RESUMO
The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in Caldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C5 and C6 sugars. In addition, C. bescii grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. C. bescii natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, C. bescii was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in C. bescii, the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of C. bescii.IMPORTANCELignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacterium Caldicellulosiruptor bescii was successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling of C. bescii was applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.
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Butileno Glicóis , Caldicellulosiruptor , Lactatos , Engenharia Metabólica , Xilanos , Biomassa , Acetoína , Composição de Bases , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA , Celulose/metabolismo , Clostridiales/metabolismo , Bactérias/metabolismo , Plantas/metabolismo , AçúcaresRESUMO
A genome-scale metabolic model, encompassing a total of 623 genes, 727 reactions, and 865 metabolites, was developed for Pyrococcus furiosus, an archaeon that grows optimally at 100°C by carbohydrate and peptide fermentation. The model uses subsystem-based genome annotation, along with extensive manual curation of 237 gene-reaction associations including those involved in central carbon metabolism, amino acid metabolism, and energy metabolism. The redox and energy balance of P. furiosus was investigated through random sampling of flux distributions in the model during growth on disaccharides. The core energy balance of the model was shown to depend on high acetate production and the coupling of a sodium-dependent ATP synthase and membrane-bound hydrogenase, which generates a sodium gradient in a ferredoxin-dependent manner, aligning with existing understanding of P. furiosus metabolism. The model was utilized to inform genetic engineering designs that favor the production of ethanol over acetate by implementing an NADPH and CO-dependent energy economy. The P. furiosus model is a powerful tool for understanding the relationship between generation of end products and redox/energy balance at a systems-level that will aid in the design of optimal engineering strategies for production of bio-based chemicals and fuels. IMPORTANCE The bio-based production of organic chemicals provides a sustainable alternative to fossil-based production in the face of today's climate challenges. In this work, we present a genome-scale metabolic reconstruction of Pyrococcus furiosus, a well-established platform organism that has been engineered to produce a variety of chemicals and fuels. The metabolic model was used to design optimal engineering strategies to produce ethanol. The redox and energy balance of P. furiosus was examined in detail, which provided useful insights that will guide future engineering designs.
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Pyrococcus furiosus , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Etanol/metabolismo , Fermentação , Engenharia Genética , Acetatos/metabolismoRESUMO
Caldicellulosiruptor species are proficient at solubilizing carbohydrates in lignocellulosic biomass through surface (S)-layer bound and secretomic glycoside hydrolases. Tapirins, surface-associated, non-catalytic binding proteins in Caldicellulosiruptor species, bind tightly to microcrystalline cellulose, and likely play a key role in natural environments for scavenging scarce carbohydrates in hot springs. However, the question arises: If tapirin concentration on Caldicellulosiruptor cell walls increased above native levels, would this offer any benefit to lignocellulose carbohydrate hydrolysis and, hence, biomass solubilization? This question was addressed by engineering the genes for tight-binding, non-native tapirins into C. bescii. The engineered C. bescii strains bound more tightly to microcrystalline cellulose (Avicel) and biomass compared to the parent. However, tapirin overexpression did not significantly improve solubilization or conversion for wheat straw or sugarcane bagasse. When incubated with poplar, the tapirin-engineered strains increased solubilization by 10% compared to the parent, and corresponding acetate production, a measure of carbohydrate fermentation intensity, was 28% higher for the Calkr_0826 expression strain and 18.5% higher for the Calhy_0908 expression strain. These results show that enhanced binding to the substrate, beyond the native capability, did not improve C. bescii solubilization of plant biomass, but in some cases may improve conversion of released lignocellulose carbohydrates to fermentation products.
Assuntos
Celulose , Saccharum , Celulose/metabolismo , Biomassa , Saccharum/metabolismo , Caldicellulosiruptor/metabolismo , Clostridiales/metabolismo , Plantas , Archaea/metabolismoRESUMO
Extremely thermophilic Caldicellulosiruptor species solubilize carbohydrates from lignocellulose through glycoside hydrolases (GHs) that can be extracellular, intracellular, or cell surface layer (S-layer) associated. Caldicellulosiruptor genomes sequenced so far encode at least one surface layer homology domain glycoside hydrolase (SLH-GH), representing six different classes of these enzymes; these can have multiple binding and catalytic domains. Biochemical characterization of a representative from each class was done to determine their biocatalytic features: four SLH-GHs from Caldicellulosiruptor kronotskyensis (Calkro_0111, Calkro_0402, Calkro_0072, and Calkro_2036) and two from Caldicellulosiruptor hydrothermalis (Calhy_1629 and Calhy_2383). Calkro_0111, Calkro_0072, and Calhy_2383 exhibited ß-1,3-glucanase activity, Calkro_0402 was active on both ß-1,3/1,4-glucan and ß-1,4-xylan, Calkro_2036 exhibited activity on both ß-1,3/1,4-glucan and ß-1,4-glucan, and Calhy_1629 was active only on arabinan. Caldicellulosiruptor bescii, the only species with molecular genetic tools as well as already a strong cellulose degrader, contains only one SLH-GH, Athe_0594, a glucanase that is a homolog of Calkro_2036; the other 5 classes of SLH-GHs are absent in C. bescii. The C. bescii secretome, supplemented with individual enzymes or cocktails of SLH-GHs, increased in vitro sugar release from sugar cane bagasse and poplar. Expression of non-native SLH-GHs in vivo, either associated with the S-layer or as freely secreted enzymes, improved total carbohydrate solubilization of sugar cane bagasse and poplar by up to 45% and 23%, respectively. Most notably, expression of Calkro_0402, a xylanase/glucanase, improved xylose solubilization from poplar and bagasse by over 70% by C. bescii. While Caldicellulosiruptor species are already prolific lignocellulose degraders, they can be further improved by the strategy described here. IMPORTANCE Caldicellulosiruptor species hold promise as microorganisms that can solubilize the carbohydrate portion of lignocellulose and subsequently convert fermentable sugars into bio-based chemicals and fuels. Members of the genus have surface layer (S-layer) homology domain-associated glycoside hydrolases (SLH-GHs) that mediate attachment to biomass as well as hydrolysis of carbohydrates. Caldicellulosiruptor bescii, the most studied member of the genus, has only one SLH-GH. Expression of SLH-GHs from other Caldicellulosiruptor species in C. bescii significantly improved degradation of sugar cane bagasse and poplar. This suggests that this extremely thermophilic bacterium can be engineered to further improve its ability to degrade specific plant biomasses by inserting genes encoding SLH-GHs recruited from other Caldicellulosiruptor species.
Assuntos
Glicosídeo Hidrolases , Populus , Glicosídeo Hidrolases/metabolismo , Biomassa , Xilanos/metabolismo , Xilose , Clostridiales/metabolismo , Celulose/metabolismo , Plantas/microbiologiaRESUMO
Caldicellulosiruptor species scavenge carbohydrates from runoff containing plant biomass that enters hot springs and from grasses that grow in more moderate parts of thermal features. While only a few Caldicellulosiruptor species can degrade cellulose, all known species are hemicellulolytic. The most well-characterized species, Caldicellulosiruptor bescii, decentralizes its hemicellulase inventory across five different genomic loci and two isolated genes. Transcriptomic analyses, comparative genomics, and enzymatic characterization were utilized to assign functional roles and determine the relative importance of its six putative endoxylanases (five glycoside hydrolase family 10 [GH10] enzymes and one GH11 enzyme) and two putative exoxylanases (one GH39 and one GH3) in C. bescii. Two genus-wide conserved xylanases, C. bescii XynA (GH10) and C. bescii Xyl3A (GH3), had the highest levels of sugar release on oat spelt xylan, were in the top 10% of all genes transcribed by C. bescii, and were highly induced on xylan compared to cellulose. This indicates that a minimal set of enzymes are used to drive xylan degradation in the genus Caldicellulosiruptor, complemented by hemicellulolytic inventories that are tuned to specific forms of hemicellulose in available plant biomasses. To this point, synergism studies revealed that the pairing of specific GH family proteins (GH3, -11, and -39) with C. bescii GH10 proteins released more sugar in vitro than mixtures containing five different GH10 proteins. Overall, this work demonstrates the essential requirements for Caldicellulosiruptor to degrade various forms of xylan and the differences in species genomic inventories that are tuned for survival in unique biotopes with variable lignocellulosic substrates. IMPORTANCE Microbial deconstruction of lignocellulose for the production of biofuels and chemicals requires the hydrolysis of heterogeneous hemicelluloses to access the microcrystalline cellulose portion. This work extends previous in vivo and in vitro efforts to characterize hemicellulose utilization by integrating genomic reconstruction, transcriptomic data, operon structures, and biochemical characteristics of key enzymes to understand the deployment and functionality of hemicellulases by the extreme thermophile Caldicellulosiruptor bescii. Furthermore, comparative genomics of the genus revealed both conserved and divergent mechanisms for hemicellulose utilization across the 15 sequenced species, thereby paving the way to connecting functional enzyme characterization with metabolic engineering efforts to enhance lignocellulose conversion.
Assuntos
Regulon , Xilanos , Celulose/metabolismo , Clostridiales/metabolismo , AçúcaresRESUMO
The production of volatile industrial chemicals utilizing metabolically engineered extreme thermophiles offers the potential for processes with simultaneous fermentation and product separation. An excellent target chemical for such a process is acetone (Tb = 56°C), ideally produced from lignocellulosic biomass. Caldicellulosiruptor bescii (Topt 78°C), an extremely thermophilic fermentative bacterium naturally capable of deconstructing and fermenting lignocellulose, was metabolically engineered to produce acetone. When the acetone pathway construct was integrated into a parent strain containing the bifunctional alcohol dehydrogenase from Clostridium thermocellum, acetone was produced at 9.1 mM (0.53 g/L), in addition to minimal ethanol 3.3 mM (0.15 g/L), along with net acetate consumption. This demonstrates that C. bescii can be engineered with balanced pathways in which renewable carbohydrate sources are converted to useful metabolites, primarily acetone and H2 , without net production of its native fermentation products, acetate and lactate.
Assuntos
Acetona/metabolismo , Biomassa , Caldicellulosiruptor/metabolismo , Hidrogênio/metabolismo , Lignina/metabolismo , Engenharia Metabólica , Caldicellulosiruptor/genéticaRESUMO
Terrestrial hot springs near neutral pH harbor extremely thermophilic bacteria from the genus Caldicellulosiruptor, which utilize the carbohydrates of lignocellulose for growth. These bacteria are technologically important because they produce novel, multi-domain glycoside hydrolases that are prolific at deconstructing microcrystalline cellulose and hemicelluloses found in plant biomass. Among other interesting features, Caldicellulosiruptor species have successfully adapted to bind specifically to lignocellulosic substrates via surface layer homology (SLH) domains associated with glycoside hydrolases and unique binding proteins (tapirins) present only in these bacteria. They also utilize a parallel pathway for conversion of glyceraldehyde-3-phosphate into 3-phosphoglycerate via a ferredoxin-dependent oxidoreductase that is conserved across the genus. Advances in the genetic tools for Caldicellulosiruptor bescii, including the development of a high-temperature kanamycin-resistance marker and xylose-inducible promoter, have opened the door for metabolic engineering applications and some progress along these lines has been reported. While several species of Caldicellulosiruptor can readily deconstruct lignocellulose, improvements in the amount of carbohydrate released and in the production of bio-based chemicals are required to successfully realize the biotechnological potential of these organisms.
Assuntos
Clostridiales , Biomassa , Biotecnologia , Glicosídeo Hidrolases , Fontes TermaisRESUMO
Reported here is the complete genome sequence (2,191,724 bp) for the thermoacidophilic archaeon Sulfuracidifex (f. Sulfolobus) metallicus DSM 6482 (Topt 65°C, pHopt 2.0). This obligately chemolithoautotrophic microorganism is a prolific metal and sulfur oxidizer and has application in metal bioleaching operations. A multi-assembly reconciliation approach enabled closure of the genome.
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The complete genome sequence of the extremely thermophilic bacterium Anaerocellum (f. Caldicellulosiruptor) danielii (DSM:8977) is reported here. A. danielii is a fermentative anaerobe and capable of lignocellulose degradation with potential applications in biomass degradation and production of chemicals and fuels from renewable feedstocks.
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The complete genome sequence of the thermoacidophilic archaeon Metallosphaera sedula (DSM 5348) is reported here. M. sedula, originally isolated from a volcanic field in Italy, is a prolific iron-oxidizing archaeon with applications in bioleaching of sulfide minerals.
RESUMO
Certain members of the family Sulfolobaceae represent the only archaea known to oxidize elemental sulfur, and their evolutionary history provides a framework to understand the development of chemolithotrophic growth by sulfur oxidation. Here, we evaluate the sulfur oxidation phenotype of Sulfolobaceae species and leverage comparative genomic and transcriptomic analysis to identify the key genes linked to sulfur oxidation. Metabolic engineering of the obligate heterotroph Sulfolobus acidocaldarius revealed that the known cytoplasmic components of sulfur oxidation alone are not sufficient to drive prolific sulfur oxidation. Imaging analysis showed that Sulfolobaceae species maintain proximity to the sulfur surface but do not necessarily contact the substrate directly. This indicates that a soluble form of sulfur must be transported to initiate cytoplasmic sulfur oxidation. Conservation patterns and transcriptomic response implicate an extracellular tetrathionate hydrolase and putative thiosulfate transporter in a newly proposed mechanism of sulfur acquisition in the Sulfolobaceae.IMPORTANCESulfur is one of the most abundant elements on earth (2.9% by mass), so it makes sense that the earliest biology found a way to use sulfur to create and sustain life. However, beyond evolutionary significance, sulfur and the molecules it comprises have important technological significance, not only in chemicals such as sulfuric acid and in pyritic ores containing critical metals but also as a waste product from oil and gas production. The thermoacidophilic Sulfolobaceae are unique among the archaea as sulfur oxidizers. The trajectory for how sulfur biooxidation arose and evolved can be traced using experimental and bioinformatic analyses of the available genomic data set. Such analysis can also inform the process by which extracellular sulfur is acquired and transported by thermoacidophilic archaea, a phenomenon that is critical to these microorganisms but has yet to be elucidated.
Assuntos
Oxirredução , Sulfolobaceae , Enxofre , Enxofre/metabolismo , Sulfolobaceae/metabolismo , Sulfolobaceae/genética , Fenótipo , Filogenia , Perfilação da Expressão Gênica , Genoma ArquealRESUMO
The order Thermoanaerobacterales currently consists of fermentative anaerobic bacteria, including the genus Caldicellulosiruptor. Caldicellulosiruptor are represented by thirteen species; all, but one, have closed genome sequences. Interest in these extreme thermophiles has been motivated not only by their high optimal growth temperatures (≥70°C), but also by their ability to hydrolyze polysaccharides including, for some species, both xylan and microcrystalline cellulose. Caldicellulosiruptor species have been isolated from geographically diverse thermal terrestrial environments located in New Zealand, China, Russia, Iceland and North America. Evidence of their presence in other terrestrial locations is apparent from metagenomic signatures, including volcanic ash in permafrost. Here, phylogeny and taxonomy of the genus Caldicellulosiruptor was re-examined in light of new genome sequences. Based on genome analysis of 15 strains, a new order, Caldicellulosiruptorales, is proposed containing the family Caldicellulosiruptoraceae, consisting of two genera, Caldicellulosiruptor and Anaerocellum. Furthermore, the order Thermoanaerobacterales also was re-assessed, using 91 genome-sequenced strains, and should now include the family Thermoanaerobacteraceae containing the genera Thermoanaerobacter, Thermoanaerobacterium, Caldanaerobacter, the family Caldanaerobiaceae containing the genus Caldanaerobius, and the family Calorimonaceae containing the genus Calorimonas. A main outcome of ANI/AAI analysis indicates the need to reclassify several previously designated species in the Thermoanaerobacterales and Caldicellulosiruptorales by condensing them into strains of single species. Comparative genomics of carbohydrate-active enzyme inventories suggested differentiating phenotypic features, even among strains of the same species, reflecting available nutrients and ecological roles in their native biotopes.
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The genome sequences of three extremely thermophilic, lignocellulolytic Caldicellulosiruptor species were closed, improving previously reported multiple-contig assemblies. All 14 classified Caldicellulosiruptor spp. now have closed genomes. Genome closure will enhance bioinformatic analysis of the species, including identification of carbohydrate-active enzymes (CAZymes) and comparison against other Caldicellulosiruptor species and lignocellulolytic microorganisms.
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Reported here are complete genome sequences for two anaerobic, thermophilic bacteria isolated from wheat straw, i.e., the (hemi)cellulolytic Thermoclostridium stercorarium subspecies strain RKWS1 (3,029,933 bp) and the hemicellulolytic Thermoanaerobacter species strain RKWS2 (2,827,640 bp). Discovery of indigenous thermophiles in plant biomass suggests that high-temperature microorganisms are more ubiquitous than previously thought.
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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.
Assuntos
Clostridium thermocellum , Lignina , Biomassa , Fermentação , Lignina/química , Clostridium thermocellum/metabolismo , Plantas/metabolismoRESUMO
The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits.
Assuntos
Edição de Genes , Lignina , Populus , Madeira , Carboidratos/análise , Lignina/genética , Madeira/genética , Sistemas CRISPR-Cas , Populus/genética , Papel , Crescimento SustentávelRESUMO
A variety of chemical and biological processes have been proposed for conversion of sustainable low-cost feedstocks into industrial products. Here, a biorefinery concept is formulated, modeled, and analyzed in which a naturally (hemi)cellulolytic and extremely thermophilic bacterium, Caldicellulosiruptor bescii, is metabolically engineered to convert the carbohydrate content of lignocellulosic biomasses (i.e., soybean hulls, transgenic poplar) into green hydrogen and acetone. Experimental validation of C. bescii fermentative performance demonstrated 82% carbohydrate solubilization of soybean hulls and 55% for transgenic poplar. A detailed technical design, including equipment specifications, provides the basis for an economic analysis that establishes metabolic engineering targets. This robust industrial process leveraging metabolically engineered C. bescii yields 206 kg acetone and 25 kg H2 per metric ton of soybean hull, or 174 kg acetone and 21 kg H2 per metric ton transgenic poplar. Beyond this specific case, the model demonstrates industrial feasibility and economic advantages of thermophilic fermentation.
Assuntos
Acetona , Lignina , Biomassa , Caldicellulosiruptor , Fermentação , Hidrogênio , Lignina/químicaRESUMO
The potential to convert renewable plant biomasses into fuels and chemicals by microbial processes presents an attractive, less environmentally intense alternative to conventional routes based on fossil fuels. This would best be done with microbes that natively deconstruct lignocellulose and concomitantly form industrially relevant products, but these two physiological and metabolic features are rarely and simultaneously observed in nature. Genetic modification of both plant feedstocks and microbes can be used to increase lignocellulose deconstruction capability and generate industrially relevant products. Separate efforts on plants and microbes are ongoing, but these studies lack a focus on optimal, complementary combinations of these disparate biological systems to obtain a convergent technology. Improving genetic tools for plants have given rise to the generation of low-lignin lines that are more readily solubilized by microorganisms. Most focus on the microbiological front has involved thermophilic bacteria from the genera Caldicellulosiruptor and Clostridium, given their capacity to degrade lignocellulose and to form bio-products through metabolic engineering strategies enabled by ever-improving molecular genetics tools. Bioengineering plant properties to better fit the deconstruction capabilities of candidate consolidated bioprocessing microorganisms has potential to achieve the efficient lignocellulose deconstruction needed for industrial relevance.
Assuntos
Lignina , Plantas , Biomassa , Lignina/química , Plantas/metabolismoRESUMO
Metabolic modeling was used to examine potential bottlenecks that could be encountered for metabolic engineering of the cellulolytic extreme thermophile Caldicellulosiruptor bescii to produce bio-based chemicals from plant biomass. The model utilizes subsystems-based genome annotation, targeted reconstruction of carbohydrate utilization pathways, and biochemical and physiological experimental validations. Specifically, carbohydrate transport and utilization pathways involving 160 genes and their corresponding functions were incorporated, representing the utilization of C5/C6 monosaccharides, disaccharides, and polysaccharides such as cellulose and xylan. To illustrate its utility, the model predicted that optimal production from biomass-based sugars of the model product, ethanol, was driven by ATP production, redox balancing, and proton translocation, mediated through the interplay of an ATP synthase, a membrane-bound hydrogenase, a bifurcating hydrogenase, and a bifurcating NAD- and NADP-dependent oxidoreductase. These mechanistic insights guided the design and optimization of new engineering strategies for product optimization, which were subsequently tested in the C. bescii model, showing a nearly 2-fold increase in ethanol yields. The C. bescii model provides a useful platform for investigating the potential redox controls that mediate the carbon and energy flows in metabolism and sets the stage for future design of engineering strategies aiming at optimizing the production of ethanol and other bio-based chemicals. IMPORTANCE The extremely thermophilic cellulolytic bacterium, Caldicellulosiruptor bescii, degrades plant biomass at high temperatures without any pretreatments and can serve as a strategic platform for industrial applications. The metabolic engineering of C. bescii, however, faces potential bottlenecks in bio-based chemical productions. By simulating the optimal ethanol production, a complex interplay between redox balancing and the carbon and energy flow was revealed using a C. bescii genome-scale metabolic model. New engineering strategies were designed based on an improved mechanistic understanding of the C. bescii metabolism, and the new designs were modeled under different genetic backgrounds to identify optimal strategies. The C. bescii model provided useful insights into the metabolic controls of this organism thereby opening up prospects for optimizing production of a wide range of bio-based chemicals.
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Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. IMPORTANCE To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.