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

开发有用的代谢物超利用原生质体功能的高速生产系统。

基本信息

批准号：
15360435
负责人：
AOYAGI Hideki
金额：
$ 9.86万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15360435/
关键词：
Artificial cell wall plant protoplast microbial protoplast DNA microarrays enzymes production useful metabolites bioreactor cell wall components 制癌剤酵索生産遺伝子発現

项目摘要

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.

虽然原生质体激活的功能不同于细胞，但细节尚未澄清。利用DNA微阵列技术对培养的酵母原生质体和细胞的基因表达进行了比较。培养的原生质体比培养的细胞对氧化胁迫更敏感。细胞周期阻滞在G1期，细胞壁合成得到促进。在培养的原生质体中，碳水化合物代谢被激活，而氨基酸的生物合成被抑制。此外，一些与代谢物分泌途径相关的基因被激活，导致这些代谢物主动分泌到肉汤中。作为DNA微阵列分析应用的一个例子，我们根据原生质体的特性开发了一种新的转化酶和α-葡萄糖苷酶的生产系统。通过将脆弱的原生质体固定在琼脂糖凝胶中（高传质和非电荷），保护它们免受物理和渗透胁迫。该系统的产酶能力远高于细胞的产酶能力。该系统也可用于利用重组S.酿酒酵母原生质体。红豆杉原生质体被用作植物原生质体的模型。建立了一种从红豆杉叶片细胞和愈伤组织中分离高活力原生质体的方法。将红豆杉原生质体固定在专用凝胶（具有高清除活性和传质功能的人工细胞壁）中，在某些信号转导物的存在下，能产生比细胞更多的紫杉醇和紫杉烷类化合物。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，开发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。新系统可以解决使用细胞的传统生产系统的问题。