Abstract
BackgroundThe filamentous fungus Trichoderma reesei is widely utilized in industry for cellulase production, but its xylanase activity must be improved to enhance the accessibility of lignocellulose to cellulases. Several transcription factors play important roles in this progress; however, nearly all the reported transcription factors typically target both cellulase and hemi-cellulase genes. Specific xylanase transcription factor would be useful to regulate xylanase activity directly.ResultsIn this study, a novel zinc binuclear cluster transcription factor (jgi|Trire2|123881) was found to repress xylanase activity, but not cellulase activity, and was designated as SxlR (specialized xylanase regulator). Further investigations using real-time PCR and an electrophoretic mobility shift assay demonstrated that SxlR might bind the promoters of GH11 xylanase genes (xyn1, xyn2, and xyn5), but not those of GH10 (xyn3) and GH30 (xyn4) xylanase genes, and thus regulate their transcription and expression directly. We also identified the binding consensus sequence of SxlR as 5′- CATCSGSWCWMSA-3′. The deletion of SxlR in T. reesei RUT-C30 to generate the mutant ∆sxlr strain resulted in higher xylanase activity as well as higher hydrolytic efficiency on pretreated rice straw.ConclusionsOur study characterizes a novel specific transcriptional repressor of GH11 xylanase genes, which adds to our understanding of the regulatory system for the synthesis and secretion of cellulase and hemi-cellulase in T. reesei. The deletion of SxlR may also help to improve the hydrolytic efficiency of T. reesei for lignocellulose degradation by increasing the xylanase-to-cellulase ratio.
Highlights
The filamentous fungus Trichoderma reesei is widely utilized in industry for cellulase production, but its xylanase activity must be improved to enhance the accessibility of lignocellulose to cellulases
In this study, using bioinformatics analysis and gene deletion with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system [19], we identified a gene encoding a protein designated as specialized xylanase regulator (SxlR)
According to the results of real-time PCR and electrophoretic mobility shift assay (EMSA) analyses, we demonstrated that SxlR plays a critical role in the inhibition of the expression of xylanases belonging to the glycoside hydrolase 11 family (GH11) in T. reesei through binding to the promoter regions of target genes directly
Summary
The filamentous fungus Trichoderma reesei is widely utilized in industry for cellulase production, but its xylanase activity must be improved to enhance the accessibility of lignocellulose to cellulases. Lignocellulosic biomass, consisting mostly of cellulose, hemi-cellulose, and lignin, is the most abundant and renewable energy source on earth [1]. Degradation of lignocellulosic biomass and continuation of the carbon cycle in nature is maintained mainly by microbial action, including different fungal species, such as Trichoderma, Aspergillus, and Penicillium. The biomass-degrading enzymes produced by these organisms have applications in various fields of industry including food, fodder, Cellulosic biofuel production continues to increase world-wide every year with a consequent rapidly increasing requirement for cellulase production from T. reesei. The production and optimization of enzyme formulations for lignocellulose degradation are still the major barriers to its extensive application. Due to the complex constitution and structure of native lignocellulose, the enzyme
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