2-37: Cell Factory Engineering of Oleaginous Bacteria for Production of Lipid-based Biofuel from Pyrolytic Sugars

Oleaginous strains of Rhodococcus including R. opacus PD630 and R. jostii RHA1 have attracted considerable attention because of their ability to accumulate significant levels of triacylglycerol (TAG), nutritional versatility, robust growth properties, and genetic tractability. We enabled the strains to unitize C5 sugars in our previous studies. Neither, however, is able to utilize the anhydrosugar of glucose, levoglucosan, as the carbon source for their growth and lipid accumulation. Levoglocosan is the major molecule produced from the pyrolysis of cellulose. To broaden their substrate utilization range, a novel metabolic pathway was introduced into the strains. This was accomplished by heterogenous expression of levoglucosan kinase (lgk) with an E. coli-Rhodococcus shuttle vector. The results showed that recombinants bearing lgk could grow on levoglucosan as the sole carbon source. The consumption rate of levoglucosan and enzymatic activity of LGK were determined. To further enhance lipid yield of the strains by metabolic engineering, the genes, acetyl-CoA carboxylase (acc) and diacylglycerol acyltransferase (dga1) for the initiation of fatty acid synthesis and final step of TAG assembly were overexpressed. Under nitrogen limited conditions, the fatty acid composition and lipid produced from levoglucosan were studied. This work demonstrated that it was feasible to produce lipids from pyrolytic sugars derived from lignocellulosic feedstock by genetic modification of the rhodococci strains. The outcome of this work combined with recent studies enhancing the selectivity of pyrolysis process to produce low cost bio-oils enriched in levoglucosan, will be transformative in the way biomass is converted into lipid-derived “drop-in” fuels and chemicals.