High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus Trichoderma reesei Requires a Carrier Polypeptide with an Intact Domain Structure

A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides. By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures. The carriers tested were the T. reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T. reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof. The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide. The recombinant Xyn11A produced had properties similar to those of the native xylanase. It constituted 6 to 10% of the total proteins secreted by the transformants. About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed. Even in the best Xyn11A producers, the levels of the fusion mRNAs were only ~10% of the level of cel7A (cbh1) mRNA in the untransformed host strain.     

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.     

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.     

Production in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> of three xylanases from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>: a recombinant thermoxylanase for biobleaching of kraft pulp

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5–7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.     

Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var. thermoidea in Trichoderma reesei

AIMS: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. METHODS AND RESULTS: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source. Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.     

Molecular and biochemical characterization of two xylanase-encoding genes from Cellulomonas pachnodae.

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.     

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.     

The modular xylanase Xyn10A from Rhodothermus marinus is cell-attached, and its C-terminal domain has several putative homologues among cell-attached proteins within the phylum Bacteroidetes

Until recently, the function of the fifth domain of the thermostable modular xylanase Xyn10A from Rhodothermus marinus was unresolved. A putative homologue to this domain was however identified in a mannanase (Man26A) from the same microorganism which raised questions regarding a common function. An extensive search of all accessible data-bases as well as the partially sequenced genomes of R. marinus and Cytophaga hutchinsonii showed that homologues of this domain were encoded by multiple genes in microorganisms in the phylum Bacteroidetes. Moreover, the domain occurred invariably at the C-termini of proteins that were predominantly extra-cellular/cell attached. A primary structure motif of three conserved regions including structurally important glycines and a proline was also identified suggesting a conserved 3D fold. This bioinformatic evidence suggested a possible role of this domain in mediating cell attachment. To confirm this theory, R. marinus was grown, and activity assays showed that the major part of the xylanase activity was connected to whole cells. Moreover, immunocytochemical detection using a Xyn10A-specific antibody proved presence of Xyn10A on the R. marinus cell surface. In the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the phylum Bacteroidetes.     

Molecular and Biochemical Characterization of Two Xylanase-Encoding Genes from Cellulomonas pachnodae

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.     

Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain.

A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei. Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant. The hybrid was purified to homogeneity by affinity chromatography on cellulose. The relative association constants (Kr) for the binding of Cex, CexCBDCbhI, the catalytic domain of Cex (p33), and CbhI to bacterial microcrystalline cellulose (BMCC) were 14.9, 7.8, 0.8, and 10.6 liters g-1, respectively. Cex and CexCBDCbhI had similar substrate specificities and similar activities on crystalline and amorphous cellulose. Both released predominantly cellobiose and cellotriose from amorphous cellulose. CexCBDCbhI was two to three times less active than Cex on BMCC, but significantly more active than Cex on soluble cellulose and on xylan. Unlike Cex, the hybrid protein neither bound to alpha-chitin nor released small particles from dewaxed cotton fibers.     

Effects of dockerin domains on Neocallimastix frontalis xylanases

Two xylanase genes were cloned from the anaerobic fungus Neocallimastix frontalis. Xyn11A had a modular structure of two catalytic domains and two dockerin domains, while Xyn11B had one catalytic domain and two dockerin domains. The characteristics of the xylanases with and without dockerin domains were investigated. The deletion of dockerin domains had little influence on the optimal pH of xylanases, while it significantly affected the optimal temperatures. The optimal temperatures increased from 55 to 60 degrees C for Xyn11A and 60 to 65 degrees C for Xyn11B after the deletion of dockerin domains. The increase of optimal temperatures was attributed to the lower stability of the second structure in full length xylanase than that in the truncated one as evidenced by the circular dichroism spectroscopy. The specific activity of Xyn11A and Xyn11B increased about 64% and 330%, respectively, after the deletion of the dockerin domains. The removal of dockerin domains appeared to increase the overall efficiency of Xyn11A' (1.2-) and Xyn11B' (2.9-) fold with oat spelts xylan as reflected by the values of k(cat)/K(m). The results suggest that the dockerin domain might play an important role in the characteristics of xylanases from anaerobic fungi.     

里氏木霉双基因同步缺失菌株的构建与甘露聚糖酶表达研究

在里氏木霉中建立了一个快速的双基因位点同步同源重组新方法,较好解决了里氏木霉基因逐个敲除周期长等问题。研究以里氏木霉自身甘露聚糖酶基因（man5A）为重组表达的报告基因,通过一步转化,将该基因定点整合入纤维二糖水解酶Ⅰ（cbh1）基因位点,同时缺失主要的两个纤维素酶基因（cbh1、cbh2）,得到重组工程菌Man12。将重组工程菌Man12与出发菌株Tu6Δku70进行摇瓶发酵,结果显示,重组菌株的甘露聚糖酶产量比出发菌株提高10倍,而纤维素酶产量降低了60%,胞外总蛋白分泌水平降低了40%。Real-time PCR检测甘露聚糖酶基因（man5A）的转录水平,发现重组菌株较出发菌株提高了25倍。在里氏木霉中首次报道了通过一步转化实现两个基因同步定点整合的方法,对利用基因工程手段构建高效表达重组蛋白的里氏木霉工程菌株具有一定的指导意义。     

Probing the stability of the modular family 10 xylanase from<Emphasis Type="Italic"> Rhodothermus marinus</Emphasis>

The thermophilic bacterium Rhodothermus marinus produces a modular xylanase (Xyn10A) consisting of two N-terminal carbohydrate-binding modules (CBMs), followed by a domain of unknown function, and a catalytic module flanked by a fifth domain. Both Xyn10A CBMs bind calcium ions, and this study explores the effect of these ions on the stability of the full-length enzyme. Xyn10A and truncated forms thereof were produced and their thermostabilities were evaluated under different calcium loads. Studies performed using differential scanning calorimetry showed that the unfolding temperature of the Xyn10A was significantly dependent on the presence of Ca²⁺, and that the third domain of the enzyme binds at least one Ca²⁺. Thermal inactivation studies confirmed the role of tightly bound Ca²⁺ in stabilizing the enzyme, but showed that the presence of a large excess of this ion results in reduced kinetic stability. The truncated forms of Xyn10A were less stable than the full-length enzyme, indicative of module/domain thermostabilizing interactions. Finally, possible roles of the two domains of unknown function are discussed in the light of this study. This is the first report on the thermostabilizing role of calcium on a modular family 10 xylanase that displays multiple calcium binding in three of its five domains/modules.Communicated by G. Antranikian     

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, or carboxymethylcellulose.     

Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6

Paenibacillus curdlanolyticus B-6 showed effective degradation activities for xylan and cellulose and produced an extracellular multienzyme complex (approximately 1,450 kDa) containing several xylanases and cellulases. To characterize the multienzyme complex, we purified the complex from culture supernatants by four kind of chromatography. The purified multienzyme complex was composed of a 280-kDa protein with xylanase activity, a 260-kDa protein that was a truncated form on the C-terminal side of the 280-kDa protein, two xylanases of 40 and 48 kDa, and 60 and 65 kDa proteins having both xylanase and carboxymethyl cellulase activities. The 280-kDa protein resembled the scaffolding proteins of cellulosomes based on its migratory behavior in polyacrylamide gels and as a glycoprotein. Cloning of the 40-kDa major xylanase subunit named Xyn11A revealed that Xyn11A contained two functional domains which belonged to glycosyl hydrolase family-11 and to carbohydrate-binding module family-36, respectively, and a glycine- and asparagine-rich linker. However, an amino acid sequence similar to a dockerin domain, which is crucial to cellulosome assembly, was not found in Xyn11A. These results suggest that the multienzyme complex produced by P. curdlanolyticus B-6 should assemble by a mechanism distinct from the cohesin-dockerin interactions known in cellulosomes.     

High-level heterologous expression of Bacillus halodurans putative xylanase xyn11a (BH0899) in Kluyveromyces lactis

The putative xyn11A structural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus halodurans strain C-125 was heterologously expressed in the yeast Kluyveromyces lactis CBS 1065 and secreted to a level of 156 microg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactis CBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or beta-xylosidase activity.     

Xyn11A, a multidomain multicatalytic enzyme fromPseudobutyrivibrio xylanivorans Mz5T

The rumen bacterium Pseudobutyrivibrio xylanivorans Mz5T has a potent xylanolytic enzyme system. A small native peptide (approximately 30-kDa, designated Xyn11A) from the bacterium was first isolated and characterized by Edman degradation. The gene coding for Xyn11A was identified using PCR amplification with consensus primers. It was then fully sequenced to reveal an open reading frame of 1809 bp. The predicted N-terminal domain exhibited xylanolytic activity and was classed to the family 11 of glycosyl hydrolases; it is followed by a region with homology to a family 6 cellulose binding module. The C-terminal domain codes for a putative NodB-like polysaccharide deacetylase which is predicted to be an acetyl esterase implicated in debranching activity in the xylan backbone. As similar domain organization was also found in several other xylanases from a diverse range of bacteria, a common ancestor of such a xylanase is considered to be present and spread, possibly by horizontal gene transfer, to other microorganisms from different ecological niches.     

Xyn11E from Paenibacillus barcinonensis BP-23: a LppX-chaperone-dependent xylanase with potential for upgrading paper pulps

A new xylanase from Paenibacillus barcinonensis BP-23, Xyn11E, has been identified and characterized. Xyn11E has been cloned and heterologously expressed in Escherichia coli. It is a single-domain xylanase belonging to the family 11 of glycosyl hydrolases (GH11) with a predicted molecular weight of 20.652 kDa and an isoelectric point (pI) of 8.7. Substrate specificity, kinetic properties, and mode of action of the purified xylanase were characterized. Xyn11E exhibited high activity toward branched xylans, being beechwood xylan the preferred substrate. The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. Catalytic constants were determined on beechwood xylan, on which Xyn11E showed a Km of 12.98 mg/ml and a Vmax of 3,023 U/mg. The enzyme hydrolyzed long xylooligosaccharides, while oligomers shorter than xylotetraose were not degraded. Products released from glucuronoxylans were shorter than those liberated from cereal arabinoxylans. The xylanase was dependent on P. barcinonensis BP-23 LppX for its expression in an active form. Coexpression of Xyn11E with E. coli chaperones could not replace the need of LppX, which seems to act as a specific chaperone for Xyn11E correct folding. Activity of the enzyme on bleached pulps was evaluated. Xyn11E liberated reducing sugars from ECF and TCF pulps from eucalyptus, sisal, and flax, which makes it a good candidate for the enzymatic-assisted production of high-cellulose-content pulps from paper-grade pulps.