Abstract
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 (q methanol) 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.
Highlights
The methanol utilization (Mut) − phenotype of P. pastoris has been overlooked in the past years for secreted protein production
There have been attempts to utilize this phenotype by several researchers (Chiruvolu et al, 1997; Inan & Meagher, 2001), but the results were nonconclusive and failed to elucidate the main advantages of the Mut− phenotype
Experiments previously undertaken on the Mut− phenotype were done with a focus on intracellularly produced proteins, which are subjected to degradation (Zhang, Liu, & Wu, 2007), and failed to show the potential of these strains for secreted protein production
Summary
Necessary for the first step of the methanol metabolism. Deletion of Pichia pastoris The me- duction because of the lower growth rate and methanol uptake rate thylotrophic properties of P. pastoris are exploited to induce, control, (Krainer et al, 2012; Looser et al, 2015). Protein production in these and produce recombinant proteins in high quantities. The use of a methanol metabolism deficient strain would, on the other hand, solve the above‐mentioned disadvantages. This led us to further investigate if the Mut− strain is able to produce secreted proteins when induced with methanol. We further investigated whether an additional carbon and energy source is necessary for protein production (Chiruvolu, Cregg, & Meagher, 1997)
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