Engineering ethanologenicity into the extremely thermophilic bacterium Anaerocellum (f. Caldicellulosiriuptor) bescii.

The anaerobic bacterium Anaerocellum (f. Caldicellulosiruptor) bescii natively ferments the carbohydrate content of plant biomass (including microcrystalline cellulose) into predominantly acetate, H2, and CO2, and smaller amounts of lactate, alanine and valine. While this extreme thermophile (growth Topt 78 °C) is not natively ethanologenic, it has been previously metabolically engineered with this property, albeit initially yielding low solvent titers (∼15 mM). Herein we report significant progress on improving ethanologenicity in A. bescii, such that titers above 130 mM have now been achieved, while concomitantly improving selectivity by minimizing acetate formation. Metabolic engineering progress has benefited from improved molecular genetic tools and better understanding of A. bescii growth physiology. Heterologous expression of a mutated thermophilic alcohol dehydrogenase (AdhE) modified for co-factor requirement, coupled with bioreactor operation strategies related to pH control, have been key to enhanced ethanol generation and fermentation product specificity. Insights gained from metabolic modeling of A. bescii set the stage for its further improvement as a metabolic engineering platform.

Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers.

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.

Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus.

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.

Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation.

Genetic engineering of hyperthermophilic organisms for the production of fuels and other useful chemicals is an emerging biotechnological opportunity. In particular, for volatile organic compounds such as ethanol, fermentation at high temperatures could allow for straightforward separation by direct distillation. Currently, the upper growth temperature limit for native ethanol producers is 72°C in the bacterium Thermoanaerobacter ethanolicus JW200, and the highest temperature for heterologously-engineered bioethanol production was recently demonstrated at 85°C in the archaeon Pyrococcus furiosus. Here, we describe an engineered strain of P. furiosus that synthesizes ethanol at 95°C, utilizing a homologously-expressed native alcohol dehydrogenase, termed AdhF. Ethanol biosynthesis was compared at 75°C and 95°C with various engineered strains. At lower temperatures, the acetaldehyde substrate for AdhF is most likely produced from acetate by aldehyde ferredoxin oxidoreductase (AOR). At higher temperatures, the effect of AOR on ethanol production is negligible, suggesting that acetaldehyde is produced by pyruvate ferredoxin oxidoreductase (POR) via oxidative decarboxylation of pyruvate, a reaction known to occur only at higher temperatures. Heterologous expression of a carbon monoxide dehydrogenase complex in the AdhF overexpression strain enabled it to use CO as a source of energy, leading to increased ethanol production. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes. This work opens the door to the potential for 'bioreactive distillation' since fermentation can be performed well above the normal boiling point of ethanol. IMPORTANCE Previously, the highest temperature for biological ethanol production was 85°C. Here, we have engineered ethanol production at 95°C by the hyperthermophilic archaeon Pyrococcus furiosus. Using mutant strains, we showed that ethanol production occurs by different pathways at 75°C and 95°C. In addition, by heterologous expression of a carbon monoxide dehydrogenase complex, ethanol production by this organism was driven by the oxidation of carbon monoxide. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes.

Biochemical and Regulatory Analyses of Xylanolytic Regulons in Caldicellulosiruptor bescii Reveal Genus-Wide Features of Hemicellulose Utilization.

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.

Genome-Scale Metabolic Model of Caldicellulosiruptor bescii Reveals Optimal Metabolic Engineering Strategies for Bio-based Chemical Production.

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.

Transcriptional Regulation of Plant Biomass Degradation and Carbohydrate Utilization Genes in the Extreme Thermophile Caldicellulosiruptor bescii.

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.