Acetivibrio thermocellus (formerly Clostridium thermocellum) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PPi-dependent 6-phosphofructokinase (PPi-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PPi-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable in vitro ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PPi can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PPi-generating mechanism to drive glycolysis, as PPi-Pfk can simply use whatever PPi is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPPi-Pfk strain, unable to valorize PPi, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PPi. Together with other mutations, this resulted in an A. thermocellus strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/gx/h. These findings are both relevant for fundamental insight into dual ATP/PPi Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of A. thermocellus for consolidated bioprocessing.

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.

To mitigate climate change, greenhouse gas emissions must be reduced to net-zero in 2050 requiring a drastic transition in today´s energy sector. To achieve this goal, the use of biofuels produced from lignocellulosic feedstocks, including agricultural and forestry residues, is expected to play an important role. The native ability of the anaerobic thermophile Clostridium thermocellum to efficiently degrade lignocellulose makes this microorganism a promising candidate for consolidated bioprocessing of lignocellulosic feedstocks into the biofuel ethanol. However, improvements in ethanol yield, titre, and tolerance are required for industrial implementation. The aim of this thesis was to increase understanding of the central metabolism of C. thermocellum and thereby aid future metabolic engineering and process optimization efforts focused on improving ethanol production from lignocellulosic material. 

The atypical glycolysis of C. thermocellum uses pyrophosphate (PP_i) instead of ATP as phosphoryl donor. This alteration is hypothesized to increase energetic efficiency but simultaneously decrease thermodynamic driving force resulting in lower achievable ethanol titres. As such, improved understanding of the PP_i metabolism has both fundamental and applied importance. Knockout studies combined with physiological characterization of four predicted metabolic PP_i sources provided valuable insights into the PP_i metabolism and demonstrated that the energetic benefits of PP_i usage are likely limited. Furthermore, biochemical characterization of the ATP-Pfk from C. thermocellum and other bacteria demonstrated that PP_i might be a key allosteric regulator in bacteria with a PP_i-dependent glycolysis. 

The low thermodynamic driving force of the ethanol formation pathway combined with a flexible redox network are key factors that impact ethanol titre, yield, and tolerance in C. thermocellum. Apart from dominant thermodynamic limitations, physiological characterization of wild-type and a non-ethanol producing mutant at various exogenous ethanol concentrations and temperatures demonstrated that biophysical limitations also impact ethanol tolerance. Lowering the cultivation temperature decreased chaotropic effects of ethanol and improved ethanol tolerance. 

By-product formation and incomplete substrate utilization decrease obtained ethanol yields. To minimize formation of one specific class of by-products, the mechanism behind amino acid secretion in C. thermocellum was investigated. Cellobiose- or ammonium-limited chemostats of wild-type and knockout strains of NADPH-supplying and NADPH-consuming pathways identified catabolic oversupply of NADPH as the main driver behind amino acid secretion. The malate shunt and the ammonium-regulated shift between nitrogen assimilation pathways with differing cofactor specificities were shown to play key roles in NADPH metabolism and amino acid secretion. 

To improve substrate utilization, laboratory evolution combined with reverse metabolic engineering was used as a tool to provide insights into increased utilization of glucose and fructose. Reproducible and constitutive growth on these hexose sugars was achieved for evolved mutant strains. Additionally, two mutations were identified that are involved in (regulation of) transport or metabolism of these hexose sugars.

Together these findings provide valuable insights into the central metabolism of C. thermocellum and aid future optimizations of this organism for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. 

För att mildra klimatförändringarna måste utsläppen av växthusgaser minskas till nettonoll år 2050 vilket kräver en drastisk förändring i dagens energisektor. För att uppnå detta mål förväntas användningen av biobränslen producerade från lignocellulosabaserade råmaterial, såsom jordbruks- och skogsrester, spela en viktig roll. Den naturliga förmågan att effektivt bryta ned lignocellulosa hittas hos den anaerobiska termofilen Clostridium thermocellum och gör denna mikroorganism till en lovande kandidat för konsoliderad bioprocessering av lignocellulosabaserade råmaterial till biobränslet etanol. För industriell implementering krävs dock förbättringar av etanolutbyte, -titer och -tolerans. Syftet med denna avhandling var att öka förståelsen av C. thermocellums centrala metabolism och därigenom vägleda framtida insatser inom metabol ingenjörskonst och processoptimering för att förbättra etanolproduktionen från lignocellulosabaserade råvaror.

Den atypiska glykolysen hos C. thermocellum använder pyrofosfat (PP_i) i stället för ATP som fosforyldonator. Denna skillnad har hypotiserats öka energieffektiviteten men samtidigt minska den termodynamiska drivkraften, och resultera i en lägre uppnåbar etanoltiter. Därför är en förbättrad förståelse av PP_i-metabolismen viktig ur fundamentala och applicerbara perspektiv. Knockoutstudier tillsammans med fysiologisk karaktärisering av fyra predikterade metaboliska PP_i-källor gav värdefulla insikter i PP_i-metabolismen och visade att energifördelarna med användningen av PP_i sannolikt är begränsade. Vidare visade biokemisk karaktärisering av ATP-Pfk från C. thermocellum och andra bakterier att PP_i kan vara en viktig allosterisk regulator för bakterier med en PP_i-beroende glykolys.

Den låga termodynamiska drivkraften hos etanolproduktionsvägen kombinerat med ett flexibelt redoxnätverk är nyckelfaktorer som påverkar etanoltitern, -utbytet och -toleransen hos C. thermocellum. Förutom övervägande termodynamiska begränsningar visade fysiologisk karaktärisering av vildtypen och en modifierad icke-etanolproducerande stam, vid olika extracellulära etanolkoncentrationer och temperaturer, att även biokemiska begränsningar påverkar etanoltoleransen. Att sänka odlingstemperaturen reducerade de kaotropiska effekterna av etanol och förbättrade etanoltoleransen.

Produktion av biprodukter och ofullständigt substratutnyttjande minskar erhållna etanolutbyten. För att minimera produktionen av en specifik klass av biprodukter undersöktes mekanismen bakom aminosyrasekretion hos C. thermocellum. Cellobios- eller ammoniumbegränsade kemostater av vildtypen och knockout-stammar av NADPH-producerande och -konsumerande reaktionsvägar identifierade ett katabolisk överskott av NADPH som den främsta drivkraften bakom aminosyrasekretion. Malatshunten samt det ammoniumreglerade skiftet mellan olika assimileringvägar av kväve med specificitet för olika kofaktorer visade sig spela en nyckelroll i NADPH-metabolismen och aminosyrasekretionen. 

För att öka substratutnyttjandet användes laboratorieevolution kombinerat med reverse metabolic engineering som verktyg för att ge insikter i hur utnyttjande av glukos och fruktos kan förbättras. Laboratorieevolutionen resulterade i stammar med reproducerbar och konstitutiv tillväxt på dessa hexoser. Sedan identifierades två mutationer involverade i (reglering av) transport eller metabolism av dessa hexoser.

Tillsammans ger dessa forskningsrön värdefulla insikter i C. thermocellums centrala metabolism och underlättar framtida optimeringar av denna organism för konsoliderad bioprocessering av lignocellulosabaserat råmaterial till bränslen och kemikalier.

Cereal arabinoxylans (AXs) are complex polysaccharides in terms of their pattern of arabinose and ferulic acid substitutions, which influence their properties in structural and nutritional applications. We have evaluated the influence of the molecular structure of three AXs from wheat and rye with distinct substitutions on the activity of β-xylanases from different glycosyl hydrolase families (GH 5_34, 8, 10 and 11). The arabinose and ferulic acid substitutions influence the accessibility of the xylanases, resulting in specific profiles of arabinoxylan-oligosaccharides (AXOS). The GH10 xylanase from Aspergillus aculeatus (AcXyn10A) and GH11 from Thermomyces lanuginosus (TlXyn11) showed the highest activity, producing larger amounts of small oligosaccharides in shorter time. The GH8 xylanase from Bacillus sp. (BXyn8) produced linear xylooligosaccharides and was most restricted by arabinose substitution, whereas GH5_34 from Gonapodya prolifera (GpXyn5_34) required arabinose substitution and produced longer (A)XOS substituted on the reducing end. The complementary substrate specificity of BXyn8 and GpXyn5_34 revealed how arabinoses were distributed along the xylan backbones. This study demonstrates that AX source and xylanase specificity influence the production of oligosaccharides with specific structures, which in turn impacts the growth of specific bacteria (Bacteroides ovatus and Bifidobacterium adolescentis) and the production of beneficial metabolites (short-chain fatty acids).

The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and maximum activity (Vmax > 156 U mg-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and maximum activity (14.5 U mg-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.

Clostridium thermocellum is a cellulolytic thermophile that is considered for the consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but the incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated via gene deletions in ammonium-regulated, nicotinamide adenine dinucleotide phosphate (NADPH)-supplying and NADPH-consuming pathways as well as via physiological characterization in cellobiose-limited or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Δppdk) or a redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased from 46 to 60%, suggesting that the secretion of these amino acids balances the NADPH surplus from the malate shunt. The unchanged amino acid secretion in Δppdk falsified a previous hypothesis on an ammonium-regulated PEP-to-pyruvate flux redistribution. The possible involvement of another NADPH-supplier, namely, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, the deletion of glutamate synthase (gogat) in ammonium assimilation resulted in the upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, a claim which is supported by the product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of the NADH needed for ethanol formation. This suggests that metabolic engineering strategies that simplify the redox metabolism and ammonium assimilation can contribute to increased ethanol yields.

BackgroundClostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures.ResultsTo separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (P-clo1313_2638::ldh* adhE) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE, in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 degrees C for ethanol concentrations >= 10 g L-1 and was able to reach higher maximum OD600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD600 and relative growth rates were higher at 45 and 50 degrees C, compared to 55 degrees C, for ethanol concentrations >= 15 g L-1. For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L-1 at 45 and 50 degrees C compared to 55 degrees C.ConclusionPositive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions.

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 g(biomass) g(substrate)-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 PP, 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.

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 g(biomass) g(substrate)(-1), respectively, compared to 0.15 +/- 0.01 g(biomass) g(substrate)(-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. IMPORTANCE C. 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.

In this study, we compared the activity of four different xylanases (GH 5_34, 8, 10 and 11) for the production of arabinoxylan-oligosaccharide or (A)XOS from cereal arabinoxylans as potential prebiotics. In terms of efficiency, the AcXyn10A and TlXyn11 produced larger quantities of small (A)XOS of mainly X1-X3 in a shorter period of time. The BXyn8 was most restricted by high arabinose substitution but could still accommodate them at the +2 subsite. The GpXyn5_34 preferred high arabinose substitution, requiring an arabinose at the -1 subsite for cleavage. Both the BXyn8 and GpXyn5_34 produced longer (A)XOS that were mostly linear and substituted, respectively. During fermentation of (A)XOS on selected gut bacteria, the long-substituted products of GpXyn5_34 stunted the growth of B. ovatus and B. adolescentis, while the mid-length, linear products of BXyn8 favoured the growth of both bacteria.