Increased CO₂ fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast

CO₂ fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO₂ fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO₂, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO₂ fixation strategies pave the way for CO₂ being used as the sole carbon source.