The project aimed to construct and establish a photoreceptor-based, light-inducible plasmid system for fine-tuning protein expression in cyanobacteria, specifically to develop a cyanobacterial equivalent of the plasmid pREDusk. This was initially achieved by transferring the optogenetic cassette from pDusk into a cyanobacterial replicative plasmid, pVZ322. To optimize functionality, the reporter gene DsRed in the resulting plasmid was replaced with EGFP to avoid the overlap of DsRed’s emission spectra with the intrinsic fluorescence of cyanobacterial chlorophyll. The modified plasmid was then introduced into the model cyanobacterium Synechocystis sp. PCC 6803. Unexpectedly, EGFP fluorescence was observed in both red-illuminated and dark-incubated cultures, indicating regulatory crosstalk between the cyanobacterial cellular machinery and the bacteriophytochrome (BphP)-based plasmid tool. To address this, the BphP-based optogenetic cassette (photosensory module of bacteriophytochrome BphP, histidine kinase FixL, response regulator FixJ, and promoter PfixK2) was replaced with the novel Rfp regulatory system (phytochrome RfpA, response regulators RfpB and RfpC, and promoter PchlF) from Synechococcus sp. PCC 7335. Analysis of the intermediate plasmid containing the fusion of the RfpA regulatory system and pREDusk in E. coli confirmed its in vivo activity, establishing a new cyanobacterial phytochrome-based optogenetic plasmid for E. coli. Currently, the Rfp regulatory system fusion is being transferred into pVZ322 for transformation into Synechocystis sp. PCC 6803. In parallel, prior to full implementation, the spectral activity of the RfpA phytochrome was characterized. The purified protein’s spectral response to red, far-red, and dark conditions was measured, and crystallization efforts have begun to explore the structure-function relationship of RfpA, with initial success in obtaining crystal hits. As the red light is essential for photosynthesis and influences various cyanobacterial metabolic processes, we identified a novel cyanobacteriochrome, DPCF2-CBCR, from Tolypothrix sp. PCC 7910, which operates in the blue and teal spectrum. If red light negatively impacts recombinant cyanobacterial metabolism, we will replace the RfpA photosensor with DPCF2-CBCR, enabling control by blue and teal wavelengths and minimizing potential metabolic disruptions.
