Abstract
Limited information on transcription factor (TF)-mediated regulation exists for most filamentous fungi, specifically for regulation of the production of plant-biomass-degrading enzymes (PBDEs). The filamentous fungus, Talaromyces pinophilus, can secrete integrative cellulolytic and amylolytic enzymes, suggesting a promising application in biotechnology. In the present study, the regulatory roles of a Zn2Cys6 protein, TP05746, were investigated in T. pinophilus through the use of biochemical, microbiological and omics techniques. Deletion of the gene TP05746 in T. pinophilus led to a 149.6% increase in soluble-starch-degrading enzyme (SSDE) production at day one of soluble starch induction but an approximately 30% decrease at days 2 to 4 compared with the parental strain ΔTpKu70. In contrast, the T. pinophilus mutant ΔTP05746 exhibited a 136.8–240.0% increase in raw-starch-degrading enzyme (RSDE) production, as well as a 90.3 to 519.1% increase in cellulase and xylanase production following induction by culturing on wheat bran plus Avicel, relative to that exhibited by ΔTpKu70. Additionally, the mutant ΔTP05746 exhibited accelerated mycelial growth at the early stage of cultivation and decreased conidiation. Transcriptomic profiling and real-time quantitative reverse transcription-PCR (RT-qPCR) analyses revealed that TP05746 dynamically regulated the expression of genes encoding major PBDEs and their regulatory genes, as well as fungal development-regulated genes. Furthermore, in vitro binding experiments confirmed that TP05746 bound to the promoter regions of the genes described above. These results will contribute to our understanding of the regulatory mechanism of PBDE genes and provide a promising target for genetic engineering for improved PBDE production in filamentous fungi.
Highlights
Soil filamentous fungi play crucial roles in terrestrial carbon cycling because they can decay organic matter, including plant biomass (Gougoulias et al, 2014) via secretion of plant-biomassdegrading enzymes (PBDEs), such as cellulase, hemicellulose, and amylase (Bornscheuer et al, 2014)
We found that TP05746 regulated the production of various PBDEs including soluble starch-degrading enzymes (SSDEs), raw starch-degrading enzymes (RSDEs), cellulase and xylanase of T. pinophilus, as well as mycelial growth and conidiation
TP05746 shared 87% and 43% of identities with putative C6 transcription factor PMAA_081800 in Talaromyces marneffei ATCC 18224 (XP_002147682.1) and AN8177.2 in Aspergillus nidulans FGSC A4 (XP 022399897.1), respectively, with the coverage of 100% and 83%, respectively, whereas most of the aligned proteins were selected based on the conserved Zn2Cys6 zinc finger domain at the C-terminus
Summary
Soil filamentous fungi play crucial roles in terrestrial carbon cycling because they can decay organic matter, including plant biomass (Gougoulias et al, 2014) via secretion of plant-biomassdegrading enzymes (PBDEs), such as cellulase, hemicellulose, and amylase (Bornscheuer et al, 2014). Genetic improvement of natural fungal producers such as enzymes needs to be further explored, with an aim of developing strains capable of high production of PBDEs. In the enzyme market, cellulases and amylases account for the majority of PBDEs. Cellulases are the complexes comprising cellobiohydrolases (EC 3.2.1.91; CBHs), endo-β-1,4-glucanases (EC 3.2.1.4; EGs), and β-1,4-glucosidases (EC 3.2.1.21; BGLs). Cellulases are the complexes comprising cellobiohydrolases (EC 3.2.1.91; CBHs), endo-β-1,4-glucanases (EC 3.2.1.4; EGs), and β-1,4-glucosidases (EC 3.2.1.21; BGLs)1 Synergistic action among these enzymes is required for cellulose hydrolysis into glucose. CBHs act on both ends of glucooligosaccharide chains to produce mainly cellobiose. Both soluble glucooligosaccharide and cellobiose are hydrolyzed by BGLs to yield glucose (Bornscheuer et al, 2014)
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