The oxygen tolerant reductive glycine pathway in eukaryotes – a native methanol, formate and CO₂ assimilation pathway in the yeast Komagataella phaffii

Abstract

The current climate change is mainly driven by excessive anthropogenic CO2 emissions. As industrial bioprocesses depend mostly on food competing organic feedstocks or fossil raw materials, we regard CO2 co-assimilation or the use of CO2-derived methanol or formate as carbon source as pathbreaking contribution to the solution of this global problem. The number of industrially relevant microorganisms that can use these two carbon sources is limited, and even less can concurrently co-assimilate CO2. Hence, we searched for alternative native methanol and native formate assimilation pathways which co-assimilate CO2 in the industrially relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using 13C-tracer-based metabolomics techniques and metabolic engineering approaches we discovered and confirmed a natively active pathway that can perform all three assimilations: the oxygen tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO2 utilisation for the production of proteins, biomass or chemicals in yeast.

A native phosphoglycolate salvage pathway of the synthetic autotrophic yeastKomagataella phaffii

“…The metabolomics workflow was performed as described by Mitic et al (Mitic et al, 2022) based on the method of Mairinger et al, 2015.…”

“…For the evaluation of 13 C incorporation into the metabolites isotopologue distribution analysis was conducted. The extracted ion chromatograms of all carbon isotopologues of a target analyte were integrated (evaluated mass/charge ratios listed in (Mitic et al, 2022)). The peak areas were corrected with the software ICT correction toolbox v.0.04 for the contribution of other heavy isotopes except 13 C, as well as the contribution of the natural 13 C abundance of the derivatization agent.…”

“…The metabolomics workflow was performed as described by Mitic et al (Mitic et al, 2022) based on the method of Mairinger et al, 2015. In brief, the cell suspension was rapidly quenched in a 4-fold volume of 60% methanol, 125 mmol L -1 TRIS-HCl, 55 mmol L -1 NaCl at pH 8.2 and -27°C (Mattanovich et al, 2017) and filtered through a cellulose acetate filter after vortexing the mixture for 4 s. After washing the cells on the filter with 60% methanol, the filters were stored at -70°C. The consecutive boiling ethanol extraction was performed with 4 mL 75% ethanol at 85°C.…”

Carbon efficient production of chemicals with yeasts

“…However, the activity of this pathway for production of glycine is not high enough to support growth without further engineering of cell metabolism (Mitic et al, 2022).…”

“…Furthermore, a native oxygen tolerant reductive glycine pathway has been recently discovered in the yeast K. phaffii . However, the activity of this pathway for production of glycine is not high enough to support growth without further engineering of cell metabolism (Mitic et al, 2022 ).…”