Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria

Cyanobacteria have
raised great interest in biotechnology due to
their potential for a sustainable, photosynthesis-driven production
of fuels and value-added chemicals. This has led to a concomitant
development of molecular tools to engineer the metabolism of those
organisms. In this regard, however, even cyanobacterial model strains
lag behind compared to their heterotrophic counterparts. For instance,
replicative shuttle vectors that allow gene transfer independent of
recombination into host DNA are still scarce. Here, we introduce the
pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive
genetic engineering of Synechocystis sp. PCC 6803.
The series is based on the small endogenous plasmids pCA2.4 and pCB2.4,
each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular
cloning and the insulated introduction of gene expression cassettes.
We made use of genes encoding green fluorescent protein (GFP) and
a Baeyer–Villiger monooxygenase (BVMO) to demonstrate functional
gene expression from the pSOMA plasmids in vivo.
Moreover, we demonstrate the expression of distinct heterologous genes
from individual plasmids maintained in the same strain and thereby
confirmed compatibility between the two pSOMA subseries as well as
with derivatives of the broad-host-range plasmid RSF1010. We also
show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging
to further explore the range of cyanobacterial host species that could
be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector
series displays an attractive alternative to existing plasmid series
and thus meets the current demand for the introduction of complex
genetic setups and to perform extensive metabolic engineering of cyanobacteria.