Development of a high-speed production system for useful metnbolites hyper-utilizing functions of protoplasts.

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Though the protoplast activates the function unlike the cell, the details have not yet been clarified. The global gene expression of cultured yeast protoplasts was compared with that of cells using DNA microarray. The cultured protoplasts were more sensitive to oxidative stress than the cultured cells. Their cell cycles were arrested at the G1 phase and cell wall synthesis was promoted. Carbohydrate metabolism was activated in cultured protoplasts, while amino acid biosynthesis was inhibited. Furthermore, some genes associated with the secretory pathway of metabolites were activated, leading to active secretion of these metabolites into the broth. As an example of the application of DNA microarray analysis, we developed novel system for the production of invertase and alpha-glucosidase based on the characteristics of protoplasts. The fragile protoplasts were protected against the physical and osmotic stresses by immobilizing them in agarose gel (high mass transfer and non-electric charge). The productivities of the enzymes by the system were much higher than those of the cells. This system was also useful for the efficient production of cutinase using recombinant S. cerevisiae protoplasts. Taxus protoplasts were used as a model for plant protoplasts. A method for efficient isolation of the protoplast with high activities from the leaf cells and callus of Taxus was developed. Taxus protoplasts, which were immobilized in the specialized gel (artificial cell wall having both high scavenging activity and mass transfer) in the presence of some signal transducers, produced much paclitaxel and taxoids than those of the cells. This system was also useful for the production of cell wall accumulative enzymes in plant protoplasts. By these studies, high-speed production systems for useful metabolites with hyper-utilizing functions of microbial and plant protoplasts were developed. The novel systems could solve the problems of conventional production systems using cells.