Microbial production of high octane and high sensitivity olefinic ester biofuels

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BackgroundAdvanced spark ignition engines require high performance fuels with improved resistance to autoignition. Biologically derived olefinic alcohols have arisen as promising blendstock candidates due to favorable octane numbers and synergistic blending characteristics. However, production and downstream separation of these alcohols are limited by their intrinsic toxicity and high aqueous solubility, respectively. Bioproduction of carboxylate esters of alcohols can improve partitioning and reduce toxicity, but in practice has been limited to saturated esters with characteristically low octane sensitivity. If olefinic esters retain the synergistic blending characteristics of their alcohol counterparts, they could improve the bioblendstock combustion performance while also retaining the production advantages of the ester moiety.ResultsOptimization of Escherichia coli isoprenoid pathways has led to high titers of isoprenol and prenol, which are not only excellent standalone biofuel and blend candidates, but also novel targets for esterification. Here, a selection of olefinic esters enhanced blendstock performance according to their degree of unsaturation and branching. E. coli strains harboring optimized mevalonate pathways, thioester pathways, and heterologous alcohol acyltransferases (ATF1, ATF2, and SAAT) were engineered for the bioproduction of four novel olefinic esters. Although prenyl and isoprenyl lactate titers were limited to 1.48 ± 0.41 mg/L and 5.57 ± 1.36 mg/L, strains engineered for prenyl and isoprenyl acetate attained titers of 176.3 ± 16.0 mg/L and 3.08 ± 0.27 g/L, respectively. Furthermore, prenyl acetate (20% bRON = 125.8) and isoprenyl acetate (20% bRON = 108.4) exhibited blend properties comparable to ethanol and significantly better than any saturated ester. By further scaling cultures to a 2-L bioreactor under fed-batch conditions, 15.0 ± 0.9 g/L isoprenyl acetate was achieved on minimal medium. Metabolic engineering of acetate pathway flux further improved titer to attain an unprecedented 28.0 ± 1.0 g/L isoprenyl acetate, accounting for 75.7% theoretical yield from glucose.ConclusionOur study demonstrated novel bioproduction of four isoprenoid oxygenates for fuel blending. Our optimized E. coli production strain generated an unprecedented titer of isoprenyl acetate and when paired with its favorable blend properties, may enable rapid scale-up of olefinic alcohol esters for use as a fuel blend additive or as a precursor for longer-chain biofuels and biochemicals.