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Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications
Author(s) -
Zavec Domen,
Gasser Brigitte,
Mattanovich Diethard
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27303
Subject(s) - pichia pastoris , methanol , yeast , recombinant dna , bioreactor , phenotype , pichia , biochemistry , chemistry , alcohol oxidase , biology , gene , organic chemistry
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 (P AOX1 ) was active in the Mut − phenotype and showed adequate eGFP fluorescence levels and protein yields (Y P/X ) in small‐scale screenings. Different bioreactor cultivation scenarios with methanol excess concentrations were tested using P AOX1 HSA and P AOX1 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 ( q methanol ) in the methanol phase were reduced by more than 80% compared with the Mut S 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.