Conversion of Aromatic Compounds to Lipids By Engineered Rhodococcus Strains

Lignocellulosic biomass is a renewable feedstock that can be converted to biofuels or high-value products using a biorefinery. However, pretreatment of lignocellulosic biomass often releases toxic inhibitors for the downstream fermentation. In addition, a major component of lignocellulosic biomass, lignin, remains an untapped carbon resource due to its recalcitrance and toxic monomeric units (phenolics). Our work focuses on developing Rhodococcus opacus as a new chassis for conversion of phenolics to triacylglycerol (TAG), a biodiesel precursor. R. opacus is a promising host for bioconversion of phenolics due to its high tolerance to aromatics and its ability to grow on phenolics as a sole carbon source. In addition, R. opacus can accumulate TAGs up to ~80% of cell dry weight (CDW) under nitrogen-limiting conditions. To enhance its innate phenolic-degrading capacity, we applied adaptive evolution, a growth-based strain selection method, by sequentially sub-culturing cells in aromatic compounds as sole carbon sources. After >200 generations, our top adapted strains demonstrated a >50% increase in final optical density (OD₆₀₀) and >30% increase in growth rate as well as high lipid accumulation compared to the wild type strain. Whole genome sequencing, RNA-seq, and ¹³C-fingerprinting analysis identified possible phenolic tolerance and utilization mechanisms such as phenolic degradation pathway upregulation. We will present progress towards development of R. opacus as a microbial cell factory.