Arginine methyltransferases PRMT2 and PRMT3 are essential for biosynthesis of plant-polysaccharide-degrading enzymes in Penicillium oxalicum.

Many filamentous fungi produce plant-polysaccharide-degrading enzymes (PPDE); however, the regulatory mechanism of this process is poorly understood. A Gal4-like transcription factor, CxrA, is essential for mycelial growth and PPDE production in Penicillium oxalicum. Its N-terminal region, CxrAΔ207-733 is required for the regulatory functions of whole CxrA, and contains a DNA-binding domain (CxrAΔ1-16&Δ59-733) and a methylated arginine (R) 94. Methylation of R94 is mediated by an arginine N-methyltransferase, PRMT2 and appears to induce dimerization of CxrAΔ1-60. Overexpression of prmt2 in P. oxalicum increases PPDE production by 41.4-95.1% during growth on Avicel, compared with the background strain Δku70;hphR+. Another arginine N-methyltransferase, PRMT3, appears to assist entry of CxrA into the nucleus, and interacts with CxrAΔ1-60 in vitro under Avicel induction. Deletion of prmt3 resulted in 67.0-149.7% enhanced PPDE production by P. oxalicum. These findings provide novel insights into the regulatory mechanism of fungal PPDE production.

Transcriptome-wide analysis of a superior xylan degrading isolate Penicillium oxalicum 5-18 revealed active lignocellulosic degrading genes.

Lignocellulose biomass raw materials have a high value in energy conversion. Recently, there has been growing interest in using microorganisms to secret a series of enzymes for converting low-cost biomass into high-value products such as biofuels. We previously isolated a strain of Penicillium oxalicun 5-18 with promising lignocellulose-degrading capability. However, the mechanisms of lignocellulosic degradation of this fungus on various substrates are still unclear. In this study, we performed transcriptome-wide profiling and comparative analysis of strain 5-18 cultivated in liquid media with glucose (Glu), xylan (Xyl) or wheat bran (WB) as sole carbon source. In comparison to Glu culture, the number of differentially expressed genes (DEGs) induced by WB and Xyl was 4134 and 1484, respectively, with 1176 and 868 genes upregulated. Identified DEGs were enriched in many of the same pathways in both comparison groups (WB vs. Glu and Xly vs. Glu). Specially, 118 and 82 CAZyme coding genes were highly upregulated in WB and Xyl cultures, respectively. Some specific pathways including (Hemi)cellulose metabolic processes were enriched in both comparison groups. The high upregulation of these genes also confirmed the ability of strain 5-18 to degrade lignocellulose. Co-expression and co-upregulated of genes encoding CE and AA CAZy families, as well as other (hemi)cellulase revealed a complex degradation strategy in this strain. Our findings provide new insights into critical genes, key pathways and enzyme arsenal involved in the biomass degradation of P. oxalicum 5-18.

Developing endophytic Penicillium oxalicum as a source of lignocellulolytic enzymes for enhanced hydrolysis of biorefinery relevant pretreated rice straw.

Endophytic fungi, as plant symbionts, produce an elaborate array of enzymes for efficient disintegration of lignocellulosic biomass into constituent monomeric sugars, making them novel source of lignocellulolytic CAZymes with immense potential in future biorefineries. The present study reports lignocellulolytic enzymes production potential of an endophytic halotolerant Penicillium oxalicum strain isolated from Citrus limon, under submerged and solid-state fermentation (SmF & SSF, respectively), in the presence and absence of salt (1 M NaCl). The comparative QTOF-LC/MS-based exoproteome analysis of the culture extracts unveiled differential expression of CAZymes, with the higher abundance of GH6 and GH7 family cellobiohydrolase in the presence of 1 M salt. The strain improvement program, employing cyclic mutagenesis and diploidization, was utilized to develop hyper-cellulase producing mutant strains of P. oxalicum. The enzyme production of the developed strain (POx-M35) was further enhanced through statistical optimization of the culture conditions utilizing glucose mix disaccharides (GMDs) as an inducer. This optimization process resulted in the lignocellulolytic cocktail that contained high titers (U/mL) of endoglucanase (EG) (146.16), cellobiohydrolase (CBHI) (6.99), ß-glucosidase (ß-G) (26.21), xylanase (336.05) and FPase (2.02 U/mL), which were 5.47-, 5.54-, 8.55-, 4.96-, and 4.39-fold higher when compared to the enzyme titers obtained in wild HP1, respectively. Furthermore, the lignocellulolytic cocktails designed by blending secretome produced by mutant POx-M35 with xylanases (GH10 and GH11) derived from Malbranchea cinnamomea resulted in efficient hydrolysis of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw. This study underscores the potential of bioprospecting novel fungus and developing an improved strain for optimized production and constitution of lignocellulolytic cocktails that can be an important determinant in advancing biomass conversion technologies.

Lignocellulose-degrading fungi newly isolated from central Morocco are potent biocatalysts for olive pomace valorization.

The investigation of lignocellulolytic catalysts is an important feature to face the challenges of lignocellulosic biomass valorization. In central Morocco, fungi were isolated from decaying wood, soil, olive crushing by-products and their compost. One hundred fifty-five isolates were submitted to a selective screening, which served to distinguish 83% of lignocellulolytic isolates. Then, a collection of 56 fungi was subjected to morphological and molecular identification with the ITS5 and ITS4 primers. This approach showed that 45% of the fungal population belonged to the genus Penicillium, followed by Aspergillus 14%, and Fusarium 11%. Alternaria, Trichoderma, Paecilomyces, Cladosporium, Trichocladium, Circinella, and Doratomyces genera are founded with a minority occurrence. Finally, validation of the enzymatic profile was done for 20 isolates, by testing their enzymatic performance on a liquid medium in the presence of cellulose, lignin, and olive pomace. The maximum protein production of 788 µg ml-1 was reached by an Alternaria strain, which produced also 10.6 IU ml-1 of endoglucanase. Thus, a ß-glucosidase activity of 5.1 IU ml-1 was obtained by a Penicillium strain isolated from decaying wood. Regarding ligninolytic activities, olive pomace was the most suitable substrate to detect these activities. Decaying wood strains presented the most remarkable results with 1.1 IU ml-1, 0.7 IU ml-1 et 0.3 IU ml-1 for laccase, LiP and MnP, respectively. The use of the selected fungi and olive pomace as local biomass are important factors for the development of green processes targeting the valorization of this by-product into high-value molecules.

Various effects of the expression of the xyloglucanase gene from Penicillium canescens in transgenic aspen under semi-natural conditions.

BACKGROUND: Recombinant carbohydrases genes are used to produce transgenic woody plants with improved phenotypic traits. However, cultivation of such plants in open field is challenging due to a number of problems. Therefore, additional research is needed to alleviate them. RESULTS: Results of successful cultivation of the transgenic aspens (Populus tremula) carrying the recombinant xyloglucanase gene (sp-Xeg) from Penicillium canescens in semi-natural conditions are reported in this paper for the first time. Change of carbohydrate composition of wood was observed in transgenic aspens carrying the sp-Xeg gene. The transformed transgenic line Xeg-2-1b demonstrated accelerated growth and increased content of cellulose in wood of trees growing in both greenhouse and outside in comparison with the control untransformed line Pt. The accelerated growth was observed also in the transgenic line Xeg-1-1c. Thicker cell-wall and longer xylem fiber were also observed in both these transgenic lines. Undescribed earlier considerable reduction in the wood decomposition rate of the transgenic aspen stems was also revealed for the transformed transgenic lines. The decomposition rate was approximately twice as lower for the transgenic line Xeg-2-3b in comparison with the control untransformed line Pt. CONCLUSION: A direct dependence of the phenotypic and biochemical traits on the expression of the recombinant gene sp-Xeg was demonstrated. The higher was the level of the sp-Xeg gene expression, the more pronounced were changes in the phenotypic and biochemical traits. All lines showed phenotypic changes in the leave traits. Our results showed that the plants carrying the recombinant sp-Xeg gene do not demonstrate a decrease in growth parameters in semi-natural conditions. In some transgenic lines, a change in the carbohydrate composition of the wood, an increase in the cell wall thickness, and a decrease in the rate of decomposition of wood were observed.

The Lipomyces starkeyi gene Ls120451 encodes a cellobiose transporter that enables cellobiose fermentation in Saccharomyces cerevisiae.

Processed lignocellulosic biomass is a source of mixed sugars that can be used for microbial fermentation into fuels or higher value products, like chemicals. Previously, the yeast Saccharomyces cerevisiae was engineered to utilize its cellodextrins through the heterologous expression of sugar transporters together with an intracellular expressed ß-glucosidase. In this study, we screened a selection of eight (putative) cellodextrin transporters from different yeast and fungal hosts in order to extend the catalogue of available cellobiose transporters for cellobiose fermentation in S. cerevisiae. We confirmed that several in silico predicted cellodextrin transporters from Aspergillus niger were capable of transporting cellobiose with low affinity. In addition, we found a novel cellobiose transporter from the yeast Lipomyces starkeyi, encoded by the gene Ls120451. This transporter allowed efficient growth on cellobiose, while it also grew on glucose and lactose, but not cellotriose nor cellotetraose. We characterized the transporter more in-depth together with the transporter CdtG from Penicillium oxalicum. CdtG showed to be slightly more efficient in cellobiose consumption than Ls120451 at concentrations below 1.0 g/L. Ls120451 was more efficient in cellobiose consumption at higher concentrations and strains expressing this transporter grew slightly slower, but produced up to 30% more ethanol than CdtG.

The Role of Cross-Pathway Control Regulator CpcA in the Growth and Extracellular Enzyme Production of Penicillium oxalicum.

CpcA is a conserved transcriptional activator for the cross-pathway control of amino acid biosynthetic genes in filamentous fungi. Previous studies of this regulator mainly revealed its function under amino acid starvation condition, where amino acid biosynthetic inhibitors were added in the culture. In this study, the biological function of CpcA in Penicillium oxalicum was investigated under different cultivation conditions. Disruption of cpcA led to decreased cell growth either in the presence or absence of histidine biosynthetic inhibitor, and the phenotype could be rescued by the addition of exogenous amino acid sources. In addition, CpcA was required for the rapid production of cellulase when cells were cultured on cellulose. Transcript abundance measurement showed that a set of amino acid biosynthetic genes as well as two major cellulase genes were significantly down-regulated in cpcA deletion mutant relative to wild type. Taken together, the results revealed the biological role of CpcA in supporting normal growth and extracellular enzyme production of P. oxalicum under amino acid non-starvation condition.

Transcription Factor Atf1 Regulates Expression of Cellulase and Xylanase Genes during Solid-State Fermentation of Ascomycetes.

Transcriptional regulation of cellulolytic and xylolytic genes in ascomycete fungi is controlled by specific carbon sources in different external environments. Here, comparative transcriptomic analyses of Penicillium oxalicum grown on wheat bran (WB), WB plus rice straw (WR), or WB plus Avicel (WA) as the sole carbon source under solid-state fermentation (SSF) revealed that most of the differentially expressed genes (DEGs) were involved in metabolism, specifically, carbohydrate metabolism. Of the DEGs, the basic core carbohydrate-active enzyme-encoding genes which responded to the plant biomass resources were identified in P. oxalicum, and their transcriptional levels changed to various extents depending on the different carbon sources. Moreover, this study found that three deletion mutants of genes encoding putative transcription factors showed significant alterations in filter paper cellulase production compared with that of a parental P. oxalicum strain with a deletion of Ku70 (ΔPoxKu70 strain) when grown on WR under SSF. Importantly, the ΔPoxAtf1 mutant (with a deletion of P. oxalicumAtf1, also called POX03016) displayed 46.1 to 183.2% more cellulase and xylanase production than a ΔPoxKu70 mutant after 2 days of growth on WR. RNA sequencing and quantitative reverse transcription-PCR revealed that PoxAtf1 dynamically regulated the expression of major cellulase and xylanase genes under SSF. PoxAtf1 bound to the promoter regions of the key cellulase and xylanase genes in vitro This study provides novel insights into the regulatory mechanism of fungal cellulase and xylanase gene expression under SSF.IMPORTANCE The transition to a more environmentally friendly economy encourages studies involving the high-value-added utilization of lignocellulosic biomass. Solid-state fermentation (SSF), that simulates the natural habitat of soil microorganisms, is used for a variety of applications such as biomass biorefinery. Prior to the current study, our understanding of genome-wide gene expression and of the regulation of gene expression of lignocellulose-degrading enzymes in ascomycete fungi during SSF was limited. Here, we employed RNA sequencing and genetic analyses to investigate transcriptomes of Penicillium oxalicum strain EU2101 cultured on medium containing different carbon sources and to identify and characterize transcription factors for regulating the expression of cellulase and xylanase genes during SSF. The results generated will provide novel insights into genetic engineering of filamentous fungi to further increase enzyme production.

Three glycoside hydrolase family 12 enzymes display diversity in substrate specificities and synergistic action between each other.

PoCel12A, PoCel12B, and PoCel12C are genes that encode glycoside hydrolase family 12 (GH12) enzymes in Penicillium oxalicum. PoCel12A and PoCel12B are typical GH12 enzymes that belong to fungal subfamilies 12-1 and 12-2, respectively. PoCel12C contains a low-complexity region (LCR) domain, which is not found in PoCel12A or PoCel12B and independent of fungal subfamily 12-1 or 12-2. Recombinant enzymes (named rCel12A, rCel12B and rCel12C) demonstrate existing diversity in the substrate specificities. Although most members in GH family 12 are typical endoglucanases and preferentially hydrolyze ß-1,4-glucan (e.g., carboxymethylcellulose), recombinant PoCel12A is a non-typical endo-(1-4)-ß-glucanase; it preferentially hydrolyzes mix-linked ß-glucan (barley ß-glucan, ß-1,3-1,4-glucan) and slightly hydrolyzes ß-1,4-glucan (carboxymethylcellulose). Recombinant PoCel12B possesses a significantly high activity against xyloglucan. A specific activity of rCel12B toward xyloglucan (239 µmol/min/mg) is the second-highest value known. Recombinant PoCel12C shows low activity toward ß-glucan, carboxymethylcellulose, or xyloglucan. All three enzymes can degrade phosphoric acid-swollen cellulose (PASC). However, the hydrolysis products toward PASC by enzymes are different: the main hydrolysis products are cellotriose, cellotetraose, and cellobiose for rCel12A, rCel12B, and rCel12C, correspondingly. A synergistic action toward PASC among rCel12A and rCel12B is observed, thereby suggesting a potential application for preparing enzyme cocktails used in lignocellulose hydrolysis.

Introduction of heterologous transcription factors and their target genes into Penicillium oxalicum leads to increased lignocellulolytic enzyme production.

Genetic engineering of transcription factors is an efficient strategy to improve lignocellulolytic enzyme production in fungi. In this study, the xylanase transcriptional regulators of Trichoderma reesei (Xyr1) and Neurospora crassa (XLR-1), as well as their constitutively active mutants (Xyr1A824V and XLR-1A828V), were heterologously expressed in Penicillium oxalicum. The two heterologous regulators were identified to be able to activate lignocellulolytic enzyme gene expression in P. oxalicum. Particularly, expression of T. reesei Xyr1 resulted in a higher cellulase production level compared with the expression of native xylanase transcriptional regulator XlnR using the same promoter. Xyr1A824V and XLR-1A828V were found to be able to confer P. oxalicum more enhanced lignocellulolytic abilities than wild-type regulators Xyr1 and XLR-1. Furthermore, introduction of regulatory modules containing Xyr1A824V/XLR-1A828V and their target cellulase genes resulted in greater increases in cellulase production than alone expression of transcriptional regulators. Through the cumulative introduction of three regulatory modules containing regulator mutants and their corresponding target cellulase genes from P. oxalicum, T. reesei, and N. crassa, a 2.8-fold increase in cellulase production was achieved in P. oxalicum.

Transcription Factor NsdD Regulates the Expression of Genes Involved in Plant Biomass-Degrading Enzymes, Conidiation, and Pigment Biosynthesis in Penicillium oxalicum.

Soil fungi produce a wide range of chemical compounds and enzymes with potential for applications in medicine and biotechnology. Cellular processes in soil fungi are highly dependent on the regulation under environmentally induced stress, but most of the underlying mechanisms remain unclear. Previous work identified a key GATA-type transcription factor, Penicillium oxalicum NsdD (PoxNsdD; also called POX08415), that regulates the expression of cellulase and xylanase genes in P. oxalicum PoxNsdD shares 57 to 64% identity with the key activator NsdD, involved in asexual development in Aspergillus In the present study, the regulatory roles of PoxNsdD in P. oxalicum were further explored. Comparative transcriptomic profiling revealed that PoxNsdD regulates major genes involved in starch, cellulose, and hemicellulose degradation, as well as conidiation and pigment biosynthesis. Subsequent experiments confirmed that a ΔPoxNsdD strain lost 43.9 to 78.8% of starch-digesting enzyme activity when grown on soluble corn starch, and it produced 54.9 to 146.0% more conidia than the ΔPoxKu70 parental strain. During cultivation, ΔPoxNsdD cultures changed color, from pale orange to brick red, while the ΔPoxKu70 cultures remained bluish white. Real-time quantitative reverse transcription-PCR showed that PoxNsdD dynamically regulated the expression of a glucoamylase gene (POX01356/Amy15A), an α-amylase gene (POX09352/Amy13A), and a regulatory gene (POX03890/amyR), as well as a polyketide synthase gene (POX01430/alb1/wA) for yellow pigment biosynthesis and a conidiation-regulated gene (POX06534/brlA). Moreover, in vitro binding experiments showed that PoxNsdD bound the promoter regions of the above-described genes. This work provides novel insights into the regulatory mechanisms of fungal cellular processes and may assist in genetic engineering of Poxalicum for potential industrial and medical applications.IMPORTANCE Most filamentous fungi produce a vast number of extracellular enzymes that are used commercially for biorefineries of plant biomass to produce biofuels and value-added chemicals, which might promote the transition to a more environmentally friendly economy. The expression of these extracellular enzyme genes is tightly controlled at the transcriptional level, which limits their yields. Hitherto our understanding of the regulation of expression of plant biomass-degrading enzyme genes in filamentous fungi has been rather limited. In the present study, regulatory roles of a key regulator, PoxNsdD, were further explored in the soil fungus Penicillium oxalicum, contributing to the understanding of gene regulation in filamentous fungi and revealing the biotechnological potential of Poxalicum via genetic engineering.

Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum.

Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular ß-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a "seesaw model" in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.

N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity.

Cellobiohydrolase I from Penicillium verruculosum (PvCel7A) has four potential N-glycosylation sites at its catalytic module: Asn45, Asn194, Asn388, and Asn430. In order to investigate how the N-glycosylation influences the activity and other properties of the enzyme, the wild type (wt) PvCel7A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens PCA10 (niaD-) strain, a fungal host for production of heterologous proteins. The rPvCel7A-wt and N45A, N194A, N388A mutants were successfully expressed and purified for characterization, whereas the expression of N430A mutant was not achieved. The MALDI-TOF mass spectrometry fingerprinting of peptides, obtained as a result of digestion of rPvCel7A forms with specific proteases, showed that the N-linked glycans represent variable high-mannose oligosaccharides and the products of their sequential enzymatic trimming, according to the formula (Man)0-13 (GlcNAc)2 , or a single GlcNAc residue. Mutations had no notable effect on pH-optimum of PvCel7A activity and enzyme thermostability. However, the mutations influenced both the enzyme adsorption ability on Avicel and its activity against natural and synthetic substrates. In particular, the N45A mutation led to a significant increase in the rate of Avicel and milled aspen wood hydrolysis, while the substrate digestion rates in the case of N194A and N388A mutants were notably lower relative to rPvCel7A-wt. These data, together with data of 3D structural modeling of the PvCel7A catalytic module, indicate that the N-linked glycans are an important part of the processive catalytic machinery of PvCel7A.

Proteomics Insights into the Biomass Hydrolysis Potentials of a Hypercellulolytic Fungus Penicillium funiculosum.

The quest for cheaper and better enzymes needed for the efficient hydrolysis of lignocellulosic biomass has placed filamentous fungi in the limelight for bioprospecting research. In our search for efficient biomass degraders, we identified a strain of Penicillium funiculosum whose secretome demonstrates high saccharification capabilities. Our probe into the secretome of the fungus through qualitative and label-free quantitative mass spectrometry based proteomics studies revealed a high abundance of inducible CAZymes and several nonhydrolytic accessory proteins. The preferential association of these proteins and the attending differential biomass hydrolysis gives an insight into their interactions and clues about possible roles of novel hydrolytic and nonhydrolytic proteins in the synergistic deconstruction of lignocellulosic biomass. Our study thus provides the first comprehensive insight into the repertoire of proteins present in a high-performing secretome of a hypercellulolytic Penicillium funiculosum, their relative abundance in the secretome, and the interaction dynamics of the various protein groups in the secretome. The gleanings from the stoichiometry of these interactions hold a prospect as templates in the design of cost-effective synthetic cocktails for the optimal hydrolysis of biomass.

Extraction protease expressed by Penicillium fellutanum from the Brazilian savanna using poly(ethylene glycol)/sodium polyacrylate/NaCl aqueous two-phase system.

The partitioning of protease expressed by Penicillium fellutanum from the Brazilian savanna in a novel inexpensive and stable aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied in this work using factorial design. The ATPS is formed by mixing both polymers with a salt (NaCl) and fermented broth of P. fellutanum. The effects of molar mass (2,000, 4,000, and 6,000 g ⋅ mol(-1)) and concentration (6, 8, and 10 wt%) of PEG and that of NaPA concentration (6, 8, and 10 wt%) on protease partitioning (K) at 25 °C were studied. A two-level factorial design (2(3)) was implemented. The effect of Na2 SO4 concentration (5, 10, and 15 wt%) on the reextraction of the enzyme was also analyzed. The partition coefficient K ranged from 77.51 to 1.21, indicating the versatility of the method. The reextraction was achieved with the addition of 5% Na2 SO4 , allowing the partitioning of the protease to the upper phase, whereas total proteins were directed to the bottom phase. The results of partitioning using the PEG/NaPA/NaCl system and that of the subsequent reextraction with Na2 SO4 suggest that this method can be used to purify proteases from fermented broth of P. fellutanum.

Newly isolated Penicillium oxalicum A592-4B secretes enzymes that degrade milled rice straw with high efficiency.

An enzyme producing micro-organism, which can directly saccharify rice straw that has only been crushed without undergoing the current acid or alkaline pretreatment, was found. From the homology with the ITS, 28S rDNA sequence, the strain named A592-4B was identified as Penicillium oxalicum. Activities of the A592-4B enzymes and commercial enzyme preparations were compared by Novozymes Cellic CTec2 and Genencore GC220. In the present experimental condition, activity of A592-4B enzymes was 2.6 times higher than that of CTec2 for degrading milled rice straw. Furthermore, even when a quarter amount of A592-4B enzyme was applied to the rice straw, the conversion rate was still higher than that by CTec2. By utilizing A592-4B enzymes, improved lignocellulose degradation yields can be achieved without pre-treatment of the substrates; thus, contributing to cost reduction as well as reducing environmental burden.

Efficient production and evaluation of lignocellulolytic enzymes using a constitutive protein expression system in Penicillium oxalicum.

Native lignocellulolytic enzyme systems secreted by filamentous fungi can be further optimized by protein engineering or supplementation of exogenous enzyme components. We developed a protein production and evaluation system in cellulase-producing fungus Penicillium oxalicum. First, by deleting the major amylase gene amy15A, a strain Δ15A producing few extracellular proteins on starch was constructed. Then, three lignocellulolytic enzymes (BGL4, Xyn10B, and Cel12A) with originally low expression levels were successfully expressed with selected constitutive promoters in strain Δ15A. BGL4 and Cel12A overexpression resulted in increased specific filter paper activity (FPA), while the overexpression of Xyn10B improved volumetric FPA but not specific FPA. By switching the culture medium, this platform is convenient to produce originally low-expressed lignocellulolytic enzymes in relatively high purities on starch and to evaluate the effect of their supplementation on the performance of a complex cellulase system on cellulose.

Molecular cloning and expression of thermostable glucose-tolerant ß-glucosidase of Penicillium funiculosum NCL1 in Pichia pastoris and its characterization.

A partial peptide sequence of ß-glucosidase isoform (Bgl4) of Penicillium funiculosum NCL1 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cDNA (bgl4) encoding Bgl4 protein was cloned from P. funiculosum NCL1 RNA by consensus RT-PCR. The bgl4 gene encoded 857 amino acids that contained catalytic domains specific for glycoside hydrolase family 3. The cDNA was over-expressed in Pichia pastoris KM71H and the recombinant protein (rBgl4) was purified with the specific activity of 1,354.3 U/mg. The rBgl4 was a glycoprotein with the molecular weight of ~130 kDa and showed optimal activity at pH 5.0 and 60 °C. The enzyme was thermo-tolerant up to 60 °C for 60 min. The rBgl4 was highly active on aryl substrates with ß-glucosidic, ß-xylosidic linkages and moderately active on cellobiose and salicin. It showed remarkably high substrate conversion rate of 3,332 and 2,083 µmol/min/mg with the substrates p-nitrophenyl ß-glucoside and cellobiose respectively. In addition, the rBgl4 showed tolerance to glucose concentration up to 400 mM. It exhibited twofold increase in glucose yield when supplemented with crude cellulase of Trichoderma reesei Rut-C30 in cellulose hydrolysis. These results suggested that rBgl4 is a thermo- and glucose-tolerant ß-glucosidase and is a potential supplement for commercial cellulase in cellulose hydrolysis and thereby assures profitability in bioethanol production.

[Use of Endoglucanase IV from Trichoderma reesei to Enhance the Hydrolytic Activity of a Cellulase Complex from the Fungus Penicillium verruculosum].

The effect of polysaccharide monooxygenase (endoglucanase IV) from the fungus Trichoderma reesei on the hydrolysis of polysaccharide substrates by cellulases secreted by the fungus Penicillium verruculosum has been investigated. Supplementation of the enzyme complex from P. verruculosum by endoglucanase IV from T. reesei has been shown to elevate the efficiency of cellulose hydrolysis by 45%.

The role of intracellular ß-glucosidase in cellulolytic response induction in filamentous fungus Penicillium verruculosum.

In this study, CRISPR/Cas9 genome editing was used to knockout the bgl2 gene encoding intracellular ß-glucosidase filamentous fungus Penicillium verruculosum. This resulted in a dramatic reduction of secretion of cellulolytic enzymes. The study of P. verruculosum Δbgl2 found that the transcription of the cbh1 gene, which encodes cellobiohydrolase 1, was impaired when induced by cellobiose and cellotriose. However, the transcription of the cbh1 gene remains at level of the host strain when induced by gentiobiose. This implies that gentiobiose is the true inducer of the cellulolytic response in P. verruculosum, in contrast to Neurospora crassa where cellobiose acts as an inducer.