Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications.

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 PAOX1 HSA and PAOX1 vHH 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.