Removal of the product from the culture medium strongly enhances free fatty acid production by genetically engineered Synechococcus elongatus

Cyanobacterial mutants engineered for production of free fatty acids (FFAs) secrete the products to the medium and hence are thought to be useful for biofuel production. The dAS1T mutant constructed from Synechococcus elongatus PCC 7942 has indeed a large capacity of FFA production, which is comparable to that of triacylglycerol production in green algae, but the yield of secreted FFAs is low because the cells accumulate most of the FFAs intracellularly and eventually die of their toxicity. To increase the FFA productivity, enhancement of FFA secretion is required. Growth of dAS1T cells but not WT cells was inhibited in a liquid medium supplemented with 0.13 g L−1 of palmitic acid. This suggested that when FFA accumulates in the medium, it would inhibit the release of FFA from the cell, leading to FFA accumulation in the cell to a toxic level. To remove FFAs from the medium during cultivation, an aqueous-organic two-phase culture system was developed. When the dAS1T culture was overlaid with isopropyl myristate (IM), the final cell density, cellular chlorophyll content, and the photosynthetic yield of PSII were greatly improved. The total amount of extracellular FFA was more than three times larger than that in the control culture grown without IM, with most of the secreted FFAs being recovered in the IM layer. The cellular FFA content was decreased by more than 85% by the presence of the IM layer. Thus, the two-phase culture system effectively facilitated FFA secretion out of the cell. An average FFA excretion rate of 1.5 mg L−1 h−1 was attained in the 432 h of cultivation, with a total amount of excreted FFA being 0.64 g L−1 of culture. These figures were more than three times higher than those reported previously for the cyanobacteria-based FFA production systems. Removal of FFA from the culture medium is important for improving the productivity of the FFA production system using cyanobacteria. Further increase in productivity would require an increase in both the rates of FFA production in the cell and active FFA export across the plasma membrane.