The fitness landscape of a form II rubisco in a photosynthetic bacterium guides engineering of oxygen tolerance
Abstract Rubisco is an important but challenging protein engineering target. Fast and selective rubiscos could enhance photosynthesis in plants and accelerate biobased production processes. To facilitate engineering of rubisco, we applied an in vivo screen that couples rubisco activity to growth rate of the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803. We screened a barcoded mutagenesis library of the form II rubisco from Gallionella sp. containing 15,000 single-site and multi-site variants. Exchanges in loop 6 near the active site, at the dimer interface, and in potential gas tunnels improved rubisco fitness. The dataset also informed protein engineering, using recombination and a trained transformer model. In vitro characterisation of two high-fitness variants showed reduced catalytic efficiency for oxygenation (k cat /K o ) in both and an increased carboxylation turnover ( k cat C ) in one. This large labeled fitness dataset, containing examples of epistasis, can be useful for benchmarking computational models of rubisco. Teaser Evolving a foreign rubisco to photosynthesis leads to reduced oxygen sensitivity.
Authors
- Ute A. Hoffmann (ORCID: https://orcid.org/0000-0002-9658-2695)
- Emil Sporre (ORCID: https://orcid.org/0000-0003-4105-7567)
- Jeroni Galmés (ORCID: https://orcid.org/0000-0002-7299-9349)
- Elton P. Hudson (ORCID: https://orcid.org/0000-0003-1899-7649)
- Karen Schriever (ORCID: https://orcid.org/0000-0002-3677-5508)
- Sebastià Capó‐Bauçà (ORCID: https://orcid.org/0000-0002-1679-7910)
- Per‐Olof Syrén (ORCID: https://orcid.org/0000-0002-4066-2776)
- Pere Aguiló‐Nicolau (ORCID: https://orcid.org/0000-0002-6503-6691)
- Mami Matsuda (ORCID: https://orcid.org/0000-0002-3731-8915)
- Rui Miao (ORCID: https://orcid.org/0000-0003-2911-6886)
- Michele Russo (ORCID: https://orcid.org/0000-0001-9958-682X)
- Tomohisa Hasunuma (ORCID: https://orcid.org/0000-0002-8382-2362)
- Axel Knave (ORCID: https://orcid.org/0009-0002-4143-9979)
- Anna Karlsson (ORCID: https://orcid.org/0009-0003-1867-5698)
- Anna Zoey Schuppe (ORCID: https://orcid.org/0009-0005-8564-1636)
Institutions
- Uppsala University (SE)
- Fundació Universitat-Empresa de les Illes Balears (ES)
- Universitat de les Illes Balears (ES)
- Kobe University (JP)
- KTH Royal Institute of Technology (SE)
Publication Details
- Journal
- Science Advances
- Published
- 2026-09-04
- DOI
- https://doi.org/10.1126/sciadv.aee9222
- Citations
- 2
- Primary Topic
- Photosynthetic Processes and Mechanisms
- Type
- article
- Field-Weighted Citation Impact
- 5.75
Funders
- Novo Nordisk Fonden
- Stiftelsen för Strategisk Forskning
- HORIZON EUROPE Framework Programme