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.