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

甲基营养酵母作为一种异源基因表达系统在学术和应用领域得到了广泛的应用。当细胞在甲醇上生长时，大量细胞（高达80%）被单膜结合的细胞器，过氧化物酶体所占据。这项研究是为了在甲基营养酵母的过氧化物酶体中产生有毒氧化酶，因为许多氧化酶对临床诊断有用。为此，对蛋白质的转运进行操纵，以实现高效的蛋白质转运，这种策略（或概念）被命名为“分选工程”（即后来的生物运输工程）。由于已知过氧化物酶体组装缺陷的遗传疾病，蛋白质转运到过氧化物酶体的分子机制在医学领域具有重要意义，并以甲基营养酵母为模式生物进行了研究。本研究将基础研究所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。在糖尿病患者中，糖化蛋白的水平很高。利用(i)对过氧化物酶体的有效靶向信号和（ii）酒精氧化酶缺失菌株，以及（iii）优化密码子的使用和培养条件，我们可以将酶的产量提高约47倍。产生的蛋白质在膜结合的过氧化物酶体中积累了近20%的可溶性蛋白质。这是基于“分选工程”（“生物交通工程”）概念进行的第一个成功的例子，类似的方法将可能在任何其他真核细胞中用于异种基因表达，特别是用于生产有毒或膜蛋白。

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.