Efficient yeast surface-display of novel complex synthetic cellulosomes.

BACKGROUND: The self-assembly of cellulosomes on the surface of yeast is a promising strategy for consolidated bioprocessing to convert cellulose into ethanol in one step. RESULTS: In this study, we developed a novel synthetic cellulosome that anchors to the endogenous yeast cell wall protein a-agglutinin through disulfide bonds. A synthetic scaffoldin ScafAGA3 was constructed using the repeated N-terminus of Aga1p and displayed on the yeast cell surface. Secreted cellulases were then fused with Aga2p to assemble the cellulosome. The display efficiency of the synthetic scaffoldin and the assembly efficiency of each enzyme were much higher than those of the most frequently constructed cellulosome using scaffoldin ScafCipA3 from Clostridium thermocellum. A complex cellulosome with two scaffoldins was also constructed using interactions between the displayed anchoring scaffoldin ScafAGA3 and scaffoldin I ScafCipA3 through disulfide bonds, and the assembly of secreted cellulases to ScafCipA3. The newly designed cellulosomes enabled yeast to directly ferment cellulose into ethanol. CONCLUSIONS: This is the first report on the development of complex multiple-component assembly system through disulfide bonds. This strategy could facilitate the construction of yeast cell factories to express synergistic enzymes for use in biotechnology.

Effects of gas condition on acetic acid fermentation by Clostridium thermocellum and Moorella thermoacetica (C. thermoaceticum).

Fermentation with acetogens can be affected by cultivation gas phase, but to date, there is not enough evidence on that matter for Clostridium thermocellum and Moorella thermoacetica. In this work, the effects of sparged CO2 as well as sparged and non-sparged N2 on these microorganisms were studied using glucose and cellobiose as substrates. It was revealed that sparged CO2 and non-sparged N2 supported growth and acetic acid production by C. thermocellum and M. thermoacetica, while sparged N2 inhibited both of the microorganisms. Notably, part of the sparged CO2 was fermented by the co-culture system and contributed to an overestimation of the products from the actual substrate as well as an erring material balance. The best condition for the co-culture was concluded to be N2 without sparging. These results demonstrate the importance of cultivation conditions for efficient fermentation by anaerobic clostridia species.

Pentose sugars inhibit metabolism and increase expression of an AgrD-type cyclic pentapeptide in Clostridium thermocellum.

Clostridium thermocellum could potentially be used as a microbial biocatalyst to produce renewable fuels directly from lignocellulosic biomass due to its ability to rapidly solubilize plant cell walls. While the organism readily ferments sugars derived from cellulose, pentose sugars from xylan are not metabolized. Here, we show that non-fermentable pentoses inhibit growth and end-product formation during fermentation of cellulose-derived sugars. Metabolomic experiments confirmed that xylose is transported intracellularly and reduced to the dead-end metabolite xylitol. Comparative RNA-seq analysis of xylose-inhibited cultures revealed several up-regulated genes potentially involved in pentose transport and metabolism, which were targeted for disruption. Deletion of the ATP-dependent transporter, CbpD partially alleviated xylose inhibition. A putative xylitol dehydrogenase, encoded by Clo1313_0076, was also deleted resulting in decreased total xylitol production and yield by 41% and 46%, respectively. Finally, xylose-induced inhibition corresponds with the up-regulation and biogenesis of a cyclical AgrD-type, pentapeptide. Medium supplementation with the mature cyclical pentapeptide also inhibits bacterial growth. Together, these findings provide new foundational insights needed for engineering improved pentose utilizing strains of C. thermocellum and reveal the first functional Agr-type cyclic peptide to be produced by a thermophilic member of the Firmicutes.

CO2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum.

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO2 This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO2 to formate serves as a CO2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO2 uptake, and provided physical evidence of a distinct in vivo "rPFOR-PFL shunt" to reduce CO2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO2 fixation via the reductive C1 metabolic pathway in C. thermocellum These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO2 as well.

Enhanced biohydrogen production from sugarcane bagasse by Clostridium thermocellum supplemented with CaCO3.

Clostridium thermocellum ATCC 27405 was used to degrade sugarcane bagasse (SCB) directly for hydrogen production, which was significantly enhanced by supplementing medium with CaCO3. The effect of CaCO3 concentration on the hydrogen production was investigated. The hydrogen production was significantly enhanced with the CaCO3 concentration increased from 10mM to 20mM. However, with the CaCO3 concentration further increased from 20mM to 100mM, the hydrogen production didn't increase further. Under the optimal CaCO3 concentration of 20mM, the hydrogen production reached 97.83±5.19mmol/L from 2% sodium hydroxide-pretreated SCB, a 116.72% increase over the control (45.14±1.03mmol/L), and the yield of hydrogen production reached 4.89mmol H2/g SCBadded. Additionally, CaCO3 promoted the biodegradation of SCB and the growth of C. thermocellum. The stimulatory effects of CaCO3 on biohydrogen production are mainly attributed to the buffering capacity of carbonate. The study provides a novel strategy to enhance biohydrogen production from lignocellulose.

Optimization of influential nutrients during direct cellulose fermentation into hydrogen by Clostridium thermocellum.

Combinatorial effects of influential growth nutrients were investigated in order to enhance hydrogen (H2) production during direct conversion of cellulose by Clostridium thermocellum DSM 1237. A central composite face-centered design and response surface methodology (RSM) were applied to optimize concentrations of cellulose, yeast extract (YE), and magnesium chloride (Mg) in culture. The overall optimum composition generated by the desirability function resulted in 57.28 mmol H2/L-culture with 1.30 mol H2/mol glucose and 7.48 mmol/(g·cell·h) when cultures contained 25 g/L cellulose, 2 g/L YE, and 1.75 g/L Mg. Compared with the unaltered medium, the optimized medium produced approximately 3.2-fold more H2 within the same time-frame with 50% higher specific productivity, which are also better than previously reported values from similar studies. Nutrient composition that diverted carbon and electron flux away from H2 promoting ethanol production was also determined. This study represents the first investigation dealing with multifactor optimization with RSM for H2 production during direct cellulose fermentation.

Recovery and purification of oligosaccharides from copra meal by recombinant endo-ß-mannanase and deciphering molecular mechanism involved and its role as potent therapeutic agent.

Production of manno-oligosaccharides (MOSs) from pretreated and defatted copra meal (dFCO) hydrolysis was achieved by endo-mannanase. Structural characterization of dFCO by FT-IR and NMR exhibited resemblance with galactomannan. The time-dependent hydrolysis of dFCO by recombinant endo-ß-(1 → 4)-mannanase of Clostridium thermocellum by TLC and HPAEC displayed the release of mannose and MOSs mannobiose and mannotriose. Purified MOSs yielded 40 % mannobiose and 18 % mannotriose confirmed by mass spectroscopy which showed mannobiose (m/z = 365) and mannotriose (m/z = 527). The homology based structural analysis of catalytic endo-mannanase (CtManT) showed the catalytic core composed of Glu181 and Glu300 acting as acid/base and Glu288 as a nucleophile during galactomannan hydrolysis. Sub-site mapping of CtManT exhibited two aglycone and four glycone sites at cleavage sites existing on either side of ß-(1 → 4)-linkage of galactomannan. Isolated MOSs displayed potential prebiotic characteristics and supported higher growth of probiotic Lactobacillus acidophilus and Bifidobacterium infantis than with standard inulin. Moreover, MOSs displayed over 97 % tolerance to simulated gastric juice, intestinal fluid, and α-amylase proving its potential as a stable prebiotic over inulin. In vitro cytotoxicity assay of MOSs (500 µg/mL) on human epithelial colorectal adenocarcinoma cell line (HT-29) demonstrated 60 % decreased viability of cells after 48 h displaying anti-tumorigenic property.

Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain.

BACKGROUND: The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. RESULTS: In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell defense mechanisms. CONCLUSION: These results suggest the mechanisms of tolerance for the Populus hydrolysate-tolerant mutant strain of C. thermocellum are based on increased cellular efficiency caused apparently by downregulation of non-critical genes and increasing the expression of genes in energy production and conversion rather than tolerance to specific hydrolysate components. The wild type, conversely, responds to hydrolysate media by down-regulating growth genes and up-regulating stress response genes.

Industrial robustness: understanding the mechanism of tolerance for the Populus hydrolysate-tolerant mutant strain of Clostridium thermocellum.

BACKGROUND: An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v). CONCLUSION/SIGNIFICANCE: The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially robust strains of consolidated bioprocessing microorganisms.

Metabolic adaption of ethanol-tolerant Clostridium thermocellum.

Clostridium thermocellum is a major candidate for bioethanol production via consolidated bioprocessing. However, the low ethanol tolerance of the organism dramatically impedes its usage in industry. To explore the mechanism of ethanol tolerance in this microorganism, systematic metabolomics was adopted to analyse the metabolic phenotypes of a C. thermocellum wild-type (WT) strain and an ethanol-tolerant strain cultivated without (ET0) or with (ET3) 3% (v/v) exogenous ethanol. Metabolomics analysis elucidated that the levels of numerous metabolites in different pathways were changed for the metabolic adaption of ethanol-tolerant C. thermocellum. The most interesting phenomenon was that cellodextrin was significantly more accumulated in the ethanol-tolerant strain compared with the WT strain, although cellobiose was completely consumed in both the ethanol-tolerant and wild-type strains. These results suggest that the cellodextrin synthesis was active, which might be a potential mechanism for stress resistance. Moreover, the overflow of many intermediate metabolites, which indicates the metabolic imbalance, in the ET0 cultivation was more significant than in the WT and ET3 cultivations. This indicates that the metabolic balance of the ethanol-tolerant strain was adapted better to the condition of ethanol stress. This study provides additional insight into the mechanism of ethanol tolerance and is valuable for further metabolic engineering aimed at higher bioethanol production.

A novel anaerobic co-culture system for bio-hydrogen production from sugarcane bagasse.

A novel co-culture of Clostridium thermocellum and Thermoanaerobacterium aotearoense with pretreated sugarcane bagasse (SCB) under mild alkali conditions for bio-hydrogen production was established, exhibiting a cost-effective and synergetic advantage in bio-hydrogen production over monoculture of C. thermocellum or T. aotearoense with untreated SCB. The optimized pretreatment conditions were established to be 3% NaOH, and a liquid to solid ratio of 25:1 at 80°C for 3h. A final hydrogen production of 50.05±1.51 mmol/L was achieved with 40 g/L pretreated SCB at 55°C. The established co-culture system provides a novel consolidated bio-processing strategy for bioconversion of SCB to bio-hydrogen.

Formation and characterization of non-growth states in Clostridium thermocellum: spores and L-forms.

BACKGROUND: Clostridium thermocellum is an anaerobic thermophilic bacterium that exhibits high levels of cellulose solublization and produces ethanol as an end product of its metabolism. Using cellulosic biomass as a feedstock for fuel production is an attractive prospect, however, growth arrest can negatively impact ethanol production by fermentative microorganisms such as C. thermocellum. Understanding conditions that lead to non-growth states in C. thermocellum can positively influence process design and culturing conditions in order to optimize ethanol production in an industrial setting. RESULTS: We report here that Clostridium thermocellum ATCC 27405 enters non-growth states in response to specific growth conditions. Non-growth states include the formation of spores and a L-form-like state in which the cells cease to grow or produce the normal end products of metabolism. Unlike other sporulating organisms, we did not observe sporulation of C. thermocellum in low carbon or nitrogen environments. However, sporulation did occur in response to transfers between soluble and insoluble substrates, resulting in approximately 7% mature spores. Exposure to oxygen caused a similar sporulation response. Starvation conditions during continuous culture did not result in spore formation, but caused the majority of cells to transition to a L-form state. Both spores and L-forms were determined to be viable. Spores exhibited enhanced survival in response to high temperature and prolonged storage compared to L-forms and vegetative cells. However, L-forms exhibited faster recovery compared to both spores and stationary phase cells when cultured in rich media. CONCLUSIONS: Both spores and L-forms cease to produce ethanol, but provide other advantages for C. thermocellum including enhanced survival for spores and faster recovery for L-forms. Understanding the conditions that give rise to these two different non-growth states, and the implications that each has for enabling or enhancing C. thermocellum survival may promote the efficient cultivation of this organism and aid in its development as an industrial microorganism.

Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress.

BACKGROUND: Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. However, C. thermocellum is relatively sensitive to ethanol compared to ethanologenic microorganisms such as yeast and Zymomonas mobilis that are used in industrial fermentations but do not possess native enzymes for industrial cellulose hydrolysis. RESULTS: In this study, C. thermocellum was grown to mid-exponential phase and then treated with ethanol to a final concentration of 3.9 g/L to investigate its physiological and regulatory responses to ethanol stress. Samples were taken pre-shock and 2, 12, 30, 60, 120, and 240 min post-shock, and from untreated control fermentations for systems biology analyses. Cell growth was arrested by ethanol supplementation with intracellular accumulation of carbon sources such as cellobiose, and sugar phosphates, including fructose-6-phosphate and glucose-6-phosphate. The largest response of C. thermocellum to ethanol shock treatment was in genes and proteins related to nitrogen uptake and metabolism, which is likely important for redirecting the cells physiology to overcome inhibition and allow growth to resume. CONCLUSION: This study suggests possible avenues for metabolic engineering and provides comprehensive, integrated systems biology datasets that will be useful for future metabolic modeling and strain development endeavors.

Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405).

Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, ≥92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products--ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively.

Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum.

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

Factors influencing cellulosome activity in consolidated bioprocessing of cellulosic ethanol.

The cellulosome, a multi-subunit protein complex catalyzing cellulose degradation in cellulolytic Clostridium thermocellum, plays a crucial role in Consolidated Bioprocessing (CBP) of lignocellulose into ethanol. Here, activity of cellulosome was tested under varying concentrations of chemical compounds derived from lignocellulose pretreatment and fermentation. We found that, firstly, the cellulolytic activity of cellulosome was actually promoted by formate, acetate and lactate; secondly, cellulosome was tolerant up to 5mM furfural, 50mM p-hydroxybenzoic acid and 1mM catechol. Furthermore, the cellulosome exhibited higher ethanol tolerance and thermostability than commercialized fungal (Trichoderma reesei) cellulase. To probe the implication of these unique enzyme-features, C. thermocellum JYT01 was cultured under conditions optimal for cellulosome activity. This CBP system yielded 491 mM ethanol, the highest level reported thus far for C. thermocellum monocultures. These findings demonstrate the potential advantages of bacterial cellulosome, and provide a novel strategy for design, selection and optimization of the cellulosome-ethanologen partnership.

Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum.

Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.

Molecular and phase toxicity of compressed and supercritical fluids in biphasic continuous cultures of Clostridium thermocellum.

A novel continuous high-pressure biphasic bioreactor was designed to investigate the toxicity of compressed and supercritical fluids on the thermophilic bacterium Clostridium thermocellum. Cultures were conducted at 1.8 and 7.0 MPa hydrostatic pressure and in the presence of compressed N(2) (7.0 MPa), gaseous (1.8 MPa) and supercritical ethane (7.0 MPa), and gaseous (1.8 MPa) and liquid (7.0 MPa) propane at a single dilution rate. No significant changes in metabolism or growth were observed in the presence of compressed N(2) relative to 7.0 MPa hydrostatic pressure, indicating that it acted as an inert fluid. However, dramatic inhibitions of growth and metabolism occurred in the presence of ethane and propane at 7.0 MPa. These inhibitions were reversed by depressurization from the supercritical (ethane) or liquid (propane) to gaseous state. Solvent toxicity by compressed and supercritical fluids was attributed to phase toxicity and was correlated with fluid density rather than conventional measures of toxicity (log P(o/w)). This biphasic reactor system facilitates investigations of solvent toxicity and dissolved gas effects on whole cells under elevated pressures.