Overexpression of endogenous galactolipases and an efflux transporter enhances the secretion of extracellular free fatty acids by Synechococcus elongatus PCC 7942.

backgroundCyanobacterial mutants engineered for production of free fatty acids (FFAs) are potentially useful sources of biofuels, since the FFAs are secreted into the extracellular medium. The exploitation of such mutants circumvents intracellular storage limits and minimizes energy-intensive downstream extraction processes. However, in addition to the inactivation of the endogenous aas gene (encoding acyl-ACP synthetase) to prevent recycling of FFAs, construction of the FFA-producing mutants requires introduction of a transgene for a thioesterase that generates FFAs from acyl-ACP. For exploitation of such mutants for biofuel production at the industrial level, in particular in countries with strict rules about dissemination of genetically modified organisms, it is essential to avoid introduction of foreign transgenes.

resultsUsing the markerless mutagenesis method, we replaced an internal 3.0-kb segment of the aas gene of Synechococcus elongatus PCC 7942 with a DNA fragment that included the ORFs of the endogenous genes for galactolipases (lipB and/or lipC) and the RND-type FFA efflux transporter (rndA1B1), which were fused, respectively, to the endogenous psbAII promoter. Under strong light (200 µmol photons·m

conclusionsCo-overexpression of endogenous galactolipases and the RND transporter resulted in efficient extracellular secretion of FFAs in engineered S. elongatus. Our approach should facilitate investigations directed toward industrial applications, particularly in countries with strict regulations related to the dissemination of genetically modified organisms, and it provides a promising platform for the sustainable production of biofuel precursors.