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.

Characterization of novel roles of a HMG-box protein PoxHmbB in biomass-degrading enzyme production by Penicillium oxalicum.

High-mobility group (HMG)-box proteins are involved in chromatin organization in eukaryotes, especially in sex determination and regulation of mitochondrial DNA compaction. Although a novel HMG-box protein, PoxHmbB, had been initially identified to be required for filter paper cellulase activity by Penicillium oxalicum, the biological roles of HMG-box proteins in biomass-degrading enzyme production have not been systematically explored. The P. oxalicum mutant ∆PoxHmbB lost 34.7-86.5% of cellulase (endoglucanase, p-nitrophenyl-ß-cellobiosidase, and p-nitrophenyl-ß-glucopyranosidase) activities and 60.3% of xylanase activity following Avicel induction, whereas it exhibited about onefold increase in amylase activity following soluble corn starch induction. Furthermore, ∆PoxHmbB presented delayed conidiation and hyphae growth. Transcriptomic profiling and real-time quantitative reverse transcription-PCR revealed that PoxHmbB regulated the expression of major genes encoding plant biomass-degrading enzymes such as PoxCel7A-2, PoxCel5B, PoxBgl3A, PoxXyn11B, and PoxGA15A, as well as those involved in conidiation such as PoxBrlA. In vitro binding experiments further confirmed that PoxHmbB directly binds to the promoter regions of these major genes. These results further indicate the diversity of the biological functions of HMG-box proteins and provide a novel and promising engineering target for improving plant biomass-degrading enzyme production in filamentous fungi.

Cellulase with high ß-glucosidase activity by Penicillium oxalicum under solid state fermentation and its use in hydrolysis of cassava residue.

In this study, we investigated cellulase production by Penicillium oxalicum EU2106 under solid-state fermentation (SSF) and its hydrolysis efficiency toward NaOH-H2O2-pretreated cassava residue (NHCR) produced after bioethanol fermentation. Optimization of SSF cultivation conditions for P. oxalicum EU2106 using a Box-behnken design-based response-surface methodology resulted in maximal cellulase activity of 34.0 ± 2.8 filter-paper units/g dry substrate, exhibiting a ~ twofold increase relative to activities obtained under non-optimized conditions. Furthermore, SSF-derived cellulase converted 94.3 ± 1.5% of NHCR cellulose into glucose within 96 h. Interestingly, P. oxalicum EU2106 produced higher ß-glucosidase activity under SSF conditions than that under submerged-state fermentation conditions, resulting in the elimination of cellobiose inhibition during the early stages of NHCR cellulose hydrolysis. Overall, this work provided an alternative for a potential cellulase source and a preferred option for cassava residue biotechnological application.

Differential transcriptomic profiling of filamentous fungus during solid-state and submerged fermentation and identification of an essential regulatory gene PoxMBF1 that directly regulated cellulase and xylanase gene expression.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.