Cellulase Gene Research Articles

Man/Cel5B, a Bifunctional Enzyme Having the Highest Mannanase Activity in the Hyperthermic Environment.

Thermotoga maritima (Tma) contains genes encoding various hyperthermophilic enzymes with great potential for industrial applications. The gene TM1752 in Tma genome has been annotated as cellulase gene encoding protein Cel5B. In this work, the gene TM1752 was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Interestingly, the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxy methyl cellulose, which is the highest among thermophilic mannanases. However, the putative enzyme did not show sequence homology with any of the previously reported mannanases; therefore, the enzyme Cel5B was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B. Man/Cel5B exhibited maximum activity at 85°C and pH 5.5. This enzyme retained more than 50% activity after 5 h of incubation at 85°C, and retained up to 80% activity after incubated for 1 h at pH 5–8. The Km and Vmax of Man/Cel5B were observed to be 4.5 mg/mL galactomannan and 769 U/mg, respectively. Thin layer chromatography depicted that locust bean gum could be efficiently degraded to mannobiose, mannotriose, and mannooligosaccharides by Man/Cel5B. These characteristics suggest that Man/Cel5B has attractive applications for future food, feed, and biofuel industries.

PoxCbh, a novel CENPB-type HTH domain protein, regulates cellulase and xylanase gene expression in Penicillium oxalicum.

The essential transcription factor PoxCxrA is required for cellulase and xylanase gene expression in the filamentous fungus Penicillium oxalicum that is potentially applied in biotechnological industry as a result of the existence of the integrated cellulolytic and xylolytic system. However, the regulatory mechanism of cellulase and xylanase gene expression specifically associated with PoxCxrA regulation in fungi is poorly understood. In this study, the novel regulator PoxCbh (POX06865), containing a centromere protein B-type helix-turn-helix domain, was identified through screening for the PoxCxrA regulon under Avicel induction and genetic analysis. The mutant ∆PoxCbh showed significant reduction in cellulase and xylanase production, ranging from 28.4% to 59.8%. Furthermore, PoxCbh was found to directly regulate the expression of important cellulase and xylanase genes, as well as the known regulatory genes PoxNsdD and POX02484, and its expression was directly controlled by PoxCxrA. The PoxCbh-binding DNA sequence in the promoter region of the cellobiohydrolase 1 gene cbh1 was identified. These results expand our understanding of the diverse roles of centromere protein B-like protein, the regulatory network of cellulase and xylanase gene expression, and regulatory mechanisms in fungi.

Screening, cloning, enzymatic properties of a novel thermostable cellulase enzyme, and its potential application on water hyacinth utilization.

Cellulose is the cheapest, natural, renewable organic substance that is used as a carbon source in various fields. Water hyacinth, an aquatic plant rich in cellulose, is often used as a raw material in fuel production. However, natural cellulase can be hardly used in industrial production on account of its low thermal stability and activity. In this study, a metagenomic library was constructed. Then, a new cellulase gene, cel1029, was screened by Congo red staining and expressed in the prokaryotic system. Enzymatic properties of Cel1029 were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions, and salt solutions. Finally, its ability of degrading water hyacinth was identified and evaluated. Cel1029 displayed high homology with endoglucanase in the glycoside hydrolase family 5 (GH5) and had high stability across a broad temperature range. More than 86% of its enzymatic activities were retained between 4 and 60 °C after 24 h of incubation. Single-factor analysis and orthogonal design were further conducted to determine the optimal conditions for the highest reducing sugar yield of water hyacinth. Interestingly, Cel1029 efficiently transformed water hyacinth with a reducing sugar yield of 430.39 mg/g in 22 h. These findings may open the door for significant industrial applications of a novel GH5 cellulase (NCBI Reference Sequence: MK051001, Cel1029) and help identify more efficient methods to degrade cellulose-rich plants.

Interdependent recruitment of CYC8/TUP1 and the transcriptional activator XYR1 at target promoters is required for induced cellulase gene expression in Trichoderma reesei.

Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei.

Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose

BackgroundTrichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose.ResultsWe found that deletion of an α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (ΔtubB) strain had high enzyme production and the same enzyme profile even if the strain was cultured in a glucose-containing medium. From transcriptome analysis, the ΔtubB strain exhibited upregulation of both cellulase and hemicellulase genes including some that were not originally induced by cellulose. Moreover, cellobiose transporter genes and the other sugar transporter genes were highly upregulated, and simultaneous uptake of glucose and cellobiose was also observed in the ΔtubB strain. These results suggested that the ΔtubB strain was released from CCR.ConclusionTrichoderma reesei α-tubulin is involved in the transcription of cellulase and hemicellulase genes, as well as in CCR. This is the first report of overcoming CCR by disrupting α-tubulin gene in T. reesei. The disruption of α-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.

Influences of genetically perturbing synthesis of the typical yellow pigment on conidiation, cell wall integrity, stress tolerance, and cellulase production in Trichoderma reesei.

The prominent protein producing workhorse Trichoderma reesei secretes a typical yellow pigment that is synthesized by a gene cluster including two polyketide synthase encoding genes sor1 and sor2. Two transcription factors (YPR1 and YPR2) that are encoded in the same cluster have been shown to regulate the expression of the sor genes. However, the physiological relevance of the yellow pigment synthesis in T. reesei is not completely clear. In this study, a yellow pigment hyper-producer OEypr1 and three yellow pigment non-producers, OEypr1-sor1, Δypr1, and OEypr2, were constructed. Their phenotypic features in mycelial growth, conidiation, cell wall integrity, stress tolerance, and cellulase production were determined. Whereas hyperproduction of the yellow pigment caused significant defects in all the physiological aspects tested, the non-producers showed similar colony growth, but improved conidiation, maintenance of cell wall integrity, and stress tolerance compared to the control strain. Moreover, in contrast to the severely compromised extracellular cellobiohydrolase production in the yellow pigment hyperproducer, loss of the yellow pigment hardly affected induced cellulase gene expression. Our results demonstrate that interfering with the yellow pigment synthesis constitutes an engineering strategy to endow T. reesei with preferred features for industrial application.

A component of the septation initiation network complex, AaSepM, is involved in multiple cellulose-responsive signaling pathways in Aspergillus aculeatus.

Various carbohydrate-active enzymes in Aspergillus are produced in response to physiological inducers, which is regulated at the transcriptional level. To elucidate the induction mechanisms in Aspergillus, we screened for new regulators involved in cellulose-responsive induction from approximately 10,000 Aspergillus aculeatus T-DNA-inserted mutants. We constructed the T-DNA-inserted mutant library using the host strain harboring the orotidine 5'-monophosphate decarboxylase gene (pyrG) under the control of the FIII-avicelase gene (cbhI) promoter. Thus, candidate mutants deficient in cellulose-responsive induction were positively screened via counter selection against 5-fluoroorotic acid (5-FOA). Among less than two hundred 5-FOA-resistant mutants, one mutant that the T-DNA inserted into the AasepM locus reduced the cbhI expression in response to cellulose. Since AaSepM is similar to Schizosaccharomyces pombe Cdc14p (E-value, 2e-20; identities, 33%), which is a component of the septation initiation network (SIN)-complex, we constructed an AasepM deletion mutant (ΔAasepM). We analyzed the expression of cellulase and xylanase genes in response to cellulose, septation, and conidiation in ΔAasepM. The AasepM deletion leads to delayed septation and decreased formation of the conidium chain in A. aculeatus but does not affect hyphal growth on minimal media. We also confirmed AaSepM's involvement in multiple cellulose-responsive signaling pathways of cellulase and xylanase genes under the control of the ManR-dependent, XlnR-dependent, and ManR- and XlnR-independent signaling pathways. KEY POINTS : • A new regulator for cellulolytic gene expression has been identified. • AaSepM is involved in septation and conidiation in A. aculeatus. • AasepM is involved in multiple cellulose-responsive signaling pathways.

Soluble expression of recombinant active cellulase in E.coli using B.subtilis (natto strain) cellulase gene

BackgroundCellulases are well known for their various industrial applications. They are naturally produced by different species of bacteria and fungi. Fermentation process of cellulase producers has limitation due to the high substrate cost required for cellulase induction and challenges to maintain the suitable condition for the respective cellulase production. Recombinant cellulase production could be the potential solution to these problems. In the current study, we investigated recombinant cellulase expression in Escherichia coli using cellulase gene from Bacillus subtilis. ResultsExtracellular cellulase production from B. subtilis strain was first confirmed on CMC agar and then the cellulase gene (1500 bp) was amplified from this strain and was further cloned in pET21a expression vector. In initial experimental studies, recombinant cellulase expression was achieved in inclusion bodies through shake flask level fermentation of transformed E. coli expression host BL21DE3. Attempts were made to express this 55 KDa His tagged recombinant cellulase into soluble form by modifications in fermentation conditions. Partially purified recombinant cellulase was obtained using Ni-NTA affinity chromatography. The activity of the purified enzyme was confirmed by 3,5-dinitrosalicylic acid (DNS) qualitative assay. ConclusionSoluble expression of active recombinant cellulase can be achieved by subtle alteration in the upstream process.

Involvement of phospholipase PLA2 in production of cellulase and xylanase by Penicillium oxalicum.

Phospholipases play vital roles in immune and inflammatory responses in mammals and plants; however, knowledge of phospholipase functions in fungi is limited. In this study, we investigated the effects of deleting predicted phospholipase genes on cellulase and xylanase production, and morphological phenotype, in Penicillium oxalicum. Individual deletion of nine of the ten predicted phospholipase genes resulted in alteration of cellulase and xylanase production, and the morphological phenotypes, to various degrees. The mutant ∆POX07277 lost 22.5 to 82.8% of cellulase (i.e., filter paper cellulase, carboxymethylcellulase, and p-nitrophenyl-β-cellobiosidase) and xylanase production, whereas p-nitrophenyl-β-glucopyranosidase production increased by 5.8-127.8 fold. POX07277 (P. oxalicum gene No. 07277) was predicted to encode phospholipase A2 and was found to negatively affect the sporulation of P. oxalicum. Comparative transcriptomic and quantitative reverse transcription-PCR analysis indicated that POX07277 dynamically affected the expression of cellulase and xylanase genes and the regulatory genes for fungal sporulation, under micro-crystalline cellulose induction. POX07277 was required for the expression of the known regulatory gene PoxCxrB (cellulolytic and xylanolytic regulator B in P. oxalicum), which is involved in cellulase and xylanase gene expression in P. oxalicum. Conversely, POX07277 expression was regulated by PoxCxrB. These findings will aid the understanding of phospholipase functions and provide novel insights into the mechanism of fungal cellulase and xylanase gene expression. KEY POINTS : • The roles of phospholipases were investigated in Penicillium oxalicum. • POX07277 (PLA2) is required for the expression of cellulase and xylanase genes. • PoxCxrB dynamically regulated POX07277 expression.

Molecular Characterization of Purified Bacterial Cellulase Enzyme Immobilized in Magnetic Nanoparticle

Background: Plants were rich in lignocellulose and cellulosic content which forms highly complex structure and this needs effective enzymatic catalyzes reaction for useful product development. Various industries like textiles, paper, agriculture and biofuel need enzymatic treatment (cellulase) which costs up to the total cost production of 40% to convert into the useful product. Methods: To get the better of this economical contingency, the cellulase cost was reduced by immobilizing with Magnetic Nanoparticle (MNP’s). It is hypothesized that cellulose enzyme immobilization with magnetic nanoparticle can meet the economically feasible cellulase production. Present study describes the cellulase molecular characterization isolated from cellulolytic bacteria and its growth parameters were optimized for its enzyme production. The cellulolytic bacteria with efficient substrate hydrolyzing property were chosen and its cellulase activity was evaluated. The cellulase kinetics was calculated using Michaelis - Menten Kinetics (MM kinetics) to find the Vmax and Km. Results: The purified cellulase was immobilized with magnetic nanoparticle and its efficiency and yield was calculated and characterized with FTIR, SEM and XRD. The peptide mass finger printing of cellulase was evaluated using MALDI-TOF-TOF analysis. The free enzyme and immobilized enzyme was checked for its temperature optimization and stability, pH optimization and stability, substrate specificity, storage stability. The reusability of the immobilized enzyme was also evaluated. Conclusion: In future, the enzymatically and molecularly characterized cellulase gene from cellulolytic bacteria will be over expressed in E.coli to make the cellulase cost feasible.

Molecular Cloning and Expression Analysis of the Endogenous Cellulase Gene MaCel1 in Monochamus alternatus

The purpose of this study was to characterize the endogenous cellulase gene MaCel1 of Monochamus alternatus, which is an important vector of Bursaphelenchus xylophilus, a pine wood nematode, which causes pine wilt disease (PWD). In this study, MaCel1 was cloned by rapid amplification of cDNA end (RACE), and its expression analyzed by RT-qPCR (real-time quantitative PCR detecting). A total of 1778 bp of cDNA was obtained. The encoding region of this gene was 1509 bp in length, encoding a protein containing 502 amino acids with a molecular weight of 58.66 kDa, and the isoelectric point of 5.46. Sequence similarity analysis showed that the amino acids sequence of MaCel1 had high similarity with the β-Glucosinolate of Anoplophoraglabripennis and slightly lower similarity with other insect cellulase genes (GH1). The β-D-Glucosidase activity of MaCel1 was 256.02 ± 43.14 U/L with no β-Glucosinolate activity. MaCel1 gene was widely expressed in the intestine of M. alternatus. The expression level of MaCel1 gene in male (3.46) and female (3.51) adults was significantly higher than that in other developmental stages, and the lowest was in pupal stage (0.15). The results will help reveal the digestive mechanism of M. alternatus and lay the foundation for controlling PWD by controlling M. alternatus.

Generation of Multi-Transgenic Pigs Using PiggyBac Transposons Co-expressing Pectinase, Xylanase, Cellulase, β-1.3-1.4-Glucanase and Phytase.

The current challenges facing the pork industry are to maximize feed efficiency and minimize fecal emissions. Unlike ruminants, pigs lack several digestive enzymes such as pectinase, xylanase, cellulase, β-1.3-1.4-glucanase, and phytase which are essential to hydrolyze the cell walls of grains to release endocellular nutrients into their digestive tracts. Herein, we synthesized multiple cellulase and pectinase genes derived from lower organisms and then codon-optimized these genes to be expressed in pigs. These genes were then cloned into our previously optimized XynB (xylanase)- EsAPPA (phytase) bicistronic construct. We then successfully generated transgenic pigs that expressed the four enzymes [Pg7fn (pectinase), XynB (xylanase), EsAPPA (phytase), and TeEGI (cellulase and β-glucanase)] using somatic cell cloning. The expression of these genes was parotid gland specific. Enzymatic assays using the saliva of these founders demonstrated high levels of phytase (2.0∼3.4 U/mL) and xylanase (0.25∼0.42 U/mL) activities, but low levels of pectinase (0.06∼0.08 U/mL) activity. These multi-transgenic pigs are expected to contribute to enhance feed utilization and reduce environmental impact.

The Copper-Controlled RNA Interference System in Trichoderma reesei.

Trichoderma reesei is capable of secreting large amounts of lignocellulose-degrading enzymes. Although the genome sequence of T. reesei has been available, the molecular mechanisms of the hyper-production of cellulases, including the transcriptional regulation and the protein secretion, have not been completely elucidated yet. This is partially due to the lack of genetic manipulation approaches. RNA interference (RNAi) is a powerful tool for functional genomic studies in eukaryotes. Some successful examples of RNAi have already been reported; however, these systems were either uncontrolled or relied on a nutrient source inducible promoter. Here, we present a copper-controlled RNAi system in T. reesei for reversible silencing of different target genes. As the proof of concept, T.reesei xyr1, the key transcriptionalactivator of cellulase genes, has been knocked down using this method.

The G-protein Coupled Receptor GPR8 Regulates Secondary Metabolism in Trichoderma reesei.

Changing environmental conditions are of utmost importance for regulation of secondary metabolism in fungi. Different environmental cues including the carbon source, light and the presence of a mating partner can lead to altered production of compounds. Thereby, the heterotrimeric G-protein pathway is of major importance for sensing and adjustment of gene regulation. Regulation of secondary metabolism is crucial in the biotechnological workhorse Trichoderma reesei for knowledge-based adjustment in industrial fermentations, but also with respect to the potential use as a host for heterologous compound production. We investigated the function of the class VII G-protein coupled receptor (GPCR) gene gpr8 that is localized in the vicinity of the SOR cluster, which is responsible for biosynthesis of sorbicillinoids. GPR8 positively impacts regulation of the genes in this cluster in darkness. Accordingly, abundance of trichodimerol and dihydrotrichotetronine as well as other secondary metabolites is decreased in the deletion mutant. Transcriptome analysis moreover showed the major role of GPR8 being exerted in darkness with a considerable influence on regulation of secondary metabolism. Genes regulated in Δgpr8 overlap with those regulated directly or indirectly by the transcription factor YPR2, especially concerning genes related to secondary metabolism. The predicted FAD/FMN containing dehydrogenase gene sor7, one of the positive targets of the cascade triggered by GPR8, has a positive effect on secondary metabolite production, but also cellulase gene expression. Hence SOR7 has some overlapping, but also additional functions compared to GPR8. The G-protein coupled receptor GPR8 exerts a light dependent impact on secondary metabolism, which is in part mediated by the transcription factor YPR2 and the function of SOR7. Hence, T. reesei may apply GPR8 to adjust production of secondary metabolites and hence chemical communication to signals from the environment.

Sophorolipid Production Using Lignocellulosic Biomass by Co-culture of Several Recombinant Strains of Starmerella bombicola with Different Heterologous Cellulase Genes from Penicillum oxalicum.

One of the reasons hindering large-scale application of sophorolipids (SLs) is high production cost. In this study, six recombinant strains of Starmerella bombicola, sbEG1, sbEG2, sbCBH1, sbCBH1-2, sbBGL1, and sbCBH2 expressing cellulase genes eg1, eg2, cbh, cbh1-2, bgl1, and cbh2 from Penicillium oxalicum were respectively constructed. Four strains showed cellulase activities and were co-cultivated in fermentation media containing 2% glucose, 1% Regenerated Amorphous Cellulose (RAC), 2% glucose, and 1% RAC, respectively. After 7days' cultivation, concentration of SLs in medium with 1% RAC (g/L) reached 1.879g/L. When 2% glucose and 1% of RAC were both contained, the titer of SLs increased by 39.5% than that of control strain and increased by 68.8% than that in the medium with only 2% glucose. Results demonstrated that cellulase genes from filamentous fungi in S. bombicola can function to degrade lignocellulosic cellulose to produce SLs.

Host-Endosymbiont Genome Integration in a Deep-Sea Chemosymbiotic Clam.

Endosymbiosis with chemosynthetic bacteria has enabled many deep-sea invertebrates to thrive at hydrothermal vents and cold seeps, but most previous studies on this mutualism have focused on the bacteria only. Vesicomyid clams dominate global deep-sea chemosynthesis-based ecosystems. They differ from most deep-sea symbiotic animals in passing their symbionts from parent to offspring, enabling intricate coevolution between the host and the symbiont. Here, we sequenced the genomes of the clam Archivesica marissinica (Bivalvia: Vesicomyidae) and its bacterial symbiont to understand the genomic/metabolic integration behind this symbiosis. At 1.52 Gb, the clam genome encodes 28 genes horizontally transferred from bacteria, a large number of pseudogenes and transposable elements whose massive expansion corresponded to the timing of the rise and subsequent divergence of symbiont-bearing vesicomyids. The genome exhibits gene family expansion in cellular processes that likely facilitate chemoautotrophy, including gas delivery to support energy and carbon production, metabolite exchange with the symbiont, and regulation of the bacteriocyte population. Contraction in cellulase genes is likely adaptive to the shift from phytoplankton-derived to bacteria-based food. It also shows contraction in bacterial recognition gene families, indicative of suppressed immune response to the endosymbiont. The gammaproteobacterium endosymbiont has a reduced genome of 1.03 Mb but retains complete pathways for sulfur oxidation, carbon fixation, and biosynthesis of 20 common amino acids, indicating the host’s high dependence on the symbiont for nutrition. Overall, the host–symbiont genomes show not only tight metabolic complementarity but also distinct signatures of coevolution allowing the vesicomyids to thrive in chemosynthesis-based ecosystems.

Alleviating product inhibition of Trichoderma reesei cellulase complex with a product-activated mushroom endoglucanase

Product inhibition of cellulase is a challenging issue in industrial processes. Here, we introduced a product-activated mushroom cellulase, PaCel3A from Polyporus arcularius, into Trichoderma reesei. The filter paper activity, carboxymethyl cellulase activity, and saccharification efficiency (substrate: pretreated rice straw, PRS) of transformants increased significantly with this enzyme (by 18.4–26.8%, 13.8–22.8%, and 17.0%, respectively). A mutant of PaCel3A, PaCel3AM, obtained based on B-factor analysis, saturated mutagenesis, and residual activity assay, showed improved thermostability. The PRS saccharification efficiency using the cellulase complex from T. reesei transformants overexpressing pacel3am increased by 56.4%–63.0%. In addition, the T. reesei cellulase complex obtained by adding the purified recombinant PaCel3AM from T. reesei (rCel3aM-tr) to hydrolyze PRS resulted in increased reducing sugar yields at all sampling points, outperforming the cellulase complexes without rCel3aM-tr. These results suggest that introducing product-activated cellulase genes is a simple and feasible method to alleviate the product inhibition of cellulase.

Trichoderma reesei XYR1 activates cellulase gene expression via interaction with the Mediator subunit TrGAL11 to recruit RNA polymerase II.

The ascomycete Trichoderma reesei is a highly prolific cellulase producer. While XYR1 (Xylanase regulator 1) has been firmly established to be the master activator of cellulase gene expression in T. reesei, its precise transcriptional activation mechanism remains poorly understood. In the present study, TrGAL11, a component of the Mediator tail module, was identified as a putative interacting partner of XYR1. Deletion of Trgal11 markedly impaired the induced expression of most (hemi)cellulase genes, but not that of the major β-glucosidase encoding genes. This differential involvement of TrGAL11 in the full induction of cellulase genes was reflected by the RNA polymerase II (Pol II) recruitment on their core promoters, indicating that TrGAL11 was required for the efficient transcriptional initiation of the majority of cellulase genes. In addition, we found that TrGAL11 recruitment to cellulase gene promoters largely occurred in an XYR1-dependent manner. Although xyr1 expression was significantly tuned down without TrGAL11, the binding of XYR1 to cellulase gene promoters did not entail TrGAL11. These results indicate that TrGAL11 represents a direct in vivo target of XYR1 and may play a critical role in contributing to Mediator and the following RNA Pol II recruitment to ensure the induced cellulase gene expression.

Biodegradation of Cellulose by New Recombinants of Rhizobium Harboring Cellulase Genes

Biodegradation of Cellulose by New Recombinants of Rhizobium Harboring Cellulase Genes

Penicillium oxalicum putative methyltransferase Mtr23B has similarities and differences with LaeA in regulating conidium development and glycoside hydrolase gene expression

Putative methyltranferase LaeA and LaeA-like proteins, which are conserved in many filamentous fungi, regulate the sporogenesis and biosynthesis of secondary metabolites. In this study, we reported the biological function of a LaeA-like methyltransferase, Penicillium oxalicum Mtr23B, which contains a methyltransf_23 domain and an S-adenosylmethionine binding domain, in controlling spore pigment formation and in the expression of secondary metabolic gene cluster and glycoside hydrolase genes. Additionally, we compared Mtr23B and LaeA, and determined their similarities and differences in terms of their roles in regulating the above biological processes. mtr23B had the highest transcriptional level among the 12 members of the methyltransf_23 family in P. oxalicum. The colony color of Δmtr23B (deletion of mtr23B) was lighter than that of ΔlaeA, although Δmtr23B produced ~ 19.2-fold more conidia than ΔlaeA. The transcriptional levels of abrA, abrB/yA, albA/wA, arpA, arpB, and aygA, which are involved in the dihydroxynaphtalene–melanin pathway, decreased in Δmtr23B. However, Mtr23B had a little effect on brush-like structures and conidium formation, and had a different function from LaeA. Mtr23B extensively regulated glycoside hydrolase gene expression. The absence of Mtr23B remarkably repressed prominent cellulase- and amylase-encoding genes in the whole culture period, while the effect of LaeA mainly occurred in the later phases of prolonged batch cultures. Similar to LaeA, Mtr23B was involved in the expression of 10 physically linked regions containing secondary metabolic gene clusters; the highest regulatory activities of Mtr23B and LaeA were observed in BrlA-dependent cascades. Although LaeA interacted with VeA, Mtr23B did not interact with VeA directly. We assumed that Mtr23B regulates cellulase and amylase gene transcription by interacting with the CCAAT-binding transcription factor HAP5 and chromatin remodeling complex.