Orsi, E.; Kabuth, K.; Cusimano, S.; Herlov-Wagner, M.; Verbakel, R.; Luppino, F.; Partipilo, M.; de Pins, B.; Noor, E.; Hümmler, L. M.; Mülleder, M.; Lindner, S. N.; Ralser, M.; Nikel, P. I.

Rubisco catalyzes the primary CO2-fixing reaction of the biosphere, yet its competing oxygenation reaction reduces net global carbon fixation and has resisted direct exploration in living cells. Here, we engineer an auxotrophic Escherichia coli strain in which 2-phosphoglycolate, the direct product of Rubisco oxygenation, becomes essential for growth, making bacterial fitness a quantitative proxy for oxygenation flux in vivo. This provides direct access to catalytic selectivity, something previously inaccessible to carboxylation-coupled assays. The platform enables screening of phylogenetically diverse Form II Rubisco and phosphoribulokinase (Prk) variants circumventing protein purification and extensive in vitro characterization. Adaptive laboratory evolution under oxygenation-selective pressure identified two mutations: Rubisco M115I genetically rebalances the in vivo carboxylation/oxygenation trade-off (resulting in 6-fold reduction in kcat,C), while Prk N216T improves overall flux without altering selectivity. This platform makes Rubisco's least-studied catalytic function selectable and evolvable in vivo, opening the carboxylation/oxygenation trade-off to systematic genetic dissection and engineering.