Rhodococcus opacus: metabolic evolution and genetic tools for production of biofuels and value-added products from lignocellulosic biomass

Lignocellulosic biomass (LCB) is a renewable feedstock that contains hexose and pentose sugars that can be used by microorganisms for conversion to biofuels and other high value products. However, pretreatment of LCB also releases lignin subunits (phenolics) that lower microbial growth rates and product yields. Additionally, lignin is not used as a carbon source for fermentation even though common feedstocks such as wheat grass and switchgrass contain 15-30% lignin by weight. To increase tolerance to and utilize phenolics, new microbial hosts have been investigated, but genetic tools for metabolic engineering are often unavailable. Our work focuses on developing genetic tools and metabolic engineering in the lipid-accumulating, phenolic-degrading Rhodococcus opacus PD630. R. opacus has been shown to accumulate 62% of its cell dry weight (CDW) in lipids under nitrogen limiting conditions using hexose, and up to 68% of its CDW in lipids in engineered pathways using pentose. Through growth-based selection, we increased the concentrations of phenol (> five-fold) at which R. opacus can tolerate and grow using it as a sole carbon source. The selection also increased the growth rate on glucose as a sole carbon source by ~2-fold. We are currently developing genetic tools to modify the cell’s metabolism for lipid accumulation and high value product synthesis. R. opacus demonstrates its potential for metabolic engineering to convert LCB into high value products.