Title: Harnessing Microbial Potential for Biomass Degradation

Abstract: Lignocellulose, also known as plant biomass, is the most abundant renewable carbon source on Earth. It is a promising feedstock for sustainable biomanufacturing, yet its recalcitrant structure limits its industrial applications. Understanding how microorganisms naturally deconstruct plant biomass can provide strategies for improving lignocellulose valorization. In this study, we investigated anaerobic gut fungi (AGF) and termite gut microbiomes, two highly specialized systems for biomass breakdown, to better understand their enzymatic capabilities and ecological adaptations.

AGF possess an extensive repertoire of carbohydrate-active enzyme (CAZymes) genes encoded in their genomes, but incomplete genome annotation and limited genetic tools have hindered the biochemical characterization of these proteins. To address this, we screened 176 AGF CAZyme genes expressed in Escherichia coli and combined heterologous expression with protein sequence and structural analyses for functional predictions. This approach led to the identification and characterization of a novel xylanase with promising industrial properties, including favorable kinetics, substrate specificity, and stability. We further investigated the interaction between AGF and lignin-rich substrates and observed that lignin and its degradation products suppress fungal growth by limiting nutrient accessibility and inducing cytotoxic effects. To overcome this barrier, adaptive laboratory evolution was applied to engineer Neocallimastix californiae strain for improved growth on woody biomass, such as eucalyptus.

To complement these fungal studies, we examined biomass deconstruction in the termite gut microbiome. Using nanostructure-initiator mass spectrometry (NIMS) and metagenomic sequencing, we profiled the hydrolytic activities and enzymatic potential across termite gut compartments. Distinct gut regions exhibit specialized functions in cellulose, hemicellulose, and lignin degradation, revealing a spatially organized biomass-deconstruction system. Overall, this thesis integrates high-throughput screening, heterologous expression systems, and multi-omics analyses to uncover the enzymatic mechanisms underlying lignocellulose biodegradation.