(1,3;1,4)-β-D-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-D-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIBT. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-D-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-D-glucans are produced with structures similar to those of the (1,3;1,4)-β-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-D-glucans in these species.

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 societal goal of reaching net-zero CO₂ emissions requires development of integrated biorefineries to produce biomass-derived fuels and chemicals. For sustainable second-generation bioethanol production, consolidated bioprocessing with the thermophile Clostridium thermocellum is regarded as a promising concept in view of the microorganism’s native ability to efficiently degrade plant cell wall material. However, for industrial implementation, improvements in ethanol titer and yield are needed. The aim of this thesis was to increase knowledge on the metabolism of C. thermocellum and thereby guide future metabolic engineering strategies to maximize the ethanol yield and titer.

Yield improvements and fundamental studies into the metabolism of C. thermocellum would benefit from higher utilization of hexose monomers as well as minimized byproduct formation. To investigate underlying mechanisms for growth on glucose and fructose, laboratory evolution in chemostats together with genome sequence-based reverse engineering was applied. This successfully revealed two underlying mutations with (regulatory) roles in metabolism or transport of the monosaccharides. Together, these mutations enable reproducible and constitutive growth and are relevant for follow-up studies into transport and upper glycolysis. Separately, the mechanism behind the surprising byproduct formation of secreted amino acids was investigated by knock-out studies in NADPH-supplying and -consuming pathways. Physiological characterization in cellobiose- or ammonium-limited chemostats of mutant strains, with deletions in the NADPH-forming malate shunt or in the putatively ferredoxin-dependent ammonium assimilation, demonstrated a central role of NADPH in driving amino acid secretion. The findings indicated that electron availability will be crucial for further yield improvements in the NADH-dependent ethanol pathway.

Fundamental mechanisms that might contribute to improved ethanol titer were addressed by studying thermodynamic and biophysical limitations. The pyrophosphate (PP_i)-dependent glycolysis of C. thermocellum has been hypothesized to increase the overall ATP yield at the expense of the overall driving force. Knock-out studies combined with functional annotation of potential PP_i-sources questioned this trade-off and increased knowledge of the PP_i metabolism. The chaotropic effect (biophysical toxicity) of ethanol is commonly counteracted by lowering the cultivation temperature. Here, physiological characterization at varying ethanol titers demonstrated improved growth and fermentation at lower temperature. Comparisons to a non-ethanol producing mutant indicated both thermodynamic and biophysical limitations specifically in the ethanol pathway.

Overall, these findings suggest that improvements in ethanol yield and titer would benefit from a simplified glycolysis that is engineered for a high driving force. While this work is beneficial for second-generation ethanol production, these findings can also be broadly applicable in the research and development of C. thermocellum as a cell factory for sustainable production of other fuels and chemicals. 

Samhällsmålet att nå nettonoll CO₂ utsläpp kräver att integrerade bioraffinaderier utvecklas för att producera bränslen och kemikalier baserade på biomassa. För hållbar andra-generationens bioetanol-produktion betraktas konsoliderad bioprocessering med termofilen Clostridium thermocellum som ett lovande koncept, utifrån dess naturliga förmåga att effektivt bryta ner växtcellväggar. Emellertid krävs ökad titer och utbyte av etanol för att nå industriell implementering. Målet med denna avhandling var att öka kunskapen om C. thermocellums metabolism och därmed vägleda framtida strategier för att maximera utbytet och titern av etanol genom metabolic engineering.

Förbättringar i utbytet samt fundamentala studier på metabolism hos C. thermocellum skulle gynnas av ett större utnyttjande av C₆-mono-sackarider samt minskad produktion av biprodukter. Underliggande mekanismer för tillväxt på glukos och fruktos undersöktes med laboratory evolution i kemostater samt genomsekvensbaserad reverse engineering. I denna studie avslöjades två underliggande mutationer med (regulatoriska) roller i metabolismen eller transporten av dessa monosackarider. Tillsammans möjliggjorde dessa mutationer reproducerbar och konstitutiv tillväxt. Mutationerna är även relevanta för uppföljningsstudier av sockertransport och den övre glykolysen. Därutöver studerades den oväntade biproduktgruppen, aminosyror, genom knockoutstudier på NADPH-producerande och -konsumerande reaktionsvägar. Stammar med knockouts i den NADPH-producerande malatshunten eller i den potentiellt ferredoxin-kopplade ammoniumassimileringen karaktäriserades fysio-logiskt i cellobios- och ammoniumbegränsande kemostater. Detta visade att NADPH har en central roll i att driva aminosyrautsöndring. Dessa upptäckter indikerade att elektrontillgänglighet är kritiskt för att öka utbytet i den NADH-beroende etanolproduktionen. 

Fundamentala mekanismer som skulle kunna bidra till förbättrad titer av etanol studerades från termodynamiska och biofysiska perspektiv. En rådande hypotes har varit att den pyrofosfat (PP_i)-beroende glykolysen hos C. thermocellum ökar ATP-utbytet på bekostnad av den totala termodynamiska drivkraften. Knockoutstudier kombinerat med funktionell annotering av potentiella PP_i-källor ifrågasatte denna hypotes och ökade förståelsen av PP_i metabolismen. Den kaotropiska effekten (biofysisk toxicitet) av etanol dämpas ofta i industriella processer genom att sänka odlingstemperaturen. Här demonstrerade fysiologisk karaktärisering vid olika etanoltiter att tillväxt och fermentering förbättras vid lägre temp-eraturer. En jämförelse mellan en modifierad icke-etanolproducerande stam och vildtypen indikerade att etanolproduktionen är begränsad av både termodynamiska och biofysiska faktorer. 

I helhet antyder dessa forskningsresultat att förbättringar i utbytet och titern av etanol skulle gynnas av en förenklad glykolys, konstruerad för att ge en hög termodynamisk drivkraft. Fastän denna avhandling fokuserar på andra-generationens etanolproduktion, kan dessa forskningsrön även appliceras mer brett i forskning och utveckling av C. thermocellum som en cellfabrik för hållbar produktion av andra bränslen och kemikalier. 

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.

BACKGROUND Clostridium 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 chaotropicinduced stress resulting in increased membrane uidization 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.

RESULTS To 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 °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 OD600and relative growth rates were higher at 45 and 50 °C, compared to 55 °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 °C compared to 55 °C.

CONCLUSION Positive 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

Clostridium thermocellum is a cellulolytic thermophile considered for consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated by gene deletions in ammonium-regulated NADPH-supplying and -consuming pathways and physiological characterization in cellobiose- 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 redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::P_eno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased 46-60%, suggesting that secretion of these amino acids balances NADPH surplus from the malate shunt. Unchanged amino acid secretion in Δppdk falsified a previous hypothesis on ammonium-regulated PEP-to-pyruvate flux redistribution. Possible involvement of another NADPH-supplier, namely NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, deletion of glutamate synthase (gogat) in ammonium assimilation resulted in upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, which is supported by 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 NADH needed for ethanol formation. This suggests that metabolic engineering strategies on simplifying redox metabolism and ammonium assimilation can contribute to increased ethanol yields.

Importance. Improving the ethanol yield of C. thermocellum is important for industrial implementation of this microorganism in consolidated bioprocessing. A central role of NADPH in driving amino acid byproduct formation was demonstrated, by eliminating the NADPH-supplying malate shunt and separately by changing the cofactor specificity in ammonium assimilation. With amino acid secretion diverting carbon and electrons away from ethanol, these insights are important for further metabolic engineering to reach industrial requirements on ethanol yield. This study also provides chemostat data relevant for training genome-scale metabolic models and improving the validity of their predictions, especially considering the reduced degree-of-freedom in redox metabolism of the strains generated here. In addition, this study advances fundamental understanding on mechanisms underlying amino acid secretion in cellulolytic Clostridia as well as regulation and cofactor specificity in ammonium assimilation. Together, these efforts aid development of C. thermocellum for sustainable consolidated bioprocessing of lignocellulose to ethanol with minimal pretreatment.