Date of Award
Doctor of Philosophy (PhD)
Lignocellulosic biomass is a potential renewable feedstock for the microbial production of fuels and chemicals. For microbial utilization, lignocellulosic biomass must undergo pretreatment (e.g., thermochemical and enzymatic processing) to release fermentable sugars.
Lignin comprises ~15-30% of lignocellulosic biomass, and lignin-derived aromatic compounds released during pretreatment can inhibit the microbial conversion of lignocellulosic sugars to bioproducts. Additionally, cost-effective separation of lignin and lignin by-products from lignocellulosic sugars remains a challenge, and current processes generate large waste streams that are typically burned or discarded. Thus, efforts are underway to improve microbial tolerance to lignin by-products and to develop approaches for valorizing waste lignin. Rhodococcus opacus PD630 is a promising biofuel production strain that can 1) consume lignocellulosic sugars, 2) accumulate large amounts of triacylglycerols (biodiesel precursors), and 3) tolerate and consume lignin-derived aromatic compounds. Understanding R. opacus aromatic tolerance and utilization mechanisms could lead to the development of this strain for cost-effective fuel and chemical production from lignocellulosic biomass. To this end, the aromatic tolerance and utilization mechanisms of R. opacus were explored by combining adaptive evolution and -omics approaches. R. opacus was adaptively evolved on both individual and mixtures of lignin model compounds, and multiple mutants were identified with improved aromatic tolerance and utilization compared to the ancestral (wild-type) strain. Whole genome sequencing of adapted strains revealed genes with mutations across multiple adaptive evolutionary lineages that could affect aromatic tolerance and utilization. Transcriptomics of adapted strains using aromatic compounds as carbon sources elucidated degradation pathways for five lignin model compounds. Furthermore, knockout studies of upregulated aromatic transporters suggest that aromatic transport is an integral part of aromatic tolerance and utilization. Lipidomic analysis of one adapted strain showed that adaptation affected the outer membrane composition during growth using a lignin model compound as a sole carbon source, which could also affect aromatic tolerance and utilization. Finally, adapted strains demonstrated improved conversion of lignin model compounds into lipids, suggesting that R. opacus has promise as a biofuel production host. Together, these results provide new insight into aromatic tolerance and utilization mechanisms and demonstrate the potential of R. opacus for biofuel production and lignin valorization applications.
Tae Seok Moon
Gautam Dantas, Marcus Foston, Fong Fu Hsu, Fuzhong Zhang,