Synthetic autotrophic yeast enables high itaconic acid production from CO ₂ <i>via</i> integrated pathway and process design

Single carbon (C1) substrates are gaining importance as future feedstocks for the production of bio-based chemicals. Carbon dioxide, a major greenhouse gas, offers a promising alternative to the traditional feedstocks to shift towards C1-based, sustainable processes. Here, we present a synthetic autotrophic <i>Komagataella phaffii</i> (<i>Pichia pastoris</i>) that is able to produce itaconic acid by the direct conversion of CO₂, achieving final titers of approximately 12 g L^-1 in bioreactor cultivations. We show that a combined approach that integrates balancing the flux between the Calvin-Benson-Bassham (CBB) cycle and itaconic acid metabolism with process design was essential to enhance the production. Our study demonstrates the potential of <i>K. phaffii</i> as a microbial platform using CO₂ as the direct carbon source, aligning with the future goals of establishing sustainable bioprocesses.