Date of Award
Doctor of Philosophy (PhD)
The discovery of fossil fuels facilitated a new era in human history and allowed many firsts, such as the mass production of goods, the ability to travel and communicate long distances, the formation of population dense cities, and unprecedented improvements in quality of life. Alternative sources of energy and chemicals are needed, however, as hydrocarbon reserves continue to deplete and the effects of burning fossils on the planet become better understood. Lignocellulosic biomass is the most abundant raw material in the world and a viable alternative to petroleum-derived products. The pre-treatment of lignocellulose (e.g., thermocatalytic depolymerization, enzymatic hydrolysis, pyrolysis, etc.) generates a range of products, including readily available sugars for microbial fermentation. One of the typically unused fractions of biomass is the structural component, referred to as lignin, that makes up 15 to 30% of the material and when depolymerized generates a heterogeneous mixture of toxic aromatic compounds. Generally, lignin is separated from the carbohydrate fraction and burned, but its utilization has been identified as a key factor in biorefinery profitability. One possible option for lignin valorization is to find a microbe that not only ferments lignocellulose-derived sugars into a valuable commodity, but also the lignin-derived aromatics.Rhodococcus opacus PD630 (hereafter R. opacus) is a non-model, gram-positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for both lignocellulose-derived sugars and aromatic compounds, significant accumulation of the biodiesel precursor triacylglycerol, a relatively rapid growth rate, and genetic tractability. Few genetic elements and molecular biology techniques, however, have been directly characterized in R. opacus, limiting its application for lignocellulose bioconversion. The goal of this dissertation is to greatly expand the genetic toolbox available in R. opacus in order to provide insight into its aromatic catabolism and to promote its use as a microbial chassis for the conversion of biomass-derived products into biofuels or other value-added products. The contributions developed as part of this dissertation include 1) the development of strong constitutive promoters for the overexpression of heterologous genes, 2) the development of chemical and metabolite sensors for tunable gene expression, 3) the characterization of native and endogenous plasmid backbones and resistance markers, 4) a heterologous T7 RNA polymerase expression platform for gene expression, 5) the demonstration of genetic logic circuits for programable gene expression, 6) a recombinase-based recombineering platform for gene knockouts and insertions, 7) a CRISPR interference (CRISPRi) platform for targeted gene repression, 8) the identification of stable reference genes for RT-qPCR applications, 9) insight into aromatic degradation through the β-ketoadipate pathway via gene knockouts, and 10) insight into the role of aromatic transporters via gene knockouts. Taken together, this work greatly advances the ability to engineer R. opacus for any desired application, in addition to providing understanding into its catabolism of aromatic compounds.
Tae Seok Moon
Gautam Dantas, Marcus Foston, Himadri Pakrasi, Fuzhong Zhang,