The biosynthesis of acetate from CO2 and light was optimised through metabolic engineering of the cyanobacterium Synechocystis PCC 6803. Insertion of a phosphoketolase (PK) in the acs gene resulted in an enhanced Calvin-Benson-Bassham cycle and 40-fold higher acetate production. Further overexpression of a phosphotransacetylase (Pta) led to an increase of 80-fold, reaching an acetate production of 2.3 g/L. A synthetic consortium, based on the Synechocystis PCC 6803 strain that secretes acetate and a phototrophic bacterium Rhodopseudomonas palustris growing on the formed acetate, enabled the production of biohydrogen and fatty acids through nitrogen and carbon dioxide fixation. Elemental balance confirmed carbon capture and nitrogen fixation into the consortium. Proteomic analysis indicated acetate exchange and light-dependent regulation of metabolic activities.
We aimed to obtain a superior Farmer strain able to provide high level of sucrose from CO2 and light as feedstock for Labour heterotrophic modules. Starting with the sucrose overproducer Synechococcus strain SBG363 (Patent WO 2021/148693 A1), we designed a further optimisation strategy, by applying strain-designing algorithms and the metabolic model of Synechococcus iJB7852, based on deletion of key genes involved in sucrose competing pathways. A total of three genes were identified and deleted in wild-type Synechococcus, which cause growth impairments, especially the glycogen synthase knock-out (∆glgA). Finally, the sucrose overproducing strategy was implemented in the triple mutant and sucrose production was assayed. Preliminary results indicated that a deeper understanding of sucrose and glycogen metabolism in Synechococcus is needed to further optimise sucrose production.
Using a rational, model-driven metabolic engineering approach, we have designed a Pseudomonas putida KT2440 biocatalyst capable of efficiently utilising sucrose-produced by cyanobacteria from CO₂ and light- while enhancing acetyl-CoA levels. These metabolic improvements enable the seamless conversion of acetyl-CoA into bioplastics—specifically, medium-chain-length polyhydroxyalkanoates (PHAs)—without nutrient limitation constraints. Supplementing the growth medium with 6-acetyl-thiohexanoic acid (6-ATH) facilitates the accumulation of up to 70% (g/g CDW) of a high-value functionalised PHA, known as PHACOS, which exhibits antimicrobial activity against pathogens such as Staphylococcus aureus.
Pseudomonas putida biocatalyst accumulating PHACOS
References
- Roussou, S., Pan, M., Krömer, J. O. & Lindblad, P. (2025). Exploring increased acetate biosynthesis in Synechocystis PCC 6803 through insertion of a heterologous phosphoketolase and overexpressing phosphotransacetylase. Metabolic Engineering, 88, 250–260. https://doi.org/10.1016/j.ymben.2025.01.008
- Pan, M., Colpo, R. A., Roussou, S., Ding, C., Lindblad, P. & Krömer, J. O. (2025). Engineering a photoautotrophic microbial coculture toward enhanced biohydrogen production. Environmental Science & Technology, 59(1), 337–348. https://doi.org/10.1021/acs.est.4c08629
- Gómez-Luengo, A. & Nogales, J. (2023). Sucrose overproducer Synechococcus strain based on deletion of competing pathways and population homogenization. Deliverable D3.1 EU Horizon 2020 PROMICON Project, Grant agreement No 101000733.
- Manoli, M.-T., Gargantilla-Becerra, A., del Cerro Sánchez, C., Rivero-Buceta, V., Prieto, M. A. & Nogales, J. (2024). A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation. Cell Reports, 43, 113979. https://doi.org/10.1016/j.celrep.2024.113979
- Rodríguez, M., Durante-Rodríguez, G., Gómez-Álvarez, H., Prieto, M. A. & Díaz, E. (2024). A set of optimized P. putida strains overproducing PHACOS from sucrose and 6-ATH. Deliverable D3.4 EU Horizon 2020 PROMICON Project, Grant agreement No 101000733.