• Directed evolution
• Pyrenoid
• Biomolecular condensate
• CO2 fixation
• RuBisCO
• High-throughput protein purification

To overcome the natural limitations in terms of carboxylation/oxygenation trade-off of RuBisCO, nature evolved carbon concentrating mechanisms (CCMs).
In the alga Chlamydomonas reinhardtii, the CCMs consist of diverse components, such as carbonate pumps through the thylakoid membranes and, most importantly, the pyrenoid. The pyrenoid is a non-membrane-bound organelle assembled by the targeted interaction of RuBisCO with the intrinsically disordered protein EPYC1 (essential pyrenoid component 1) through liquid-liquid phase separation. In this work, we develop a protocol for the in vitro directed evolution of protein condensates in order to engineer a functional phase separating RuBisCO-IDP fusion mimicking the pyrenoid. By providing an example of the engineering of two traits in a single protein, we show the potential of this approach in reducing complex phenotypes to a minimal number of independent polypeptides, to facilitate their implementation in vivo.