IMPROVED PROTEIN-SYNTHESIS AND SECRETION THROUGH MEDIUM ENRICHMENT INA STABLE RECOMBINANT YEAST-STRAIN

Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a reco mbinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid-k genes. The combination of these three mu tations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex me dium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the furl and urid-k mutations. The p lasmid utilized contains the yeast invertase gene under the control of the MFalpha 1 promoter and leader sequence. The expression and secret ion of invertase for the autoselection strain were examined in batch c ulture for three media: a minimal medium (SD), a semidefined medium (S DC), and a rich complex medium (YPD). Biomass yields and invertase pro ductivity (volumetric activity) increased with the complexity of the m edium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lo west in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80-90% of the invertase was secreted into the periplasmic space and/or culture medium. A gluc ose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, i nvertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped fr om 54% to 34% during one batch fermentation. During long-term sequenti al batch culture in YPD, invertase activity decreased by 90% and the p lasmid-containing fraction dropped from 56% to 8.8% over 44 generation s of growth. The expression level for the autoselection strain, howeve r, remained high and constant over this time period, and no reversion at the furl or urid-k locus was observed.