Research Institute for Innovations in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Yeast. 2010 Nov;27(11):905-17. doi: 10.1002/yea.1794.
The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast.
南极假丝酵母（Pseudozyma antarctica）产生大量的糖脂生物表面活性剂，称为甘露糖基赤藓糖醇脂（MELs），其不仅具有优异的表面活性，而且具有多种生化作用。为了研究酵母中 MEL 的生物合成，我们最近报道了表达序列标签（EST）分析，并估计了在 MEL 生产条件下表达的基因。在这些基因中，一个 938bp 的连续序列 PA_004 与编码 Ustilago maydis 赤藓糖醇/甘露糖转移酶的基因 emt1 具有高度的序列同一性，该基因对于 MEL 生物合成是必需的。包含两个内含子和一个终止密码子的扩展 PA_004 的预测翻译产物与 Ustilago maydis 的 Emt1 对齐。预测的氨基酸序列与 Ustilago maydis 的 Emt1 具有高度的同一性（72%），尽管氨基端不完整。为了确定该基因是编码南极假丝酵母赤藓糖醇/甘露糖转移酶的 PaEMT1，我们通过靶向基因敲除的方法开发了基因敲除菌株，以潮霉素 B 抗性作为选择标记。获得的ΔPaEMT1 菌株未能产生 MELs，而其生长与亲本菌株相同。在培养物中添加额外的甘露糖基赤藓糖醇，使ΔPaEMT1 菌株能够形成 MELs，而不管提供的碳源如何，这表明赤藓糖醇/甘露糖转移酶活性存在缺陷。此外，我们发现 MEL 的形成与酵母的形态和耐低温性有关。
