Diversity of fungal sugar transporters (STs) revealed through integrative omics approaches

The conversion of plant biomass into biofuels, biochemicals, and other bioproducts holds great potential, with fungi being highly effective in breaking down complex carbohydrates into soluble sugars. In fungal plant biomass conversion (FPBC), sugar transporters (STs) are essential for absorbing sugars from enzymatic breakdown of polysaccharide for fungal growth. This thesis integrates bioinformatics methods and omics data to analyze the gene content and diversity of STs across fungal species, providing insights to optimize biomass conversion processes. The first chapter introduces the study, outlining the structure, mechanism, and distribution of STs in fungi, then summarizing the biochemical traits and functions of various STs. It highlights the diversity in ST function, affinity, and specificity. Chapter 2 presents a comparative analysis of genome diversity and transcriptome dynamics among four filamentous fungi: Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens and Trichoderma reesei. Using a domain search with PFAM PF00083, 90, 83, 117, and 52 STs were predicted in each species, respectively, for a total of 342 STs classified into ten clades. The predicted ST specificity was inferred from the known STs in the same phylogenetic clades. Comparative transcriptomics on different sugars revealed complex expression patterns. Moreover, STs often co-expressed with carbohydrate-active enzymes (CAZymes), sugar catabolic enzymes (SCEs), and some transcription factors (TFs). Co-expression analysis confirmed known functions of D-galacturonic acid and D-xylose STs and suggested roles for newly predicted L-rhamnose STs. Chapter 3 investigates the evolutionary genomic origin of fungal STs by phylogenetically analyzing STs across species in Ascomycota, Basidiomycota, Mucoromycota, and Zoopagomycota. Our results showed the total number of STs differed significantly among the studied fungi. Each ST clade had its own unique evolutionary pattern, with expansion of maltose/sucrose STs (clade B) in Ascomycetes. I also detected the co-expansion of maltose/sucrose STs with intracellular α-1,4-glucosidases and invertases in certain fungal lineages. The presence of STs and glycoside hydrolases related to maltose/sucrose utilization partially determines fungal growth on these disaccharides. RNA-sequencing technology has been applied to the studies of fungal genetic and molecular mechanisms, such as gene expression analyses of STs. Using stably expressed reference genes is a useful approach to normalizing gene expression from heterologous RNA-seq datasets. However, some traditional reference genes can be unstable across varying conditions. Chapter 4 focuses on identifying stable genes in A. niger, using three normalization methods. From extensive transcriptome datasets, 98 optimal reference genes were identified, including both known and newly identified genes. Further evaluation found that many of the newly identified reference genes showed greater expression stability compared to the traditionally and commonly used reference genes. Chapter 5 concludes the thesis by discussing the genomic and transcriptomic diversity of fungal STs, as well as their co-expression and co-evolution relationships with other sugar utilization genes. I also introduced other STs families that are involved in fungal sugar import and export. Given the current limitations in our understanding of STs, more computational approaches and integrating diverse datasets are recommended in fungal ST research.