Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered this prototypical bacterium into an efficient producer of free fatty acids. The genetic alterations include: (a) knocking out the endogenous fadD gene, which encodes an acyl-CoA synthetase, to block fatty acid degradation; (b) heterologous expression of a plant thioesterase to increase the abundance of shorter chain fatty acids with an eye towards improving fuel quality; (c) increasing the supply of malonyl-CoA by over-expressing acetyl-CoA carboxylase; and (d) releasing feedback inhibition caused by long-chain fatty acyl-ACPs through over-expression of an endogenous thioesterase.

To evaluate the performance of the above biocatalyst in a process that mimics large-scale industrial fermentation processes, fed-batch fermentation runs were performed at a 6 L scale. Under defined media fermentation conditions, using glycerol as the primary carbon source, the metabolically engineered E. coli strain produced a final titer of about 2.5 g/L and a linear productivity of about 0.17 mg/ml/h sustained for 12 h post-induction. During this linear period, a carbon mass yield of about 4.8% was achieved. At least 50% of the fatty acids produced were present in the free acid form. The observation that E. coli can efficiently convert a cheap carbon source such as glycerol into free fatty acid without apparent toxicity opens the door for developing microbial biodiesel as an alternative to petroleum-based long-chain alkane fuels.