Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube, 755-8505, Japan.
Antonie Van Leeuwenhoek. 2013 Apr;103(4):933-45. doi: 10.1007/s10482-012-9874-0. Epub 2013 Jan 22.
By random integrative mutagenesis with a kanMX4 cassette in Kluyveromyces marxianus DMKU 3-1042, we obtained three mutants of COX15, ATP25 and CYC3 encoding a cytochrome oxidase assembly factor (singleton), a transcription factor required for assembly of the Atp9p subunit of mitochondrial ATP synthase and cytochrome c heme lyase, respectively, as mutants lacking growth capability on xylose and/or arabinose. They exhibited incapability of growth on non-fermentable carbon sources, such as acetate or glycerol, and thermosensitiveness. Their biomass formation in glucose medium was reduced, but ethanol yields were increased with a high ethanol level in the medium, compared to those of the parental strain. Experiments with respiratory inhibitors showed that cox15 and cyc3, but not atp25, were able to grow in glucose medium containing antimycin A and that the atp25 mutant was KCN-resistant. Activities of NADH and ubiquinol oxidases in membrane fractions of each mutant became a half of that of the parent and negligible, respectively, and their remaining NADH oxidase activities were found to be resistant to KCN. Absolute absorption spectral analysis revealed that the peak corresponding to a + a 3 was very small in atp25 and negligible in cox15 and cyc3. These findings suggest that the K. marxianus strain possesses an alternative KCN-resistant oxidase that is located between primary dehydrogenases and the ubiquinone pool and that the respiratory activity is essential for utilization of pentoses.
通过在 Kluyveromyces marxianus DMKU 3-1042 中用卡那霉素抗性基因盒进行随机整合诱变，我们得到了三个编码细胞色素氧化酶组装因子（单体）、线粒体 ATP 合酶 Atp9p 亚基组装所需的转录因子和细胞色素 c 血红素裂合酶的 COX15、ATP25 和 CYC3 的突变体，它们作为突变体缺乏在木糖和/或阿拉伯糖上生长的能力。它们表现出在非发酵碳源（如乙酸盐或甘油）上生长的能力下降，以及热敏性。与亲本菌株相比，它们在葡萄糖培养基中的生物量形成减少，但在培养基中乙醇水平较高时，乙醇产量增加。与呼吸抑制剂的实验表明，cox15 和 cyc3，但不是 atp25，能够在含有抗霉素 A 的葡萄糖培养基中生长，并且 atp25 突变体对 KCN 具有抗性。每个突变体的膜部分中的 NADH 和泛醌氧化酶的活性分别变为亲本的一半和可忽略不计，并且它们剩余的 NADH 氧化酶活性被发现对 KCN 具有抗性。绝对吸收光谱分析表明，在 atp25 中，与 + a 3 对应的峰非常小，在 cox15 和 cyc3 中可忽略不计。这些发现表明，K. marxianus 菌株具有一种替代的 KCN 抗性氧化酶，它位于初级脱氢酶和泛醌库之间，呼吸活性对于戊糖的利用是必需的。
