Expression of <Emphasis Type="Italic">Ruminococcus albus</Emphasis> Xylanase Gene (<Emphasis Type="Italic">xyn</Emphasis>A) in <Emphasis Type="Italic">Streptococcus bovis</Emphasis> 12-U-1

The objective of this study was to ligate the xylanase gene A (xynA) isolated from Ruminococcus albus 7 into the promoter and signal-peptide region of the lichenase [β-(1,3-1,4)-glucanase] gene of Streptococcus bovis JB1. This fusion gene was inserted into the pSBE11 vector, and the resulting recombinant, plasmid pXA, was used to transform S. bovis 12-U-1 cells. The transformant, S. bovis 12UXA, secreted the xylanase, which was stable against freeze-thaw treatment and long-time incubation at 37°C. The introduction of pXA and production of xylanase did not affect cell growth, and the xylanase produced degraded xylan from oat-spelt and birchwood. Received: 24 June 2002 / Accepted: 7 October 2002     

Molecular study on cloned endoglucanase gene from rumen bacterium

An endoglucanase gene was subcloned from anaerobic rumen bacterium Ruminococcus flavefaciens strain 17. To express endoglucanase gene in Escherichia coli and Streptococcus bovis JB1, an endoglucanase gene fragment was inserted into pVA838-based shuttle vectors. Removal of endoglucanase gene promoter and expression of endoglucanase by promoter of S. bovis JB1 alpha-amylase gene (pACMCS) was also achieved. Survival of constructs pVACMCI, pTACMC and pACMCS, which carry endoglucanase gene, and stability of endoglucanase gene in S. bovis JB1, were observed. Maximal endoglucanase activities from S. bovis JB1/pVACMCI were 2- to 3-fold higher than from E. coli/pVACMCI. Specific cell activity of E. coli/pACMCS was found to be approximately 2- to -3 fold higher than the both E. coli/pVACMCI and E. coli/pTACMC. Specific cell activity of S. bovis JB1/pACMCS was also found to be approximately 2-fold higher than the both S. bovis/pVACMCI and S. bovis JB1/pTACMC.     

Heterologous expression of an endoglucanase gene (endA) from the ruminai anaerobe Ruminococcus flavefaciens 17 in Streptococcus bovis and Streptococcus sanguis

Abstract The heterologous expression of a cloned endoglucanase gene ( endA ) from the ruminai bacterium Ruminococcus flavefaciens 17 was demonstrated in the Streptococcus species S. bovis JB1 and S. sanguis DLL The endA gene was introduced into S. bovis and S. sanguis using the Escherichia coli/Streptococcus shuttle vector pVA838. Expression of the gene was detected by clearing zones around the recombinant colonies on agar plates containing carboxymethylcellulose stained with Congo red. S. bovis JB1 containing the endA gene was capable of utilizing cellotetraose at a faster rate than the parent strain. This is the first demonstration that Streptococcus species can express a gene from a Ruminococcus flavefaciens strain.     

Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1.

Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.     

Molecular breeding of transgenic rice expressing a xylanase domain of the<Emphasis Type="Italic"> xynA</Emphasis> gene from<Emphasis Type="Italic"> Clostridium thermocellum</Emphasis>

The gene encoding the catalytic domain of thermostable xylanase from Clostridium thermocellum F1 was expressed in rice plants under the control of a constitutive promoter. The gene encoding Xylanase A was modified to encode the catalytic domain of family 11 xylanase without the signal sequence (xynA1), and was introduced into rice plants and expressed under the control of a modified cauliflower mosaic virus 35S promoter. Zymogram analysis indicated that the recombinant xylanase was produced in rice plants. The xynA1 gene was stably expressed in rice straw and seed grains. No phenotypic effect of xylanase expression was noted. The enzyme was detected in the desiccated grain. High levels of enzyme activity were maintained in the cell-free extract during incubation at 60 degrees C for 24 h. The results indicated that high levels of xylanase can be produced in rice plants.     

Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering

Xylanase is the enzyme complex that is responsible for the degradation of xylan; however, novel xylanase producers remain to be explored in marine environment. In this study, a Streptomyces strain M11 which exhibited xylanase activity was isolated from marine sediment. The 16S rDNA sequence of M11 showed the highest identity (99 %) to that of Streptomyces viridochromogenes. The xylanase produced from M11 exhibited optimum activity at pH 6.0, and the optimum temperature was 70 °C. M11 xylanase activity was stable in the pH range of 6.0–9.0 and at 60 °C for 60 min. Xylanase activity was observed to be stable in the presence of up to 5 M NaCl. Antibiotic-resistant mutants of M11 were isolated, and among the various antibiotics tested, streptomycin showed the best effect on obtaining xylanase overproducer. Mutant M11-1(10) isolated from 10 μg/ml streptomycin-containing plate showed 14 % higher xylanase activities than that of the wild-type strain. An analysis of gene rpsL (encoding ribosomal protein S12) showed that rpsL from M11-1(10) contains a K88R mutation. This is the first report to show that marine-derived S. viridochromogenes strain can be used as a xylanase producer, and utilization of ribosome engineering for the improvement of xylanase production in Streptomyces was also first successfully demonstrated.     

Transcriptional repression of ADH2-regulated beta-xylanase production by ethanol in recombinant strains of Saccharomyces cerevisiae

The regulation of endo-beta-(1,4)-xylanase production by two different strains of Saccharomyces cerevisiae, each transformed with the XYN2 gene from Trichoderma reesei under control of the promoter of the alcohol dehydrogenase II (ADH2) gene of S. cerevisiae, was investigated. In batch culture, the rate of xylanase production was severely reduced by the pulse addition of 390 mmol ethanol l(-1). Pulses of 190-630 mmol ethanol l(-1) into aerobic glucose-limited steady-state continuous cultures reduced the xylanase activity about five-fold and showed that ethanol repressed the ADH2 promoter, as was evident from Northern blot analyses. Derepression of the ADH2-regulated xylanase gene occurred at ethanol concentrations below approximately 50 mmol l(-1).     

Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans"

The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.     

耐碱性木聚糖酶基因在巨大芽孢杆菌中的表达及其酶学性质

摘要：【目的】从耐碱性木聚糖酶高产短小芽孢杆菌中克隆得到带有自身启动子的木聚糖酶基因,将其在巨大芽孢杆菌中进行表达,并对表达产物进行性质分析。【方法】将克隆得到的木聚糖酶基因xynA以及带有自身启动子序列的结构基因, 构建在芽孢杆菌表达载体pWH1520和改造后的载体pWG03中,得到重组质粒pWTEJX和pWGXYN,分别转化到巨大芽孢杆菌BM70中,获得重组巨大芽孢杆菌BMJXH9和BMGpp12;经过诱导产酶培养,均得到分泌表达。【结论】重组巨大芽孢杆菌BMGpp12比BMJXH9产酶活力提高了三倍     

Purification and characterization of a thermostable xylanase from Saccharopolyspora pathumthaniensis S582 isolated from the gut of a termite

An extracellular thermostable xylanase produced by Saccharopolyspora pathumthaniensis S582 was purified 167-fold to homogeneity with a recovery yield of 12%. The purified xylanase appeared as a single protein band on SDS-PAGE, with a molecular mass of 36 kDa. The optimal temperature and pH of the xylanase were 70 °C and 6.5. The enzyme was stable within a pH range of 5.5-10.0. It retained its activity after incubation at 50 °C for 2 h. Its half lives at temperatures of 60 and 70 °C were 180 and 120 min respectively. Hydrolysis of beechwood xylan by the xylanase yielded xylobiose and xylose as major products. The enzyme acted specifically on xylan as an endo-type xylanase, and exhibited a K(m) value of 3.92 mg/mL and a V(max) value of 256 μmol/min/mg. Enzyme activity was completely inhibited by Hg(2+), and was stimulated by Rb(+) and Cs(+). The xylanase gene was cloned from genomic DNA of Saccharopolyspora pathumthaniensis S582 and sequenced. The ORF consisted of 1,107 bp and encoded 368 amino acid residues containing a putative signal peptide of 23 residues. This xylanase is a new member of family (GH) 10 that shows highest identity, of 63.4%, with a putative xylanase from Nocardiopsis dassonvillei subsp. dassonvillei.     

Molecular characterization and expression of pyruvate formate-lyase-activating enzyme in a ruminal bacterium,Streptococcus bovis

To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe(2+) for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.     

Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli

A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, lambda gtWES lambda B, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated lambda FSX101 and lambda FSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from lambda FSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-beta-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the beta-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of beta-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan.     

The physiological and genetic diversity of bovine Streptococcus bovis strains(1)

Laboratory Streptococcus bovis strains and isolates obtained from a steer fed increasing amounts of grain had similar growth characteristics, but they differed in their sensitivity to 2-deoxyglucose (2DG), a non-metabolizable glucose analog. The addition of 2DG decreased both growth rate (0.92+/-0.34 h(-1)) and growth yield (ranging from 25 to 63%), but these differences could not be correlated with diet. However, isolates from a steer fed a 90% grain diet were more prone to 2DG-dependent lysis than those from a hay diet (P<0.001). All S. bovis laboratory strains and isolates had an identical restriction fragment length polymorphism pattern, when their 16S rDNA was digested with HaeIII and HhaI. However, when genomic BOX elements were amplified, 5-12 bands were observed, and the S. bovis isolates and laboratory strains could be grouped into 13 different BOX types. Strains 26 and 581AXY2 had the same BOX type, but the remaining laboratory strains did not form closely related clusters. Strains JB1 and K27FF4 were most closely related to each other. Most of the fresh isolates (24 out of 30) could be grouped into a single cluster (>90% Dice similarity). This cluster contained isolates from all three diets, but it did not have any of the laboratory strains. The majority (90%) of the isolates obtained from the hay-fed steer exhibited the same BOX type. Because more BOX types were observed if grain was added to the diet, it appears that ruminal S. bovis diversity may be a diet-dependent phenomenon.     

Bacterial competition between a bacteriocin-producing and a bacteriocin-negative strain of Streptococcus bovis in batch and continuous culture

A bacteriocin-producing Streptococcus bovis strain (HC5) outcompeted a sensitive strain (JB1) before it reached stationary phase (pH 6.4), even though it grew 10% slower and cell-free bovicin HC5 could not yet be detected. The success of bacteriocin-negative S. bovis isolates was enhanced by the presence of another sensitive bacterium (Clostridium sticklandii SR). PCR based on repetitive DNA sequences indicated that S. bovis HC5 was not simply transferring bacteriocin genes to S. bovis JB1. When the two S. bovis strains were coinoculated into minimal medium, bacteriocin-negative isolates predominated, and this effect could be explained by the longer lag time (0.5 vs. 1.5 h) of S. bovis HC5. If the glucose concentration of the minimal medium was increased from 2 to 7 mg mL(-1), the effect of lag time was diminished and bacteriocin-producing isolates once again dominated the coculture. When the competition was examined in continuous culture, it became apparent that batch culture inocula were never able to displace a strain that had already reached steady state, even if the inoculum was large. This result indicated that bacterial selection for substrate affinity was even more important than bacteriocin production.     

A truncated bacterial xylanase expressed in mammalian cells exhibits increased sensitivity to proteases

A truncated xylanase gene from the ruminal bacterium Fibrobacter succinogenes S85 was expressed in Chinese hamster ovary (CHO) cells and pancreatic acinar 266–6 cells using the SV40 early promoter and the mouse Amy-2.2 promoter/enhancer, respectively. The enzyme produced in both systems was active and secreted into the medium. The xylanase secreted from CHO cells was highly glycosylated and became sensitive to protease digestion compared to the unglycosylated form. © Rapid Science Ltd. 1998     

极端耐热木聚糖酶基因在枯草芽孢杆菌中的整合表达

目的:为了在枯草芽孢杆菌中整合表达极端耐热木聚糖酶。方法:将嗜热网球菌(Dictyoglomus thermophilum)Rt46B.1的极端耐热木聚糖酶基因xynB通过穿梭载体pDL整合到B.subtilis168染色体上,使其实现表达。结果:极端耐热木聚糖基因在枯草芽孢杆菌中成功整合并表达。结论:基因工程菌B.subtilis168-xynB能外泌表达极端耐热木聚糖酶,且表达水平为0.732IU/mL,比在大肠杆菌中的高。酶学性质表明,此酶分子量约为24kD,其最适反应温度为85℃,最适反应pH值为6.5,且在弱碱性条件下稳定。