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How an essential Zn2Cys6 transcription factor PoxCxrA regulates cellulase gene expression in ascomycete fungi?

ABSTRACT: Background:Soil ascomycete fungi produce plant-biomass-degrading enzymes to facilitate nutrient and energy uptake in response to exogenous stress. This is controlled by a complex signal network, but the regulatory mechanisms are poorly understood. An essential Zn2Cys6 transcription factor (TF) PoxCxrA was identified to be required for cellulase and xylanase production in Penicillium oxalicum. The genome-wide regulon and DNA binding sequences of PoxCxrA were further identified through RNA-Sequencing, DNase I footprinting experiments and in vitro electrophoretic mobility shift assays. Moreover, a minimal DNA-binding domain in PoxCxrA was recognised. Results:A PoxCxrA regulon of 1970 members was identified in P. oxalicum, and it was displayed that PoxCxrA regulated the expression of genes encoding major plant cell wall-degrading enzymes, as well as important cellodextrin and/or glucose transporters. Interestingly, PoxCxrA positively regulated the expression of a known important TF PoxClrB. DNase I footprinting experiments and in vitro electrophoretic mobility shift assays further revealed that PoxCxrA directly bound the promoter regions of PoxClrB and a cellobiohydrolase gene cbh1 (POX05587/Cel7A-2) at different nucleic acid sequences. Remarkably, PoxCxrA autoregulated its own PoxCxrA gene expression. Additionally, a minimal 42-amino-acid PoxCxrA DNA-binding domain was identified. Conclusion:PoxCxrA could directly regulate the expression of cellulase genes and the regulatory gene PoxClrB via binding their promoters at different nucleic acid sequences. This work expands the diversity of DNA-binding motifs known to be recognised by Zn2Cys6 TFs, and demonstrates novel regulatory mechanisms of fungal cellulase gene expression.

SUBMITTER: Liao LS

PROVIDER: S-EPMC6498484 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:BackgroundPerfect and low cost of fungal amylolytic and cellulolytic enzymes are prerequisite for the industrialization of plant biomass biorefinergy to biofuels. Genetic engineering of fungal strains based on regulatory network of transcriptional factors (TFs) and their targets is an efficient strategy to achieve the above described aim. Talaromyces pinophilus produces integrative amylolytic and cellulolytic enzymes; however, the regulatory mechanism associated with the expression of amylase and cellulase genes in T. pinophilus remains unclear. In this study, we screened for and identified novel TFs regulating amylase and/or cellulase gene expression in T. pinophilus 1-95 through comparative transcriptomic and genetic analyses.ResultsComparative analysis of the transcriptomes from T. pinophilus 1-95 grown on media in the presence and absence of glucose or soluble starch as the sole carbon source screened 33 candidate TF-encoding genes that regulate amylase gene expression. Thirty of the 33 genes were successfully knocked out in the parental strain T. pinophilus ∆TpKu70, with seven of the deletion mutants firstly displaying significant changes in amylase production as compared with the parental strain. Among these, ∆TpRfx1 (TpRfx1: Talaromyces pinophilus Rfx1) showed the most significant decrease (81.5%) in amylase production, as well as a 57.7% reduction in filter paper cellulase production. Real-time quantitative reverse transcription PCR showed that TpRfx1 dynamically regulated the expression of major amylase and cellulase genes during cell growth, and in vitro electrophoretic mobility shift assay revealed that TpRfx1 bound the promoter regions of genes encoding α-amylase (TP04014/Amy13A), glucoamylase (TP09267/Amy15A), cellobiohydrolase (TP09412/cbh1), β-glucosidase (TP05820/bgl1), and endo-β-1,4-glucanase (TP08514/eg1). TpRfx1 protein containing a regulatory factor X (RFX) DNA-binding domain belongs to RFX family.ConclusionWe identified a novel RFX protein TpRFX1 that directly regulates the expression of amylase and cellulase genes in T. pinophilus, which provides new insights into the regulatory mechanism of fungal amylase and cellulase gene expression.

Project description: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.

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How an essential Zn2Cys6 transcription factor PoxCxrA regulates cellulase gene expression in ascomycete fungi?

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