Characterization of neutral sites to use Synechocystis sp. PCC 6803 as a photoautotrophic chassis

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Filipe Pinto, Catarina C. Pacheco, Paulo Oliveira, Arnau Montagud, Javier F. Urchuegua and Paula Tamagnini

IBMC - Instituto de Biologia Molecular e Celular & Faculdade de Ciências, Portugal

Cyanobacteria are promising “low-cost” cell factories since they have minimal nutritional requirements and high metabolic plasticity. The unicellular non-N2-fixing cyanobacterium Synechocystis sp. PCC 6803 is the best studied strain and the vast amount of physiological and molecular data available already allowed the construction of computational genome-scale metabolic models [1]. Moreover, a previous work from our group reported the construction of a Synechocystis chassis prone to receive synthetic devices primarily designed for hydrogen production [2]. Within this work, five neutral sites were identified and characterized to stably integrate ectopic DNA into the chromosome, foreseeing the use of Synechocystis as a photoautotrophic chassis in biotechnological applications. The neutrality of the sites was evaluated by producing and analyzing deletion/insertion mutants, and their functionality was assessed introducing a synthetic device expressing the reporter green fluorescent protein. Moreover, integrative vectors including a multiple cloning site compatible with the BioBrick RFC[10] standard and insulated by transcription terminators were constructed, constituting robust cloning interfaces for synthetic biology purposes. Synechocystis mutants (chassis) ready to receive purpose-built synthetic modules/circuits are also available. References: [1] Montagud A et al. (2010) BMC Syst Biol 4:156. [2] Pinto F et al. (2012) Microbiology 158:448-464.