Identification of Novel Transcription Factors Involved in Regulating the Production of Lignocellulolytic Enzymes in Myceliophthora thermophila
Fossil fuels such as crude oil, coal, and natural gas are non-renewable energy resources that are increasingly depleted due to heavy exploitation driven by global population and industrialisation. Their combustion also contributes to global warming and environmental pollution, posing significant threats to human health and the environment. Therefore, the exploitation of renewable and environmentally friendly alternatives to fossil fuel is urgently required. Lignocellulosic biomass, the most abundant renewable resource on Earth, can be converted into biochemicals such as glycolic acid, xylitol, xylonic acid, and xylo-oligosaccharides, and into fermentable sugars for the production of biofuels, making it a potential substitute for fossil fuels. The thermophilic filamentous fungus Myceliophthora thermophila secretes large amounts of thermostable lignocellulolytic enzymes, including cellulases and xylanases, which enable efficient degradation of lignocellulose. However, the native production levels of these enzymes in this fungus are generally lower than those of mesophilic fungi such as Trichoderma and Aspergillus species. Lignocellulose degradation in filamentous fungi is regulated by a complex transcriptional network involving various transcriptional activators and repressors. To date, only a limited number of transcription factors have been identified in M. thermophila that regulate cellulase and xylanase biosynthesis, and the underlying mechanisms remain unclear, impeding rational strain engineering. The aim of this thesis was to identify and characterise transcription factors involved in cellulase and xylanase regulation in M. thermophila and to elucidate their molecular mechanisms. Comparative transcriptomic analysis of M. thermophila grown in Avicel and glucose was firstly performed to identify candidate transcription factors. To facilitate functional studies, a CRISPR/Cas9-based gene editing tool and a constitutive promoter (Ppdc)-driven overexpression system were developed. Using these approaches, three transcription factors, MtFKH1, MtHAC-1, and MtCLR-2, were shown to regulate cellulase and/or xylanase production. Through molecular genetic analyses and comparative transcriptomic profiling, the regulatory roles of these transcription factors were elucidated. MtFKH1 is the first forkhead transcription factor identified in M. thermophila, which negatively regulates the expression of major cellulase and xylanase genes and plays an important role in fungal sporulation. The EMSAs indicated that MtFKH1 directly binds to the promoter regions of bgl1, cbh1, and xyn1. The bZIP transcription factor MtHAC-1 exerts dual regulatory effects on cellulase and xylanase production. It plays a positive role during the early phase of growth on Avicel (48 h) but acts as a repressor in the middle and later stages (72 and 96 h). The EMSAs suggested that MtHAC-1 can bind to the promoter regions of both bgl1, cbh1, egl2, xyn1, and xyr1. Thus, it regulates cellulase and xylanase gene expression through two mechanisms: direct binding, and modulation of the crucial xylanolytic activator Mtxyr1. MtHAC-1 was also found to be associated with the expression of genes of the 26S proteasome system. The Zn2Cys6-type transcription factor MtCLR-2 acts as a pivotal activator of cellulase production. The EMSAs and RT-qPCR analyses indicated that MtCLR-2 regulates cellulase gene expression by directly binding to the promoter regions of major cellulase genes egl2 and bgl1. Moreover, MtCLR-2 is involved in the transcriptional regulation of approximately half of the ribosomal protein genes. This thesis contributes to a deeper understanding of the transcriptional regulation associated with lignocellulose degradation in M. thermophila. The findings expand the repertoire of known transcription factors involved in cellulase and xylanase expression and provide novel targets for engineering of M. thermophila to elevate lignocellulolytic enzyme production.
Myceliophthora thermophila, cellulase, xylanase, gene expression, transcription factor, MtFKH1, MtHAC-1, MtCLR-2, CRISPR/Cas9, comparative transcriptomic analysis
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