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“…Bioreactors were prepared for C. thermocellum culture by autoclaving cellobiose, water, and resazurin (total volume, 800 ml) in 1-liter Biostat Q-Plus bioreactors (Sartorius Stedim, Gottingen, Germany) for 30 min. Reactors were sparged with nitrogen gas for 1 h, and a sterile cocktail of the remaining medium for thermophilic clostridia (MTC) components was prepared (45). A cocktail (100 ml) was added to each reactor, and sparging with nitrogen continued overnight with observable loss of color from reduced resazurin.…”

Section: Methodsmentioning

confidence: 99%

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

Appl Environ Microbiol

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Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ␤-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5-to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and ␣-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters. IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

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Section: Methodsmentioning

confidence: 99%

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

Appl Environ Microbiol

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“…Peptide concentrations in the combined filtrate were measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). Acidified aliquots of combined filtrate containing ϳ150 g of the digest were purified by loading onto a 1-by-100-mm C 18 column (5-m Luna C18 [2]; Phenomenex, Torrance, CA) and eluted using 80% (vol/vol) acetonitrile. Purified aliquots were lyophilized and redissolved in buffer A (0.1% formic acid in water) for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis.…”

Section: Methodsmentioning

confidence: 99%

“…T he use of designer cocultures is a strategy that is receiving increased attention for its effectiveness at achieving improved biofuel yields and conversion efficiencies from lignocellulosic biomass through consolidated bioprocessing (CBP) (1)(2)(3). In a CBP platform, which involves concomitant enzyme production, biomass hydrolysis, and biofuel production (4), an ideal consortium would achieve (i) efficient and complete biomass hydrolysis, (ii) simultaneous, rather than sequential, utilization of cellulose, and hemicellulose constituent saccharides, and (iii) industrially relevant biofuel yields.…”

mentioning

confidence: 99%

“…However, its inability to grow and produce biofuels from hemicellulose constituent saccharides, most notably pentoses (8,9), has often provided a rationale for the identification and investigation of suitable coculture partners. Previous C. thermocellum cocultures with bacteria possessing more diverse substrate utilization capabilities have resulted in improved rates of biomass degradation and biofuel yield (1,2,10,11).…”

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confidence: 99%

“…Since C. thermocellum downregulates expression of its own xylanases when grown on xylan containing substrates (6) in comparison to growth on cellulose alone, the xylan-hydrolyzing ability of T. thermohydrosulfuricus WC1 suggests it may be an effective C. thermocellum coculture partner. The value of this phenotype in C. thermocellum-Caldicellulosiruptor bescii cocultures has recently been reported as increased rates of biomass hydrolysis were directly attributed to the xylanolytic capabilities of Caldicellulosiruptor bescii (2). Constructing cocultures whereby both members contribute to lignocellulose hydrolysis may be particularly valuable given that biomass hydrolysis is a major limitation toward achieving industrially viable lignocellulosic biofuels (24,25).…”

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confidence: 99%

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Thermoanaerobacterthermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates

Verbeke

Spicer

Krokhin

et al. 2014

Appl Environ Microbiol

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e Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using "omic"-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain, Thermoanaerobacter thermohydrosulfuricus WC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species "omic" comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverse Thermoanaerobacter spp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.

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Overflow metabolism and growth cessation in Clostridium thermocellum DSM1313 during high cellulose loading fermentations

Thompson

Trinh

2017

Biotech & Bioengineering

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As a model thermophilic bacterium for the production of second-generation biofuels, the metabolism of Clostridium thermocellum has been widely studied. However, most studies have characterized C. thermocellum metabolism for growth at relatively low substrate concentrations. This outlook is not industrially relevant, however, as commercial viability requires substrate loadings of at least 100 g/L cellulosic materials. Recently, a wild-type C. thermocellum DSM1313 was cultured on high cellulose loading batch fermentations and reported to produce a wide range of fermentative products not seen at lower substrate concentrations, opening the door for a more in-depth analysis of how this organism will behave in industrially relevant conditions. In this work, we elucidated the interconnectedness of overflow metabolism and growth cessation in C. thermocellum during high cellulose loading batch fermentations (100 g/L). Metabolic flux and thermodynamic analyses suggested that hydrogen and formate accumulation perturbed the complex redox metabolism and limited conversion of pyruvate to acetyl-CoA conversion, likely leading to overflow metabolism and growth cessation in C. thermocellum. Pyruvate formate lyase (PFL) acts as an important redox valve and its flux is inhibited by formate accumulation. Finally, we demonstrated that manipulation of fermentation conditions to alleviate hydrogen accumulation could dramatically alter the fate of pyruvate, providing valuable insight into process design for enhanced C. thermocellum production of chemicals and biofuels. Biotechnol. Bioeng. 2017;114: 2592-2604. © 2017 Wiley Periodicals, Inc.

show abstract

Nitrogen and sulfur requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on cellulosic substrates in minimal nutrient media

Cited by 31 publications

References 31 publications

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Thermoanaerobacterthermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates

Overflow metabolism and growth cessation in Clostridium thermocellum DSM1313 during high cellulose loading fermentations

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