Kato Tatsuya, Park Enoch Y
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, 422-8529, Japan.
Appl Microbiol Biotechnol. 2006 Jun;71(1):46-52. doi: 10.1007/s00253-005-0124-5. Epub 2005 Sep 13.
Two plasmids containing an autonomously replicating sequence from Saccharomyces cerevisiae were constructed. Using these vectors, the AGX1 gene encoding alanine:glyoxylate aminotransferase (AGT) from S. cerevisiae, which converts glyoxylate into glycine but is not present in Ashbya gossypii, was expressed in A. gossypii. Geneticin-resistant transformants with the plasmid having the kanamycin resistance gene under the control of the translation elongation factor 1 alpha (TEF) promoter and terminator from A. gossypii were obtained with a transformation efficiency of approximately 10-20 transformants per microgram of plasmid DNA. The specific AGT activities of A. gossypii pYPKTPAT carrying the AGX1 gene in glucose- and rapeseed-oil-containing media were 40 and 160 mU mg-1 of wet mycelial weight, respectively. The riboflavin concentrations of A. gossypii pYPKTPAT carrying AGX1 gene in glucose- and rapeseed-oil-containing media were 20 and 150 mg l-1, respectively. In the presence of 50 mM glyoxylate, the riboflavin concentration and the specific riboflavin concentration of A. gossypii pYPKTPAT were 2- and 1.3-fold those of A. gossypii pYPKT without the AGX1 gene.
构建了两个含有来自酿酒酵母自主复制序列的质粒。利用这些载体，将酿酒酵母中编码丙氨酸：乙醛酸转氨酶（AGT）的AGX1基因（该基因可将乙醛酸转化为甘氨酸，但在棉阿舒囊霉中不存在）在棉阿舒囊霉中进行表达。在翻译延伸因子1α（TEF）启动子和来自棉阿舒囊霉的终止子的控制下，获得了带有卡那霉素抗性基因的质粒的遗传霉素抗性转化体，转化效率约为每微克质粒DNA产生10 - 20个转化体。在含葡萄糖和菜籽油的培养基中，携带AGX1基因的棉阿舒囊霉pYPKTPAT的特异性AGT活性分别为每毫克湿菌丝体重量40和160 mU。在含葡萄糖和菜籽油的培养基中，携带AGX1基因的棉阿舒囊霉pYPKTPAT的核黄素浓度分别为20和150 mg l-1。在50 mM乙醛酸存在下，携带AGX1基因的棉阿舒囊霉pYPKTPAT的核黄素浓度和特异性核黄素浓度分别是不含AGX1基因的棉阿舒囊霉pYPKT的2倍和1.3倍。
