The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO 2 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.