Expanding the toolbox for sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters.

Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO as energy and carbon sources. The unicellular sp. PCC 6803, already considered the 'green' , is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform . Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into using one of the SEVA plasmids. The presence of this device restored the production of the GG in a deficient mutant validating the functionality of the tools/device developed in this study.