Dictyoglomus Research Articles

Transglycosylation Activity ofDictyoglomus thermophilumAmylase A

Amylase A from Dictyoglomus thermophilum is a thermophilic enzyme and has about 40% identity with 4-alpha-glucanotransferase (GTase) from Thermococcus litoralis, and both of these enzymes belong to family 57 glycosyl hydrolase. Since the transglycosylation activity of T. litoralis GTase has been well characterized, the substrate specificity and reaction products of amylase A from D. thermophilum were examined. alpha-1,4 Glucan was produced from maltooligosaccharides, and glucoamylase-resistant molecules (cycloamyloses) were produced from longer chain amylose (average molecular mass 200 kDa). It has been reported that amylase A from D. thermophilum hydrolyzes starch, but in this study it was found that the enzyme was also able to use maltooligosaccharides and long chain amylose as substrate and has transglycosylation activity.

Bacillus pumilus TX703 유래 Xylanase 유전자(xynK)의 Cloning과 염기서열 분석

Xylanase를 생산하는 내열성 Bacillus pumilus TX703의 chromosomal DNA로부터 xylanase 유전자를 cloning하여 그 염기배열 순서를 결정한 다음 이로부터 유전자 발현에 관련된 구조를 분석하였다. Xylanase 유전자의 cloning을 위해 제한효소 HindIII로 절단한 B. pumilus TX703의 chromosomal DNA와 pUC19을 ligation시켜 E. coli DH5 <TEX>$\alpha$</TEX>에 형질전환시킨 후 형질전환체 중에서 xylanase 활성을 나타내는 재조합 plasmid pXES106을 분리하였다. 재조합 plasmid pXES106은 pUC19의 HindIII 부위 내에 2.24 kb의 외래 DNA가 삽입되었고, 이 plasmid DNA를 분리하여 E. coli DH5 <TEX>$\alpha$</TEX>에 재형질전환시킨 결과 vector 내에 xylanase 유전자가 cloning되었음을 확인하였다. Cloning된 유전자의 염기배열을 분석한 결과 이 유전자의 총 크기는 2,187 bp였고 이는 409개기 아미노산을 coding 하는 open reading frame 1,227 bp를 포함하고 있었다. 이 염기배열은 ATG개시 codon으로부터 각각 193과 216 base 상류에 TTTAAT의 -10 box와 TCGAAA인 -35 box로 추정되는 염기배열이 존재하였고 -10 box로부터 7 bp하류에 전사개시점인 A가 위치하고 있었다. 또한, 개시 codon으로부터 432 bp 상류에 공통염기배열과 14개의 염기 중 11개의 염기가 일치하는 TGATGGCGTCGGCA의 catabolite responsive element (CRE)가 존재하였다. B. pumilus TX703의 xylanase와 아미노산배열의 유사성이 가장 높은 xylanase는 Hordeum vulgare의 isozyme X-I이었고 본 xylanase는 208번째와 322번째에 glutamic acid 잔기를 가지고 있어 Clostridium thermocellum, Dictyoglomus thermophilum, Thermotoga neapolitana 등에서 밝혀진 바와 같이 glutamic acid 부위가 xylanase의 활성부위라 여겨진다. A gene coding for xylanase from thermo-tolerant Bacillus pumilus TX703 was cloned into Escherichia coli DH5 <TEX>$\alpha$</TEX> using pUC19. Among 7,400 transformants, four transformants showed clear zones on the detection agar plates containing oat-spells xylan. One of them which showed highest xylanase activity was selected and its recombinant plasmid, named pXES106, was found to carry 2.24 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynK) was determined, xynK gene was found to consist of 1,227 base-pair open reading frame coding for a polypeptide of 409 amino acids with a deduced molecular weight of 48 kDa. The coding sequence was preceded by a putative ribosome binding site, the transcription initiation signals, and cia-acting catabolite responsive element. The deduced amino acids sequence of xylanase is similar to those of the xylanases from Hordeum vulgare (barley) and Clostridium thermocellum, with 39 and 31% identical residues, respectively. The amino acids sequence of this xylanase was quite different from those of the xylanases from other Bacillus species.

Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts.

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.

β-Mannanases from Thermotoga species

Thermostable mannanases have been identified from Thermotoga neapolitana , Rhodothermus marinus , Bacillus stearotherrnophilus , Thermo- anaerobacterium polysaccharolyticum , Caldocellosiruptor saccharolyticus ," and Dictyoglomus thermophilum . The β -mannanases from T. Neapolitana (family 5) and R. marinus (family 26) are the most thermostable of these. The mannanase from T. neapolitana is less stable at 85°, but more stable at 90°, than the corresponding enzyme from R. marinus . The former has a half-life of 34 hr at 85° and 13 hr at 90°, whereas the latter has a half life of 45.3 and 4.5 hr at the respective temperatures. Hyperthermophilic mannanases are useful in several industrial applications where thermostability and thermoactivity are desirable. These include coffee extraction, oil/gas well stimulation, and wood pulp treatment. This chapter describes the purification and characterization of a β -mannanase from T. neapolitana , as well as cloning and sequencing of this enzyme from T. neapolitana and T. maritima .

Structure of XynB, a highly thermostable beta-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 A resolution.

Microorganisms employ a large array of enzymes to break down the cellulose and hemicelluloses of plant biomass. These enzymes, especially those with high thermal stability, have many uses in biotechnology. We have solved the crystal structure of a beta-1, 4-xylanase, XynB, from the extremely thermophilic bacterium Dictyoglomus thermophilum, isolate Rt46B.1. The protein crystallized from 1.6 M ammonium sulfate, 0.2 M HEPES pH 7.2 and 10% glycerol, with unit-cell parameters a = b = 91.3, c = 44.9 A and space group P4(3). The structure was solved at high resolution (1.8 A) by X-ray crystallography, using the method of isomorphous replacement with a single mercury derivative, and refined to a final R factor of 18.3% (R(free) = 22.1%). XynB has the single-domain fold typical of family 11 xylanases, comprising a jelly roll of two highly twisted beta-sheets that create a deep substrate-binding cleft. The two catalytic residues, Glu90 and Glu180, occupy this cleft. Compared with other family 11 xylanases, XynB has a greater proportion of polar surface and has a slightly extended C-terminus that, combined with the extension of beta-strand A5, gives additional hydrogen bonding and hydrophobic packing. These factors may account for the enhanced thermal stability of the enzyme.

Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei.

The catalytic domain of the xynB (xylanase) gene from the thermophilic bacterium Dictyoglomus thermophilum was reconstructed by PCR to match the codon preference of Trichoderma reesei. The 0.6-kb DNA fragment encoding the enzyme was first amplified by primer extension with a mixture of eight overlapping oligonucleotides, followed by PCR with outside primers containing restriction enzyme sites for directional cloning into Escherichia coli and T. reesei vectors. The synthetic gene was expressed in both organisms, producing a clearing halo around transformant colonies in plate assay utilizing an overlay of oat spelts xylan. Effective transcription of xyn B in T. reesei was obtained after changing 20 codons.

Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei

The catalytic domain of the xynB (xylanase) gene from the thermophilic bacterium Dictyoglomus thermophilum was reconstructed by PCR to match the codon preference of Trichoderma reesei. The 0.6-kb DNA fragment encoding the enzyme was first amplified by primer extension with a mixture of eight overlapping oligonucleotides, followed by PCR with outside primers containing restriction enzyme sites for directional cloning into Escherichia coli and T. reesei vectors. The synthetic gene was expressed in both organisms, producing a clearing halo around transformant colonies in plate assay utilizing an overlay of oat spelts xylan. Effective transcription of xynB in T. reesei was obtained after changing 20 codons.

Sequencing and expression of a beta-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme.

A beta-mannanase gene (manA) was isolated from the extremely thermophilic bacterium Dictyoglomus thermophilum Rt46B.1. ManA is a single-domain enzyme related to one group of beta-mannanases (glycosyl hydrolase family 26). The manA gene was expressed in the heat-inducible vector pJLA602 and the expression product, ManA, purified to homogeneity. The recombinant ManA is a monomeric enzyme with a molecular mass of 40 kDa and an optimal temperature and pH for activity of 80 degrees C and 5.0. In the absence of substrate, the enzyme showed no loss of activity at 80 degrees C over 16 h, while at 90 degrees C the enzyme had a half-life of 5.4 min. Hydrolysis of the galactomannan locust bean gum (LBG) by purified ManA released mainly mannose, mannobiose, and mannotriose, confirming that ManA is an endo-acting beta-mannanase. Sequence comparisons with related beta-mannanases has allowed the design of consensus PCR primers for the identification and isolation of related genes.

Purification and properties of the pyrophosphate-dependent phosphofructokinase from Dictyoglomus thermophilum Rt46 B.1.

The distribution of phosphofructokinase phosphoryl donor subtypes (ATP-, ADP-, and pyrophosphate) in the deeply rooted phylogenetic lineages of thermophiles is of interest with regard to the evolution of phosphofructokinase activity and of the Embden-Meyerhof pathway. In this article we present the first biochemical description of a thermostable pyrophosphate-dependent phosphofructokinase from the hyperthermophilic bacterium Dictyoglomus thermophilum. The enzyme was not allosterically controlled by traditional modulators of phosphofructokinases and has significant activity with tripolyphosphate and polyphosphate. Kinetic parameters of the enzyme suggest it plays primarily a glycolytic role. The enzyme required Mg2+ for optimal activity, was partially activated by some monovalent and divalent cations, and was strongly inhibited by Cu2+. The sequence of the 21 N-terminal residues suggests that the enzyme is most similar to the pyrophosphate-dependent phosphofructokinases from Amycolatopsis methanolica and the hyperthermophilic crenarchaeon Thermoproteus tenax, enzymes which have been suggested to represent an ancient lineage of phosphofructokinases (Siebers et al. 1998). The unexpected finding of a pyrophosphate-dependent phosphofructokinase in Dictyoglomus thermophilum, which is phylogenetically related to Thermotoga maritima, previously shown to possess an ATP-dependent phosphofructokinase activity, is discussed.

Family 10 and 11 xylanase genes from Caldicellulosiruptor sp. strain Rt69B.1.

Three family 10 xylanase genes (xynA, xynB, and xynC) and a single family 11 xylanase gene (xynD) were identified from the extreme thermophile Caldicellulosiruptor strain Rt69B.1 through the use of consensus PCR in conjunction with sequencing and polyacrylamide gel electrophoresis. These genes appear to comprise the complete endoxylanase system of Rt69B.1. The xynA gene was found to be homologous to the xynA gene of the closely related Caldicellulosiruptor strain Rt8B.4, and primers designed previously to amplify the Rt8B.4 xynA gene could amplify homologous full-length xynA gene fragments from Rt69B.1. The complete nucleotide sequences of the Rt69B.1 xynB, xynC, and xynD genes were obtained using genomic walking PCR. The full-length xynB and xynC genes are more than 5 kb in length and encode highly modular enzymes that are the largest xylanases reported to date. XynB has an architecture similar to the family 10 xylanases from Thermoanaerobacterium saccharolyticum (XynA) and Clostridium thermocellum (XynX) and may be cell wall associated, while XynC is a bifunctional enzyme with an architecture similar to the bifunctional beta-glycanases from Caldicellulosiroptor saccharolyticus. The xynD gene encodes a two-domain family 11 xylanase that is identical in architecture to the XynB family 11 xylanase from the unrelated extreme thermophile Dictyoglomus thermophilum strain Rt46B.1. The sequence similarities between the Rt69B.1 xylanases with respect to their evolution are discussed.

Aminobacterium colombiensegen. nov. sp. nov., an Amino Acid-degrading Anaerobe Isolated from Anaerobic Sludge

A new Gram-negative, non-sporulating, mesophilic, amino acid fermenting bacterium, designated strain ALA-1T(T=type strain), was isolated from an anaerobic lagoon of a dairy wastewater treatment plant. The strain is curved (3-4μm×0.2-0.3μm) and occurs singly or in pairs. Optimum growth occurs at 37°C and pH 7.3. The G+C content of the DNA is 46mol %. The strain requires yeast extract for growth, grows poorly on casamino acids, peptones, cystein, and α-ketoglutarate, but readily grows on serine, threonine, glycine and pyruvate. When cocultured with the hydrogenotrophic methanogenMethanobacterium formicicum, strain ALA-1Toxidized alanine, glutamate, leucine, isoleucine, valine, aspartate, and methionine. Phylogenetic analysis revealed that it forms a distinct and independent line of descent in the vicinity ofDethiosulfovibrio peptidovorans, Dictyoglomus thermophilum, andAnaerobaculum thermoterrenumwhich are members of the low G+C containing Gram-positive bacteria. The phylogenetic results concur with the phenotypic and genomic data which reveal that it is a novel strain. Based on these findings, we designate strain ALA-1TasAminobacterium colombiense(DSM 12261) gen. nov., sp. nov.

Expression and secretion of a thermostable bacterial xylanase in Kluyveromyces lactis.

The xynA structural gene from the extremely thermophilic anaerobe Dictyoglomus thermophilum Rt46B.1 was fused in frame with the secretion signal of the Kluyveromyces lactis killer toxin in episomal expression vectors based on the Kluyveromyces plasmid pKD1. XynA was secreted predominantly as an unglycosylated 35-kDa protein which comprised up to 90% of the total extracellular proteins and reached a concentration of 130 micrograms/ml in shake-flask cultures grown under selective conditions.

Cloning and expression of the 4-α-glucanotransferase gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1, and characterization of the enzyme

The 4-α-glucanotransferase gene ( gtpK) of a hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and the nucleotide sequence analysis showed that the gtpK gene is composed of 1,973 bp, which encodes a protein of 653 amino acids with a calculated molecular weight of 76,693 Da. The gtpK was then overexpressed under the control of the lac promoter in Escherichia coli. The enzyme was purified by heat treatment, hydrophobic interaction chromatography, and anion-exchange chromatography. GtpK could react each maltooligosaccharides (from maltose to maltoheptaose) as the effective substrate to form glucose and various maltooligosaccharides containing higher polymeric molecules than the substrate. These results showed that GtpK is a 4-α-glucanotransferase which transfers the glucosyl and maltooligosyl units to a new C-4 position in another maltooligosaccharides and glucose. The optimum temperature and pH for the enzyme activity were found to be 100°C and 6–8, respectively, and the enzyme was considerably stable even after heating at 100°C for 30 min. When the deduced amino acid sequence of GtpK was compared with the protein sequences available in the databases, only two significant similar enzymes (α-amylases of Pyrococcus furiosus and Dictyoglomus thermophilum exhibited an identity of 68.3% and 36.0%, respectively) were found. Interestingly, only these three homologous enzymes did not preserve the conserved regions of α-amylase family, but the other α-amylases and 4-α-glucanotransferase are classified into the α-amylase family since all enzymes have the conserved homologous regions. Therefore, we consider that these enzymes, including GtpK, are classified into a new family of starch-degrading enzymes.

Close evolutionary relatedness among functionally distantly related members of the ( α/ β) 8-barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region

A short conserved sequence equivalent to the fifth conserved sequence region of α-amylases (173_LPDLD, Aspergillus oryzae α-amylase) comprising the calcium-ligand aspartate, Asp-175, was identified in the amino acid sequences of several members of the family of ( α/ β) 8-barrel glycosyl hydrolases. Despite the fact that the aspartate is not invariantly conserved, the stretch can be easily recognised in all sequences to be positioned 26–28 amino acid residues in front of the well-known catalytic aspartate (Asp-206, A. oryzae α-amylase) located in the β4-strand of the barrel. The identification of this region revealed remarkable similarities between some α-amylases (those from Bacillus megaterium, Bacillus subtilis and Dictyoglomus thermophilum) on the one hand and several different enzyme specificities (such as oligo-1,6-glucosidase, amylomaltase and neopullulanase, respectively) on the other hand. The most interesting example was offered by B. subtilis α-amylase and potato amylomaltase with the regions LYDWN and LYDWK, respectively. These observations support the idea that all members of the family of glycosyl hydrolases adopting the structure of the α-amylase-type ( α/ β) 8-barrel are mutually closely related and the strict evolutionary borders separating the individual enzyme specificities can be hardly defined.

Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1 and activity of the enzyme on fiber-bound substrate.

A genomic library of the Dictyoglomus sp. strain Rt46B.1 was constructed in the phage vector lambda ZapII and screened for xylanase activity. A plaque expressing xylanase activity, designated B6-77, was isolated and shown to contain a genomic insert of 5.3 kb. Subcloning revealed that the xylanase activity was restricted to a internal 1,507-bp PstI-HindIII fragment which was subsequently sequenced and shown to contain a single complete open reading frame coding for a single-domain xylanase, XynA, with a putative length of 352 amino acids. Homology comparisons show that XynA is related to the family F group of xylanases. The temperature and pH optima of the recombinant enzyme were determined to be 85 degrees C and pH 6.5, respectively. However, the enzyme was active across a broad pH range, with over 50% activity between pH 5.5 and 9.5. XynA was shown to be a true endo-acting xylanase, being capable of hydrolyzing xylan to xylotriose and xylobiose, but it could not hydrolyze xylobiose to monomeric xylose. XynA was also shown to hydrolyze xylan present in Pinus radiata kraft pulp, indicating that it may be of use as an aid in pulp bleaching. The equivalent xylanase gene was also isolated from the related bacterium Dictyoglomus thermophilum, and DNA sequencing showed these genes to be identical, which, together with the 16S small-subunit rRNA gene sequencing data, indicates that Rt46B.1 and D. thermophilum are very closely related.

Alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli.

A gene encoding a highly thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 649-amino acid protein with a calculated molecular mass of 76.3 kDa, which corresponds well with the value obtained from purified enzyme using denaturing polyacrylamide gel electrophoresis. The NH2 terminus of the deduced amino acid sequence corresponds precisely to that obtained from the purified enzyme, excluding the NH2-terminal methionine. The amylase expressed in E. coli exhibits temperature-dependent activation characteristic of of the original enzyme from P. furiosus, but has a higher apparent molecular weight which is attributed to the improper formation of the native quaternary structure. No homology was found with previously characterized promotor or termination sequences. The deduced amino acid sequence displayed strong homology to the alpha-amylase A of Dictyoglomus thermophilum, an obligately anaerobic, extremely thermophilic bacterium. Evolutionary implications of this homology are discussed.

The phylogenetic position of Dictyoglomus thermophilum based on 16S rRNA sequence analysis

The phylogenetic position of Dictyoglomus thermophilum has been determined by comparative sequence analysis of in vitro amplified 16S rRNA genes from the type strain as well as from a Dictyoglomus isolate. Results indicate that it forms a deep branch within the phylum of Thermotogales or may even represent its own phylum. It does not contain signature sequences within the 16S rRNA which could relate it to the Thermotogales group.

The phylogenetic position ofDictyoglomus thermophilumbased on 16S rRNA sequence analysis

The phylogenetic position of Dictyoglomus thermophilum has been determined by comparative sequence analysis of in vitro amplified 16S rRNA genes from the type strain as well as from a Dictyoglomus isolate. Results indicate that it forms a deep branch within the phylum of Thermotogales or may even represent its own phylum. It does not contain signature sequences within the 16S rRNA which could relate it to the Thermotogales group.

Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine‐plus‐cytosine contents

An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and characterized, we have cloned two additional genes, amyB and amyC, coding for amylases of this thermophile, into Escherichia coli and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coli promoters. Almost all activity was detected in the intracellular fraction in the E. coli cells. The molecular mass and NH2-terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coli transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67,000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59,000). The optimal temperatures for AmyB and AmyC activities on soluble starch were 80 degrees C and 70 degrees C, respectively. Both AmyB and AmyC showed a pH optimum of 5.5. AmyB and AmyC showed a different pattern of starch hydrolysis when examined by thin-layer chromatography. Some homology in the amino acid sequences with the functional regions of Taka-amylase A was found in both AmyB and AmyC. The codon usage in the amyA, amyB and amyC genes was highly biased, which reflects the fact that the guanine-plus-cytosine (G + C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the codons was non-random; the G + C content of the first position of codons is significantly high, whereas that of the third position is somewhat low. In addition, codons consisting only of A and T were preferentially used in this thermophile.

Cloning and nucleotide sequence of a heat‐stable amylase gene from an anaerobic thermophile, Dictyoglomus thermophilum

A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81,200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.