Engineering Rhodococcus to convert biomass to triacylglycerides

Effective lignolytic biocatalysts would facilitate the economic and sustainable development of biomass-derived products. Rhodococci and related Actinobacteria are of particular interest for their ability to convert biomass to triacylglycerides (TAGs), a biodiesel precursor. Indeed, rhodococci accumulate over 50% of their dry weight in TAGs under certain conditions of nutrient limitation. We are elucidating and engineering the TAG biosynthetic pathways in Rhodococcus jostii RHA1, a soil bacterium that catabolizes a range of lignin-derived aromatic compounds. Transcriptomic profiling revealed a number of putative TAG biosynthetic genes to be strongly up-regulated under conditions of nitrogen-limitation. These genes included atf8 and atf10, two of the 14 atf homologs in RHA1 that are predicted to encode diglyceride acyltransferases. Construction and analysis of an atf8 deletion mutant together with biochemical analysis of the purified Atf8 enzyme established its role in catalyzing the last step of TAG biosynthesis. Lipid profiling using GC-MS revealed that TAG composition varied as a function of growth substrate. However, atf8 was strongly upregulated regardless of growth substrate. Moreover, overexpression of atf8 in RHA1 augmented TAG biosynthesis under a variety of conditions. Similar analyses involving a combination of targeted gene deletion, lipid profiling and enzymatic assays are establishing the role of other enzymes in TAG biosynthesis, particularly under different growth conditions. These efforts are being matched by the engineering of RHA1 to secrete lignolytic enzymes. Overall, our studies are helping to establish RHA1 as a platform for the development of lignolytic biocatalysts