MOLECULAR MECHANISM OF ASSEMBLY OF SINGLE MEMBRANE-BOUND ORGANELLS AND "SORTING ENGINEERING"

单膜结合细胞器组装的分子机制和“分选工程”

基本信息

批准号：
09460156
负责人：
SAKAI Yasuyoshi
金额：
$ 8.83万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

The methylotrophic yeast is widely used as a heterologous gene expression system in academic and applied fields. When cells are grown on methanol, a large volume of cells (up to 80%) was occupied by a single-membrane bound organelle, the peroxisome. The study was conducted to produce toxic oxidases within peroxisomes of the methylotrophic yeast, since many oxidases are useful for clinical diagnosis. To this end, the protein traffic was manipulated for efficient protein transport, and this strategy (or concept) was named "sorting engineering" (later biotraffic engineering). Since genetic diorders deficient in peroxisome assembly are known, the molecular mechanism for protein traffic to peroxisomes was in important in medical field and investigated using the methylotrophic yeast as a model organism. In this study, the information obtained from the basic research was applied to production of fungal fructosyl amino acid oxidase, which is useful for determination of glycated proteins. In diabetic patients, the level of glycated proteins is high. Using (i) an efficient targeting signal to peroxisomes and (ii) an alcohol-oxidase depleted strain, and (iii) optimizing codon usage and culture conditions, we could improve the enzyme productivity approximately 47-fold from the orginal transformant. The produced protein accumulated within membrane-bound peroxisomes up to nearly 20% of the toal soluble protein. This is the first successful example which was conducted based on the concept "sorting engineering" ("biotraffic engineering"), and a similar approach will be possible in any other eukaryotic cells for heterologous gene expression, especially for the production of toxic or membrane proteins.

甲基营养酵母作为异源基因表达系统广泛应用于学术和应用领域。当细胞在甲醇中生长时，大量细胞（高达 80%）被单膜结合的细胞器（过氧化物酶体）占据。该研究的目的是在甲基营养酵母的过氧化物酶体内产生有毒的氧化酶，因为许多氧化酶可用于临床诊断。为此，操纵蛋白质运输以实现高效的蛋白质运输，这种策略（或概念）被命名为“分选工程”（后来的生物运输工程）。由于过氧化物酶体组装缺陷的遗传缺陷是已知的，因此蛋白质运输到过氧化物酶体的分子机制在医学领域非常重要，并使用甲基营养酵母作为模式生物进行研究。在本研究中，将从基础研究中获得的信息应用于真菌果糖基氨基酸氧化酶的生产，该酶可用于糖化蛋白的测定。糖尿病患者的糖化蛋白水平很高。使用 (i) 过氧化物酶体的有效靶向信号和 (ii) 乙醇氧化酶耗尽的菌株，以及 (iii) 优化密码子使用和培养条件，我们可以将酶生产率比原始转化体提高约 47 倍。产生的蛋白质在膜结合过氧化物酶体中积累，高达总可溶性蛋白质的近 20%。这是第一个基于“分选工程”（“生物流量工程”）概念进行的成功例子，类似的方法将可能在任何其他真核细胞中用于异源基因表达，特别是用于有毒或膜蛋白的生产。