Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria

Cyanobacteria have raised great interest in biotechnology due to theirpotential for a sustainable, photosynthesis-driven production of fuels and value-addedchemicals. This has led to a concomitant development of molecular tools to engineerthe metabolism of those organisms. In this regard, however, even cyanobacterial modelstrains lag behind compared to their heterotrophic counterparts. For instance,replicative shuttle vectors that allow gene transfer independent of recombination intohost DNA are still scarce. Here, we introduce the pSOMA shuttle vector seriescomprising 10 synthetic plasmids for comprehensive genetic engineering ofSynechocystissp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 andpCB2.4, each combined with a replicon fromEscherichia coli,different selection markersas well as features facilitating molecular cloning and the insulated introduction of geneexpression cassettes. We made use of genes encoding greenfluorescent protein (GFP) and a Baeyer-Villiger monooxygenase(BVMO) to demonstrate functional gene expression from the pSOMA plasmidsin vivo. Moreover, we demonstrate the expression ofdistinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between thetwo pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into thefilamentous model strainAnabaenasp. PCC 7120 is generally possible, which is encouraging to further explore the range ofcyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays anattractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups andto perform extensive metabolic engineering of cyanobacteria