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

甲基营养型酵母作为外源基因表达系统在学术和应用领域有着广泛的应用。当细胞在甲醇上生长时，大体积的细胞（高达80%）被单膜结合的细胞器（过氧化物酶体）占据。进行该研究以在甲基营养型酵母的过氧化物酶体内产生毒性氧化酶，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有效的蛋白质运输，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双序群是已知的，蛋白质运输到过氧化物酶体的分子机制在医学领域是重要的，并且使用甲基营养型酵母作为模式生物来研究。本研究将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，该酶可用于糖基化蛋白的测定。在糖尿病患者中，糖化蛋白的水平很高。使用（i）针对过氧化物酶体的有效靶向信号和（ii）醇氧化酶耗尽的菌株，以及（iii）优化密码子使用和培养条件，我们可以将酶产率从原始菌株提高约47倍。所产生的蛋白质积累在膜结合过氧化物酶体内，达到总可溶性蛋白质的近20%。这是基于“分选工程”（“生物交通工程”）的概念进行的第一个成功的例子，并且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，特别是用于产生毒性或膜蛋白。

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.