Comparative genomic, transcriptomic and secretomic profiling of Penicillium oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106, and identification of two novel regulatory genes of cellulase and xylanase gene expression

Abstract

BackgroundThe filamentous fungus Penicillium oxalicum is a potential alternative to Trichoderma reesei for industrial production of a complete cellulolytic enzyme system for a bio-refinery. Comparative omics approaches can support rational genetic engineering and/or breeding of filamentous fungi with improved cellulase production capacity. In this study, comparative genomic, transcriptomic and secretomic profiling of P. oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106 were employed to screen for novel regulators of cellulase and xylanase gene expression.ResultsThe 30.62 Mb P. oxalicum HP7-1 genome was sequenced, and 9834 protein-coding genes were annotated. Re-sequencing of the mutant EU2106 genome identified 274 single nucleotide variations and 12 insertion/deletions. Comparative genomic, transcriptomic and secretomic profiling of HP7-1 and EU2106 revealed four candidate regulators of cellulase and xylanase gene expression. Deletion of these candidate genes and measurement of the enzymatic activity of the resultant mutants confirmed the identity of three regulatory genes. POX02484 and POX08522, encoding a putative Zn(II)2Cys6 DNA-binding domain and forkhead protein, respectively, were found to be novel, while PoxClrB is an ortholog of ClrB, a key transcriptional regulator of cellulolytic enzyme gene expression in filamentous fungi. ΔPOX02484 and ΔPOX08522 mutants exhibited significantly reduced β-glucosidase activity, increased carboxymethylcellulose cellulase and xylanase activities, and altered transcription level of cellulase and xylanase genes compared with the parent strain ΔPoxKu70, with Avicel as the sole carbon source.ConclusionsTwo novel genes, POX02484 and POX08522, were found and characterized to regulate the expression of cellulase and xylanase genes in P. oxalicum. These findings are important for engineering filamentous fungi to improve cellulase and xylanase production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0616-9) contains supplementary material, which is available to authorized users.

Transcriptome analysis of fungicide-responsive gene expression profiles in two Penicillium italicum strains with different response to the sterol demethylation inhibitor (DMI) fungicide prochloraz

“…Construction of mfs1-knockout mutant (Δmfs1) from Pi-R The mutant Δmfs1 was derived from its parental strain Pi-R by introduction of mfs1-knockout cassette into the DMI-resistant P. italicum protoplasts that were prepared according to the method of Zhao et al [104]. Doublejoint PCR was performed to construct the desirable knockout cassette, also according to the method of Zhao et al [104].…”

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

“…All T. pinophilus strains were maintained on potato-dextrose agar (PDA) plates at 4 • C or stored in 25% glycerol at -80 • C. Cultivation of Penicillium oxalicum strains was consistent with the T. pinophillus strains. Mutant PoxKu70 (#3.15650, CGMCC) was derived from the wild-type HP7-1 via deleting gene PoxKu70 (Zhao et al, 2016).…”

“…These fragments were amplified using PCR with specific primer pairs ( Supplementary Table S1). The constructed overexpression cassette was introduced into the fresh protoplasts of the parental strain PoxKu70 based on the methods previously described (Zhao et al, 2016). The overexpressed transformants were isolated and confirmed via corresponding antibiotics G418 and hygromycin, and PCR with specific confirmation primers ( Supplementary Table S1).…”

“…RT-qPCR, real-time quantitative reverse transcription PCR; WA, wheat bran plus Avicel. over-expressed in filamentous fungus P. oxalicum parental strain PoxKu70 (Supplementary Figure S4) derived from the wild-type HP7-1 via deleting gene PoxKu70 (Zhao et al, 2016), and then its production of PBDEs was measured when cultivated on Avicel for 1-3 days. The results indicated the obtained overexpressed strain OXTP05746_POX lost 54.4-84.6% of cellulase (FPase, pNPCase and CMCase) and xylanase production, 15.6-49.9% of SSDE and RSDE production, while increased 131.1-275.1% of pNPGase production, compared with the parental strain PoxKu70 (p < 0.01, Student's t test; Figures 10A-G).…”

“…Transcriptional expression of genes encoding PBDEs is strictly controlled by specific TFs in filamentous fungi. Since XlnR was first identified to be involved in regulating the expression of genes encoding enzymes involved in degrading plant cell walls in Aspergillus (van Peij et al, 1998), several TFs have been found to be expressed in the presence of recalcitrant carbon sources, such as the key activators CLR-2 (Coradetti et al, 2012;Zhao et al, 2016), AmyR , and PoxCxrA (Yan et al, 2017). Among them, CLR-2 and PoxCxrA encode Gal4-like Zn2Cys6 proteins, and play essential roles in the regulation of fungal cellulase gene expression in the presence of cellulose.…”