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The use of genetic engineering tools to produce industrial strains, especially from non-model microorganisms such as cyanobacteria, is always subject to limitations. In this research, a system-oriented method was used to design a culture medium instead of strain designing and its ability to increase ethanol production by Synechocystis sp. PCC 6803 was experimentally evaluated. In this method, compounds are added to the medium to regulate the activity of target enzymes not for the purpose of being consumed by the cells, and thus, the designed culture medium eliminates the intracellular constraints on the production. A metabolic model was used to determine the minimum level of ethanol production and to identify genes that increase or decrease of their expression increase this minimum level. Then, regulators of the enzyme expressed by the target genes were extracted from the Brenda database and their effect on the production was evaluated experimentally and design of experiment was performed to optimize the concentration of the selected compounds. Among the compounds identified, two inhibitors (salicylic acid and mercuric chloride) and one activator (pyruvate) were selected to be added to the medium and their concentration was optimized using the central composite design method. The proposed regulatory medium increased the production of ethanol from 352 to 1116 mg/l, indicating the effectiveness of the added regulatory compounds on the cyanobacteria metabolism. The proposed system-oriented method can be used to design medium culture for other important bio-products such as recombinant proteins.

Keywords: Regulatory culture medium, bio-fuel, activator and inhibitor, Brenda database, design of experiment, genom-scale metabolic model, Synechocystis

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