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

甲醇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。当细胞在甲醇中生长时，大量的细胞(高达80%)被单膜结合的细胞器--过氧化物体占据。这项研究是为了在甲基营养酵母的过氧化物体中产生有毒的氧化物酶，因为许多氧化物酶对临床诊断有用。为此，为了有效地运输蛋白质而操纵蛋白质交通，这一策略(或概念)被命名为分类工程(后来的生物交通工程)。由于缺乏过氧化体组装的遗传二级体是已知的，因此蛋白质转运到过氧化酶体的分子机制在医学领域具有重要的意义，并以甲基营养酵母为模式生物进行了研究。本研究将从基础研究中获得的信息应用到真菌果糖氨基酸氧化酶的生产中，这对糖化蛋白的测定是有用的。在糖尿病患者中，糖化蛋白水平很高。使用(I)有效的针对过氧化物体的靶向信号和(Ii)酒精氧化酶耗竭的菌株，以及(Iii)优化密码子的使用和培养条件，我们可以将原转化子的酶产量提高约47倍。产生的蛋白质在膜结合的过氧化物体中积累，占总可溶性蛋白质的近20%。这是基于“分类工程”(“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表达异源基因，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.