Cloning, expression, and cell surface localization of Paenibacillus sp. strain W-61 xylanase 5, a multidomain xylanase

We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa. Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp. strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5. xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence. Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains. Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101. Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa. Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer. Thus, Paenibacillus sp. strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains. Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5.     

Cloning, expression, and transglycosylation reaction of Paenibacillus sp. strain W-61 xylanase 1

A xylanase gene, xyn1, which encodes Paenibacillus sp. strain W-61 xylanase 1 (Xyn1), was cloned in Escherichia coli. xyn1 encodes 211 amino acid residues, including 28 amino acid residues of a signal peptide. The deduced amino acid sequence of the mature Xyn1 showed 95.7%, 84.0%, and 83.7% identity to family 11 xylanases of Aeromonas caviae ME-1, Paenibacillus sp., and Bacillus stearothermophilus respectively. The physico-chemical properties of recombinant Xyn1 were very similar to those of intact Xyn1, except for the molecular mass. The pattern of xylooligosaccharides generated by rXyn1 was investigated by fluorophore-assisted carbohydrate electrophoresis (FACE). The degradation rate of xylohexaose by rXyn1 increased markedly as compared with that of xylopentaose. Xylohexaose had a single preferential point of cleavage by rXyn1. On the basis of the pattern of action of xylooligosaccharides, the number of subsites was estimated to be six. The catalytic site was located between the third and the fourth subsites from non-reducing end.     

Purification, characterization and amplification of a 1.8 kbp fragment of xylanase 5 from Aeromonas caviae W-61

Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1, 2, 3, 4, and 5. In this study, we purified and characterized the xylanase 5 of A. caviae W-61, and amplified a part of xylanase 5 gene (xyn5). The purified xylanase 5 was found to be a single polypeptide with molecular mass of 140 kDa. It was an endo-beta-1,4-xylanase showing optimum temperature 40 degrees C and optimum pH 6.0. Xylobiose, xylotriose, xylotetrose, xylopentose, xylohexose and a small amount of xylose were detected as the hydrolysis products. The N-terminal amino acid sequence and several internal amino acid sequences of xylanases 5 were determined. From the sequence, a 1.8 kbp fragment was amplified by PCR using forward and reverse primers. DNA sequencing confirmed the presence of nucleotide sequences corresponding to the N-terminal amino acid sequence and the internal amino acid sequences of xylanase 5.     

Cloning, functional expression and promoter analysis of xylanase III gene from Trichoderma reesei

In this study, the xyn3 gene from the filamentous mesophilic fungus Trichoderma reesei (Hypocrea jecorina) PC-3-7 was cloned and sequenced. Analysis of the deduced amino acid sequence of XYN III revealed considerable homology with xylanases belonging to glycoside hydrolase family 10. These results show that XYN III is distinguishable from XYN I and XYN II, two other T. reesei xylanases that belong to the glycosidase family 11. When xyn3 was expressed in Escherichia coli, significant activity was observed in the cell-free extract, and higher activity (13.2 U/ml medium) was recovered from the inclusion bodies in the cell debris. The sequence of the 5′-upstream region of the gene in the parent strain QM9414 is identical to that of PC-3-7, although the expression level of xyn3 in PC-3-7 has been reported to be at least 1,000 times greater than in QM9414. These results suggest that xyn3 expression in T. reesei QM9414 is silenced. The consensus sequences for ACEI, ACEII, CREI, and the Hap2/3/5 protein complex are all present in the upstream region of xyn3. Deletion analysis of the upstream region revealed that two regions containing consensus sequences for the known regulatory elements play important roles for xyn3 expression.     

Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus β-glucosidase 1 for efficient biomass conversion

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.     

Increased production of xylanase by expression of a truncated version of the xyn11A gene from Nonomuraea flexuosa in Trichoderma reesei

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3' to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter-1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.     

Protein phosphorylation and binding of a 14-3-3 protein in Vicia guard cells in response to ABA

Under drought stress, ABA promotes stomatal closure to prevent water loss. Although protein phosphorylation plays an important role in ABA signaling, little is known about these processes at the biochemical level. In this study, we searched for substrates of protein kinases in ABA signaling through the binding of a 14-3-3 protein to phosphorylated proteins using Vicia guard cell protoplasts. ABA induced binding of a 14-3-3 protein to proteins with molecular masses of 61, 43 and 39 kDa, with the most remarkable signal for the 61 kDa protein. The ABA-induced binding to the 61 kDa protein occurred only in guard cells, and reached a maximum within 3 min at 1 microM ABA. The 61 kDa protein localized in the cytosol. ABA induced the binding of endogenous vf14-3-3a to the 61 kDa protein in guard cells. Autophosphorylation of ABA-activated protein kinase (AAPK), which mediates anion channel activation, and ABA-induced phosphorylation of the 61 kDa protein showed similar time courses and similar sensitivities to the protein kinase inhibitor K-252a. AAPK elicits the binding of the 14-3-3 protein to the 61 kDa protein in vitro when AAPK in guard cells was activated by ABA. The phosphorylation of the 61 kDa protein by ABA was not affected by the NADPH oxidase inhibitor, H(2)O(2), W-7 or EGTA. From these results, we conclude that the 61 kDa protein may be a substrate for AAPK and that the 61 kDa protein is located upstream of H(2)O(2) and Ca(2+), or on Ca(2+)-independent signaling pathways in guard cells.