Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism.

Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. 13C and 2H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of 2H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to 13C-tracers. Furthermore, isotope tracer studies in industrially relevant bacteria that metabolize complex substrates such as cellulose, hemicellulose, or lignocellulosic biomass, are challenging given the difficulty in obtaining these as isotopically labeled substrates. In this study, we examine the potential of deuterated water (2H2O) as an affordable, substrate-neutral isotope tracer for studying central carbon metabolism. We apply 2H2O labeling to investigate the reversibility of glycolytic reactions across three industrially relevant bacterial species -C. thermocellum, Z. mobilis, and E. coli-harboring distinct glycolytic pathways with unique thermodynamics. We demonstrate that 2H2O labeling recapitulates previous reversibility and thermodynamic findings obtained with established 13C and 2H labeled nutrient substrates. Furthermore, we exemplify the utility of this 2H2O labeling approach by applying it to high-substrate C. thermocellum fermentations -a setting in which the use of conventional tracers is impractical-thereby identifying the glycolytic enzyme phosphofructokinase as a major bottleneck during high-substrate fermentations and unveiling critical insights that will steer future engineering efforts to enhance ethanol production in this cellulolytic organism. This study demonstrates the utility of deuterated water as a substrate-agnostic isotope tracer for examining flux and reversibility of central carbon metabolic reactions, which yields biological insights comparable to those obtained using costly 2H-labeled nutrient substrates.

Mechanism of furfural toxicity and metabolic strategies to engineer tolerance in microbial strains.

Lignocellulosic biomass represents a carbon neutral cheap and versatile source of carbon which can be converted to biofuels. A pretreatment step is frequently used to make the lignocellulosic carbon bioavailable for microbial metabolism. Dilute acid pretreatment at high temperature and pressure is commonly utilized to efficiently solubilize the pentose fraction by hydrolyzing the hemicellulose fibers and the process results in formation of furans-furfural and 5-hydroxymethyl furfural-and other inhibitors which are detrimental to metabolism. The presence of inhibitors in the medium reduce productivity of microbial biocatalysts and result in increased production costs. Furfural is the key furan inhibitor which acts synergistically along with other inhibitors present in the hydrolysate. In this review, the mode of furfural toxicity on microbial metabolism and metabolic strategies to increase tolerance is discussed. Shared cellular targets between furfural and acetic acid are compared followed by discussing further strategies to engineer tolerance. Finally, the possibility to use furfural as a model inhibitor of dilute acid pretreated lignocellulosic hydrolysate is discussed. The furfural tolerant strains will harbor an efficient lignocellulosic carbon to pyruvate conversion mechanism in presence of stressors in the medium. The pyruvate can be channeled to any metabolite of interest by appropriate modulation of downstream pathway of interest. The aim of this review is to emphasize the use of hydrolysate as a carbon source for bioproduction of biofuels and other compounds of industrial importance.

Ethanol tolerance in engineered strains of Clostridium thermocellum.

Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75-80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.

Characterization and Amelioration of Filtration Difficulties Encountered in Metabolomic Studies of Clostridium thermocellum at Elevated Sugar Concentrations.

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.

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 (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.

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 (PPi) instead of ATP as the phosphate donor for the PFK reaction. The reduced thermodynamic driving force of the PPi-PFK reaction shifts the entire pathway closer to thermodynamic equilibrium, which has been predicted to limit product titers. Here, we replace the PPi-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 13C labeling) decreases. The final titer and distribution of fermentation products, however, do not change, demonstrating that the thermodynamic constraints of the PPi-PFK reaction are not the sole factor limiting product titer. IMPORTANCE The ability to control the distribution of thermodynamic driving force throughout a metabolic pathway is likely to be an important tool for metabolic engineering. The phosphofructokinase reaction is a key enzyme in Embden-Mayerhof-Parnas glycolysis and therefore improving the thermodynamic driving force of this reaction in C. thermocellum is believed to enable higher product titers. Here, we demonstrate switching from pyrophosphate to ATP does in fact increases the thermodynamic driving force of the phosphofructokinase reaction in vivo. This study also identifies and overcomes a physiological hurdle toward expressing an ATP-dependent phosphofructokinase in an organism that utilizes an atypical glycolytic pathway. As such, the method described here to enable expression of ATP-dependent phosphofructokinase in an organism with an atypical glycolytic pathway will be informative toward engineering the glycolytic pathways of other industrial organism candidates with atypical glycolytic pathways.

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. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. IMPORTANCE Cellulosic biofuels are a promising approach to decarbonize the heavy-duty-transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of cellulose to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum, are promising candidates for cellulosic biofuel production; however, they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially relevant organism, which can reduce the time needed for adaptive evolution experiments.

Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model.

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13C-metabolic flux analysis (13C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13C labeling data were used with 13C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.

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.

Functional Analysis of H+-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum.

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 ± 0.01 h-1) and the biomass yield by 38% (0.18 ± 0.00 gbiomass gsubstrate-1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk-malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PPi stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi, or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi- and ATP-dependent glycolysis.

Inhibition of Pyruvate Kinase From Thermoanaerobacterium saccharolyticum by IMP Is Independent of the Extra-C Domain.

The pyruvate kinase (PYK) isozyme from Thermoanaerobacterium saccharolyticum (TsPYK) has previously been used in metabolic engineering for improved ethanol production. This isozyme belongs to a subclass of PYK isozymes that include an extra C-domain. Like other isozymes that include this extra C-domain, we found that TsPYK is activated by AMP and ribose-5-phosphate (R5P). Our use of sugar-phosphate analogs generated a surprising result in that IMP and GMP are allosteric inhibitors (rather than activators) of TsPYK. We believe this to be the first report of any PYK isozyme being inhibited by IMP and GMP. A truncated protein that lacks the extra C-domain is also inhibited by IMP. A screen of several other bacterial PYK enzymes (include several that have the extra-C domain) indicates that the inhibition by IMP is specific to only a subset of those isozymes.

Laboratory Evolution and Reverse Engineering of Clostridium thermocellum for Growth on Glucose and Fructose.

The native ability of Clostridium thermocellum to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of C. thermocellum, as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of C. thermocellum on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 ± 0.00 and 0.18 ± 0.00 gbiomass gsubstrate-1, respectively, compared to 0.15 ± 0.01 gbiomass gsubstrate-1 for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, while wild type showed 42 ± 4 h on glucose and >80 h on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 h with a growth rate of 0.11 ± 0.01 h-1 and resulted in immediate growth on glucose at 0.24 ± 0.01 h-1 Additional introduction of a G-to-V mutation at position 148 in cbpA resulted in immediate growth on fructose at 0.32 ± 0.03 h-1 These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings.IMPORTANCEC. thermocellum is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that C. thermocellum can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing, and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with C. thermocellum using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of C. thermocellum can be engineered to (i) reduce glucose accumulation, (ii) study cellodextrin transport systems in vivo, (iii) allow experiments at >120 g liter-1 soluble substrate concentration, or (iv) reduce costs for labeling studies.

Methods for Metabolic Engineering of Thermoanaerobacterium saccharolyticum.

In this work, we describe genetic tools and techniques for engineering Thermoanaerobacterium saccharolyticum. In particular, the T. saccharolyticum transformation protocol and the methods for selecting for transformants are described. Methods for determining strain phenotypes are also presented.

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

BACKGROUND: 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. RESULTS: Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. CONCLUSIONS: Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.

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

Conversion of phosphoenolpyruvate to pyruvate in Thermoanaerobacterium saccharolyticum.

Thermoanaerobacterium saccharolyticum is an anaerobic thermophile that can ferment hemicellulose to produce biofuels, such as ethanol. It has been engineered to produce ethanol at high yield and titer. T. saccharolyticum uses the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis. However, the genes and enzymes used in each step of the EMP pathway in T. saccharolyticum are not completely known. In T. saccharolyticum, both pyruvate kinase (PYK) and pyruvate phosphate dikinase (PPDK) are highly expressed based on transcriptomic and proteomic data. Both enzymes catalyze the formation of pyruvate from phosphoenolpyruvate (PEP). PYK is typically the last step of EMP glycolysis pathway while PPDK is reversible and is found mostly in C4 plants and some microorganisms. It is not clear what role PYK and PPDK play in T. saccharolyticum metabolism and fermentation pathways and whether both are necessary. In this study we deleted the ppdk gene in wild type and homoethanologen strains of T. saccharolyticum and showed that it is not essential for growth or ethanol production.

Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose.

BACKGROUND: Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. RESULTS: We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase-hydroxybutyryl-CoA dehydrogenase-crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. CONCLUSIONS: Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h.