Synthetic autotrophic yeast enables high itaconic acid production from CO2 via 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 Komagataella phaffii (Pichia pastoris) 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 K. phaffii as a microbial platform using CO2 as the direct carbon source, aligning with the future goals of establishing sustainable bioprocesses.