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BackgroundEthanol is a very important clean energy and it has many applications in medical and chemical fields. Large-scale production of ethanol has mainly been carried out through the fermentation of crops such as grain but its output and cost issues have attracted widespread attention. ResultsWith the ability to fix carbon dioxide directly, cyanobacteria have been used as a photosynthetic microbial cell factory to generate biofuels and chemicals. Here, we constructed the biosynthetic pathway of ethanol in cyanobacterium Synechocystis sp. PCC 6803 through the following approaches. (1) We used homologous substitution to introduce pyruvate decarboxylase (pdc) gene from Zymomonas mobilis and NADPH-dependent aldehyde reductase (yqhD) gene from Escherichia coli into the neutral site of Synechocystis sp. PCC 6803. (2) The native superpromoter Pcpc560, consisting of two promoters from the cpcB gene and 14 predicted transcription factor binding sites (TFBSs) from Synechocystis sp. PCC6803 genome, was used to drive the over-expression of ethanol-producing genes. (3) To further increase ethanol production, we used molecular biotechnology to inhibit the metabolic pathway that direct the carbon flux of intermediate pyruvate metabolism to phosphoenolpyruvate (PEP) through disrupting the cyanobacterial endogenous PEP synthase. These approaches led to the production of 2.79g/g dry cell weight ethanol directly from light and greenhouse gas CO2 in Synechocystis after cultivating for 9 days. ConclusionOur study provides insights into the biosynthetic pathway for ethanol production in Synechocystis indicating that knocking out the competitive pathway of the initial precursor and enhancing the expression of exogenous genes can effectively increase the amount of the targeted chemicals.