The transcription factor TpRfx1 is an essential regulator of amylase and cellulase gene expression in Talaromyces pinophilus

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1276-8) contains supplementary material, which is available to authorized users.

Diversity of Cellulase-Producing Filamentous Fungi From Tibet and Transcriptomic Analysis of a Superior Cellulase Producer Trichoderma harzianum LZ117

“…Filamentous fungi, particularly, Penicillium sp., Trichoderma sp., Aspergillus sp., are of great interest due to their great capacity to produce plant cell wall-degrading enzymes (PCWDEs) used to convert lignocellulosic biomass to fermentable sugars (Passos et al, 2018). In addition, the Talaromyces genus, early regarded as a teleomorph state of Penicillium, forms a monophyletic clade distinct from the other Penicillium subgenera (Yilmaz et al, 2014), and Talaromyces strains have also been studied as cellulase producers (Liao et al, 2018;Méndez-Líter et al, 2019). Thus, 23 fungal strains that belongs to the genera of Penicillium, Trichoderma, Aspergillus and Talaromyces were selected for evaluation of PCWDEs production.…”

Isolation and Characterization of Cellulolytic Marine Bacteria for Litopenaeus Vannamei Aquaculture Using Sugarcane Bagasse as Carbon Source

“…The cost of cellulolytic enzymes is a major factor in the hydrolysis of lignocellulosic materials to fermentable sugars [47]. Currently, various cellulolytic microorganisms were from fungi, such as Aspergillus [48,49], Penicillium [50,51], Trichoderma [30,[52][53][54], and Talaromyces [55][56][57], which required long production cycles. Compared with fungi, bacteria not only have a higher growth rate, but also produce cellulase in a short time.…”

Identification of a Novel Transcription Factor TP05746 Involved in Regulating the Production of Plant-Biomass-Degrading Enzymes in Talaromyces pinophilus

“…Talaromyces pinophilus wild-type strain 1-95 (#2645, China General Microbiological Culture Collection, Beijing, China; CGMCC) was isolated from a dry ploughed field in China (Xian et al, 2015). Deletion mutants TpKu70 and TP05746 were constructed by knocking out the TpKu70 gene in strain 1-95 and the TP05746 gene in TpKu70, respectively Liao et al, 2018). The TpKu70 has no apparent difference in vegetative growth and enzyme production when compared with wild-type strain 1-95 .…”

“…Asexual spores (conidia) were collected from fungal cells cultured on PDA plates at 28 • C for 6 days, resuspended in aqueous 0.2% (v/v) Tween-80, and adjusted to a concentration of 1 × 10 8 spores mL −1 . For mycelial growth for DNA extraction, the fungus was cultured on liquid complete medium (LCM; Liao et al, 2018). Culture conditions for enzyme activity analysis, RNA sequencing, and real-time quantitative reverse transcription-PCR (RT-qPCR) analysis were as described previously .…”

“…For mycelial growth and observation by light microscopy, standard liquid medium (SLM; Liao et al, 2018) containing 1% (w/v) D-glucose, 1% (w/v) SCS, 2% (w/v) wheat bran plus Avicel [WA; wheat bran: Avicel = 1: 1; (w/w); Sigma-Aldrich, St.] or 2% (w/v) Avicel and/or 0.2% (w/v) 2-deoxy-glucose (2-DG) was inoculated with T. pinophilus spores and cultured at 28 • C for 12 to 120 h. For phenotypic analyses, 1% (w/v) D-glucose, 1% (w/v) SCS or 2% (w/v) WA were added to solid low-salt minimal medium (LsMM) plates, with PDA being used as a positive control. For measurement of enzymatic activity, P. oxalicum strains were cultivated in Avicel medium according to the descried by Yan et al (2017).…”