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.
Keywords: Komagataella phaffii; Mut-; bioprocess; growth decoupled; methanol induction.
© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.