Periodic Reporting for period 1 - PhotoCyX (Seeing in the New Light: Photoreceptor Expression System for Cyanobacteria)
Okres sprawozdawczy: 2022-09-01 do 2024-08-31
Cyanobacteria, the green microbial factories and photosynthetic microorganisms, have shown significant promise in addressing critical global issues such as air pollution and fuel security. These organisms can produce alternative and cleaner biofuels (e.g. bioethanol and hydrogen) and biodegradable plastics (e.g. polyhydroxy-butyrate) with minimal inputs, relying primarily on sunlight, atmospheric carbon dioxide, and basic nutrients. Such capabilities offer a timely and sustainable solution to environmental challenges. However, despite their potential, the commercial application of these biochemicals has been limited by low production yields and technological challenges. The stability and yield of production depend on the balanced and sufficient synthesis of enzymes that direct metabolic flux toward desired compounds; without this balance, excess production of one compound could strain cellular resources and reduce overall efficiency. To overcome these limitations, developing technologies that enable customizable, tunable control over enzyme production within cyanobacteria has become essential. This research aimed to create a cyanobacterial expression tool responsive to red light, using a phytochrome photoreceptor to enable precise, light-inducible gene expression. This tool would facilitate highly adjustable control of cyanobacterial gene expression through two wavelengths of red light, presenting a promising approach for optimizing enzyme levels. The project began by constructing expression plasmids and applying them to the cyanobacterium Synechocystis sp. PCC 6803. The light responses of the resulting expression strains were then tested in illumination experiments, where the increased expression of a reporter protein was measured through fluorescence spectroscopy. Although still in progress, this project aims to pave the way for more efficient and targeted protein expression in cyanobacteria, ultimately breaking production barriers and advancing cyanobacterial contributions to sustainable industrial solutions.
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.
The project has made significant strides toward advancing optogenetics and cyanobacterial biotechnology, with achievements that include the development and optimization of novel optogenetic tools like the RfpA-based regulatory system. This system has been transformed into E. coli and analyzed for functionality, expanding the toolkit for optogenetic control in cyanobacteria and enhancing the understanding of two-component signaling systems. These innovations hold promise for applications in synthetic biology and industrial biotechnology, especially where controlled gene expression is crucial, such as in biofuel production. The Rfp signaling system, part of the unique Far-Red Light Photoacclimation (FaRLiP) mechanism in specific cyanobacteria, enables photosynthesis under far-red light, offering an advantage in shaded environments. This project’s demonstration of the RfpA-based system’s functionality in E. coli introduces a promising optogenetic tool, while insights into RfpA’s regulatory mechanisms support further customization for broader synthetic biology applications.