UC San Diego

In recent decades, cyanobacteria have garnered interest as promising biological platforms for producing valuable biofuel and feedstock molecules. Genetic tools are instrumental in conducting genetic modifications in cyanobacteria, but tools are generally limited to a few domesticated laboratory strains. Here, we present new genetic tools and techniques using broad-host range, RSF1010-based plasmids to facilitate genetic engineering across diverse cyanobacterial strains. First, we developed a set of orthogonal constitutive promoters constructed in RSF1010-based plasmids. The promoter library generated a wide range of gene expression levels in five diverse cyanobacterial strains and allowed for strict transcriptional control. Next, we used an RSF1010-based plasmid to conduct biparental conjugal mating experiments with wild cyanobacteria. Serving as a bioprospecting technique, we isolated new, genetically tractable strains with high salt and alkalinity tolerance from a soda lake. Finally, we constructed a plasmid harboring the ptxD gene derived from Pseudomonas stutzeri, which codes for a phosphite oxidoreductase. We engineered three cyanobacterial strains to express the enzyme, which allows them to convert phosphite to phosphate and grow in media containing phosphite as the sole source of phosphorus. We propose that this plasmid can be used to facilitate research on engineering strains to metabolize phosphite as a strategy for reducing biological contaminants in outdoor cultivation ponds. Overall, these RSF1010-based genetic tools are useful additions to the biotechnological toolkit to enable genetic engineering across diverse cyanobacterial strains.