Enhanced productivity of extracellular free fatty acids by gene disruptions of acyl-ACP synthetase and S-layer protein in Synechocystis sp. PCC 6803

Based on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl-acyl carrier protein synthetase), and surface layer protein (encoded by sll1951). Three Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient conditions, BG.sub.11-half N and BG.sub.11-N conditions, with the exception of strain KS. Under BG.sub.11-N condition at day 10, strain KAS strikingly secreted FFAs products up to 40%w/DCW or 238.1 mg/L, with trace amounts of PHB. Unexpectedly, strain KS, with S-layer disruption, appeared to have endured longer in BG.sub.11-N growth medium. This strain KS significantly acclimated to the BG.sub.11-N environment by accumulating a greater glycogen pool with lower FFA production, whereas strain KA favored higher PHB and intracellular lipid accumulations with moderate FFA secretion. Mutations of both aas and sll1951 genes in Synechocystis sp. PCC 6803 significantly improved the productivity of secreted FFAs, especially under nitrogen deprivation.