Directly converting CO2 into biofuels is regarded as a promising strategy for producing carbon-neutral renewable energy (Atsumi et al., 2009). Fatty acyl-ACP is the important precursor for the biosynthesis of fatty-acid based biofuel molecules, such as free fatty acids, fatty alcohols and alkanes (Liu et al., 2011). Herein, gene overexpression targets were analyzed for overproducing fatty acyl-ACP in the cyanobacterium Synechocystis sp. PCC 6803. As the dominant component of fatty acyl-ACP, palmitoyl-ACP (C16:0 ACP, palmACP[c]) was chosen as the Target product in the UP Finder. Fig. 4 shows the output results. A total of 30 metabolic reactions, involving 6 genes (accABCD, fabD, fabF, fabG, fabZ, fabI) were identified as the key pathways/genes for palmitoyl-ACP overproduction. 28 out of the 30 reactions are catalyzed by fatty acid synthases in Synechocystis sp. PCC 6803, in which 4 key genes (fabF, fabG, fabZ, fabI) are involved. The reactions were ordered by their associated Ratio values, and the different Ratio values of the 30 identified up-regulated pathways represent different up-regulation levels to achieve the same Yield (theoretical maximum yield). Thus, the higher Ratio values indicated the higher demands of metabolic flux for up-regulation toward palmitoyl-ACP overproduction. On the other hand, the completed fatty acid synthesis pathways of the constraint-based model enabled the detailing of fluxes for each single reaction in multiple-repeated fatty acid biosynthesis. It was found that the reactions with same Ratio values reflected the similar level of metabolic flux for going through, which might be used as a quantitative standard for identifying metabolic modules in complex metabolic pathways.Fig. 4Analysis results from the UP Finder for fatty acyl-ACP (palmitoyl-ACP) overproduction in Synechocystis sp. PCC 6803. Identified gene targets are also shown in the metabolic pathway of fatty acyl-ACP biosynthesis presented with gene overexpression preference toward fatty acyl-ACP overproduction. RB15BP, ribulose 1,5-bisphosphate; 3PG, 3-phosphoglycerate; α-KG, α-ketoglutarate; Mal-CoA, Malonyl-CoA; Mal-ACP, Malonyl-ACP. See Supplementary Table S1–S2 for the abbreviations of reaction and metabolite names shown in the UP Finder results.Fig. 4