P154: Metabolic engineering for a malonyl-CoA-dependent pathway for fatty acid production in Escherichia coli

Fatty acids (FAs) are attractive intermediates for conversion to a wide diversity of products, ranging from chemical precursors to biofuels.  Production of FAs from sustainable carbon sources is further desirable from societal and economic viewpoints.  However, FA metabolism in Escherichia coli, both anabolic and catabolic, is complex and regulated at multiple levels.  Thus we have chosen to develop a novel pathway for FA production in this host that is independent of the intrinsic FA biosynthetic machinery.

Our approach utilizes enzymes that are capable of catalyzing a 2-carbon extension of an acyl-CoA from malonyl-CoA.  In addition to the heterologous expression of these enzyme genes, we have engineered hosts to: 1) eliminate production of lactate, acetate and ethanol; 2) increase flux from sugar to acetyl-CoA; 3) conditionally decrease flux through the host acyl carrier protein-dependent FA biosynthetic pathway resulting in increased malonyl-CoA availability; 4) eliminate thioesterases with preference for short chain FAs while increasing thioesterase activity on acyl-CoAs of medium-long chain length; and 5) eliminate utilization of free FA.  With these biocatalysts, we have demonstrated production of FAs at rates exceeding 0.1 g/gDCW.hr from glucose.  Further improvement of this pathway, as well as its implementation in a microbe that is capable of growth on CO₂ and H₂ as the sole carbon and energy sources, is in progress.