Identification of the Butyrivibrio fibrisolvens xylosidase gene (xylB) coding region and its expression in Escherichia coli

The gene encoding the principal Butyrivibrio fibrisolvens xylosidase (xylB) has been cloned and expressed in Escherichia coli under the control of the lac promoter. The coding region for this gene was localized within a 3.2-kilobase B. fibrisolvens DNA fragment in pUC18. A new protein band was observed in recombinant E. coli containing xylB. This protein (approximately 60,000 molecular weight) was presumed to be the xylosidase monomer. The optimal pH (5.5) and substrate range for the recombinant and native xylosidases appeared identical. Both enzymes hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 5 and both were inactive on xylan.     

Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities.

A single gene (xylB) encoding both beta-D-xylosidase (EC 3.2.1.37) and alpha-L-arabinofuranosidase (EC 3.2.1.55) activities was identified and sequenced from the ruminal bacterium Butyrivibrio fibrisolvens. The xylB gene consists of a 1.551-bp open reading frame (ORF) encoding 517 amino acids. A subclone containing a 1.843-bp DNA fragment retained both enzymatic activities. Insertion of a 10-bp NotI linker into the EcoRV site within the central region of this ORF abolished both activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytoplasmic proteins from recombinant Escherichia coli confirmed the presence of a 60,000-molecular-weight protein in active subclones and the absence of this protein in subclones lacking activity. With p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside as substrates, the specific activity of arabinosidase was found to be approximately 1.6-fold higher than that of xylosidase. The deduced amino acid sequence of the xylB gene product did not exhibit a high degree of identity with other xylan-degrading enzymes or glycosidases. The xylB gene was located between two incomplete ORFs within the 4,200-bp region which was sequenced. No sequences resembling terminators were found within this region, and these three genes are proposed to be part of a single operon. Based on comparison with other glycosidases, a conserved region was identified in the carboxyl end of the translated xylB gene which is similar to that of glucoamylase from Aspergillus niger.     

Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities

A single gene (xylB) encoding both beta-D-xylosidase (EC 3.2.1.37) and alpha-L-arabinofuranosidase (EC 3.2.1.55) activities was identified and sequenced from the ruminal bacterium Butyrivibrio fibrisolvens. The xylB gene consists of a 1.551-bp open reading frame (ORF) encoding 517 amino acids. A subclone containing a 1.843-bp DNA fragment retained both enzymatic activities. Insertion of a 10-bp NotI linker into the EcoRV site within the central region of this ORF abolished both activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytoplasmic proteins from recombinant Escherichia coli confirmed the presence of a 60,000-molecular-weight protein in active subclones and the absence of this protein in subclones lacking activity. With p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside as substrates, the specific activity of arabinosidase was found to be approximately 1.6-fold higher than that of xylosidase. The deduced amino acid sequence of the xylB gene product did not exhibit a high degree of identity with other xylan-degrading enzymes or glycosidases. The xylB gene was located between two incomplete ORFs within the 4,200-bp region which was sequenced. No sequences resembling terminators were found within this region, and these three genes are proposed to be part of a single operon. Based on comparison with other glycosidases, a conserved region was identified in the carboxyl end of the translated xylB gene which is similar to that of glucoamylase from Aspergillus niger.     

Cloning, sequencing, and expression of a xylanase gene from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens.

A gene coding for xylanase activity, xynA, from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens 49 was cloned into Escherichia coli JM83 by using plasmid pUC19. The gene was located on a 2.3-kilobase (kb) DNA insert composed of two adjacent EcoRI fragments of 1.65 and 0.65 kb. Expression of xylanase activity required parts of both EcoRI segments. In E. coli, the cloned xylanase enzyme was not secreted and remained cell associated. The enzyme exhibited no arabinosidase, cellulase, alpha-glucosidase, or xylosidase activity. The isoelectric point of the cloned protein was approximately 9.8, and optimal xylanase activity was obtained at pH 5.4. The nucleotide sequence of the 1,535-base-pair EcoRV-EcoRI segment from the B. fibrisolvens chromosome that included the xynA gene was determined. An open reading frame was found that encoded a 411-amino-acid-residue polypeptide of 46,664 daltons. A putative ribosome-binding site, promoter, and leader sequence were identified. Comparison of the XynA protein sequence with that of the XynA protein from alkalophilic Bacillus sp. strain C-125 revealed considerable homology, with 37% identical residues or conservative changes. The presence of the cloned xylanase gene in other strains of Butyrivibrio was examined by Southern hybridization. The cloned xylanase gene hybridized strongly to chromosomal sequences in only two of five closely related strains.     

Cloning, characterization, and functional expression of the Klebsiella oxytoca xylodextrin utilization operon (xynTB) in Escherichia coli

Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na(+)/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.     

Genetic evidence for a defective xylan degradation pathway in Lactococcus lactis

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974-3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a beta-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.     

Cloning, nucleotide sequence, and enzymatic characterization of an alpha-amylase from the ruminal bacterium Butyrivibrio fibrisolvens H17c.

A Butyrivibrio fibrisolvens amylase gene was cloned and expressed by using its own promoter on the recombinant plasmid pBAMY100 in Escherichia coli. The amylase gene consisted of an open reading frame of 2,931 bp encoding a protein of 976 amino acids with a calculated Mr of 106,964. In E. coli(pBAMY100), more than 86% of the active amylase was located in the periplasm, and TnphoA fusion experiments showed that the enzyme had a functional signal peptide. The B. fibrisolvens amylase is a calcium metalloenzyme, and three conserved putative calcium-binding residues were identified. The amylase showed high sequence homology with other alpha-amylases in the three highly conserved regions which constitute the active centers. These and other conserved regions were located in the N-terminal half, and no similarity with any other amylase was detected in the remainder of the protein. Deletion of approximately 40% of the C-terminal portion of the amylase did not result in loss of amylolytic activity. The B. fibrisolvens amylase was identified as an endo-alpha-amylase by hydrolysis of the Phadebas amylase substrate, hydrolysis of gamma-cyclodextrin to maltotriose, maltose, and glucose and the characteristic shape of the blue value and reducing sugar curves. Maltotriose was the major initial hydrolysis product from starch, although extended incubation resulted in its hydrolysis to maltose and glucose.     

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.     

半定量RT-PCR检测运动发酵单胞菌中外源基因转录水平的研究

本研究运用半定量RT-PCR法检测运动发酵单胞菌重组菌中外源基因xylB的转录水平。提取野生型运动发酵单胞菌CP4及其2个重组菌的总RNA, 检测无DNA污染后定量至同一浓度、并反转录为cDNA。观测目的基因xylB和内标基因16S rRNA的PCR扩增曲线、并确定合适的循环数, 选用相同量的cDNA为模板, PCR检测各样本中xylB相对16S rRNA的转录水平。结果表明野生型菌株CP4中xylB基因没有转录, 而两株重组菌中皆有xylB的转录本, 且转录丰度基本一致, 酶活测定也进一步证实该基因在重组菌中有效表达。该方法可用于鉴定运动发酵单胞菌中特定基因的转录水平, 是一种快速有效的检测方法。     

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a beta-glucosidase

The cloning, expression and nucleotide sequence of a 3.74 kb DNA segment on pLS215 containing a beta-glucosidase gene (bglA) from Butyrivibrio fibrisolvens H17c was investigated. The B. fibrisolvens bglA open reading frame (ORF) of 2490 bp encoded a beta-glucosidase of 830 amino acid residues with a calculated Mr of 91,800. In Escherichia coli C600(pLS215) cells the beta-glucosidase was localized in the cytoplasm and these cells produced an additional protein with an apparent Mr of approximately 94,000. The bglA gene was expressed from its own regulatory region in E. coli and a single mRNA initiation point was identified upstream of the bglA ORF and adjacent to a promoter consensus sequence. The primary structure of the beta-glucosidase showed greater than 40% similarity with a domain of 237 amino acids present in the beta-glucosidases of Kluyveromyces fragilis and Clostridium thermocellum. The B. fibrisolvens beta-glucosidase hydrolysed cellobiose to a limited extent, cellotriose to cellobiose and glucose, and cellotetraose and cellopentaose to predominantly glucose.     

Secretory expression of a β‐xylosidase gene from Thermomyces lanuginosus in Escherichia coli and characterization of its recombinant enzyme

Aims: To characterize a β‐xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results: A gene (designated TlXyl43) encoding β‐xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017‐bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β‐xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6‐fold to homogeneity with an estimated mass of 51·6 kDa by SDS‐PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a K_i value of 63 mmol l^?1. Conclusions: In this study, a GH family 43 β‐xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study: This is the first report of the cloning and functional expression of a β‐xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.     

Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46

Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into lambda gt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2.3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley beta-glucan, Avicel, filter paper and p-nitrophenyl beta-D-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48,863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.     

代谢工程改造大肠杆菌合成D-1,2,4-丁三醇

【目的】D-1,2,4-丁三醇是一种四碳的多元醇,在军事和医药领域具有广泛的应用。为实现生物法一步转化生产D-1,2,4-丁三醇,对Escherichia coli W3100的木糖代谢途径进行改造。【方法】将来源于柄杆菌的D-木糖脱氢酶基因xylB和恶臭假单胞菌的苯甲酰甲酸脱羧酶基因mdlC克隆至E.coli W3100,得到重组菌E.coli(pEtac-mdlC-tac-xylB)。在此基础上对重组菌代谢木糖合成D-1,2,4-丁三醇的能力进行考察。【结果】在30°C下,以30 g/L D-木糖为底物,重组菌E.coli(pEtac-mdlC-tac-xylB)的D-1,2,4-丁三醇产量达到了0.9 g/L,摩尔转化率为4%。【结论】实现了D-1,2,4-丁三醇的一步法发酵生产,为国内开展相关研究奠定了坚实的基础。     

Engineering of a xylose metabolic pathway in Corynebacterium glutamicum

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.     

Cloning and analysis of the xylAB operon and characterization of xylose isomerase from Thermoanaerobacter ethanolicus

Three genes, xylA-like, xylA and xylB, were cloned and sequenced from the chromosome of Thermoanaerobacter ethanolicus JW200. xylA and xylB share an operon and encode xylose isomerase and xylulokinase, respectively. The xylA-like gene locates upstream of xylAB operon and encodes a hypothetical protein that lacks xylose isomerase activity. The xylose isomerase was expressed in Escherichia coli and purified by heat treatment and an ion-exchange chromatography. The enzyme had highest activity at 85°C and pH 7.0, and a half-life for 1 h at 85°C. The K (m) and V (max) values for xylose were 11 mM and 25 U/mg, respectively. The high level of expression, easy purification, and thermostability of the XylA from T. ethanolicus JW200 suggests industrial usefulness.     

The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region.

Genes coding for three xylan-degrading activities, xylanase, xylosidase, and arabinosidase, were simultaneously cloned from the colonic anaerobic organism Bacteriodes ovatus. The genes for the three enzymes were located on a 3.8-kilobase EcoRI genomic insert and were cloned by using plasmid pUC18. All three activities were expressed in Escherichia coli JM83, and all were cell associated. Expression of the xylanase gene was independent from expression of the xylosidase and arabinosidase genes, whereas expression of the latter two genes appeared to be coordinated. Restriction endonuclease analysis of the arabinosidase and xylosidase genes and partial purification of these enzyme activities from E. coli suggested that these activities were catalyzed by a bifunctional protein or two proteins of very similar molecular weight. All three enzyme activities were regulated in B. ovatus in response to the carbon source used for growth. This is the first report of the cloning and expression of B. ovatus genes.     

Role of xylose transporters in xylitol production from engineered Escherichia coli

Escherichia coli W3110 was previously engineered to co-utilize glucose and xylose by replacing the wild-type crp gene with a crp* mutant encoding a cAMP-independent CRP variant (Cirino et al., 2006 [Cirino, P.C., Chin, J.W., Ingram, L.O., 2006. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167-1176.]). Subsequent deletion of the xylB gene (encoding xylulokinase) and expression of xylose reductase from Candida boidinii (CbXR) resulted in a strain which produces xylitol from glucose-xylose mixtures. In this study we examine the contributions of the native E. coli xylose transporters (the d-xylose/proton symporter XylE and the d-xylose ABC transporter XylFGH) and CRP* to xylitol production in the presence of glucose and xylose. The final batch xylitol titer with strain PC09 (Delta xylB and crp*) is reduced by 40% upon deletion of xylG and by 60% upon deletion of both xyl transporters. Xylitol production by the wild-type strain (W3110) expressing CbXR is not reduced when xylE and xylG are deleted, demonstrating tight regulation of the xylose transporters by CRP and revealing significant secondary xylose transport. Finally, plasmid expression of XylE or XylFGH with CbXR in PC07 (Delta xylB and wild-type crp) growing on glucose results in xylitol titers similar to that achieved with PC09 and provides an alternative strategy to the use of CRP*.     

Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity.

A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates. The gene was expressed in Escherichia coli from its own promoter. The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes. A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases. The B. fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold. The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame. The enzymatic properties of the purified enzyme were investigated. The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.