(346d) Metabolic Engineering for the Production Fuels and Chemicals From Bio-Oils

The use of oils and fats as renewable feedstocks for the production of chemicals and fuels is a promising avenue to establish biorefineries. Moreover, the highly reduced nature of carbon atoms in these compounds, as compared to carbohydrates, would ensure the production of chemicals and fuels at higher yields. However, metabolism of free fatty acids (FAs), the main constituents of fats and oils, requires the presence of an external electron acceptor, which in turn could preclude the synthesis of metabolic products. We have used micro-respiratory conditions and targeted genetic manipulations to establish a respiro-fermentative metabolic mode in which a balance between cell growth and synthesis of reduced products is simultaneously achieved.

Ethanol and succinate were chosen as model products and E. coli as model organism to illustrate the feasibility of the aforementioned approach. The maximum theoretical yields for the synthesis of ethanol and succinate from FAs are 1.33 g/g and 1.85 g/g, respectively, compared to 0.51 g/g (ethanol) and 1.12 g/g (succinate) for their production from sugars. This implies that the use of FAs would allow for a 2.6- and 1.7-fold increase in the yield of ethanol and succinate, respectively. To enhance ethanol production, we created a mutant of the enzyme acetaldehyde/alcohol dehydrogenase (r-AdhE) that is functional in the presence of oxygen. Overexpression of r-AdhE in wild-type E. coli MG1655 grown under micro-respiratory conditions led to a yield of 0.60 g ethanol/g FAs, which already surpasses the maximum theoretical from sugars. Similar improvements in the synthesis of succinate were achieved by engineering the TCA cycle and the glyoxylate shunt to prevent succinate degradation and enhance its synthesis.