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 CO₂ emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO₂ co-assimilation or the use of CO₂-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 CO₂. Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO₂ in the industrially-relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using ¹³C-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 CO₂ utilisation to produce proteins, biomass, or chemicals in yeast.