A High-Molecular-Weight, Cell-Associated Xylanase Isolated from Exponentially Growing Thermoanaerobacterium sp. Strain JW/SL-YS485

An unusual cell-associated (beta)-1,4-xylanase was purified to gel electrophoretic homogeneity from a cell extract of the bacterium Thermoanaerobacterium sp. strain JW/SL-YS485 harvested at the late exponential growth phase. The molecular mass of the xylanase was 350 kDa as determined by gel filtration and 234 kDa as determined by native gradient gel electrophoresis. The enzyme contained 6% carbohydrates. Heterosubunits of 180 and 24 kDa were observed for the xylanase on sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis gels. The xylanase had a pI of 4.37 and a half-life of 1 h at 70(deg)C. Using a 5-min assay, we observed the highest level of activity at pH 6.2 and 80(deg)C. The K(infm) and k(infcat) values when oat spelt xylan was used were 3 mg/ml and 26,680 U/(mu)mol, respectively. The Arrhenius energy was 41.8 kJ/mol. The purified enzyme differed in size, subunit structure, and location from other xylanases that have been described. The cell-associated enzyme activity appeared in the S-layer fraction.     

Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp. strain JW/SL-YS485.

Two acetyl esterases (EC 3.1.1.6) were purified to gel electrophoretic homogeneity from Thermoanaerobacterium sp. strain JW/SL-YS485, an anaerobic, thermophilic endospore former which is able to utilize various substituted xylans for growth. Both enzymes released acetic acid from chemically acetylated larch xylan. Acetyl xylan esterases I and II had molecular masses of 195 and 106 kDa, respectively, with subunits of 32 kDa (esterase I) and 26 kDa (esterase II). The isoelectric points were 4.2 and 4.3, respectively. As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases I and II occurred at pH 7.0 and 80 degrees C and at pH 7.5 and 84 degrees C, respectively. Km values of 0.45 and 0.52 mM 4-methylumbelliferyl acetate were observed for acetyl xylan esterases I and II, respectively. At pH 7.0, the temperatures for the 1-h half-lives for acetyl xylan esterases I and II were 75 degrees and slightly above 100 degrees C, respectively.     

Mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase: kinetic studies

The catalytic mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) from family 39 of glycoside hydrolases has been subjected to a detailed kinetic investigation using a range of substrates. The enzyme exhibits a bell-shaped pH dependence of k(cat)/K(m), reflecting apparent pK(a) values of 4.1 and 6.8. The k(cat) and k(cat)/K(m) values for a series of aryl xylosides have been measured and used to construct two Br?nsted plots. The plot of log(k(cat)/K(m)) against the pK(a) of the leaving group reveals a significant correlation (beta(lg) = -0.97, r(2) = 0.94, n = 8), indicating that fission of the glycosidic bond is significantly advanced in the transition state leading to the formation of the xylosyl-enzyme intermediate. The large negative value of the slope indicates that there is relatively little proton donation to the glycosidic oxygen in the transition state. A biphasic, concave-downward plot of log(k(cat)) against pK(a) provides good evidence for a two-step double-displacement mechanism involving a glycosyl-enzyme intermediate. For activated leaving groups (pK(a) < 9), the breakdown of the xylosyl-enzyme intermediate is the rate-determining step, as indicated by the absence of any effect of the pK(a) of the leaving group on log(k(cat)) (beta(lg) approximately 0). However, a strong dependence of the first-order rate constant on the pK(a) value of relatively poor leaving groups (pK(a) > 9) suggests that the xylosylation step is rate-determining for these substrates. Support for the dexylosylation chemical step being rate-determining for activated substrates comes from nucleophilic competition experiments in which addition of dithiothreitol results in an increase in turnover rates. Normal secondary alpha-deuterium kinetic isotope effects ((alpha-D)(V) or (alpha-D)(V/K) = 1.08-1.10) for three different substrates of widely varying pK(a) value (5.15-9.95) have been measured and these reveal that the transition states leading to the formation and breakdown of the intermediate are similar and both steps involve rehybridization of C1 from sp(3) to sp(2). These results are consistent only with "exploded" transition states, in which the saccharide moiety bears considerable positive charge, and the intermediate is a covalent acylal-ester where C1 is sp(3) hybridized.     

Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485

Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild‐type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo‐form wild‐type TsXylA and the xylobiose‐, xylotriose‐, and xylotetraose‐bound E146A and E251A mutants were solved by X‐ray diffraction to high resolution (1.32–1.66 Å). The protein forms a classic (β/α)₈ folding of typical GH10 xylanases. The ligands in substrate‐binding groove as well as the interactions between sugars and active‐site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering. Proteins 2013; 81:1256–1265. © 2013 Wiley Periodicals, Inc.     

Purification and Characterization of the (alpha)-Glucuronidase from Thermoanaerobacterium sp. Strain JW/SL-YS485, an Important Enzyme for the Utilization of Substituted Xylans

A cell-associated (alpha)-glucuronidase was purified to gel electrophoretic homogeneity from the thermophilic anaerobic bacterium Thermoanaerobacterium sp. strain JW/SL-YS485. This enzyme had a pI of 4.65, a molecular weight of 130,000, and two subunits; the molecular weight of each subunit was 74,000. The enzyme exhibited the highest level of activity at pH 5.4 and 60(deg)C, as determined by a 5-min assay. The K(infm) and k(infcat) values of the enzyme for 4-methylglucuronosyl xylobiose were 0.76 mM and 1,083 IU/(mu)mol, respectively. The Arrhenius energy was 26.4 kJ/mol. The specific activities of the enzyme with 4-O-methylglucuronosyl xylobiose, 4-O-methylglucuronosyl xylotriose, and 4-O-methylglucuronosyl xylotetraose were 8.4, 4.8, and 3.9 IU/mg, respectively. The purified (alpha)-glucuronidase and a (beta)-xylosidase purified from the same organism interacted synergistically to hydrolyze 4-methylglucuronosyl xylotetraose.     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

Characterization of a salt-tolerant xylanase from Thermoanaerobacterium saccharolyticum NTOU1

A xylanase gene was PCR-cloned from Thermoanaerobacterium saccharolyticum and expressed in Escherichia coli. The xylanase (XynA) consisted of a signal peptide, glycoside hydrolase family 10 domains, carbohydrate-binding modules, and surface layer homology domains. It was optimally active at 70–73°C and at pH 5–7. It had enhanced activity with NaCl with optimal activity at 0.4 M but was tolerant up to 2 M NaCl. The thermostable and salt-tolerant properties of this xylanase suggest that it may be useful for industrial applications.     

Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp. strain JW/SL YS485: a beta-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity.

The genes encoding acetyl xylan esterase 1 (axe1) and a beta-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp. strain JW/SL YS485. axe1 is located 22 nucleotides 3' of the xylB sequence. The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1. The xylB gene was identified by expression cloning and by sequence homology to known beta-xylosidases. Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or beta-xylosidase (pXylo-1.1) were constructed in Escherichia coli. Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities. Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant beta-xylosidase. Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum. Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did have strong similarity to cephalosporin C deacetylase from Bacillus subtilis. Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid.     

Characterization of a thermophilic l-arabinose isomerase from Thermoanaerobacterium saccharolyticum NTOU1

l-Arabinose isomerase (EC 5.3.1.4, l-AI) mainly catalyzes the reversible aldose–ketose isomerization between l-arabinose and l-ribulose. l-AIs can also catalyze other reactions, such as the conversion of d-galactose to d-tagatose. In this study, the araA gene encoding l-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant l-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7–7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55–65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn²⁺ or 0.05 mM Co²⁺, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 l-AI has the lowest demand for metal ions among all characterized thermophilic l-AIs. This thermophilic l-AI shows a potential to be used in industry to produce d-tagatose from d-galactose.     

Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T. saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference.     

Crystal structure of beta-D-xylosidase from Thermoanaerobacterium saccharolyticum, a family 39 glycoside hydrolase

1,4-beta-D-Xylan is the major component of plant cell-wall hemicelluloses. beta-D-Xylosidases are involved in the breakdown of xylans into xylose and belong to families 3, 39, 43, 52, and 54 of glycoside hydrolases. Here, we report the first crystal structure of a member of family 39 glycoside hydrolase, i.e. beta-D-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. This study also represents the first structure of any beta-xylosidase of the above five glycoside hydrolase families. Each monomer of T. saccharolyticum beta-xylosidase comprises three distinct domains; a catalytic domain of the canonical (beta/alpha)(8)-barrel fold, a beta-sandwich domain, and a small alpha-helical domain. We have determined the structure in two forms: D-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-alpha-D-xylosyl-enzyme intermediate complex, thus providing two snapshots in the reaction pathway. This study provides structural evidence for the proposed double displacement mechanism that involves a covalent intermediate. Furthermore, it reveals possible functional roles for His228 as the auxiliary acid/base and Glu323 as a key residue in substrate recognition.     

Regulation and Characterization of Xylanolytic Enzymes of Thermoanaerobacterium saccharolyticum B6A-RI

During growth on xylan and xylose Thermoanaerobacterium saccharolyticum B6A-RI produced endoxylanase, β-xylosidase, arabinofuranosidase, and acetyl esterase, and the first three activities appeared to be produced coordinately. During nonlimiting growth on xylan, these enzyme activities were predominantly cell associated; however, during growth on limiting concentrations of xylan, the majority of endoxylanase activity was extracellular rather than cell associated. Endoxylanase, β-xylosidase, and arabinofuranosidase activities were induced by xylan, xylose, and arabinose, respectively. Acetyl esterase activity was constitutive, and endoxylanase activity was catabolite repressed by glucose. Extracellular endoxylanase existed as a high-molecular-weight complex (molecular weight, more than 10⁶). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms, the crude endoxylanase complex was composed of at least six activity bands. Endoxylanase was purified by gel filtration with Sephacryl S-300 and affinity chromatography with xylan coupled to Sepharose CL-4B preequilibrated to 45°C with 50 mM sodium acetate buffer (pH 4.0) and eluted with 0.1% soluble xylan. A single area of endoxylanase activity was identified on the zymogram; when this activity was analyzed by SDS-PAGE, it was composed of a major protein with a molecular weight of approximately 160,000 and a minor protein with a molecular weight of approximately 130,000. The endoxylanase activity stained with Schiff's reagent, indicative of glycoproteins, displayed a specific activity of 41 U/mg of protein on xylan, and had pH and temperature optima of 6.0 and 70°C, respectively.     

Metabolic engineering of Thermoanaerobacterium saccharolyticum for n-butanol production

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.     

Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.     

A case for reverse protonation: identification of Glu160 as an acid/base catalyst in Thermoanaerobacterium saccharolyticum beta-xylosidase and detailed kinetic analysis of a site-directed mutant

The catalytic mechanism of the family 39 Thermoanaerobacterium saccharolyticum beta-xylosidase (XynB) involves a two-step double-displacement mechanism in which a covalent alpha-xylosyl-enzyme intermediate is formed with assistance from a general acid and then hydrolyzed with assistance from a general base. Incubation of recombinant XynB with the newly synthesized active site-directed inhibitor, N-bromoacetyl-beta-D-xylopyranosylamine, resulted in rapid, time-dependent inactivation of the enzyme (k(i)/K(i) = 4.3 x 10(-4) s(-1)mM(- 1)). Protection from inactivation using xylose or benzyl 1-thio-beta-xyloside suggested that the inactivation was active site-directed. Mass spectrometric analysis indicated that incubation of the enzyme with the inactivator resulted in the stoichiometric formation of a new enzyme species bearing the label. Comparative mapping of peptic digests of both the labeled and unlabeled enzyme by HPLC coupled to an electrospray ionization mass spectrometer permitted the identification of a labeled peptide. Sequencing of this peptide by tandem mass spectrometry identified Glu160 within the sequence (157)IWNEPNL(164) as the site of attachment of the N-acetyl-beta-D-xylopyranosylamine moiety. Kinetic analysis of the Glu160Ala mutant strongly suggests that this residue is involved in acid/base catalysis as follows. First, a significant difference in the dependence of k(cat)/K(m) on pH as compared to that seen for the wild-type enzyme was found, as expected for a residue that is involved in acid/base catalysis. The changes, however, were not as simple as those seen in other cases. Second, a dramatic decrease (up to 10(5)-fold) in the catalytic efficiency (k(cat)/K(m)) of the enzyme with a substrate requiring protonic assistance is observed upon such mutation. In contrast, the catalytic efficiency of the enzyme with substrates bearing a good leaving group, not requiring acid catalysis, is only moderately impaired relative to that of the wild-type enzyme (8-fold). Surprisingly, however, the glycosylation step was rate-limiting for all but the most reactive substrates. Last, the addition of azide as a competitive nucleophile resulted in the formation of a beta-xylosyl azide product and increased the k(cat) and K(m) values up to 8-fold while k(cat)/K(m) remained relatively unchanged. Such kinetic behavior is consistent with azide acting competitively with water as a nucleophile in the second step of the enzyme catalyzed reaction involving breakdown of the xylosyl-enzyme intermediate. Together, these results provide strong evidence for a role of Glu160 in acid/base catalysis but suggest that it may be partnered by a second carboxylic acid residue and that the enzyme may function through using acid catalysis involving reverse protonation of active site residues.     

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium saccharolyticum through Targeted Gene Deletions

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 C₅ and C₆ 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.     

The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum

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.     

Marker removal system for Thermoanaerobacterium saccharolyticum and development of a markerless ethanologen

Marker removal strategies were developed for Thermoanaerobacterium saccharolyticum to select against the pyrF gene and the pta and ack genes. The pta- and ack-based haloacetate selective strategy was subsequently used to create strain M0355, a markerless Δldh Δpta Δack strain that produces ethanol at a high yield.     

Gene cloning, sequencing, and biochemical characterization of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

The gene encoding endoxylanase (xynA) from Thermoanaerobacterium saccharolyticum B6A-RI was cloned and expressed in Escherichia coli. A putative 33-amino-acid signal peptide, which corresponded to the N-terminal amino acids, was encoded by xynA. An open reading frame of 3,471 bp, corresponding to 1,157 amino acid residues, was found, giving the xynA gene product a molecular mass of 130 kDa. xynA from T. saccharolyticum B6A-RI had strong similarity to genes from family F beta-glycanases. The temperature and pH optimum for the activity of the cloned endoxylanase were 70 degrees C and 5.5, respectively. The cloned endoxylanase A was stable at 75 degrees C for 60 min and displayed a specific activity of 227.4 U/mg of protein on oat spelt xylan. The cloned xylanase was an endo-acting enzyme.