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DOE OSTI2020

In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with 2 H and 13 C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grown Escherichia coli . The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellummthan in T. saccharolyticum. These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of C. thermocellum and T. saccharolyticum, two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in T. saccharolyticum is as thermodynamically favorable as that found in model organisms, such as E. coli or Saccharomyces cerevisiae , the glycolytic pathway of C. thermocellum operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype

Clostridium thermocellum is a thermophilic, anaerobic bacterium that natively ferments cellulose to ethanol and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here, we reintroduced this mutation using recently developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. Here, the ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural Characterization and Dynamics of AdhE Ultrastructures from Clostridium thermocellum Show a Containment Strategy for Toxic Intermediates

Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe; however, ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 A structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

09 BIOMASS FUELS↗
DOE OSTI2020

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum . Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum , could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + P i → ADP + PP i .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

Abstract Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners— Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum —exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

09 BIOMASS FUELS↗
DOE OSTI2022

Composition and yield of non-cellulosic and cellulosic sugars in soluble and particulate fractions during consolidated bioprocessing of poplar biomass by Clostridium thermocellum

Abstract Background Terrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed. Results The total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I. Conclusions The significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum .

09 BIOMASS FUELS↗
DOE OSTI2022

Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in Clostridium thermocellum

Glycolysis is an ancient, widespread, and highly conserved metabolic pathway that converts glucose into pyruvate. In the canonical pathway, the phosphofructokinase (PFK) reaction plays an important role in controlling flux through the pathway. Clostridium thermocellum has an atypical glycolysis and uses pyrophosphate (PP i ) instead of ATP as the phosphate donor for the PFK reaction. The reduced thermodynamic driving force of the PP i -PFK reaction shifts the entire pathway closer to thermodynamic equilibrium, which has been predicted to limit product titers. Here, in this study, we replace the PP i -PFK reaction with an ATP-PFK reaction. We demonstrate that the local changes are consistent with thermodynamic predictions: the ratio of fructose 1,6-bisphosphate to fructose-6-phosphate increases, and the reverse flux through the reaction (determined by 13 C labeling) decreases. The final titer and distribution of fermentation products, however, do not change, demonstrating that the thermodynamic constraints of the PP i -PFK reaction are not the sole factor limiting product titer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis

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 (PP i ) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PP i 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 PP i 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 PP i -generating cycles. In all cases, the metabolic cost of PP i 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 PP i could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PP i sources likely account for most PP i production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PP i levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Rewiring metabolism of Clostridium thermocellum for consolidated bioprocessing of lignocellulosic biomass poplar to produce short-chain esters

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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The effect of switchgrass plant cell wall properties on its deconstruction by thermochemical pretreatments coupled with fungal enzymatic hydrolysis or Clostridium thermocellum consolidated bioprocessing

A combination of thermochemical pretreatment and biological digestion technologies is usually required to overcome lignocellulosic recalcitrance and accomplish effective biomass deconstruction. In this study, we aimed to understand switchgrass breakdown by hydrothermal, dilute acid, dilute alkali, and co-solvent enhanced lignocellulosic fractionation (CELF) pretreatments followed by application of traditional fungal enzymatic hydrolysis (EH) and Clostridium thermocellum consolidated bioprocessing (CBP) to the resulting solids. Unpretreated and pretreated switchgrass and their EH and CBP residues were characterized by a suite of analytical techniques to understand structural changes that occurred during deconstruction. CELF pretreated solids showed the highest accessibility and digestibility by both EH and CBP followed by dilute alkali and then dilute acid/hydrothermal pretreated solids. Lignin removal from biomass had a more positive impact on substrate accessibility and digestibility than did xylan removal, while xyloglucan removal by pretreatment appeared essential for cellulose digestion by fungal enzymes. The extent of CBP digestion of cellulose and non-cellulosic glycans was larger than that by EH. Unlike dilute alkali pretreatment, cellulose crystallinity increased for acid-based pretreatments in the following order: hydrothermal, dilute acid, and CELF. Acid-based pretreatments also substantially reduced cellulose degree of polymerization. All thermochemical and biological digestion approaches increased syringyl to guaiacyl lignin (S/G) ratio and reduced β-O-4 lignin interunit linkage and hydroxycinnamates content from levels in unpretreated switchgrass. The substantial increase in S/G ratio after hydrothermal and dilute alkali preatreatments suggested that high temperatures or alkali removed a large portion of G lignin from switchgrass.

09 BIOMASS FUELS↗
DOE OSTI2023

Simultaneous expression of an endogenous spermidine synthase and a butanol dehydrogenase from Thermoanaerobacter pseudethanolicus in Clostridium thermocellum results in increased resistance to acetic acid and furans, increased ethanol production and an increase in thermotolerance

Abstract Background Sensitivity to inhibitors derived from the pretreatment of plant biomass is a barrier to the consolidated bioprocessing of these complex substrates to fuels and chemicals by microbes. Spermidine is a low molecular weight aliphatic nitrogen compound ubiquitous in microorganisms, plants, and animals and is often associated with tolerance to stress. We recently showed that overexpression of the endogenous spermidine synthase enhanced tolerance of the Gram-positive bacterium, Clostridium thermocellum to the furan derivatives furfural and HMF. Results Here we show that co-expression with an NADPH-dependent heat-stable butanol dehydrogenase from Thermoanaerobacter pseudethanolicus further enhanced tolerance to furans and acetic acid and most strikingly resulted in an increase in thermotolerance at 65 °C. Conclusions Tolerance to fermentation inhibitors will facilitate the use of plant biomass substrates by thermophiles in general and this organism in particular. The ability to grow C. thermocellum at 65 °C has profound implications for metabolic engineering.

09 BIOMASS FUELS↗
DOE OSTI2026

Biohydrogen Generation from Un-Pretreated Spent Coffee Grounds with Clostridium Thermocellum

Coffee is among the world's most widely consumed beverages, generating 18 million wet tonnes of waste spent coffee grounds annually. Disposal of SCGs is complicated by their high moisture content, their recalcitrant chemical composition, and the presence of caffeine and other bioactive compounds. Dark fermentation is a promising technology for conversion of waste cellulosic biomass into renewable hydrogen, carbon dioxide, and volatile fatty acids. Typical SCGs have high moisture content, are pre-milled to fine particle sizes, and contain 8%-19% cellulose and up to 40% hemicellulose, making them an attractive feedstock for dark fermentation. In this study, we investigate biohydrogen production from unpretreated SCGs using Clostridium thermocellum strain KJC19-9, a cellulolytic bacterium engineered to co-utilize xylose, specifically examining the inhibitory effects of residual caffeine and high solids loadings on microbial growth and hydrogen production efficiency. While SCGs were initially resistant to both cell growth and biohydrogen production, a strategy to co-ferment with low concentrations of cellobiose dramatically enhanced process performance, reducing lag phase duration and enabling cellulosome production for efficient cellulose hydrolysis. This co-substrate approach generated up to 944 mL hydrogen per L reaction from 50 g/L of spent coffee grounds over 120 h (1.34 mol H2/mol carbohydrate), validating SCGs as a promising dark fermentation feedstock and underscoring the value of mixed substrate approaches in consolidated bioprocessing.

08 HYDROGEN↗
DOE OSTI2020

Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production

Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Maximizing long-term biohydrogen production with Clostridium thermocellum for high solids conversion of lignocellulosic biomass

Biological hydrogen production from lignocellulosic biomass sustainably couples organic waste reduction with renewable energy generation. Efficient conversion is challenged by the structural complexity of lignocellulose and resulting recalcitrance to enzymatic degradation. Clostridium thermocellum natively breaks down biomass with highly effective hemi-/cellulases systems (i.e., cellulosomes) and generates hydrogen in anaerobic cultivation, creating a compelling platform for lignocellulosic biohydrogen production. Achieving commercially viable production rates requires balancing high biomass loading and throughput against uniform mixing conditions required for enzyme dispersion, pH and temperature control, and efficient hydrogen and metabolite removal in continuous operation. To address these barriers to process intensification, we implemented novel reactor and process designs for high-solids lignocellulosic biomass fermentations using the C. thermocellum KJC19-9 strain, genetically engineered for co-utilization of cellulose and hemicellulose sugars (i.e., xylose). Via computational fluid dynamics (CFD) modeling and experimental validation, we achieved a >50% improvement in biohydrogen production with an improved anchor-type impeller morphology, coupled to a threefold reduction in agitation rate. To further reduce rheological constraints and accumulation of toxic metabolites, we then transitioned the process to sequencing fed-batch operation. The resulting process generated 24.87 L H 2 L −1 from 160 g L −1 of deacetylated and mechanically refined (DMR)-pretreated corn stover biomass over 16 days while solubilizing >95% of influent cellulose and hemicellulose, setting a new performance benchmark for continuous production of biohydrogen from lignocellulose.

08 HYDROGEN↗
DOE OSTI2021

In vivo evolution of lactic acid hyper-tolerant Clostridium thermocellum

Lactic acid (LA) has several applications in the food, cosmetics and pharmaceutical industries, as well as in the production of biodegradable plastic polymers, namely polylactides. Industrial production of LA is essentially based on microbial fermentation. Recent reports have shown the potential of the cellulolytic bacterium Clostridium thermocellum for direct LA production from inexpensive lignocellulosic biomass. However, C. thermocellum is highly sensitive to acids and does not grow at pH < 6.0. Improvement of LA tolerance of this microorganism is pivotal for its application in cost-efficient production of LA. In the present study, the LA tolerance of C. thermocellum strains LL345 (wild-type fermentation profile) and LL1111 (high LA yield) was increased by adaptive laboratory evolution. At large inoculum size (10 %), the maximum tolerated LA concentration of strain LL1111 was more than doubled, from 15 g/L to 35 g/L, while subcultures evolved from LL345 showed 50–85 % faster growth in medium containing 45 g/L LA. Gene mutations (pyruvate phosphate dikinase, histidine protein kinase/phosphorylase) possibly affecting carbohydrate and/or phosphate metabolism have been detected in most LA-adapted populations. Although improvement of LA tolerance may sometimes also enable higher LA production in microorganisms, C. thermocellum LA-adapted cultures showed a yield of LA, and generally of other organic acids, similar to or lower than parental strains. Based on its improved LA tolerance and LA titer similar to its parent strain (LL1111), mixed adapted culture LL1630 showed the highest performing phenotype and could serve as a framework for improving LA production by further metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Development of a Genome-Scale Metabolic Model of Clostridium thermocellum and Its Applications for Integration of Multi-Omics Datasets and Computational Strain Design

Solving environmental and social challenges such as climate change requires a shift from our current non-renewable manufacturing model to a sustainable bioeconomy. To lower carbon emissions in the production of fuels and chemicals, plant biomass feedstocks can replace petroleum using microorganisms as biocatalysts. The anaerobic thermophile Clostridium thermocellum is a promising bacterium for bioconversion due to its capability to efficiently degrade lignocellulosic biomass. However, the complex metabolism of C. thermocellum is not fully understood, hindering metabolic engineering to achieve high titers, rates, and yields of targeted molecules. In this study, we developed an updated genome-scale metabolic model of C. thermocellum that accounts for recent metabolic findings, has improved prediction accuracy, and is standard-conformant to ensure easy reproducibility. We illustrated two applications of the developed model. We first formulated a multi-omics integration protocol and used it to understand redox metabolism and potential bottlenecks in biofuel (e.g., ethanol) production in C. thermocellum. Second, we used the metabolic model to design modular cells for efficient production of alcohols and esters with broad applications as flavors, fragrances, solvents, and fuels. The proposed designs not only feature intuitive push-and-pull metabolic engineering strategies, but also present novel manipulations around important central metabolic branch-points. We anticipate the developed genome-scale metabolic model will provide a useful tool for system analysis of C. thermocellum metabolism to fundamentally understand its physiology and guide metabolic engineering strategies to rapidly generate modular production strains for effective biosynthesis of biofuels and biochemicals from lignocellulosic biomass.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Rhamnogalacturonan I is a recalcitrant pectin domain during Clostridium thermocellum -mediated deconstruction of switchgrass biomass

Background Liquid fuels from lignocellulosic feedstocks are required for transition to a sustainable bioeconomy. However, the recalcitrance of carbon-containing feedstock cell walls to deconstruction poses a barrier to cost effective biological conversion of plant biomass to biofuels. One-step consolidated bioprocessing (CBP) in which anaerobic thermophilic bacteria convert lignocellulosic biomass into liquid fuels is a platform for overcoming the recalcitrance of plant biomass. Results The amounts of hemicellulosic and pectic polysaccharides, two complex cell wall glycans that contribute to plant biomass recalcitrance and that are partially solubilized during CBP of switchgrass aerial biomass by Clostridium thermocellum were evaluated in the liquor, solid residues and residue washate recovered during a 120-h CBP process. After 120 h, 24% of milled switchgrass was solubilized in the C. thermocellum CBP platform. Higher concentrations of arabinose, xylose, galactose, and glucose accumulated in the CBP-fermentation liquor and washate compared to fermentation controls without C. thermocellum, indicating that C. thermocellum solubilized hemicelluloses, but did not fully metabolize them. After five days of fermentation, the relative amount of rhamnose in the solid residues increased by 16% compared to controls, and CBP solid residues had more than 23% increased reactivity against RG-I reactive monoclonal antibodies, indicating that the pectic polymer rhamnogalacturonan I (RG-I) was not effectively solubilized from switchgrass biomass by C. thermocellum CBP. Similarly, the amount of mannose (Man) in the CBP solid residues increased by 7% and reactivity against galactomannan reactive antibodies increased by greater than 14%, indicating that the hemicellulosic polymer galactomannan was also resistant to degradation by C. thermocellum during CBP fermentation. Conclusions These findings show that C. thermocellum is unable to effectively degrade RG-I pectic and galactomannan hemicellulosic components in switchgrass biomass. Targeting these polymers for improved solubilization could enhance the efficiency of conversion of grass biomass to biofuels.

09 BIOMASS FUELS↗
DOE OSTI2021

Identification and characterization of proteins of unknown function (PUFs) in Clostridium thermocellum DSM 1313 strains as potential genetic engineering targets

Abstract Background Mass spectrometry-based proteomics can identify and quantify thousands of proteins from individual microbial species, but a significant percentage of these proteins are unannotated and hence classified as proteins of unknown function (PUFs). Due to the difficulty in extracting meaningful metabolic information, PUFs are often overlooked or discarded during data analysis, even though they might be critically important in functional activities, in particular for metabolic engineering research. Results We optimized and employed a pipeline integrating various “guilt-by-association” (GBA) metrics, including differential expression and co-expression analyses of high-throughput mass spectrometry proteome data and phylogenetic coevolution analysis, and sequence homology-based approaches to determine putative functions for PUFs in Clostridium thermocellum . Our various analyses provided putative functional information for over 95% of the PUFs detected by mass spectrometry in a wild-type and/or an engineered strain of C. thermocellum . In particular, we validated a predicted acyltransferase PUF (WP_003519433.1) with functional activity towards 2-phenylethyl alcohol, consistent with our GBA and sequence homology-based predictions. Conclusions This work demonstrates the value of leveraging sequence homology-based annotations with empirical evidence based on the concept of GBA to broadly predict putative functions for PUFs, opening avenues to further interrogation via targeted experiments.

09 BIOMASS FUELS↗