Cyanobacteria are a very large, diverse group of prokaryotes, which are united by their photoautotrophic metabolism which relies on oxygenic photosynthesis (1). Cyanobacteria are primary drivers of biogeochemical nutrient cycling and are primary producers in fresh water and marine ecosystems, fixing carbon and nitrogen. While beneficial in a balanced ecosystem, overgrowth of Cyanobacteria due to lake eutrophication and warming can lead to outbreaks of harmful algal blooms (HABs), which can foul water bodies due to overgrowth of Cyanobacteria species, and even the production of a variety of toxins.
Algal bloom in a lake in Oneonta, NY.
Recently, Cyanobacteria have become an emerging chassis organism for use in synthetic biology. The ability of Cyanobacteria to fix carbon, along with their ability to grow in salinated, polluted water sources, and the natural competence of some species, make them an attractive target for use in the production of biofuels, plastics, and a variety of other organic compounds, such as fatty acids and long-chain alcohols (2). This increased interest in cyanobacteria as chassis organisms has correlated with the creation of Cyanobacteria-compatible parts, and the characterization of lab strains (3).
Cyanobacteria are an emerging chassis for synthetic biology. Figure from (7).
We at SUNY Oneonta were inspired by the potential use of Cyanobacteria to replace petroleum products through production of synthetic alternatives. We decided to focus our project on the creation of resources to make engineering of Cyanobacteria easier. Not all species of Cyanobacteria are naturally able to uptake environmental DNA (4). In aquatic environments, Cyanophages contribute significantly to horizontal gene transfer within and between species of Cyanobacteria (5,6). Viral vectors have been used for genetic engineering of both prokaryotic and eukaryotic cells with in vitro and in vivo applications. These two facts inspired us to create a viral vector for the engineering of Cyanobacteria. Our strategy was to reverse engineer a phagemid that, when expressed in an E. coli chassis, would result in the synthesis of ghost phages – phage capsids devoid of any genetic material. Once complete, the phagemid could be modified by synthetic biologists to custom fit their needs. Such modifications might include:
- Addition of a DNA packaging protein and a DNA circuit to deliver the DNA to Cyanobacterial cells for engineering purposes.
- Modification of the viral capsid to enable use of the virus as a nanomaterial for delivery of other materials to Cyanobacteria – for example an antimicrobial.
- Modification of tail-fiber sequences to create viruses that can specifically recognize different species of Cyanobacteria for tagging purposes (e.g., with GFP) or for immobilization.
- Whitton, B. A., Potts, M. (2012). Ecology of Cyanobacteria II: Their diversity in space and time. (B.A. Whitton Ed.) Springer Publishing. doi=10.1007/978-94-007-3855-3.2.
- Gronenberg, L. S., Marcheschi, R. J., & Liao, J. C. (2013). Next generation biofuel engineering in prokaryotes. Current opinion in chemical biology, 17(3), 462–471. https://doi.org/10.1016/j.cbpa.2013.03.037
- Berla, B. M., Saha, R., Immethun, C. M., Maranas, C. D., Moon, T. S., & Pakrasi, H. B. (2013). Synthetic biology of cyanobacteria: unique challenges and opportunities. Frontiers in microbiology, 4, 246. https://doi.org/10.3389/fmicb.2013.00246
- Schirmacher, A. M., Hanamghar, S. S., & Zedler, J. (2020). Function and Benefits of Natural Competence in Cyanobacteria: From Ecology to Targeted Manipulation. Life (Basel, Switzerland), 10(11), 249. https://doi.org/10.3390/life10110249
- Aminov R. I. (2011). Horizontal gene exchange in environmental microbiota. Frontiers in microbiology, 2, 158. https://doi.org/10.3389/fmicb.2011.00158
- Bailey, S., Clokie, M. R., Millard, A., & Mann, N. H. (2004). Cyanophage infection and photoinhibition in marine cyanobacteria. Research in microbiology, 155(9), 720–725. https://doi.org/10.1016/j.resmic.2004.06.002
- A., Hantke, J. G., Toepel, J., Bühler, B., Nürnberg, D. J., & Klähn, S. (2022). Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria. ACS synthetic biology, 11(5), 1758–1771. https://doi.org/10.1021/acssynbio.1c00605