The transition to a more environmentally friendly economy has prompted studies of modern biorefineries, including the utilization of low-value lignocellulose. The major challenge facing the widespread application of biorefineries is the high cost of enzymes that can efficiently hydrolyze recalcitrant cellulose to sugars. Penicillium oxalicum produces large amounts of plant-cell-wall-degrading enzymes, but their production is tightly controlled by complex regulatory networks, resulting in low yields of the native enzymes. Regulatory genes have been the targets of genetic engineering to improve enzyme production in microorganisms. In this study, we used transcriptomic profiling and genetic analyses to screen for and identify novel key regulators of cellulase and xylanase gene expression in P. oxalicum. A comparative analysis of the transcriptomes of P. oxalicum HP7-1 on different carbon sources, including glucose, wheat bran, and wheat bran plus Avicel, identified 40 candidate genes regulating the expression of cellulolytic enzyme genes. Deletion mutants of 31 candidate genes were constructed in P. oxalicum ∆PoxKu70 and 11 resultant mutants showed significant changes in their filter-paper cellulase production compared with the parental strain ∆PoxKu70. Among these 11 mutants, ΔPoxCxrA, ΔPoxCxrB, and ΔPoxNsdD showed the most significant reduction in the enzyme production (96.8, 75.9, and 58.5%, respectively). Ten of these 11 genes are here reported to be involved in cellulase production for the first time. Further tests revealed that ΔPoxCxrA, ΔPoxCxrB, and ΔPoxNsdD displayed significantly reduced xylanase production, whereas ΔPoxCxrA produced negligible xylanase. Interestingly, ΔPoxCxrB and ΔPoxNsdD showed significantly increased β-glucosidase production. Real-time quantitative reverse transcription–PCR and an electrophoretic mobility shift assay (EMSA) showed that PoxCxrA, PoxCxrB, and PoxNsdD regulate the expression of one another, but the mode of regulation changes dynamically during the growth of fungal cells in the presence of cellulose. EMSA showed that PoxCxrA, PoxCxrB, and PoxNsdD directly bind the putative promoters of major cellulase and xylanase genes. We have detected and identified three key new regulatory genes, PoxCxrA, PoxCxrB, and PoxNsdD, that directly and indirectly regulate the expression of cellulase and xylanase genes in P. oxalicum. This study provides novel insights into the regulatory mechanisms of fungal cellulase and xylanase gene expression.

Background: Application of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. RSDEs are generally produced by filamentous fungi, such as Penicillium oxalicum, but with very low yields, which seriously hampers industrialization of raw starch processing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production. Results: A total of 3532 P. oxalicum colonies were generated after multiple rounds of mutagenesis, by atmospheric and room-temperature plasma (ARTP) and/or ethyl methanesulfonate (EMS). Of these, one mutant A2-13 had the highest RSDE activity of 162.7 U/mL, using raw cassava flour as substrate, a yield increase of 61.1%, compared with that of the starting strain, OXPoxGA15A. RSDE activity of A2-13 further increased to 191.0 U/mL, through optimization of culture conditions. Increased expression of major amylase genes, including the raw starch-degrading glucoamylase gene, PoxGA15A, and its regulatory gene, PoxAmyR, as well as several single-nucleotide polymorphisms in the A2-13 genome, were detected by real-time reverse transcription quantitative PCR and genomic re-sequencing, respectively. In addition, crude RSDEs produced by A2-13, combined with commercial α-amylase, could efficiently digest raw corn flour and cassava flour at 40 °C. Conclusions: Overall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperproducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with commercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing.

Solid-state fermentation (SSF) mimics the natural decay environment of soil fungi and can be employed to investigate the production of plant biomass-degrading enzymes. However, knowledge on the transcriptional regulation of fungal genes during SSF remains limited. Herein, transcriptional profiling was performed on the filamentous fungus Penicillium oxalicum strain HP7-1 cultivated in medium containing wheat bran plus rice straw (WR) under SSF (WR_SSF) and submerged fermentation (WR_SmF; control) conditions. Novel key transcription factors (TFs) regulating fungal cellulase and xylanase gene expression during SSF were identified via comparative transcriptomic and genetic analyses. Expression of major cellulase genes was higher under WR_SSF condition than that under WR_SmF, but the expression of genes involved in the citric acid cycle was repressed under WR_SSF condition. Fifty-six candidate regulatory genes for cellulase production were screened out from transcriptomic profiling of P. oxalicum HP7-1 for knockout experiments in the parental strain ∆PoxKu70, resulting in 43 deletion mutants including 18 constructed in the previous studies. Enzyme activity assays revealed 14 novel regulatory genes involved in cellulase production in P. oxalicum during SSF. Remarkably, deletion of the essential regulatory gene PoxMBF1, encoding Multiprotein Bridging Factor 1, resulted in doubled cellulase and xylanase production at 2 days after induction during both SSF and SmF. PoxMBF1 dynamically and differentially regulated transcription of a subset of cellulase and xylanase genes during SSF and SmF, and conferred stress resistance. Importantly, PoxMBF1 bound specifically to the putative promoters of major cellulase and xylanase genes in vitro. We revealed differential transcriptional regulation of P. oxalicum during SSF and SmF, and identified PoxMBF1, a novel TF that directly regulates cellulase and xylanase gene expression during SSF and SmF. These findings expand our understanding of regulatory mechanisms of cellulase and xylanase gene expression during fungal fermentation.

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 P . oxalicum via genetic engineering.