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

虽然原生质激活的功能不同于细胞，但其细节尚不清楚。利用DNA芯片技术比较了酵母原生质体与细胞的整体基因表达。原生质体对氧化应激的敏感性高于培养细胞。它们的细胞周期被阻滞在G1期，细胞壁合成被促进。原生质体碳水化合物代谢被激活，氨基酸的生物合成受到抑制。此外，与代谢物分泌途径相关的一些基因被激活，导致这些代谢物积极分泌到肉汤中。作为DNA微阵列分析应用的一个例子，我们基于原生质体的特性开发了一种新的生产转化酶和α -葡萄糖苷酶的系统。将脆弱的原生质体固定在琼脂糖凝胶中（高传质和无电荷），以保护其免受物理和渗透胁迫。该系统的酶产率远高于细胞的酶产率。该体系也可用于利用重组酿酒酵母原生质体高效生产角质酶。以红豆杉原生质体作为植物原生质体的模型。建立了一种从红豆杉叶细胞和愈伤组织中高效分离活性较高的原生质体的方法。将红豆杉原生质体固定在特定的凝胶（具有高清除活性和传质能力的人造细胞壁）中，在一些信号转导存在的情况下，比细胞产生更多的紫杉醇和类紫杉醇。该系统也可用于植物原生质体细胞壁积累酶的生产。通过这些研究，建立了具有微生物和植物原生质体超利用功能的有用代谢物的高速生产系统。这种新系统可以解决使用细胞的传统生产系统的问题。

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.