The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO2 in the yeast Komagataella phaffii

The current climatic change is predominantly driven by excessive anthropogenic CO 2 emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO 2 co-assimilation or the use of CO 2 -derived methanol or formate as carbon sources are considered pathbreaking contributions to solving this global problem. The number of industrially-relevant microorganisms that can use these two carbon sources is limited, and even fewer can concurrently co-assimilate CO 2 . Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO 2 in the industrially-relevant methylotrophic yeast Komagataella phaffii ( Pichia pastoris ). Using 13 C-tracer-based metabolomic techniques and metabolic engineering approaches, we discover and confirm a growth supporting pathway based on native enzymes that can perform all three assimilations: namely, the oxygen-tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO 2 utilisation to produce proteins, biomass, or chemicals in yeast. One carbon compounds such as CO 2 , methanol and formate are cost-effective and environmentally friendly microbial feedstocks for biomanufacturing. Here, the authors report the oxygen tolerant reductive glycine pathway in Komagataella phaffii can co-assimilate CO 2 , methanol and formate.