Search

Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd-EtfAB (BCD) complex was overexpressed, the resulting strain PRH-B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45-0.51 g/L/h and 0.46-0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin-NAD + reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Second-generation ethanol, a promising biofuel for reducing greenhouse gas emissions, faces challenges due to the inefficient metabolism of xylose, a pentose sugar. Overcoming this hurdle requires exploration of genes, pathways, and organisms capable of fermenting xylose. Thermoanaerobacterium saccharolyticum is an organism capable of naturally fermenting compounds of industrial interest, such as xylose, and understanding evolutionary adaptations may help to bring novel genes and information that can be used for industrial yeast, increasing production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations in Thermoanaerobacterium saccharolyticum that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose-binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve Thermoanaerobacterium saccharolyticum as a new microbial cell factory, which may help to solve current problems found in the biofuels' industry., (© 2024. The Author(s), under exclusive licence to Springer Nature Japan KK, part of Springer Nature.)

l-Rhamnose isomerase (l-RhI) catalyzes rare sugar isomerization between aldoses and ketoses. In an attempt to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 l-RhI (TsRhI), residue Ile102 was changed to other polar or charged amino acid residues by site-directed mutagenesis. The results of activity-screening using different substrates indicate that I102N, I102Q, and I102R TsRhIs can increase the preference against d-allose in comparison with the wild-type enzyme. The catalytic efficiencies of the purified I102N, I102Q, and I102R TsRhIs against d-allose are 148 %, 277 %, and 191 %, respectively, of that of wild-type enzyme, while those against l-rhamnose are 100 %, 167 % and 87 %, respectively. Mutant I102N, I102Q, and I102R TsRhIs were noted to have the altered substrate specificity, and I102Q TsRhI has the highest catalytic efficiency against d-allose presumably through the formation of an additional hydrogen bond with d-allose. The purified wild-type and mutant TsRhIs were further used to produce d-allose from 100 g/L d-fructose in the presence of d-allulose 3-epimerase, and the yields can reach as high as 22 % d-allulose and 12 % d-allose upon equilibrium. I102Q TsRhI takes only around half of the time to reach the same 12 % d-allose yield, suggesting that this mutant enzyme has a potential to be applied in d-allose production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Thermoanaerobacterium aotearoense strain SCUT27 is a potential industrial biofuel-producing strain because of its broad substrate spectrum, especially the ability to co-use glucose and xylose. The bottleneck hindering the development of strain SCUT27 is the lack of selective markers for polygene manipulation in this thermophilic bacterium. In this study, the endogenous type I-B clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system was developed for multiplex genome editing of strain SCUT27. The protospacer-adjacent motif was identified by in silico analysis and verified with orotidine-5'-phosphate decarboxylase ( pyrF ) or lactate dehydrogenase ( ldh ) as the editing target. The type I-B CRISPR/Cas system was functional in strain SCUT27 with 58.3% to 100% editing efficiency. A multiplex genome editing method based on thymidine kinase ( tdk ) as a negative selection marker was developed, and strain SCUT27/Δ tdk /Δ ldh /Δ argR , in which ldh and the arginine repressor ( argR ) were knocked out successively, was successfully obtained. Strain SCUT27/Δ tdk /Δ ldh /Δ argR exhibited prominent advantages over wild-type SCUT27 in ethanol production, with significantly improved ability to metabolize xylose. IMPORTANCE Thermophilic microbes have attracted great attention as potential candidates for production of biofuels and chemicals from lignocellulose because of their thermal tolerance and wide substrate spectra. The ability to edit multiple genes using the native type I-B CRISPR/Cas system would speed up engineering of Thermoanaerobacterium aotearoense strain SCUT27 for higher ethanol production from lignocellulosic hydrolysates. Here, we produced a mutant strain, T. aotearoense SCUT27/Δ tdk /Δ ldh /Δ argR , using the native CRISPR/Cas system. The engineered strain showed satisfactory performance with improved ethanol productivity from various lignocellulosic hydrolysates. Our data lay the foundations for development of this thermophilic microbe into an excellent ethanol producer using lignocellulosic hydrolysates. The methods described here may also provide a reference to develop multigene editing methods for other microorganisms.

Carbohydrate metabolism via cyclodextrins (CM-CD) is an uncommon starch-converting pathway that thoroughly depends on extracellular cyclomaltodextrin glucanotransferases (CGTases) to transform the surrounding starch substrate to α-(1,4)-linked oligosaccharides and cyclodextrins (CDs). The CM-CD pathway has emerged as a convenient microbial adaptation to thrive under extreme temperatures, as CDs are functional amphipathic toroids with higher heat-resistant values than linear dextrins. Nevertheless, although the CM-CD pathway has been described in a few mesophilic bacteria and archaea, it remains obscure in extremely thermophilic prokaryotes (T opt  ≥ 70 °C). Here, a new monophyletic group of CGTases with an exceptional three-domain ABC architecture was detected by (meta)genome mining of extremely thermophilic Thermoanaerobacterales living in a wide variety of hot starch-poor environments on Earth. Functional studies of a representative member, CldA, showed a maximum activity in a thermoacidophilic range (pH 4.0 and 80 °C) with remarkable product diversification that yielded a mixture of α:β:γ-CDs (34:62:4) from soluble starch, as well as G3-G7 linear dextrins and fermentable sugars as the primary products. Together, comparative genomics and predictive functional analysis, combined with data of the functionally characterized key proteins of the gene clusters encoding CGTases, revealed the CM-CD pathway in Thermoanaerobacterales and showed that it is involved in the synthesis, transportation, degradation, and metabolic assimilation of CDs., (© 2022. The Author(s).)

Recently, thermophilic Thermoanaerobacterium species have attracted increasing attentions in consolidated bioprocessing (CBP), which can directly utilize lignocellulosic materials for biofuels production. Compared to the mesophilic process, thermophilic process shows greater prospects in CBP due to its relatively highly efficiency of lignocellulose degradation. In addition, thermophilic conditions can avoid microbial contamination, reduce the cooling costs, and further facilitate the downstream product recovery. However, only few reviews specifically focused on the microbial applications of thermophilic Thermoanaerobacterium species in lignocellulosic biorefinery. Accordingly, this review will comprehensively summarize the recent advances of Thermoanaerobacterium species in lignocellulosic biorefinery, including their secreted xylanases and bioenergy production. Furthermore, the co-culture can significantly reduce the metabolic burden and achieve the more complex work, which will be discussed as the further perspectives. KEY POINTS: • Thermoanaerobacterium species, promising chassis for lignocellulosic biorefinery. • Potential capability of hemicellulose degradation for Thermoanaerobacterium species. • Efficient bioenergy production by Thermoanaerobacterium species through metabolic engineering., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales , Halanaerobiales , Thermoanaerobacterales , and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases., (© 2021 Stepanov and Fox; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The redox-sensing transcriptional repressor Rex (Rex) displayed diverse functions in different microbial species. Nowadays, only part function of rex has been verified in vitro and alcohol dehydrogenase gene (adhE) as the target of Rex has been widely reported. In this study, rex was knocked out in Thermoanaerobacterium aotearoense SCUT27 (GDMCC 60765) and the carbon metabolic distribution analysis was performed. Results showed that the ethanol yield (mol product/mol carbon) of SCUT27(Δrex) had increased by 75.00-90.91%, cell growth improved by 27.27-36.36%, and acetic acid and lactic acid decreased by 58.33-61.54% accompanied with the yield of hydrogen decreased by 46.15-58.35% within different carbon sources. The ability of sugar consumption of SCUT27(Δrex) had improved about 74.19-130.55% with the improvement of total ATP concentration and the cofactors NADH and NAD + concentrations. In addition, the specific activities of alcohol dehydrogenase of SCUT27(Δrex) with NADH and NADPH as cofactors were improved by 119.26-140.28% and 35.66-47.69%, respectively. After ldh was further knocked out in SCUT27(Δrex), SCUT27(ΔldhΔrex) showed higher ethanol production and yield when various carbon resources were used as substrates (including glucose, xylose, glucose/xylose mixture and three kinds of lignocellulosic hydrolysates). This study confirms that Rex is an important regulator for determining products distribution in SCUT27 and deletion of rex and ldh is a promising strategy for enhanced ethanol production.

Sucrose phosphorylase (SPase, EC 2.4.1.7) has a wide range of application in food, cosmetics, and pharmaceutical industries because of its broad substrate specificity. However, low SPase yields produced by wild-type strains cannot meet industrial requirements due to their complex metabolic regulation mechanisms. In this study, spase gene from Thermoanaerobacterium thermosaccharolyticum was cloned and expressed in Escherichia coli BL21 (DE3), leading to 7.05 U/mL (3.71 U/mg) of T. thermosaccharolyticum SPase (TtSPase) under optimum conditions. Co-expression of molecular chaperone teams pGro7 (GroES-GroEL), pG-KJE8 (DnaK-DnaJ-GrpE and GroES-GroEL), and pG-TF2 (GroES-GroEL-Tig) significantly enhanced the TtSPase activities to 18.5 U/mg (59.2 U/mL), 9.52 U/mg (28.6 U/mL), and 25.7 U/mg (64.5 U/mL), respectively. Results suggested that GroES-GroEL chaperone combination could regulate protein folding processes and protect misfolded proteins from aggregation. The enzymatic characterization results showed that TtSPase had an optimal temperature of 60 °C and optimal pH of 6.5. In particular, it had high thermostability of T 50 30  = 67 °C and half-life (t 1/2  at 70 °C) of 19 min. Furthermore, purified TtSPase was used for hydroquinone transglycosylation and 21% of molar production yield of α-arbutin was obtained. This study provides a TtSPase with high thermostability for potential industrial applications, and develops an effective strategy for improving soluble TtSPase production in E. coli., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The fiber in corn kernels, currently unutilized in the corn to ethanol process, represents an opportunity for introduction of cellulose conversion technology. We report here that Clostridium thermocellum can solubilize over 90% of the carbohydrate in autoclaved corn fiber, including its hemicellulose component glucuronoarabinoxylan (GAX). However, Thermoanaerobacterium thermosaccharolyticum or several other described hemicellulose-fermenting thermophilic bacteria can only partially utilize this GAX. We describe the isolation of a previously undescribed organism, Herbinix spp. strain LL1355, from a thermophilic microbiome that can consume 85% of the recalcitrant GAX. We sequence its genome, and based on structural analysis of the GAX, identify six enzymes that hydrolyze GAX linkages. Combinations of up to four enzymes are successfully expressed in T. thermosaccharolyticum. Supplementation with these enzymes allows T. thermosaccharolyticum to consume 78% of the GAX compared to 53% by the parent strain and increases ethanol yield from corn fiber by 24%.

β-Mannanase was expressed in Thermoanaerobacterium aotearoense SCUT27 induced by locust bean gum (LBG). The open reading frame encoding a GH26 β-mannanase was identified and encoded a preprotein of 515 amino acids with a putative signal peptide. The enzyme without a signal sequence (Man25) was overexpressed in Escherichia coli with a specific activity of 1286.2 U/mg. Moreover, a facile method for β-mannanase activity screening was established based on agar plates. The optimum temperature for the purified Man25 using LBG as a substrate was 55 °C. The catalytic activity and thermostability of Man25 displayed a strong dependence on calcium ions. Through saturation mutagenesis at the putative Ca 2+ binding sites in Man25, the best mutant ManM3-3 (D143A) presented improvements in thermostability with 3.6-fold extended half-life at 55 °C compared with that of the wild-type. The results suggest that mutagenesis at metal binding sites could be an efficient approach to increase enzyme thermostability.

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.

Novel hydrolases from hot and other extreme environments showing appropriate performance and/or novel functionalities and new approaches for their systematic screening are of great interest for developing new processes, for improving safety, health and environment issues. Existing processes could benefit as well from their properties. The workflow, based on the HotZyme project, describes a multitude of technologies and their integration from discovery to application, providing new tools for discovering, identifying and characterizing more novel thermostable hydrolases with desired functions from hot terrestrial and marine environments. To this end, hot springs worldwide were mined, resulting in hundreds of environmental samples and thousands of enrichment cultures growing on polymeric substrates of industrial interest. Using high-throughput sequencing and bioinformatics, 15 hot spring metagenomes, as well as several sequenced isolate genomes and transcriptomes were obtained. To facilitate the discovery of novel hydrolases, the annotation platform Anastasia and a whole-cell bioreporter-based functional screening method were developed. Sequence-based screening and functional screening together resulted in about 100 potentially new hydrolases of which more than a dozen have been characterized comprehensively from a biochemical and structural perspective. The characterized hydrolases include thermostable carboxylesterases, enol lactonases, quorum sensing lactonases, gluconolactonases, epoxide hydrolases, and cellulases. Apart from these novel thermostable hydrolases, the project generated an enormous amount of samples and data, thereby allowing the future discovery of even more novel enzymes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A novel thermophilic and butanogenic Thermoanaerobacterium thermosaccharolyticum M5 was successfully isolated and characterized, which could produce butanol from hemicellulose via a unique ethanol-butanol (EB) pathway through consolidated bioprocessing (CBP). This represents the first wild-type bacterium which could produce butanol from hemicellulose via CBP under thermophilic conditions. The assembled draft genome of strain M5 is 2.64 Mp, which contains 2638 genes and 2465 protein-coding sequences with 33.90% G + C content. Among these annotated proteins, xylanases, xylosidases, and bifunctional alcohol/aldehyde dehydrogenase (AdhE) play key roles in the achievement of EB production from hemicellulose through CBP.

To simplify purification and improve heat tolerance of a thermostable β-xylosidase (ThXylC), a short ELK16 peptide was attached to its C-terminus, which is designated as ThXylC-ELK. Wild-type ThXylC was normally expressed in soluble form. However, ThXylC-ELK assembled into aggregates with 98.6% of total β-xylosidase activity. After simple centrifugation and buffer washing, the ThXylC-ELK particles were collected with 92.57% activity recovery and 95% purity, respectively. Meanwhile, the wild-type ThXylC recovery yield was less than 55% after heat inactivation, affinity and desalting chromatography followed by HRV 3C protease cleavage purification. Catalytic efficiency ( K cat / K m ) was increased from 21.31 mM -1 s -1 for ThXylC to 32.19 mM -1 s -1 for ThXylC-ELK accompanied by a small increase in K m value. Heat tolerance of ThXylC-ELK at high temperatures was also increased. The ELK16 peptide attachment resulted in 6.2-fold increase of half-life at 65 °C. Released reducing sugars were raised 1.3-fold during sugar cane bagasse hydrolysis when ThXylC-ELK was supplemented into the combination of XynAΔSLH and Cellic CTec2.

Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred.

Lactose is a main by-product in the cheese industry. Many attempts have been made to convert the lactose to high value-added products, including epilactose. Epilactose is a valuable prebiotic and can be epimerized from lactose with cellobiose 2-epimerase (CEase). The objective of the present work was to construct a food-grade recombinant Bacillus subtilis that produces CEase from Thermoanaerobacterium saccharolyticum. The CEase was expressed in B. subtilis without antibiotic resistance genes. After fermentation, the maximum volumetric activity of the fermented broth was more than 7 U/mL. The activity of the recombinant B. subtilis was increased by up to 3.7 fold after ethanol permeabilization. Then, 66.9 ± 0.7 g/L of epilactose was produced from 300 g/L of whey powder solution in 1 h with 13.3 U/mL of permeabilized biocatalyst. In addition, an enzymatic route including degradation of the lactose, yeast fermentation, and cation exchange chromatography was described to further purify the produced epilactose from lactose. Finally, epilactose with a purity >98% was produced from 300 g/L of lactose with a yield of 24.0%. In conclusion, neither antibiotics nor pathogenic bacteria were used throughout the epilactose production and purification procedure., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C., Result: Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum., Conclusion: The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield.

The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para -nitrophenyl ( p NP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries., (Copyright © 2017 American Society for Microbiology.)

As a nongenetic engineering technique, adaptive evolution is an effective and easy-to-operate approach to strain improvement. In this work, a commercial Thermoanaerobacterium aotearoense SCUT27/Δldh-G58 was successfully isolated via sequential batch fermentation with step-increased carbon concentrations. Mutants were isolated under selective high osmotic pressures for 58 passages. The evolved isolate rapidly catabolized sugars at high concentrations and subsequently produced ethanol with good yield. A 1.6-fold improvement of ethanol production was achieved in a medium containing 120 g/L of carbon substrate using the evolved strain, compared to the start strain. The analysis of transcriptome and intracellular solute pools suggested that the adaptive evolution altered the synthesis of some compatible solutes and activated the DNA repair system in the two Thermoanaerobacterium sp. evolved strains. Overall, the results indicated the potential of adaptive evolution as a simple and effective tool for the modification and optimization of industrial microorganisms.

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Energy shortage and environmental pollution are two severe global problems, and biological hydrogen production from lignocellulose shows great potential as a promising alternative biofuel to replace the fossil fuels. Currently, most studies on hydrogen production from lignocellulose concentrate on cellulolytic microbe, pretreatment method, process optimization and development of new raw materials. Due to no effective approaches to relieve the inhibiting effect of inhibitors, the acid pretreated lignocellulose hydrolysate was directly discarded and caused environmental problems, suggesting that isolation of inhibitor-tolerant strains may facilitate the utilization of acid pretreated lignocellulose hydrolysate., Results: Thermophilic bacteria for producing hydrogen from various kinds of sugars were screened, and the new strain named MJ1 was isolated from paper sludge, with 99% identity to Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. The hydrogen yields of 11.18, 4.25 and 2.15 mol-H 2 /mol sugar can be reached at an initial concentration of 5 g/L cellobiose, glucose and xylose, respectively. The main metabolites were acetate and butyrate. More important, MJ1 had an excellent tolerance to inhibitors of dilute-acid (1%, g/v) pretreated sugarcane bagasse hydrolysate (DAPSBH) and could efficiently utilize DAPSBH for hydrogen production without detoxication, with a production higher than that of pure sugars. The hydrogen could be quickly produced with the maximum hydrogen production reached at 24 h. The hydrogen production reached 39.64, 105.42, 111.75 and 110.44 mM at 20, 40, 60 and 80% of DAPSBH, respectively. Supplementation of CaCO 3 enhanced the hydrogen production by 21.32% versus the control., Conclusions: These results demonstrate that MJ1 could directly utilize DAPSBH for biohydrogen production without detoxication and can serve as an excellent candidate for industrialization of hydrogen production from DAPSBH. The results also suggest that isolating unique strains from a particular environment offers an ideal way to conquer the related problems.

Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 3' end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054-1064. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95°C and could retain more than 80% of initial activity in the presence of 20mM Mn 2+ at 85°C for 48h. The K m and k cat /K m values for ToGI were 81.46mM and 21.77min -1 mM -1 , respectively. Furthermore, the maximum conversion yield of 400g/L d-glucose to d-fructose at 85°C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Strain USBA-019 T , an anaerobic and thermophilic strain, was identified as a new member of the genus Thermoanaerobacterium. USBA-019 T cells are gram-positive, strictly anaerobic, thermophilic, chemoorganotrophic, moderately acidophilic, non-motile, endospore-forming, slightly curved, and rod-shaped. Cells measure 0.4×3.0-7.0μm. Optimal growth occurs at 50-55°C (35-65°C). Optimum pH is 5.0-5.5 (4.0-8.5). Thiosulfate, elemental sulfur and nitrate were utilized as electron acceptors. Fermentation of glucose, lactose, cellobiose, galactose, arabinose, xylose, starch and xylan primarily produced acetate and butyrate. Xylan, starch and cellobiose produced ethanol and starch, cellobiose, galactose, arabinose and mannose produced lactic acid. Phylogenetic analyses based on 16S rRNA gene sequence comparison and genomic relatedness indices show the close relation of USBA-019 T to Thermoanaerobacterium thermostercoris and Thermoanaerobacterium aotearoense (similarity value: 99%). Hybridization of USBA-019 T , Th. thermostercoris DSM22141 T and Th. aotearoense DMS10170 T found DNA-DNA relatedness of 33.2% and 18.2%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic evidence, along with low identity at whole genome level, USBA-019 T is a novel species of the genus Thermoanaerobacterium which we propose to name Thermoanaerobacterium butyriciformans sp. nov. The type strain is USBA-019 T (=CMPUJ U-019 T =DSM 101588 T )., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Conversion of organic wastes to syngas is an attractive way to utilize wastes. The produced syngas can be further used to produce a variety of chemicals. In this study, a hollow-fiber membrane biofilm reactor with mix cultures was operated at 55 °C to convert syngas (H 2 /CO 2 ) into acetate. A high concentration of acetate (42.4 g/L) was reached in batch experiment while a maximum acetate production rate of 10.5 g/L/day was achieved in the continuous-flow mode at hydraulic retention time (HRT) of 1 day. Acetate was the main product in both batch and continuous-flow experiments. n-Butyrate was the other byproduct in the reactor. Acetate accounted for more than 98.5 and 99.1% of total volatile fatty acids in batch and continuous modes, respectively. Illumina Miseq high-throughput sequencing results showed that microorganisms were highly purified and enriched in the reactor. The main genus was Thermoanaerobacterium (66% of relative abundance), which was usually considered as H 2 producer in the literature, however, likely played a role as a H 2 consumer in this study. This study provides a new method to generate the high producing rate and purity of acetate from syngas.

Biohydrogen is considered as one of the most promising energy alternatives considering the climate and energy crisis. The dark fermentative hydrogen production from xylose at extreme thermophilic condition (70 °C) using mixed culture was conducted in this study. The effects of initial pH values (ranged from 5.0 to 10.0) and substrate concentrations (ranged from 2.5 to 15.0 g/L) on the hydrogen production, substrate degradation and metabolite distributions were investigated using batch-mode operations. Results showed that initial substrate pH values in the neutral region (6.0-7.0) were beneficial for hydrogen production. The fermentation at initial pH 7.0 and 7.5 g/L xylose reached an optimal hydrogen yield of 1.29 mol-H 2 /mol-xylose consumed . Ethanol, butyrate, and propionate were the major liquid metabolites. The xylose biodegradation efficiency of the mixed culture decreased sharply at high initial culture pH values. The increase of xylose concentration resulted in the accumulation of propionate and an obvious decrease in the final pH value, as well as a low hydrogen yield. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis indicated that hydrogen producing bacteria were enriched by repeated culture under extreme thermophilic conditions. Also, the mixed culture was dominated with bacterial species related to Clostridium and Thermoanaerobacterium.

Ferredoxin:NAD + oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD + This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum In wild-type T. saccharolyticum, deletion of tsac_1705 resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol., Importance: Redox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD + , a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H2 at a rate 4-12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H2 production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium.

β-1,3-Glucans, important structural components of cell wall or nutritional components of the endosperm, are extensively found in bacteria, fungi, yeast, algae, and plants. The structural complexity of β-1,3-glucans implies that the enzymatic depolymerization of polysaccharides needs combined activities of distinct enzymes. In this study, Lam16A-GH, the catalytic module of a putative glycoside hydrolase (GH) family 16 laminarinase/lichenase from thermophilic bacterium Caldicellulosiruptor sp. F32, was purified and characterized through heterologous expression in Escherichia coli. Lam16A-GH can hydrolyze both β-1,3-glucan (laminarin) and β-1,3-1,4-glucan (barley β-glucan) revealed by analysis of the products of polysaccharide degradation using thin-layer chromatography (TLC). The time required for the loss of 50 % of its activity is 45 h under the optimal condition of 75 °C and pH 6.5. Oligosaccharides degradation assay indicated that Lam16A-GH can catalyze endo-hydrolysis of the β-1,4 glycosidic linkage adjacent to a 3-O-substituted glucosyl residue in the mixed linked β-glucans, as well as the β-1,3 linkage. The survival rate of Saccharomyces cerevisiae cells depends on the addition of Lam16A-GH, and the cytoplasm protein was released from the apparently deconstructed yeast cells. These results indicate that the bi-functional thermostable Lam16A-GH exhibits unique enzymatic properties and potential for yeast lysis.

A series of standardized activity experiments were performed to characterize three different temperature ranges of hydrogen fermentation from different carbon sources. 16S rRNA sequences analysis showed that the bacteria were close to Enterobacter genus in the mesophilic mixed culture (MMC) and Thermoanaerobacterium genus in the thermophilic and hyper-thermophilic mixed cultures (TMC and HMC). The MMC was able to utilize the glucose and cellulose to produce methane gas within a temperature range between 25 and 45 °C and hydrogen gas from 35 to 60°C. While, the TMC and HMC produced only hydrogen gas at all temperature ranges and the highest activity of 521.4mlH2/gVSSd was obtained by TMC. The thermodynamic analysis showed that more energy is consumed by hydrogen production from cellulose than from glucose. The experimental results could help to improve the economic feasibility of cellulosic biomass energy using three-phase technology to produce hythane., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Chaperonins (CPNs) are megadalton sized ATP-dependent nanomachines that facilitate protein folding through complex cycles of complex allosteric articulation. They consist of two back-to-back stacked multisubunit rings. CPNs are usually classified into Group I and Group II. Here, we report the crystallization of both the AMPPNP (an ATP analogue) and ADP bound forms of a novel CPN, classified as belonging to a third Group, recently discovered in the extreme thermophile Carboxydothermus hydrogenoformans. Crystals of the two forms were grown by the vapor batch crystallization method at 295 K. Crystals of the Ch-CPN/AMPPNP complex diffracted to 3.0 Å resolution and belonged to the space group P422, with unit-cell parameters a = b = 186.166, c = 160.742 Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 60.02%. Crystals of the Ch-CPN/ADP complex diffracted to 4.0 Å resolution and belonged to the space group P4212, with unit-cell parameters a = b = 209.780, c = 169.813Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 70.19%.

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are bacteria under investigation for production of biofuels from plant biomass. Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at high yield (>90% of theoretical) and titer (>70 g/l). Efforts to engineer C. thermocellum have not, to date, been as successful, and efforts are underway to transfer the ethanol production pathway from T. saccharolyticum to C. thermocellum One potential challenge in transferring metabolic pathways is the possibility of incompatible levels of nicotinamide cofactors. These cofactors (NAD(+), NADH, NADP(+) and NADPH) and their oxidation state are important in the context of microbial redox metabolism. In this study we directly measured the concentrations and reduced oxidized ratios of these cofactors in a number of strains of C. thermocellum and T. saccharolyticum by using acid/base extraction and enzymatic assays. We found that cofactor ratios are maintained in a fairly narrow range, regardless of the metabolic network modifications considered. We have found that the ratios are similar in both organisms, which is a relevant observation in the context of transferring the T. saccharolyticum ethanol production pathway to C. thermocellum., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The strictly anaerobic, Gram-positive bacterium, Thermoanaerobacterium aotearoense SCUT27, is capable of producing ethanol, hydrogen and lactic acid by directly fermenting glucan, xylan and various lignocellulosically derived sugars. By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG). However, the xylose and mannose consumptions were not markedly affected by 2-DG at the concentration of one-tenth of the metabolizable sugar. Accordingly, T. aotearoense SCUT27 could consume xylose and mannose in the presence of glucose. The carbon catabolite repression (CCR) related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization. The highly purified Histidine-containing Protein (HPr) could be specifically phosphorylated at Serine 46 by HPr kinase/phosphatase (HPrK/P) with no need to add fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc-6-P) in the reaction mixture. The specific protein-interaction of catabolite control protein A (CcpA) and phosphorylated HPr was proved via affinity chromatography in the absence of formaldehyde. The equilibrium binding constant (KD) of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR) analysis, indicating the high affinity between these two proteins.

Thermoanaerobacterium aotearoense P8G3#4 produced β-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding β-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (β/α)8 TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na(+) or K(+). The enzyme showed strong ability to hydrolyze cellobiose with a K m and V max of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K i of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

Unlabelled: NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP(+). The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H2 formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss of nfnAB caused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnAB strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnAB strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of nfnAB caused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lost nfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from nfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity., Importance: Thermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P)(+). The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP(+) and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. We report the first deletion of nfnAB and demonstrate a role for NfnAB in metabolism and ethanol formation in Thermoanaerobacterium saccharolyticum and show that this may be an important feature among other thermophilic ethanologenic anaerobes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Caldicellulosiruptor saccharolyticus has proven itself to be an excellent candidate for biological hydrogen (H2) production, but still it has major drawbacks like sensitivity to high osmotic pressure and low volumetric H2 productivity, which should be considered before it can be used industrially. A whole genome re-annotation work has been carried out as an attempt to update the incomplete genome information that causes gap in the knowledge especially in the area of metabolic engineering, to improve the H2 producing capabilities of C. saccharolyticus. Whole genome re-annotation was performed through manual means for 2,682 Coding Sequences (CDSs). Bioinformatics tools based on sequence similarity, motif search, phylogenetic analysis and fold recognition were employed for re-annotation. Our methodology could successfully add functions for 409 hypothetical proteins (HPs), 46 proteins previously annotated as putative and assigned more accurate functions for the known protein sequences. Homology based gene annotation has been used as a standard method for assigning function to novel proteins, but over the past few years many non-homology based methods such as genomic context approaches for protein function prediction have been developed. Using non-homology based functional prediction methods, we were able to assign cellular processes or physical complexes for 249 hypothetical sequences. Our re-annotation pipeline highlights the addition of 231 new CDSs generated from MicroScope Platform, to the original genome with functional prediction for 49 of them. The re-annotation of HPs and new CDSs is stored in the relational database that is available on the MicroScope web-based platform. In parallel, a comparative genome analyses were performed among the members of genus Caldicellulosiruptor to understand the function and evolutionary processes. Further, with results from integrated re-annotation studies (homology and genomic context approach), we strongly suggest that Csac_0437 and Csac_0424 encode for glycoside hydrolases (GH) and are proposed to be involved in the decomposition of recalcitrant plant polysaccharides. Similarly, HPs: Csac_0732, Csac_1862, Csac_1294 and Csac_0668 are suggested to play a significant role in biohydrogen production. Function prediction of these HPs by using our integrated approach will considerably enhance the interpretation of large-scale experiments targeting this industrially important organism.

Background: Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation., Results: Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results., Conclusion: These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.

Unlabelled: Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase., Importance: Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of adhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In strains without adhE, we note changes in biochemical activity, product formation, and growth., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Biologically produced alcohols are of great current interest for renewable solvents and liquid transportation fuels. While bioethanol is now produced on a massive scale, butanol has superior fuel characteristics and an additional value as a solvent and chemical feedstock. Butanol production has been demonstrated at ambient temperatures in metabolically-engineered mesophilic organisms, but the ability to engineer a microbe for in vivo high-temperature production of commodity chemicals has several distinct advantages. These include reduced contamination risk, facilitated removal of volatile products, and a wide temperature range to modulate and balance both the engineered pathway and the host׳s metabolism. We describe a synthetic metabolic pathway assembled from genes obtained from three different sources for conversion of acetyl-CoA to 1-butanol, and 1-butanol generation from glucose was demonstrated near 70°C in a microorganism that grows optimally near 100°C. The module could also be used in thermophiles capable of degrading plant biomass., (Copyright © 2014 International Metabolic Engineering Society. All rights reserved.)

Hemicelluloses, the polysaccharide component of plant cell walls, represent one of the most abundant biopolymers in nature. The most common hemicellulosic constituents of softwoods, such as conifers and cycads, are mannans consisting of a 1,4-linked β-mannopyranosyl main chain with branch decorations. Efforts toward the utilization of hemicellulose for bioconversion into cellulosic biofuels have resulted in the identification of several families of glycoside hydrolases that can degrade mannan. However, effective biofermentation of manno-oligosaccharides is limited by a lack of appropriate uptake route in ethanologenic organisms. Here, we used transcriptome sequencing to gain insights into mannan degradation by the thermophilic anaerobic bacterium Caldanaerobius polysaccharolyticus. The most highly up-regulated genes during mannan fermentation occur in a cluster containing several genes encoding enzymes for efficient mannan hydrolysis as well as a solute-binding protein (CpMnBP1) that exhibits specificity for short mannose polymers but exhibited the flexibility to accommodate branched polysaccharide decorations. Co-crystal structures of CpMnBP1 in complex with mannobiose (1.4-Å resolution) and mannotriose (2.2-Å resolution) revealed the molecular rationale for chain length and oligosaccharide specificity. Calorimetric analysis of several active site variants confirmed the roles of residues critical to the function of CpMnBP1. This work represents the first biochemical characterization of a mannose-specific solute-binding protein and provides a framework for engineering mannan utilization capabilities for microbial fermentation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

This is the first report describing the cloning, expression, and characterization of a putative thermostable, cellulase-free xylanase (XYN) from the thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum DSM 571. The temperature and pH values for optimal enzyme activity of XYN were found to be 65 °C and pH 6.5, respectively. The XYN activity was apparently enhanced by Co(2+), Mn(2+), and Tween 60 and significantly inactivated by Al(3+), Cu(2+), Zn(2+), and SDS. The K m and V max values of XYN for the hydrolysis of beechwood xylan were 2.1 mg/ml and 222.1 U/mg, respectively. The k cat values of XYN for beechwood xylan at the optimal temperature and pH values were 481.4 s(-1). XYN represents an attractive candidate for use in the large-scale production of xylooligosaccharides (XOs) from forest residues because it is an endo-xylanase capable of degrading xylan.

Acidification results from the excessive accumulation of volatile fatty acids and the breakthrough of buffering capacity in anaerobic digesters. However, little is known about the identity of the acidogenic bacteria involved. Here, we identified an active fermentative bacterium during acidification in a thermophilic anaerobic digester by sequencing and phylogenetic analysis of isotopically labeled rRNA. The digestion sludge retrieved from the beginning of pH drop in the laboratory-scale anaerobic digester was incubated anaerobically at 55 °C for 4 h during which 13 C-labeled glucose was supplemented repeatedly. 13 CH 4 and 13 CO 2 were produced after substrate addition. RNA extracts from the incubated sludge was density-separated by ultracentrifugation, and then bacterial communities in the density fractions were screened by terminal restriction fragment length polymorphism and clone library analyses based on 16S rRNA transcripts. Remarkably, a novel lineage within the genus Thermoanaerobacterium became abundant with increasing the buoyant density and predominated in the heaviest fraction of RNA. The results in this study indicate that a thermoacidophilic bacterium exclusively fermented the simple carbohydrate glucose, thereby playing key roles in acidification in the thermophilic anaerobic digester., (© 2014 Federation of European Microbiological Societies.)

β-Xylosidase from Thermoanaerobacterium thermosaccharolyticum was purified by His-trap affinity chromatography giving a specific activity of 5.15 U mg(-1). From gel-filtration chromatography, the purified enzyme was a tetramer with a total molecular mass of 245 kDa. Maximal enzyme activity using o-nitrophenyl(NP)-β-D-xylopyranoside was at pH 6.5 and 60 °C, with a half-life of 50 h. The enzyme had highest activity for oNP-β-D-xylopyranoside among aryl-glycosides, and was only active for notoginsenosides R1 and R2 amongst various ginsenosides. β-Xylosidase completely converted 2 g notoginsenosides R1 and R2 l(-1) to 1.69 g ginsenoside Rg1 l(-1) and 1.63 g ginsenoside Rh1 l(-1) in 4 and 18 h, respectively, with molar conversion yields of 100 % and specific productivities of 0.21 and 0.05 g g-enzyme(-1) h(-1), respectively. To our knowledge, this is the first report on the enzymatic production of ginsenosides Rg1 and Rh1 from notoginsenosides R1 and R2.

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis.

Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum JW/SL-YS485 was investigated as a host for n-butanol production. A systematic approach was taken to demonstrate functionality of heterologous components of the clostridial n-butanol pathway via gene expression and enzymatic activity assays in this organism. Subsequently, integration of the entire pathway in the wild-type strain resulted in n-butanol production of 0.85 g/L from 10 g/L xylose, corresponding to 21% of the theoretical maximum yield. We were unable to integrate the n-butanol pathway in strains lacking the ability to produce acetate, despite the theoretical overall redox neutrality of n-butanol formation. However, integration of the n-butanol pathway in lactate deficient strains resulted in n-butanol production of 1.05 g/L from 10 g/L xylose, corresponding to 26% of the theoretical maximum., (© 2013 The Authors. Published by International Metabolic Engineering Society on behalf of International Metabolic Engineering Society All rights reserved.)

A thermostable acetylxylan esterase gene, TTE0866, which catalyzes the deacetylation of cellulose acetate, was cloned from the genome of Caldanaerobacter subterraneus subsp. tengcongensis. The pH and temperature optima were 8.0 and 60 °C. The esterase was inhibited by phenylmethylsulfonyl fluoride. A mixture of the esterase and cellulolytic enzymes efficiently degraded insoluble cellulose acetate with a higher degree of substitution.

Xylan-1,4-β-xylosidase (β-xylosidase) hydrolyses xylo-oligomers at their non-reducing ends into individual xylose units. Recently, XylC, a β-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48-2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel β-helix (residues 1-75 and 201-638) and the flanking region (residues 76-200) folds into a β-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition.

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C(5) and C(6) sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of β-xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum.