Premium
Evaluation of Mut + and Mut S Pichia pastoris Phenotypes for High Level Extracellular scFv Expression under Feedback Control of the Methanol Concentration
Author(s) -
Pla Itzcoatl A.,
Damasceno Leonardo M.,
Vannelli Todd,
Ritter Gerd,
Batt Carl A.,
Shuler Michael L.
Publication year - 2006
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp060012+
Subject(s) - pichia pastoris , bioreactor , fermentation , methanol , extracellular , pichia , chemistry , yeast , secretion , inducer , biology , recombinant dna , biochemistry , gene , botany , organic chemistry
Abstract Extracellular secretion of over 4 g·L −1 of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut + and Mut S phenotypes were compared in a bioreactor under closed‐loop PID control of the methanol level. As expected, the Mut S phenotype has a growth rate lower than that of the Mut + (0.37 vs 1.05 d −1 ) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut + (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g·L −1 for the Mut S also has a positive effect on the final product concentration. Both Mut + and Mut S phenotypes reach a maximum biomass density around 450 g·L −1 wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.