Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut– phenotype. The Mut– phenotype is commonly regarded as unable to utilize methanol. In contrast to literature, we found that the Mut– strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut– increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.

The methanol utilization (Mut) phenotype in the yeast Pichia pastoris (syn. Komagataella spp.) is defined by the deletion of the genes AOX1 and AOX2. The Mut− phenotype cannot grow on methanol as a single carbon source. We assessed the Mut− phenotype for secreted recombinant protein production. The methanol inducible AOX1 promoter (PAOX1) was active in the Mut− phenotype and showed adequate eGFP fluorescence levels and protein yields (YP/X) in small‐scale screenings. Different bioreactor cultivation scenarios with methanol excess concentrations were tested using PAOX1HSA and PAOX1vHH expression constructs. Scenario B comprising a glucose‐methanol phase and a 72‐hr‐long methanol only phase was the best performing, producing 531 mg/L HSA and 1631 mg/L vHH. 61% of the HSA was produced in the methanol only phase where no biomass growth was observed, representing a special case of growth independent production. By using the Mut− phenotype, the oxygen demand, heat output, and specific methanol uptake (qmethanol) in the methanol phase were reduced by more than 80% compared with the MutS phenotype. The highlighted improved process parameters coupled with growth independent protein production are overlooked benefits of the Mut− strain for current and future applications in the field of recombinant protein production.

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The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N‐linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O‐glycosylation. O‐glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O‐mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O‐glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, PGAP and PAOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI‐MS. In the samples expressed with PGAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with PAOX1. The results indicate that the O‐glycosylation level is markedly higher when the protein is produced in a methanol‐based expression system.

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