Effects of methanol concentration on expression levels of recombinant protein in fed‐batch cultures of Pichia methanolica

Abstract

The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol‐inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on‐line monitoring and control system were investigated in mixed glucose/methanol fed‐batch cultures of P. methanolica expressing the human transferrin N‐lobe protein. The PMAD18 P. methanolica strain utilized is a knock‐out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 291–298, 2003.