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Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.

Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on cellulose as an inducer, suggesting that clrB is controlled post-transcriptionally. In this study, using a reporter gene system in yeast, we identified the C-terminal region of ClrB/CLR-2 as a transcriptional activation domain. Expression of clrBID, encoding a ClrB derivative in which the DNA-binding and transcriptional activation domains are fused together to remove the middle region, led to cellulase production in the absence of cellulose in P. oxalicum. Strikingly, the clrBID-expressing strain produced cellulase on carbon sources that normally repress cellulase expression, including glucose and glycerol. Results from deletion of the carbon catabolite repressor gene creA in the clrBID-expressing strain suggested that the effect of clrBID is independent of CreA's repressive function. A similar modification of clrB in Aspergillus niger resulted in the production of a mannanase in glucose medium. Taken together, these results indicate that ClrB suppression under noninducing conditions involves its middle region, suggesting a potential strategy to engineer fungal strains for improved cellulase production on commonly used carbon sources.

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.

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 β-1,4-glucan (e.g., carboxymethylcellulose), recombinant PoCel12A is a non-typical endo-(1-4)-β-glucanase; it preferentially hydrolyzes mix-linked β-glucan (barley β-glucan, β-1,3-1,4-glucan) and slightly hydrolyzes β-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 β-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.

Hyphal morphology is considered to have a close relationship with the production level of secreted proteins by filamentous fungi. In this study, the gul1 gene, which encodes a putative mRNA-binding protein, was disrupted in cellulase-producing fungus Trichoderma reesei. The hyphae of Δgul1 strain produced more lateral branches than the parent strain. Under the condition for cellulase production, disruption of gul1 resulted in smaller mycelial clumps and significantly lower viscosity of fermentation broth. In addition, cellulase production was improved by 22% relative to the parent strain. Transcriptome analysis revealed that a set of genes encoding cell wall remodeling enzymes as well as hydrophobins were differentially expressed in the Δgul1 strain. The results suggest that the regulatory role of gul1 in cell morphogenesis is likely conserved in filamentous fungi. To our knowledge, this is the first report on the engineering of gul1 in an industrially important fungus.

As the universal methyl donor for methylation reactions, S-adenosylmethionine (AdoMet) plays an indispensable role in most cellular metabolic processes. AdoMet is synthesized by AdoMet synthetase. We identified the only one AdoMet synthetase (PoSasA) in filamentous fungus Penicillium oxalicum. PoSasA was widely distributed in mycelium at different growth stages. The absence of PoSasA was lethal for P. oxalicum. The misregulation of the PoSasA encoding gene affected the synthesis of extracellular cellulolytic enzymes. The expression levels of cellobiohydrolase encoding gene cbh1/cel7A, β-1-4 endoglucanase eg1/cel7B, and xylanase encoding gene xyn10A were remarkably downregulated as a result of decreased PosasA gene expression. The production of extracellular cellulases and hemicellulases was also reduced. By contrast, the overexpression of PosasA improved the production of extracellular cellulases and hemicellulases. A total of 133 putative interacting proteins with PoSasA were identified using tandem affinity purification and mass spectrometry. The results of functional enrichment on these proteins showed that they were mainly related to ATP binding, magnesium ion binding, and ATP synthetase activity. Several methyltransferases were also observed among these proteins. These results were consistent with the intrinsic feature of AdoMet synthetase. This work reveals the indispensable role of PoSasA in various biological processes.

Spent ammonium sulphite liquor (SASL) is the main effluent from the ammonium sulphite pulping process, and contains amounts of lignocellulosic oligomers, monosaccharides and ammonium salts. The effect of continuous SASL-feeding on cellulase production by Penicillium oxalicum was studied. With a rate-varying feeding strategy, the maximal filter paper enzyme (FPase) activity reached 17.66U/mL at 144h, and the specific FPase activity increased from 1.74U/mg (without SASL) to 2.40U/mg. Accordingly, the glucan hydrolysis conversion of delignified corn cob residue by the enzymes from continuous SASL-feeding fermentation was significantly higher than that without SASL at equal protein loadings. Comparative proteomic analysis demonstrated that the proteins involved in lignocellulose degradation were specifically up-regulated in the crude cellulase with SASL-feeding. The obtained crude enzyme was efficient in the hydrolysis of pulping products, with a glucan conversion of 81.87% achieved after 72h saccharification of ammonium sulphite pulp.

Efficient deconstruction of lignocellulose is achieved by the synergistic action of various hydrolytic and oxidative enzymes. However, the aldonolactones generated by oxidative enzymes have inhibitory effects on some cellulolytic enzymes. In this work, D-glucono-1,5-lactone was shown to have a much stronger inhibitory effect than D-glucose and D-gluconate on β-glucosidase, a vital enzyme during cellulose degradation. AltA, a secreted enzyme from Penicillium oxalicum, was identified as an aldonolactonase which can catalyze the hydrolysis of D-glucono-1,5-lactone to D-gluconic acid. In the course of lignocellulose saccharification conducted by cellulases from P. oxalicum or Trichoderma reesei, supplementation of AltA was able to relieve the decrease of β-glucosidase activity obviously with a stimulation of glucose yield. This boosting effect disappeared when sodium azide and ethylenediaminetetraacetic acid (EDTA) were added to the saccharification system to inhibit the activities of oxidative enzymes. In summary, we describe the first heterologous expression of a fungal secreted aldonolactonase and its application as an efficient supplement of cellulolytic enzyme system for lignocellulose biodegradation.

The current high cost of lignocellulolytic enzymes is a major bottleneck in the economic bioconversion of lignocellulosic biomass to fuels and chemicals. Fungal lignocellulolytic enzyme systems are secreted at high levels, making them the most promising starting points for further development of highly efficient lignocellulolytic enzyme systems. In this paper, recent advances in improvement of fungal lignocellulolytic enzyme systems are reviewed, with an emphasis on the achievements made using genomic approaches. A general strategy for lignocellulolytic enzyme system development is proposed, including the improvement of the hydrolysis efficiencies and productivities of current enzyme systems. The applications of genomic, transcriptomic and proteomic analysis methods in examining the composition of native enzyme systems, discovery of novel enzymes and synergistic proteins from natural sources, and understanding of regulatory mechanisms for lignocellulolytic enzyme biosynthesis are summarized. By combining systems biology and synthetic biology tools, engineered fungal strains are expected to produce high levels of optimized lignocellulolytic enzyme systems.

Cellodextrin transporters (cellodextrin permeases) have been identified in fungi in recent years. However, the functions of these transporters in cellulose utilization and cellulase expression have not been well studied. In this study, three cellodextrin transporters, namely, CdtC, CdtD, and CdtG, in the cellulolytic fungus Penicillium oxalicum (formally was classified as P. decumbens) were identified, and their functions were analyzed. The deletion of a single cellodextrin transporter gene slightly decreased cellobiose consumption, but no observable effect on cellulase expression was observed, which was attributed to the overlapping activity of isozymes. Further simultaneous deletion of cdtC and cdtD resulted in significantly decreased cellobiose consumption and poor growth on cellulose. The extracellular activity and transcription level of cellulases in the mutant without cdtC and cdtD were significantly lower than those in the wild-type strain when grown on cellulose. This result provides direct evidence of the crucial function of cellodextrin transporters in the induction of cellulase expression by insoluble cellulose.

Many Penicillium species produce enzyme systems with good performances in lignocellulose degradation. In our laboratory, lignocellulolytic enzyme-producing Penicillium oxalicum (formerly classified as Penicillium decumbens) strains have been studied for more than 30 years. High cellulase-producing mutants have been obtained through random mutagenesis and genetic engineering, and the components in the enzyme systems have been elucidated using systems biology tools. The effects of different carbon sources on the production level of lignocellulolytic enzymes have been studied, and the related molecular mechanisms have been investigated. When compared with the widely used cellulase producer Trichoderma reesei, some unique features have been found in P. oxalicum, including higher β-glucosidase activity, higher numbers of lignocellulolytic enzyme gene, and different response of cellulase gene expression to some disaccharides. To boost the economic potential of the biorefineries using lignocellulosic biomass, P....

Heterotrimeric G proteins (G proteins) have been extensively investigated for their regulatory functions in morphogenesis and development in filamentous fungi. In addition, G proteins were also shown to be involved in the regulation of cellulase expression in some fungi. Here, we report the different regulatory effects of PGA3, a group III G protein α subunit, on the expressions of amylases and cellulases in Penicillium decumbens. Deletion of pga3 resulted in impaired amylase production and significantly decreased transcription of the major amylase gene amy15A. Supplementation of exogenous cAMP or its analog dibutyryl-cAMP restored amylase production in Δpga3 strain, suggesting an essential role of PGA3 in amylase synthesis via controlling cAMP level. On the other hand, the transcription of major cellulase gene cel7A-2 increased, nevertheless cellulase activity in the medium was not affected, in Δpga3. The above regulatory effects of PGA3 are carbon source-independent, and are achieved, at least, by cAMP-mediated regulation of the expression level of transcription factor AmyR. The functions of PGA3 revealed by gene deletion were partially supported by the analysis of the mutant carrying dominantly-activated PGA3. The results provided new insights into the understanding of the physiological functions of G protein-cAMP pathway in filamentous fungi.

Cellulolytic enzyme hydrolysis of lignocellulose biomass to release fermentable sugars is one of the key steps in biofuel refining. Gene expression of fungal cellulolytic enzymes is tightly controlled at the transcriptional level. Key transcription factors such as activator ClrB/CLR2 and XlnR/XYR1, as well as repressor CreA/CRE1 play crucial roles in this process. The putative protein methyltransferase LaeA/LAE1 has also been reported to regulate the gene expression of the cellulolytic enzyme. The formation and gene expression of the cellulolytic enzyme was compared among Penicillium oxalicum wild type (WT) and seven mutants, including ΔlaeA (deletion of laeA), OEclrB (clrB overexpression), OEclrBΔlaeA (clrB overexpression with deletion of laeA), OExlnR (xlnR overexpression), OExlnRΔlaeA (xlnR overexpression with deletion of laeA), ΔcreA (deletion of creA), and ΔcreAΔlaeA (double deletion of creA and laeA). Results revealed that LaeA extensively affected the expression of glycoside hydrolase genes. The expression of genes that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA. Cellulase synthesis of four mutants ΔlaeA, OEclrBΔlaeA, OExlnRΔlaeA, and ΔcreAΔlaeA was repressed remarkably compared with their parent strains WT, OEclrB, OExlnR, and ΔcreA, respectively. The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent, suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR. In contrast to LaeA positive roles in regulating prominent cellulase and hemicellulase, the extracellular β-xylosidase formation was negatively regulated by LaeA. The extracellular β-xylosidase activities improved over 5-fold in the OExlnRΔlaeA mutant compared with that of WT, and the expression of prominent β-xylosidase gene xyl3A was activated remarkably. The cumulative effect of LaeA and transcription factor XlnR has potential applications in the production of more β-xylosidase.

A β-glucosidase from Penicillium decumbens was purified and characterized. The enzyme presented as a single band of 120kDa on SDS-PAGE, showed optimal temperature of 65-70°C and optimal pH of 4.5-5.0. The β-glucosidase showed relatively higher affinity to pNPG and the highest affinity to salicin with the Km value as 0.0064 and 0.0188mM, respectively. The gene coding for it was obtained with an ORF of 2586bp coding for 861 amino acids belonging to glycoside hydrolases family 3. The purified enzyme could improve the saccharifying ability of cellulose when it was added to the cellulase systems of Trichoderma reesei QM 9414. The several properties of it, including its pH and temperature optima, the high affinity to substrates and high specific activity, make it has great potential to be utilized as supplementation in conversion of corncob residue and other lignocellulosic biomass into simple sugars.

Two major endoglucanase genes (cel7B and cel5A) were cloned from Penicillium decumbens 114-2 using the method of modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of cel7B gene in its chromosomal DNA. The expression level of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that two genes were coordinately expressed and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimal active at 60 C and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30-fold, 5-fold higher enzyme activity towards carboxymethyl cellulose, barely beta-glucan and PASC respectively in comparison with that of Cel5A. However, their activities toward pNPC and Avicel were minor difference. The result suggested that Cel7B is a strict endoglucanase, while Cel5A show processivity because of its relative higher ability to hydrolyze the crystal cellulose.

Cellulase production in filamentous fungi is largely regulated at the transcriptional level, and several transcription factors have been reported to be involved in this process. In this study, we identified ClrC, a novel transcription factor in cellulase production in Penicillium oxalicum. ClrC and its orthologs have a highly conserved basic leucine zipper (bZIP) DNA binding domain, and their biological functions have not been explored. Deletion of clrC resulted in pleiotropic effects, including altered growth, reduced conidiation and increased sensitivity to oxidative and cell wall stresses. In particular, the clrC deletion mutant ΔclrC showed 46.1% ± 8.1% and 58.0% ± 8.7% decreases in production of filter paper enzyme and xylanase activities in cellulose medium, respectively. In contrast, 57.4% ± 10.0% and 70.9% ± 19.4% increased production of filter paper enzyme, and xylanase was observed in the clrC overexpressing strain, respectively. The transcription levels of major cellulase genes, as well as two cellulase transcriptional activator genes, clrB and xlnR, were significantly downregulated in ΔclrC, but substantially upregulated in clrC overexpressing strains. Furthermore, we observed that the absence of ClrC reduced full induction of cellulase expression even in the clrB overexpressing strain. These results indicated that ClrC is a novel and efficient engineering target for improving cellulolytic enzyme production in filamentous fungi.

Filamentous fungi can initiate vegetative growth on complex plant polysaccharides in nature through secreting a large amount of lignocellulose-degrading enzymes. These fungi develop a large amount of asexual spores to disperse and survive under harsh conditions, such as carbon and nitrogen depletion. Numerous studies report the presence of a cross-talk between asexual development and extracellular enzyme production, especially at the regulation level. This study identified and characterized a C2H2-type transcription factor called PoFlbC, which is an Aspergillus FlbC ortholog, in cellulolytic fungus Penicillium oxalicum. Results showed that the native level of PoFlbC was crucial for the normal growth and asexual development of P. oxalicum. Importantly, deletion of the PoflbC gene substantially reduced cellulase and hemicellulase productions. Comparative transcriptome analysis by RNA sequencing revealed a global downregulation of genes encoding cellulases, hemicellulases, and other proteins with functions in lignocellulose degradation. A similar defect was also observed in the OEPoflbC strain, suggesting that the production of cellulolytic enzymes was maintained by native expression of the PoflbC. In this study, an essential activator for both fungal asexual development and cellulase production was established in P. 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 β-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., Author Summary Cellulolytic fungi have evolved into sophisticated lignocellulolytic systems to adapt to their natural habitat. This trait is important for filamentous fungi, which are the main source of cellulases utilized to degrade lignocellulose to fermentable sugars. Penicillium oxalicum, which produces lignocellulolytic enzymes with more diverse components than Trichoderma reesei, has the capacity to secrete large amounts of cellulases. Meanwhile, cellulase expression is regulated by a complex network involved in many transcription factors in this organism. To better understand how cellulase genes are systematically regulated in P. oxalicum, we employed molecular genetics to uncover the cellulolytic transcription factors on a genome-wide scale. We discovered the synergistic and tunable 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.

Penicillium decumbens has been used in the industrial production of lignocellulolytic enzymes in China for more than 15 years. Conidiation is essential for most industrial fungi because conidia are used as starters in the first step of fermentation. To investigate the mechanism of conidiation in P. decumbens, we generated mutants defective in two central regulators of conidiation, FluG and BrlA. Deletion of fluG resulted in neither “fluffy” phenotype nor alteration in conidiation, indicating possible different upstream mechanisms activating brlA between P. decumbens and Aspergillus nidulans. Deletion of brlA completely blocked conidiation. Further investigation of brlA expression in different media (nutrient-rich or nutrient-poor) and different culture states (liquid or solid) showed that brlA expression is required but not sufficient for conidiation. The brlA deletion strain exhibited altered hyphal morphology with more branches. Genome-wide expression profiling identified BrlA-dependent genes in P. decumbens, including genes previously reported to be involved in conidiation as well as previously reported chitin synthase genes and acid protease gene (pepB). The expression levels of seven secondary metabolism gene clusters (from a total of 28 clusters) were drastically regulated in the brlA deletion strain, including a downregulated cluster putatively involved in the biosynthesis of the mycotoxins roquefortine C and meleagrin. In addition, the expression levels of most cellulase genes were upregulated in the brlA deletion strain detected by real-time quantitative PCR. The brlA deletion strain also exhibited an 89.1 % increase in cellulase activity compared with the wild-type strain. The results showed that BrlA in P. decumbens not only has a key role in regulating conidiation, but it also regulates secondary metabolism extensively as well as the expression of cellulase genes.

Many Penicillium species could produce extracellular enzyme systems with good lignocellulose hydrolysis performance. However, these species and their enzyme systems are still poorly understood and explored due to the lacking of genetic information. Here, we present the genomic and secretomic analyses of Penicillium decumbens that has been used in industrial production of lignocellulolytic enzymes in China for more than fifteen years. Comparative genomics analysis with the phylogenetically most similar species Penicillium chrysogenum revealed that P. decumbens has evolved with more genes involved in plant cell wall degradation, but fewer genes in cellular metabolism and regulation. Compared with the widely used cellulase producer Trichoderma reesei, P. decumbens has a lignocellulolytic enzyme system with more diverse components, particularly for cellulose binding domain-containing proteins and hemicellulases. Further, proteomic analysis of secretomes revealed that P. decumbens produced significantly more lignocellulolytic enzymes in the medium with cellulose-wheat bran as the carbon source than with glucose. The results expand our knowledge on the genetic information of lignocellulolytic enzyme systems in Penicillium species, and will facilitate rational strain improvement for the production of highly efficient enzyme systems used in lignocellulose utilization from Penicillium species.

Cellulases from fungi typically contain one catalytic domain with or without a cellulose-binding domain. We characterized an endo-acting cellulase PdCel5C from industrial cellulase-producing fungus Penicillium decumbens with distinctive domain composition. In addition to a cellulose-binding domain and a catalytic domain, PdCel5C contains two immunoglobulin (Ig)-like domains near the C-terminal end. Truncated mutation experiment reveals that the two Ig-like domains are important for the hydrolytic activity of PdCel5C. Moreover, PdCel5C releases cello-oligosaccharides from cellulosic substrates, which is different from that of most characterized cellulases in the same glycoside hydrolase family 5. To the best of our knowledge, this is the first report on the characterization of an Ig-like domain-containing cellulase in fungi. The results expand our understanding on the diversity of cellulases in fungi, and suggest possible shared catalytic mechanisms between bacterial and fungal cellulases.

The gene encoding a glycoside hydrolase (GH) family 45 endoglucanase (Cel45A) was cloned from P. decumbens 114-2 and expressed in Pichia pastoris. To our knowledge, this is the first report of characterization of a GH family 45 protein from Penicillium species. The purified recombinant enzyme showed a higher activity on konjac glucomannan (KGM) than on sodium carboxymethyl cellulose (CMC-Na) or phosphoric acid swollen cellulose (PASC). The highest hydrolytic activity was detected at pH5.0 on KGM and pH 3.5 on CMC-Na, indicating the mode of action on the two substrates may be different for Cel45A. The optimum temperatures on the two substrates were both 60 degrees C and about 90% relative activities were retained at 70 degrees C. Products released from PASC and CMC-Na were mainly cellobiose, cellotriose and cellotetraose. The protein with higher glucomannanase activity might help the efficient degradation of lignocellulose by P. decumbens in the natural state.

Aims: Improvement of cellulase production of Penicillium decumbens by genome shuffling of an industrial catabolite-repression-resistant strain JU-A10 with its mutants. Methods and Results: After two rounds of genome shuffling, three fusants, GS2-15, GS2-21 and GS2-22, were obtained, showing 100%, 109% and 94% increase in FPase activity than JU-A10 respectively. The cellulase production of the fusants on various substrates, such as corn stover, wheat straw, bagasse and the corncob residue, was studied. The maximum productivities of GS2-15, GS2-21 and GS2-22 were 92·15, 102·63 and 92·35 FPU l−1 h−1 on the corncob residue at 44 h respectively, which were 117%, 142% and 118% higher than that of JU-A10 (42·44 FPU l−1 h−1, at 90 h). The improvements of the fusants were possibly because of their enhanced growth rates, earlier cellulase synthesis and higher secretion of extracellular proteins. Conclusions: The fusants obtained after genome shuffling could produce abundant cellulase much earlier, and they could be potential candidates for bioconversion process. Significance and Impact of the Study: This is the first report on the improvement of cellulase production in fungi by genome shuffling, and this is a good technique to improve other important phenotypes in fungi.

Cel5A (endoglucanase II) of Trichoderma reesei was expressed in Saccharomyces cerevisiae then purified. Two components (C1 and C2) of recombinant Cel5A with different glycosylation were obtained. Purified C1 had a larger molecular mass (57 kDa) than that of the native Cel5A produced by T. reesei (48 kDa) due to the different extents of asparagines-linked glycosylation. There was no significant difference in enzymatic activity between the C1 and the native Cel5A from T. reesei. C1 treated with Endoglycosidase H had a molecular mass of 54 kDa and retained about 88% of its original activity. Unpurified C2 was larger form of hyperglycosylation proteins. Its molecular mass was larger than 85 kDa till up to 200 kDa. It still retained activity regardless of its magnitude molecular mass. With increased glycosylation extent of the enzyme components (C2 >C1 >native Cel5A), the pH range of activity become wider, and thermal stability become higher.