Optimization of arsB (Acr3) as a positive selection marker in the cyanobacterium Synechocystis sp. PCC 6803

Abstract

AbstractThe use of cyanobacteria as bio-factories for production of numerous compounds of interest (biofuels, bioplastics …) has attracted lots of attention mainly due to their simple nutritional requirements, coupled with the decrease in atmospheric CO2 levels. However, although cyanobacteria are easily genetically manipulated, there are few genetic tools developed and, in some cases, the modifications necessary for metabolic engineering are limited for this reason. We have developed a new positive selection marker based on the arsenic resistance system of the cyanobacterium Synechocystis sp. PCC 6803. In this cyanobacterium, resistance to arsenic is mediated by the arsBHC operon in which arsB encodes an arsenite transporter whose mutation confers hypersensitivity to the presence of both arsenite and arsenate. Using arsB mutant strain (SARSB) as recipient we introduced plasmids containing both arsB and an antibiotic resistance gene and transformants were selected using either arsenic or the antibiotic with similar efficiency. The plasmids and conditions to use the arsB gene as a selectable marker have been optimized. Furthermore, we have generated an integrative vector to delete the whole arsBHC operon that allows easy introduction of regulated genes in this locus. Analysis of this strain have shown that the ΔarsBHC mutant has a higher sensitivity to arsenite than the SARSB strain, even when they are complemented with an arsB copy. These suggest that arsH, arsC or both could have an additional role in arsenite resistance.