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.

BackgroundIn cellulolytic fungi, induction and repression mechanisms synchronously regulate the synthesis of cellulolytic enzymes for accurate responses to carbon sources in the environment. Many proteins, particularly transcription regulatory factors involved in these processes, were identified and genetically engineered in Penicillium oxalicum and other cellulolytic fungi. Despite such great efforts, its effect of modifying a single target to improve the production of cellulase is highly limited.ResultsIn this study, we developed a systematic strategy for the genetic engineering of P. oxalicum to enhance cellulase yields, by enhancing induction (by blocking intracellular inducer hydrolysis and increasing the activator level) and relieving the repression. We obtained a trigenic recombinant strain named ‘RE-10’ by deleting bgl2 and creA, along with over-expressing the gene clrB. The cellulolytic ability of RE-10 was significantly improved; the filter paper activity and extracellular protein concentration increased by up to over 20- and 10-fold, respectively, higher than those of the wild-type (WT) strain 114-2 both on pure cellulose and complex wheat bran media. Most strikingly, the cellulolytic ability of RE-10 was comparable with that of the industrial P. oxalicum strain JU-A10-T obtained by random mutagenesis. Comparative proteomics analysis provided further insights into the differential secretomes between RE-10 and WT strains. In particular, the enzymes and accessory proteins involved in lignocellulose degradation were elevated specifically and dramatically in the recombinant, thereby confirming the importance of them in biomass deconstruction and implying a possible co-regulatory mechanism.ConclusionsWe established a novel route to substantially improve cellulolytic enzyme production up to the industrial level in P. oxalicum by combinational manipulation of three key genes to amplify the induction along with derepression, representing a milestone in strain engineering of filamentous fungi. Given the conservation in the mode of cellulose expression regulation among filamentous fungi, this strategy could be compatible with other cellulase-producing fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0253-8) contains supplementary material, which is available to authorized users.

Epigenetic agents, histone deacetylase inhibitor (SAHA) and DNA methyltransferase inhibitor (5-Aza), were added to Czapek-Dox medium to trigger the chemical diversity of marine-derived fungus Aspergillus versicolor XS-20090066. By HPLC and 1 H NMR analysis, the diversity of fungal secondary metabolites was significantly increased compared with the control. With the aid of MS/MS-based molecular networking, two new nucleoside derivatives, kipukasins K (1) and L (2) were obtained. Meanwhile, the yields of four known nucleoside derivatives were significantly enhanced. In addition, one new bisabolane sesquiterpene, aspergillusene E (7), along with ten known derivatives were also isolated. The structures were elucidated by comprehensive spectroscopic methods of NMR and HRESIMS analysis. Compounds 1 and 7 displayed antibacterial activities against Staphylococcus epidermidis and Staphylococcus aureus with the MIC values of 8-16 µg/mL. Our study revealed that the fungus A. versicolor XS-20090066 has been effectively induced by chemical epigenetic manipulation with a combination of SAHA and 5-Aza to produce new metabolites.

BackgroundThe mining of high-performance enzyme systems is necessary to develop industrial lignocellulose bioconversion. Large amounts of cellulases and hemicellulases can be produced by Penicillium oxalicum. Hence, the enzyme system of this hypercellulolytic fungus should be elucidated to help design optimum enzyme systems for effective biomass hydrolysis.ResultsThe cellulolytic and xylanolytic activities of an SP enzyme system prepared from P. oxalicum JU-A10 were comparatively analyzed. Results indicated that the fungus possesses a complete cellulolytic-xylanolytic enzyme system. The cellobiohydrolase- and xylanase-specific activities of this system were higher than those of two other enzyme systems, i.e., ST from Trichoderma reesei SN1 and another commercial preparation Celluclast 1.5L. Delignified corncob residue (DCCR) could be hydrolyzed by SP to a greater extent than corncob residue (CCR). Beta-glucosidase (BG) supplemented in SP increased the ability of the system to hydrolyze DCCR and CCR, and resulted in a 64 % decrease in enzyme dosage with the same glucose yield. The behaviors of the enzyme components in the hydrolysis of CCR were further investigated by monitoring individual enzyme dynamics. The total protein concentrations and cellobiohydrolase (CBH), endoglucanase (EG), and filter paper activities in the supernatants significantly decreased during saccharification. These findings were more evident in SP than in the other enzyme systems. The comparative proteomic analysis of the enzyme systems revealed that both SP and ST were rich in carbohydrate-degrading enzymes and multiple non-hydrolytic proteins. A larger number of carbohydrate-binding modules 1 (CBM1) were also identified in SP than in ST. This difference might be linked to the greater adsorption to substrates and lower hydrolysis efficiency of SP enzymes than ST during lignocellulose saccharification, because CBM1 not only targets enzymes to insoluble cellulose but also leads to non-productive adsorption to lignin.ConclusionsPenicillium oxalicum can be applied to the biorefinery of lignocellulosic biomass. Its ability to degrade lignocellulosic substrates could be further improved by modifying its enzyme system on the basis of enzyme activity measurement and proteomic analysis. The proposed strategy may also be applied to other lignocellulolytic enzyme systems to enhance their hydrolytic performances rationally.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0477-2) contains supplementary material, which is available to authorized users.