Systems and Synthetic Biology of Rhodococcus Opacus to Enable Conversion of Lignin-Derived Aromatic Compounds into Lipids

Lignocellulosic biomass is a renewable feedstock that can be converted into biofuels and biochemicals using microorganisms. However, pretreatment of lignocellulosic biomass releases toxic compounds that inhibit microbial growth. Additionally, lignin has been an untapped carbon source due to its recalcitrance and toxic monomeric units (e.g., aromatic compounds). To achieve sustainable and economic utilization of lignocellulosic biomass, we must develop technologies that utilize the complete biomass, including lignin, which is currently considered as waste. To this end, our work focuses on developing Rhodococcus opacus as a new chassis for conversion of aromatic compounds into triacylglycerols (TAGs), biodiesel precursors. R. opacus is a promising host due to its high tolerance to and utilization of aromatics as a sole carbon source. Importantly, R. opacus can accumulate TAGs up to ~80% of cell dry weight under nitrogen-limiting conditions. To enhance its innate aromatic-degrading capacity, we applied adaptive evolution, a growth-based strain selection method, by sequentially sub-culturing cells in diverse combinations of lignin-derived aromatic compounds as sole carbon sources. Our adapted strains demonstrated higher growth rates and higher lipid accumulation compared to the wild type strain. Whole genome sequencing, RNA-seq, and ¹³C-fingerprinting analysis have identified possible aromatic tolerance and utilization mechanisms such as upregulation of degradation pathways and putative transporters for aromatic compounds. In addition, synthetic biology tools have been developed to enable engineering of the identified tolerance genes, regulators, and pathways. We will present progress towards development of R. opacus as a microbial cell factory.