Engineering Escherichia coli towards the production of branched-chain advanced biofuels

Branched-chain fatty acids (BCFAs) are key advanced biofuel precursors with improved cold-flow properties compared to straight chain fatty acids (SCFA). Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and the co-production of SCFAs, complicating efficient BCFA isolation. Through extensive strain characterization, I identified that insufficient lipoylation of 2-oxoacid dehydrogenases is a key bottleneck in BCFA production. Consequently, I complemented the lipoylation defect by engineering two lipoylation pathways, which not only restored protein lipoylation, but allowed for the highest BCFA titer reported (over 276 mg/L), with BCFA production comprising over 85% of total free fatty acids. The high percentage of BCFA achieved facilitates downstream conversions and purification. Colleagues and I then fine-tuned BCFA branch positions by engineering the upstream pathway to control the supply of various branched-chain acyl-CoAs, demonstrating tight control of chain structure, with over 93% of BCFA corresponding to the input chain structure. We then applied the same BCFA platform to generate branched-chain fatty acid ethyl esters, alcohols, and alkanes, all in high percentages- demonstrating the utility of the platform to generate high quality advanced biofuels and chemicals.