Shobayashi Megumi, Mukai Nobuhiko, Iwashita Kazuhiro, Hiraga Yoshikazu, Iefuji Haruyuki
National Research Institute of Brewing, 3-7-1, Kagamiyama, Higashi Hiroshima, Hiroshima, 739-0046, Japan.
Appl Microbiol Biotechnol. 2006 Feb;69(6):704-10. doi: 10.1007/s00253-005-0009-7. Epub 2005 Jul 12.
S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7-5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.
S-腺苷甲硫氨酸（SAM）是一种重要的代谢产物，在所有生物体中作为甲基供体参与许多反应，并且由于其改善多种疾病（如抑郁症、肝病和骨关节炎）的潜力而在临床研究中引起了广泛关注。由于这些潜在应用，需要一种更有效的方法来生产SAM。因此，在本研究中我们开发了一种阳性选择方法来分离积累SAM的酵母。在酿酒酵母中，消耗SAM的主要反应之一被认为是由Erg6p催化的麦角固醇生物合成中的甲基化反应。麦角固醇生物合成存在缺陷的突变体可能由于麦角固醇生物合成中SAM消耗的减少而积累SAM。我们已应用这种方法用制霉菌素分离积累SAM的酵母，制霉菌素已被用于选择麦角固醇生物合成存在缺陷的突变体。通过这种方法有效地从酿酒酵母K-9和X2180-1A中分离出了积累SAM的突变体。这些突变体积累的SAM比其亲本菌株多1.7至5.5倍。核磁共振（NMR）和气相色谱-质谱联用（GC-MS）分析表明，来自K-9的两个突变体在erg4基因中存在突变，实验室菌株的erg4缺失突变体积累的SAM也比其亲本菌株多。这些结果表明，在麦角固醇生物合成中，在Erg6p下游起作用的酶的基因发生突变的突变体在积累SAM方面是有效的。
