RECOMBINANT PROTEIN-PRODUCTION IN AN ALCOHOL OXIDASE-DEFECTIVE STRAINOF PICHIA-PASTORIS IN FEDBATCH FERMENTATIONS

The methylotrophic yeast Pichia pastoris synthesizes high levels of al cohol oxidase from the AOX1 gene during growth on methanol as a carbon source. We have a transcriptional fusion of the lacZ gene to the AOX1 promoter as a model system for investigating recombinant protein prod uction in an alcohol oxidase (aox1, aox2) defective strain. Growth and recombinant protein production with glycerol as the carbon source (fe d at various constant feedrates) was studied. A feedrate of 1 g l(-1) h(-1) was found to be optimum resulting in a specific activity of 8.62 x 10(4) U mg(-1) dry cell. The specific yield did not improve when gl ycerol was increased in steps. High feedings rates gave low specific y ields (U mg(-1) dry cell mass) and high cell masses. Low protein yield s at higher glycerol feedrates were due to partial repression of the A OX1 promoter by glycerol and the by-product, ethanol. In comparison, t he wild type (Mut(+)) strain gave a maximum specific yield of 5.52 x 1 0(4) U mg(-1) dry cell. (C) 1997 Elsevier Science Inc.