ABSTRACT
In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.
Subject(s)
Cellulose , Clostridium thermocellum , Saccharum , Solubility , Thermoanaerobacterium , Saccharum/chemistry , Cellulose/chemistry , Cellulose/metabolism , Thermoanaerobacterium/metabolism , Clostridium thermocellum/metabolism , Fermentation , Coculture Techniques , Ethanol/metabolismABSTRACT
Cotreatment, mechanical disruption of lignocellulosic biomass during microbial fermentation, is a potential alternative to thermochemical pretreatment as a means of increasing the accessibility of lignocellulose to biological attack. Successful implementation of cotreatment requires microbes that can withstand milling, while solubilizing and utilizing carbohydrates from lignocellulose. In this context, cotreatment with thermophilic, lignocellulose-fermenting bacteria has been successfully evaluated for a number of lignocellulosic feedstocks. Here, cotreatment was applied to sugarcane bagasse using monocultures of the cellulose-fermenting Clostridium thermocellum and cocultures with the hemicellulose-fermenting Thermoanaerobacterium thermosaccharolyticum. This resulted in 76 % carbohydrate solubilization (a 1.8-fold increase over non-cotreated controls) on 10 g/L solids loading, having greater effect on the hemicellulose fraction. With cotreatment, fermentation by wild-type cultures at low substrate concentrations increased cumulative product formation by 45 % for the monoculture and 32 % for the coculture. These findings highlight the potential of cotreatment for enhancing deconstruction of sugarcane bagasse using thermophilic bacteria.
Subject(s)
Cellulose , Coculture Techniques , Fermentation , Saccharum , Solubility , Saccharum/chemistry , Cellulose/metabolism , Cellulose/chemistry , Clostridium thermocellum/metabolism , Thermoanaerobacterium/metabolism , Lignin/metabolism , Lignin/chemistry , Bacteria, Anaerobic/metabolismABSTRACT
Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by â¼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different ß-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the ß-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.
Subject(s)
Clostridium thermocellum , Metabolic Engineering , Polysaccharides , Clostridium thermocellum/metabolism , Clostridium thermocellum/genetics , Polysaccharides/metabolism , Polysaccharides/genetics , Xylose/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cellulose/metabolism , Xylosidases/metabolism , Xylosidases/geneticsABSTRACT
Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass-sensing RsgI-type anti-σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti-σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/ß/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn-Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti-σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.
Subject(s)
Bacterial Proteins , Clostridium thermocellum , Molecular Dynamics Simulation , Proteolysis , Clostridium thermocellum/metabolism , Clostridium thermocellum/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Sigma Factor/chemistry , Sigma Factor/metabolism , Sigma Factor/genetics , Amino Acid Sequence , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Cellulosomes/metabolism , Cellulosomes/chemistry , Crystallography, X-Ray , Tandem Mass Spectrometry , Protein Binding , Protein Domains , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/geneticsABSTRACT
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components.
Subject(s)
Clostridium thermocellum , Cohesins , Clostridium thermocellum/chemistry , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Multienzyme Complexes , Bacterial Proteins/chemistryABSTRACT
Bacterial σI factors of the σ70-family are widespread in Bacilli and Clostridia and are involved in the heat shock response, iron metabolism, virulence, and carbohydrate sensing. A multiplicity of σI paralogues in some cellulolytic bacteria have been shown to be responsible for the regulation of the cellulosome, a multienzyme complex that mediates efficient cellulose degradation. Here, we report two structures at 3.0 Å and 3.3 Å of two transcription open complexes formed by two σI factors, SigI1 and SigI6, respectively, from the thermophilic, cellulolytic bacterium, Clostridium thermocellum. These structures reveal a unique, hitherto-unknown recognition mode of bacterial transcriptional promoters, both with respect to domain organization and binding to promoter DNA. The key characteristics that determine the specificities of the σI paralogues were further revealed by comparison of the two structures. Consequently, the σI factors represent a distinct set of the σ70-family σ factors, thus highlighting the diversity of bacterial transcription.
Subject(s)
Bacterial Proteins , Clostridium thermocellum , Bacterial Proteins/metabolism , Clostridium/genetics , Promoter Regions, Genetic , Bacteria/genetics , Sigma Factor/metabolismABSTRACT
Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (PNAS, 110: 7182-7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from Clostridium thermocellum (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin's phosphorolysis.
Subject(s)
Cellobiose , Clostridium thermocellum , Clostridium thermocellum/genetics , Starch , Substrate Specificity , Amylose , Cellulose/chemistry , Glucosyltransferases/metabolism , GlucoseABSTRACT
In this consortium, DSM 1313 was responsible for degrading lignocellulose by cellulosome, while the highly efficient hydrogen-producing bacterium MJ1 consumed the sugar produced by DSM 1313 to grow and produce more hydrogen. The results showed that the maximum hydrogen production of 259.57 mL/g substrate was obtained at the inoculation ratio (OD600) of 2:1 (DSM 1313:MJ1) and substrate concentration of 10 g/L, 70.84 % higher than pure culture. Furthermore, MJ1 dominated the co-culture system by using various sugars resulting from the biodegradation of substrate, thereby relieving the inhibition of sugar on DSM 1313 and leading to more hydrogen production. In the co-culture system, the value of extracellular oxidation-reduction potential and the ratio of NAD+/NADH was lower than that of pure culture. Additionally, at the gene level, [NiFe]-hydrogenase and [FeFe]-hydrogenase related enzymes were significantly up-regulated, leading to a two-fold increase in hydrogenase activity of co-culture compared with pure culture.
Subject(s)
Clostridium thermocellum , Hydrogenase , Hydrogenase/metabolism , Clostridium/metabolism , Clostridium thermocellum/metabolism , Sugars , Hydrogen/metabolismABSTRACT
Autoproteolysis has been discovered to play key roles in various biological processes, but functional autoproteolysis has been rarely reported for transmembrane signaling in prokaryotes. In this study, an autoproteolytic effect was discovered in the conserved periplasmic domain of anti-σ factor RsgIs from Clostridium thermocellum, which was found to transmit extracellular polysaccharide-sensing signals into cells for regulation of the cellulosome system, a polysaccharide-degrading multienzyme complex. Crystal and NMR structures of periplasmic domains from three RsgIs demonstrated that they are different from all known proteins that undergo autoproteolysis. The RsgI-based autocleavage site was located at a conserved Asn-Pro motif between the ß1 and ß2 strands in the periplasmic domain. This cleavage was demonstrated to be essential for subsequent regulated intramembrane proteolysis to activate the cognate SigI, in a manner similar to that of autoproteolysis-dependent activation of eukaryotic adhesion G protein-coupled receptors. These results indicate the presence of a unique prevalent type of autoproteolytic phenomenon in bacteria for signal transduction.
Subject(s)
Clostridium thermocellum , Sigma Factor , Sigma Factor/chemistry , Sigma Factor/metabolism , Signal Transduction , Clostridium thermocellum/chemistry , Clostridium thermocellum/metabolism , Receptors, G-Protein-Coupled/metabolism , Bacterial Proteins/metabolismABSTRACT
The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and maximum activity (Vmax > 156 U mg-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and maximum activity (14.5 U mg-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.
Subject(s)
Clostridium thermocellum , Phosphofructokinases , Phosphofructokinases/metabolism , Clostridium thermocellum/metabolism , Diphosphates , Amino Acid Sequence , Phosphofructokinase-1/genetics , Phosphofructokinase-1/metabolism , Bacteria/metabolism , Adenosine Triphosphate , Guanosine Triphosphate , KineticsABSTRACT
A recombinant ß-1,4 endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC27405 was explored for biochemical properties and the role of its associated CBMs in catalysis. The gene expressing full-length multi-modular ß-1,4-endoglucanase (AtGH9C-CBM3A-CBM3B) and its truncated derivatives (AtGH9C-CBM3A, AtGH9C, CBM3A and CBM3B) were independently cloned and expressed in Escherichia coli BL21(DE3) cells and purified. AtGH9C-CBM3A-CBM3B showed maximal activity at 55 °C and pH 7.5. AtGH9C-CBM3A-CBM3B exhibited highest activity against carboxy methyl cellulose (58.8 U/mg) followed by lichenan (44.5 U/mg), ß-glucan (36.2 U/mg) and hydroxy ethyl cellulose (17.9 U/mg). Catalytic module, AtGH9C showed insignificant activity against the substrates, signifying the essential requirement of CBMs in catalysis. AtGH9C-CBM3A-CBM3B displayed stability in pH range, 6.0-9.0 and thermostability up to 60 °C for 90 min with unfolding transition midpoint (Tm) of 65 °C. The generation of cellotetraose and other higher oligosaccharides by AtGH9C-CBM3A-CBM3B confirmed it as an endo-ß-1,4-glucanase. AtGH9C activity was partially recovered by the addition of equimolar concentration of CBM3A, CBM3B or CBM3A + CBM3B by 47 %, 13 % or 50 %, respectively. Moreover, the associated CBMs imparted thermostability to the catalytic module, AtGH9C. These results showed that the physical association of AtGH9C with its associated CBMs and the cross-talk between CBMs are necessary for AtGH9C-CBM3A-CBM3B in effective cellulose catalysis.
Subject(s)
Cellulase , Clostridium thermocellum , Cellulase/chemistry , Carbohydrate Binding Modules , Clostridium thermocellum/genetics , Oligosaccharides , CatalysisABSTRACT
Consolidated bioprocessing (CBP) of lignocellulosic biomass uses cellulolytic microorganisms to enable enzyme production, saccharification, and fermentation to produce biofuels, biochemicals, and biomaterials in a single step. However, understanding and redirecting metabolisms of these microorganisms compatible with CBP are limited. Here, a cellulolytic thermophile Clostridium thermocellum was engineered and demonstrated to be compatible with CBP integrated with a Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment for conversion of hardwood poplar into short-chain esters with industrial use as solvents, flavors, fragrances, and biofuels. The recombinant C. thermocellum engineered with deletion of carbohydrate esterases and stable overexpression of alcohol acetyltransferases improved ester production without compromised deacetylation activities. These esterases were discovered to exhibit promiscuous thioesterase activities and their deletion enhanced ester production by rerouting the electron and carbon metabolism. Ester production was further improved up to 80-fold and ester composition could be modulated by deleting lactate biosynthesis and using poplar with different pretreatment severity.
Subject(s)
Clostridium thermocellum , Clostridium thermocellum/genetics , Clostridium thermocellum/metabolism , Biomass , Biofuels , Lignin/chemistry , Fermentation , Solvents/metabolismABSTRACT
Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.
Subject(s)
Clostridium thermocellum , Clostridium thermocellum/genetics , Clostridium thermocellum/metabolism , Diphosphates/metabolism , Glycolysis/genetics , Fermentation , Adenosine Triphosphate/metabolismABSTRACT
Clostridium thermocellum, a promising candidate for consolidated bioprocessing, has been subjected to numerous engineering strategies for enhanced bioethanol production. Measurements of intracellular metabolites at substrate concentrations high enough (>50 g/L) to allow the production of industrially relevant titers of ethanol would inform efforts toward this end but have been difficult due to the production of a viscous substance that interferes with the filtration and quenching steps during metabolite extraction. To determine whether this problem is unique to C. thermocellum, we performed filtration experiments with other organisms that have been engineered for high-titer ethanol production, including Escherichia coli and Thermoanaerobacterium saccharolyticum. We addressed the problem through a series of improvements, including active pH control (to reduce problems with viscosity), investigation of different filter materials and pore sizes (to increase the filtration capacity), and correction for extracellular metabolite concentrations, and we developed a technique for more accurate intracellular metabolite measurements at elevated substrate concentrations. IMPORTANCE The accurate measurement of intracellular metabolites (metabolomics) is an integral part of metabolic engineering for the enhanced production of industrially important compounds and a useful technique to understand microbial physiology. Previous work tended to focus on model organisms under laboratory conditions. As we try to perform metabolomic studies with a wider range of organisms under conditions that more closely represent those found in nature or industry, we have found limitations in existing techniques. For example, fast filtration is an important step in quenching metabolism in preparation for metabolite extraction; however, it does not work for cultures of C. thermocellum at high substrate concentrations. In this work, we characterize the extent of the problem and develop techniques to overcome it.
Subject(s)
Clostridium thermocellum , Sugars , Sugars/metabolism , Clostridium thermocellum/metabolism , Metabolic Engineering , Ethanol/metabolismABSTRACT
The gram-positive bacterium Clostridium thermocellum contains a set of carbohydrate-active enzymes that can potentially be employed to generate high-value-added products from lignocellulose. In this study, the gene expression profiling of C. thermocellum B8 was provided during growth in the presence of sugarcane bagasse and straw as a carbon source in comparison to growth using microcrystalline cellulose. A total of 625 and 509 genes were up-regulated for growth in the presence of bagasse and straw, respectively. These genes were mainly grouped into carbohydrate-active enzymes (CAZymes), cell motility, chemotaxis, quorum sensing pathway and expression control of glycoside hydrolases. These results show that type of carbon source modulates the gene expression profiling of carbohydrate-active enzymes. In addition, highlight the importance of cell motility, attachment to the substrate and communication in deconstructing complex substrates. This present work may contribute to the development of enzymatic cocktails and industrial strains for biorefineries based on sugarcane residues as feedstock.
Subject(s)
Clostridium thermocellum , Saccharum , Cellulose/metabolism , Saccharum/chemistry , CarbohydratesABSTRACT
Clostridium thermocellum is a cellulolytic thermophile that is considered for the consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but the incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated via gene deletions in ammonium-regulated, nicotinamide adenine dinucleotide phosphate (NADPH)-supplying and NADPH-consuming pathways as well as via physiological characterization in cellobiose-limited or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Δppdk) or a redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased from 46 to 60%, suggesting that the secretion of these amino acids balances the NADPH surplus from the malate shunt. The unchanged amino acid secretion in Δppdk falsified a previous hypothesis on an ammonium-regulated PEP-to-pyruvate flux redistribution. The possible involvement of another NADPH-supplier, namely, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, the deletion of glutamate synthase (gogat) in ammonium assimilation resulted in the upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, a claim which is supported by the product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of the NADH needed for ethanol formation. This suggests that metabolic engineering strategies that simplify the redox metabolism and ammonium assimilation can contribute to increased ethanol yields. IMPORTANCE Improving the ethanol yield of C. thermocellum is important for the industrial implementation of this microorganism in consolidated bioprocessing. A central role of NADPH in driving amino acid byproduct formation was demonstrated by eliminating the NADPH-supplying malate shunt and separately by changing the cofactor specificity in ammonium assimilation. With amino acid secretion diverting carbon and electrons away from ethanol, these insights are important for further metabolic engineering to reach industrial requirements on ethanol yield. This study also provides chemostat data that are relevant for training genome-scale metabolic models and for improving the validity of their predictions, especially considering the reduced degree-of-freedom in the redox metabolism of the strains generated here. In addition, this study advances the fundamental understanding on the mechanisms underlying amino acid secretion in cellulolytic Clostridia as well as on the regulation and cofactor specificity in ammonium assimilation. Together, these efforts aid in the development of C. thermocellum for the sustainable consolidated bioprocessing of lignocellulose to ethanol with minimal pretreatment.
Subject(s)
Amino Acids , Ammonium Compounds , Clostridium thermocellum , NADP , Amino Acids/biosynthesis , Amino Acids/metabolism , Ammonium Compounds/metabolism , Clostridium thermocellum/genetics , Clostridium thermocellum/metabolism , Ethanol/metabolism , Ferredoxins/metabolism , Malates/metabolism , NAD/metabolism , NADP/metabolism , Pyruvates/metabolism , Oxidation-ReductionABSTRACT
The processive cellulase (CelO) is an important modular enzyme of Clostridium thermocellum. To study the effect of the carbohydrate-binding module (CBM3b) on the catalytic domain of CelO (GH5), four engineered derivatives of CelO were designed by truncation and terminal fusion of CBM3b. These are CBM at the N-terminus, native form (CelO-BC, 62 kDa); catalytic domain only (CelO-C, 42 kDa); CBM at the C-terminus (CelO-CB, 54 kDa) and CBM attached at both termini (CelO-BCB, 73 kDa). All constructs were cloned into pET22b (+) and expressed in Escherichia coli BL21 (DE3) star. The expression levels of CelO-C, CelO-CB, CelO-BC, and CelO-BCB were 35%, 35%, 30%, and 20%, respectively. The enzyme activities of CelO-C, CelO-CB, CelO-BC, and CelO-BCB against 1% regenerated amorphous cellulose (RAC) were 860, 758, 985, and 1208 units per µmole of the enzyme, respectively. The enzymes were partially purified from the lysate of E. coli cells by heat treatment followed by anion exchange FPLC purification. Against RAC, CelO-C, CelO-CB, CelO-BC, and CelO-BCB showed KM values of 32, 33, 45, and 43 mgâ mL-1 and Vmax values of 3571, 3846, 3571, and 4545 Uâ min-1 , respectively. CBM3b at the N-terminus of GH5 linked through a P/T-rich linker was found to enhance the catalytic activity and thermostability of the enzyme.
Subject(s)
Cellulase , Clostridium thermocellum , Clostridium thermocellum/genetics , Clostridium thermocellum/metabolism , Cellulase/genetics , Cellulase/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Catalytic DomainABSTRACT
Naturally occurring, microbial contaminants were found in plant biomasses from common bioenergy crops and agricultural wastes. Unexpectedly, indigenous thermophilic microbes were abundant, raising the question of whether they impact thermophilic consolidated bioprocessing fermentations that convert biomass directly into useful bioproducts. Candidate microbial platforms for biomass conversion, Acetivibrio thermocellus (basionym Clostridium thermocellum; Topt 60 °C) and Caldicellulosiruptor bescii (Topt 78 °C), each degraded a wide variety of plant biomasses, but only A. thermocellus was significantly affected by the presence of indigenous microbial populations harbored by the biomass. Indigenous microbial growth was eliminated at ≥75 °C, conditions where C. bescii thrives, but where A. thermocellus cannot survive. Therefore, 75 °C is the thermophilic threshold to avoid sterilizing pre-treatments on the biomass that prevents native microbes from competing with engineered microbes and forming undesirable by-products. Thermophiles that naturally grow at and above 75 °C offer specific advantages as platform microorganisms for biomass conversion into fuels and chemicals.
Subject(s)
Clostridium thermocellum , Lignin , Biomass , Fermentation , Lignin/chemistry , Clostridium thermocellum/metabolism , Plants/metabolismABSTRACT
The gene encoding rhamnogalacturonan acetylesterase, CtPae12B from Clostridium thermocellum was cloned, expressed, purified and biochemically characterized. Purified CtPae12B was soluble and exhibited homogenous single band. Phylogenetically it was most closely related to an RGAE, YesT from B. subtilis. CtPae12B production was maximum with LB medium. CtPae12B showed optimal temperature, 65 °C and thermostability with half-life, 5.1 h at 80 °C. CtPae12B was alkaliphilic with optimal pH, 8.0, while it displayed stability at both acidic and alkaline pH ranges. Inhibition of CtPae12B activity by PMSF showed the importance of nucleophilic serine in the catalytic triad. The metal ions, chemical or chelating agents used, did not enhance CtPae12B activity, which was also corroborated by protein melting study. The enzymatic activity of CtPae12B remained unaffected by 5 M urea. CtPae12B showed broad substrate specificity as it displayed activity against a range of synthetic substrates showing highest Vmax, 770 U/mg and Km, 1.2 mM with ß-D-gluco pentaacetate. CtPae12B could deacetylate both pectic and xylan substrates showing highest Vmax, 770 U/mg and Km, 13.4 mg/mL with potato rhamnogalacturonan and Vmax, 105 U/mg and Km, 7.1 mg/mL with acetylated birchwood xylan. The thermostability, pH stability and broad substrate specificity of CtPae12B makes it a versatile enzyme for industrial applications.
Subject(s)
Clostridium thermocellum , Esterases , Clostridium thermocellum/genetics , Substrate Specificity , Xylans , Hydrogen-Ion Concentration , Cloning, MolecularABSTRACT
BACKGROUND: Clostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures. RESULTS: To separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (Pclo1313_2638::ldh* ∆adhE) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE, in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 °C for ethanol concentrations ≥ 10 g L-1 and was able to reach higher maximum OD600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD600 and relative growth rates were higher at 45 and 50 °C, compared to 55 °C, for ethanol concentrations ≥ 15 g L-1. For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L-1 at 45 and 50 °C compared to 55 °C. CONCLUSION: Positive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions.