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To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungi Penicillium oxalicum and Trichoderma reesei. Employing the 5S rRNA promoter from Aspergillus niger for guide RNA expression, the β-glucosidase gene bgl2 in P. oxalicum was deleted using a donor DNA carrying 40-bp homology arms or a donor containing no selectable marker gene. Using a markerless donor DNA as editing template, precise replacement of a small region was achieved in the creA gene. In T. reesei, the A. niger 5S rRNA promoter was less efficient than that in P. oxalicum when used for gene editing. Using a native 5S rRNA promoter, stop codons were introduced into the lae1 coding region using a markerless donor DNA with an editing efficiency of 36.67%. Efficient genome editing systems were developed in filamentous fungi P. oxalicum and T. reesei by using heterologous or native 5S rRNA promoters for guide RNA expression.

Fungi sense environmental signals and coordinate growth, development, and metabolism accordingly. Calcium-calmodulin-calcineurin signaling is a conserved cascade pathway in fungi. One of the most important downstream targets of this pathway is the transcription factor Crz1/CrzA, which plays an essential role in various cellular processes. The putative collaborators of Penicillium oxalicum CrzA (PoCrzA) were found, through tandem affinity purification followed by mass spectrometric analysis (TAP-MS). A total of 50 protein-protein interaction collaborators of PoCrzA were observed. Among them, some collaborators, such as the catalytic subunit of calcineurin (Cna1, calcineurin A), the regulatory catalytic subunit of calcineurin (Cnb1, calcineurin B), and a 14-3-3 protein Bmh1, which were previously reported in yeast, were identified. Some putative collaborators, including two karyopherins (exportin Los1 and importin Srp1), two kinases (Fus3 and Slt2p), and a general transcriptional corepressor (Cyc8), were also found. The CrzA deletion mutant ΔPocrzA exhibited slow hyphal growth, impaired conidiogenesis, and reduced extracellular cellulase synthesis. Phenotype and transcriptome analysis showed that PoCrzA regulated fungal development in a Flbs-BrlA-dependent manner and participated in cellulase synthesis by modulating cellulolytic gene expression. On the basis of the results of TAP-MS, transcriptome, and phenotypic analysis in P. oxalicum, our study was the first to draft the calcineurin-CrzA pathway in cellulolytic fungi.

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.

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

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.

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.

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.

Casein kinase CK2 is a ubiquitous and conserved phosphate transferase that is critical for the growth and development of eukaryotic cells. In Penicillium oxalicum, one catalytic subunit (CK2A) and two regulatory subunits (CK2B1 and CK2B2) of CK2 were annotated. In this study, CK2 regulatory subunit-defective mutants Δck2B1 and Δck2B2 were constructed to investigate the biological function of CK2 in P. oxalicum. The Δck2B1 strain exhibited minimal changes in morphogenesis and conidiation, whereas the Δck2B2 strain showed delayed conidial germination and drastically reduced conidiation compared with the parent strain. The defect in conidiation in Δck2B2 could be attributed to the reduced expression of transcription factor BrlA. Both Δck2B1 and Δck2B2 showed delayed autolysis in carbon-starvation medium compared with the parent strain. Cellulase and amylase production were decreased considerably in both mutants. The transcript abundances of the main extracellular glycoside hydrolase genes cel7A-2, bgl1, and amy15A, as well as those of three related transcriptional activators (i.e., ClrB, XlnR, and AmyR), were reduced or delayed in the mutants. Epistasis analysis suggested that CK2B1 and CK2B2 might function upstream of transcription factor CreA by inhibiting its repressing activity. In summary, CK2 plays important roles in development and extracellular enzyme production in P. oxalicum, with both unique and overlapping functions performed by the two regulatory subunits.

α-l-Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α-l-arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l-arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V , is constructed to improve the production of α-l-arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V -overexpressing strain can synthesize α-l-arabinofuranosidase in the absence of an inducer and shows a 54.1-fold increase in α-l-arabinofuranosidase production and a 7.4-fold increase in α-galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α-l-arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α- l-arabinofuranosidase production.

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.

The morphological development of fungi is a complex process and is often coupled with secondary metabolite production. In this study, we assessed the function of putative methyltransferase LaeA and transcription factor CreA in controlling asexual development and secondary metabolic gene cluster expression in Penicillium oxalicum. The deletion of laeA (ΔlaeA) impaired the conidiation in P. oxalicum, with a downregulated expression of brlA. Overexpression of P. oxalicum brlA in ΔlaeA could upregulate brlA and abaA remarkably, but could not rescue the conidiation defect; therefore, brlA and abaA expression were necessary but not sufficient for conidiation. Deletion of creA in ΔlaeA background (ΔlaeAΔcreA) blocked conidiation with a white fluffy phenotype. Nutrient-rich medium could not rescue developmental defects in ΔlaeAΔcreA mutant but could rescue defects in ΔlaeA. Expression of 10 genes, namely, albA/wA, abrB/yA, arpA, aygA, arpA-like, arpB, arpB-like, rodA, rodA-like, and rodB, for pigmentation and spore wall protein genes was silenced in ΔlaeAΔcreA, whereas only six of them were downregulated in ΔlaeA. Among the 28 secondary metabolism gene clusters in P. oxalicum, four secondary metabolism gene clusters were silenced in ΔlaeA and two were also silenced in ΔbrlA mutant. A total of 10 physically linked and coregulated genes were distributed over five chromosomes in ΔlaeA. Six of these genes were located in subtelomeric regions, thus demonstrating a positional bias for LaeA-regulated clusters toward subtelomeric regions. All of silenced clusters located in subtelomeric regions were derepressed in ΔlaeAΔcreA, hence showing that lack of CreA could remediate the repression of gene clusters in ΔlaeA background. Results show that both putative methyltransferase LaeA and transcription factor CreA are necessary for proper asexual development and controlling secondary metabolic gene cluster expression.

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.

Penicillium decumbens 114-2 is a fast-growing filamentous fungus which secretes a variety of lignocellulolytic enzymes. Its catabolite-repression-resistant mutant JU-A10 with high secretion capacity of cellulolytic enzymes has been used industrially for biomass hydrolysis. Transcription levels of 6 important lignocellulolytic enzymes genes (cel5A, cel6A, cel7A, cel7B, xyn10A, and xyn11A) from both strains were determined on different carbon sources (glucose, sorbose, lactose, cellobiose, cellulose, and cellulose-wheat bran), by means of a real-time quantitative polymerase chain reaction. For both strains, the 6 genes are coordinately regulated at transcriptional level. Glucose and cellobiose repressed whereas cellulose and cellulose-wheat bran induced expression of 6 genes in both strains. Expression levels of all genes tested in the mutant strain JU-A10 were substantially higher than those in wild-type strain 114-2 on all carbon sources. On glucose repression condition, the mutant JU-A10 appeared obviously derepressed. Lactose was first proved to have an inductive effect on lignocellulolytic enzyme genes expression at lower concentration in Penicillium spp.

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.