Optimization of fungal polygalacturonase synthesis by Saccharomyces fungal polygalacturonase synthesis by Saccharomyces

To define parameters for a technical fermentation process, the production of a fungal polygalacturonase by the yeast sccharomyces crrevisiae was optimized in fed-batch culture. Synthesis of the secreted enzyme was markedly influenced by medium composition as well as by fermentation parameters. The kinetics of recombinant protein synthesis was growth-associated following an unstructured Monod kinetics under carbon-limited conditions. Productivity (as heterologous protein synthesized per time and biomass) did not remain constant during the fed-batch process and decreased after an initial peak, whereas the specific mRNA level remained high. Supplementing the medium with complex nutrients did not improve the production kinetics. However, an increased concentration of nitrogen (a C:N ratio of 6.5: 1) resulted in a high level productivity that remained constant during the entire fed-batch process. While low nitrogen concentrations (C:N ratio of 14: 1) were sufficient for optimal growth, higher nitrogen concentrations were required for maximum productivity. Using two control strains, it was shown that the recombinant plasmid per se had a negative effect on the growth rate ( p,,,,) in comparison to the wild-type strain, and that the synthesis of the recombinant protein moreover decreased the biomass yield (g cell dry weight per g sucrose). The high-level expression of the recombinant protein in the fed-batch process put metabolic stress on the cells which resulted in markedly reduced vitality