Towards sustainable production of biofuels via milking the cells of Synechococcus elongatus PCC 7942 engineered for free fatty acid excretion

Free fatty acids are considered a potential source of biodiesel because they are excreted by engineered cyanobacterial cells, eliminating the processing costs associated with cell recovery and extraction. Although the cyanobacterial mutants engineered for the production of free fatty acids do excrete significant amounts of the product, the yield per dry cell weight is low due to their rapid proliferation and the consequent substantial cell biomass, which makes it impossible to support sustainable biofuel production. Development of strategies to increase the production of free fatty acids per cell is urgently needed. To increase the per-cell production of free fatty acids, we attempted to increase the allocation of photosynthetically fixed carbon into fatty acid production by restricting cell proliferation. Growth limitation of free fatty acid-producing cells was achieved by cultivating dAS2T, a free fatty acid-producing mutant of Synechococcus elongatus PCC 7942 defective in active nitrate uptake, in media containing nitrate as the sole source of nitrogen. Although the nitrogen-limited cultivation increased the per-cell yield of free fatty acids, it significantly decreased the extracellular free fatty acid level, reducing the volumetric productivity. Overexpressing an endogenous free fatty acid-efflux pump in the dAS2T strain elevated extracellular free fatty acid level to those seen under nitrate-sufficient conditions, while maintaining slow cell growth. By decoupling the production of fatty acids from cell growth in this manner, excretion of 0.90 g of free fatty acids per g of dry cell weight was achieved in 240 h, with a free fatty acid productivity of 1.8 mg L −1 h −1 . This represents the highest yield of free fatty acids obtained to date without significantly decreasing the volumetric productivity. Growth limitation and enhancement of excretion of the product are essential to increase the per-cell yield of the free fatty acid production system using cyanobacteria. The high yield and productivity of extracellular free fatty acids achieved in this study suggest that the cyanobacteria-based production of free fatty acids provides a reliable approach for sustainable biofuel production through the “milking” strategy.