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Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 0.1560.01 gbiomass gsubstrate -1 for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, while wild type showed 4264 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 0.2460.01 h-1. Additional introduction of a G-to-V mutation at position 148 in cbpA resulted in immediate growth on fructose at 0.3260.03 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. [ABSTRACT FROM AUTHOR]

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden–Mayerhof–Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage.

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden–Mayerhof–Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage. [ABSTRACT FROM AUTHOR]

The cellulases of family 9 glycoside hydrolase with subtle difference in amino acid sequence have shown different types of catalytic activities such as endo-, exo- or processive endocellulase. However, the reason behind the different types of catalytic activities still unclear. In this study, the processive endocellulase, Ht GH9 of family 9 GH from Hungateiclostridium thermocellum was modeled by homology modeling. The catalytic module (Ht GH9t) of Ht GH9 modeled structure displayed the (α/α) 6 barrel topology and associated family 3 carbohydrate binding module (Ht CBM3c) displayed β-sandwich fold. Ramachandran plot of Ht GH9 modeled structure displayed all the amino acid residues in allowed region except Asn225 and Asp317. Secondary structure analysis of modeled Ht GH9 showed the presence of 41.3% α-helices and 11.0% β-strands which was validated through circular dichroism analysis that showed the presence of 42.6% α-helices and 14.5% β-strands. Molecular Dynamic (MD) simulation of Ht GH9 structure for 50 ns showed Root Mean Square Deviation (RMSD), 0.84 nm and radius of gyration (Rg) 3.1 nm. The Small-angle X-ray scattering of Ht GH9 confirmed the monodisperse state. The radius of gyration for globular shape (Rg) was 5.50 ± 0.15 nm and for rod shape (Rc) by Guinier plot was 2.0 nm. The loop formed by amino acid residues, 264–276 towards one end of the catalytic site of Ht GH9 forms a barrier, that blocks the non-reducing end of the cellulose chain causing the processive cleavage resulting in the release of cellotetraose. The position of the corresponding loop in cellulases of family 9 GH is responsible for different types of cleavage patterns. Image 1 • The 3-D structure of cellulase, Ht GH9 was modeled by homology modelling. • Asp75, Asp78 and Glu443 are the catalytic amino acid residues. • MD simulation of the modeled Ht GH9 enzyme for 50 ns showed stable conformation. • SAXS analysis showed that Ht GH9 enzyme is stable and in monodispersed nature. • Tower barrier formed by the loop in catalytic site, responsible for processive cleavage. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2021 Yayo et al.)

The function of CBM3c in the enzymatic catalysis varies among the members of family 9 Glycoside Hydrolases (GH). A new member of family 9 GH (Cel9W) from thermophilic anaerobic bacterium, Hungateiclostridium thermocellum was explored for elucidation of the role of CBM3c in catalysis by GH9. Cel9W is a multimodular theme B1, cellulase enzyme comprising a catalytic module of family 9 glycoside hydrolase (Ht GH9t) at N-terminal, a family 3c carbohydrate binding module (Ht CBM3c) and a dockerin domain at the C-terminal. The ORF of Cel9W encoding full length β-1,4-glucanase, Ht GH9 (containing both GH9 and CBM3c modules), the truncated GH9 catalytic module (Ht GH9t) and module CBM3c (Ht CBM3c) were cloned and over-expressed using E. coli BL21 cells. Ht GH9 showed maximum activity at pH 6.5 and 90 °C. It displayed highest activity of 64 U/mg against lichenan followed by 44.6 U/mg (β-glucan) and 22.3 U/mg (Carboxymethyl cellulose). Ht GH9 showed stability in the pH ranging from 5.0 to 9.0 and thermal stability up to 70 °C for 1.0 h. The presence of EDTA and EGTA decreased the activity of Ht GH9 and also shifted the melting curve peak from 93 °C to 88 °C indicating that the enzyme inherently possesses metal ions, which play role in catalysis and structural stability. The TLC analysis of Ht GH9 hydrolysed Avicel showed the presence cellotetraose indicating the processive endoglucanase activity in Ht GH9. The module, Ht GH9t alone exhibited very low level of activity (1.22 U/mg) against lichenan. It partially recovered (51%) the enzyme activity in presence of equimolar concentration of Ht CBM3c. Non-denaturing gel electrophoresis revealed a non-covalent binding interaction between the catalytic Ht GH9t module with Ht CBM3c module and their physical association showed the partial recovery of its endoglucanase activity. This study confirmed that the physical association of the catalytic module Ht GH9t with Ht CBM3c is necessary for Ht GH9 to efficiently hydrolyze the cellulosic substrates. Image 1 • β-1,4-glucanase (Ht GH9) expressed as a soluble protein of molecular size ~71.7 kDa. • Ht GH9 displayed maximum activity against lichenan and at 90 °C. • Ht GH9 displayed processive endoglucanase enzyme activity. • Ht CBM3c is essential for catalysis by processive endoglucanase, Ht GH9. [ABSTRACT FROM AUTHOR]