Novel Transcription Factor CXRD Regulates Cellulase and Xylanase Biosynthesis in Penicillium oxalicum under Solid-State Fermentation.

Penicillium oxalicum produces an integrated, extracellular cellulase and xylanase system, strictly regulated by several transcription factors. However, the understanding of the regulatory mechanism of cellulase and xylanase biosynthesis in P. oxalicum is limited, particularly under solid-state fermentation (SSF) conditions. In our study, deletion of a novel gene, cxrD (cellulolytic and xylanolytic regulator D), resulted in 49.3 to 2,230% enhanced production of cellulase and xylanase, except for 75.0% less xylanase at 2 days, compared with the P. oxalicum parental strain, when cultured on solid medium containing wheat bran plus rice straw for 2 to 4 days after transfer from glucose. In addition, the deletion of cxrD delayed conidiospore formation, leading to 45.1 to 81.8% reduced asexual spore production and altered mycelial accumulation to various extents. Comparative transcriptomics and real-time quantitative reverse transcription-PCR found that CXRD dynamically regulated the expression of major cellulase and xylanase genes and conidiation-regulatory gene brlA under SSF. In vitro electrophoretic mobility shift assays demonstrated that CXRD bound to the promoter regions of these genes. The core DNA sequence 5'-CYGTSW-3' was identified to be specifically bound by CXRD. These findings will contribute to understanding the molecular mechanism of negative regulation of fungal cellulase and xylanase biosynthesis under SSF. IMPORTANCE Application of plant cell wall-degrading enzymes (CWDEs) as catalysts in biorefining of lignocellulosic biomass into bioproducts and biofuels reduces both chemical waste production and carbon footprint. The filamentous fungus Penicillium oxalicum can secrete integrated CWDEs, with potential for industrial application. Solid-state fermentation (SSF), simulating the natural habitat of soil fungi, such as P. oxalicum, is used for CWDE production, but a limited understanding of CWDE biosynthesis hampers the improvement of CWDE yields through synthetic biology. Here, we identified a novel transcription factor CXRD, which negatively regulates the biosynthesis of cellulase and xylanase in P. oxalicum under SSF, providing a potential target for genetic engineering to improve CWDE production.

The Psychological Impact of COVID-19 on Chinese Individuals.

COVID-19 not only affects the physical health of Chinese people, but also their psychological health. This article mainly summarized the causes, clinical manifestations and preventive measures of COVID-19 impact on psychology of Chinese people, and presented two representative cases at the same time.

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.

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.

Transcription Factor NsdD Regulates the Expression of Genes Involved in Plant Biomass-Degrading Enzymes, Conidiation, and Pigment Biosynthesis in Penicillium oxalicum.

Soil fungi produce a wide range of chemical compounds and enzymes with potential for applications in medicine and biotechnology. Cellular processes in soil fungi are highly dependent on the regulation under environmentally induced stress, but most of the underlying mechanisms remain unclear. Previous work identified a key GATA-type transcription factor, Penicillium oxalicum NsdD (PoxNsdD; also called POX08415), that regulates the expression of cellulase and xylanase genes in P. oxalicum PoxNsdD shares 57 to 64% identity with the key activator NsdD, involved in asexual development in Aspergillus In the present study, the regulatory roles of PoxNsdD in P. oxalicum were further explored. Comparative transcriptomic profiling revealed that PoxNsdD regulates major genes involved in starch, cellulose, and hemicellulose degradation, as well as conidiation and pigment biosynthesis. Subsequent experiments confirmed that a ΔPoxNsdD strain lost 43.9 to 78.8% of starch-digesting enzyme activity when grown on soluble corn starch, and it produced 54.9 to 146.0% more conidia than the ΔPoxKu70 parental strain. During cultivation, ΔPoxNsdD cultures changed color, from pale orange to brick red, while the ΔPoxKu70 cultures remained bluish white. Real-time quantitative reverse transcription-PCR showed that PoxNsdD dynamically regulated the expression of a glucoamylase gene (POX01356/Amy15A), an α-amylase gene (POX09352/Amy13A), and a regulatory gene (POX03890/amyR), as well as a polyketide synthase gene (POX01430/alb1/wA) for yellow pigment biosynthesis and a conidiation-regulated gene (POX06534/brlA). Moreover, in vitro binding experiments showed that PoxNsdD bound the promoter regions of the above-described genes. This work provides novel insights into the regulatory mechanisms of fungal cellular processes and may assist in genetic engineering of Poxalicum for potential industrial and medical applications.IMPORTANCE 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 Poxalicum via genetic engineering.