The project can be divided into several parts:

1. Optimization of sucrose production
   To enable growth of a heterotrophic partner in the synthetic consortium, one major factor is the optimization of sucrose production by the cyanobacterium. Sucrose secretion is based on inducible, heterologous production of a sucrose transporter that allows tunable secretion of sucrose into the culture medium. This approach is often combined with salt stress and some other metabolic modifications such as overexpression of sucrose pathway genes, or the knock-out of competing pathways or carbon sinks to increase sucrose production (Santos-Merino et al. 2023, Thiel et al. 2019, Kirsch et al. 2018). Another important approach in this context is the characterization of the optimal cultivation conditions for cyanobacterial sucrose production and simultaneous growth of all co-culture partners. Major factors to consider are light intensity, light/dark cycles and carbon dioxide availability, but also temperature, pH, cell density and media compositions (e.g. salt stress).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
2. Establishment of a stable co-culture/consortium
   Based on the best sucrose-producing cyanobacterium a stable co-culture with one (or later two) heterotrophic partner with carbon dioxide as the only carbon source can be established. Previous studies already successfully established simple synthetic consortia consisting of two partners, a phototrophic cyanobacterium, engineered to secrete sucrose, feeding a heterotrophic microorganism (Hays et al. 2017, Ducat et al. 2012). For the characterization of these co-cultures different cultivation devices (such as standard shaking flasks combined with online backscatter measurement (Aquila CGQ)) and different photobioreactor setups (such as multi-cultivators, lab-scale flat bed bioreactors, membrane bioreactors) for online measurement of biomass, pH and dissolved carbon dioxide will be used. Co-cultures will be analyzed by single-cell flow cytometry (CytoFLEX S by Beckman Coulter) which enables the identification of different species based on their cell size, autofluorescence or fluorescent proteins and allows the quantification of the cell numbers of each individual population in the culture.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       
3. Biosensors in cyanobacteria
   To allow online measurement of important metabolites during cultivation, we also aim to establish biosensors in cyanobacteria. One promising candidate is a ratiometric Matryoshka biosensor combined with a sugar-binding protein for sucrose detection (Ast et al. 2017, Sadoine et al. 2021). However, due to high levels of autofluorescence caused by pigments, the selection of suitable fluorescent proteins in cyanobacteria is challenging and requires further investigation and adjustment of the available biosensors. Suitable fluorescent proteins and biosensors will be screened using single-cell flow cytometry (CytoFLEX S by Beckman Coulter) and plate reader measurements.

Key publications:

• Santos-Merino, M., Yun, L., & Ducat, D. C. (2023). Cyanobacteria as cell factories for the photosynthetic production of sucrose. Frontiers in Microbiology, 14. https://doi.org/10.3389/fmicb.2023.1126032
• Hays, S. G., Yan, L. L. W., Silver, P. A., & Ducat, D. C. (2017). Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction. Journal of Biological Engineering, 11(1), 4. https://doi.org/10.1186/s13036-017-0048-5
• Ducat, D. C., Avelar-Rivas, J. A., Way, J. C., & Silver, P. A. (2012). Rerouting carbon flux to enhance photosynthetic productivity. Applied and Environmental Microbiology, 78(8), 2660–2668. https://doi.org/10.1128/AEM.07901-11
• Thiel, K., Patrikainen, P., Nagy, C., Fitzpatrick, D., Pope, N., Aro, E.-M., & Kallio, P. (2019). Redirecting photosynthetic electron flux in the cyanobacterium Synechocystis sp. PCC 6803 by the deletion of flavodiiron protein Flv3. Microbial Cell Factories, 18(1), 189. https://doi.org/10.1186/s12934-019-1238-2
• Kirsch, F., Luo, Q., Lu, X., & Hagemann, M. (2018). Inactivation of invertase enhances sucrose production in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology, 164(10), 1220–1228. https://doi.org/10.1099/mic.0.000708

• Wang, M., Da, Y., & Tian, Y. (2023). Fluorescent proteins and genetically encoded biosensors. Chemical Society Reviews, 52(4), 1189–1214. https://doi.org/10.1039/d2cs00419d
• Ast, C., Foret, J., Oltrogge, L. M., De Michele, R., Kleist, T. J., Ho, C.-H., & Frommer, W. B. (2017). Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins. Nature Communications, 8(1), 431. https://doi.org/10.1038/s41467-017-00400-2
• Sadoine, M., Reger, M., Wong, K. M., & Frommer, W. B. (2021). Affinity Series of Genetically Encoded Förster Resonance Energy-Transfer Sensors for Sucrose. ACS Sensors, 6(5), 1779–1784. https://doi.org/10.1021/acssensors.0c02495