Jaafar Lahcen, León Maela, Zueco Jesús
Unidad de Microbiología, Facultad de Farmacia, Universidad de Valencia, Avda. Vicente Andres Estelles s/n, 46100-Burjassot, Valencia, Spain.
Yeast. 2003 May;20(7):633-44. doi: 10.1002/yea.990.
In this work we describe the isolation of the Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN9 gene, which we have named YlMNN9, and the phenotype analysis of a Y. lipolytica strain containing the disrupted YlMNN9 allele. YlMNN9 was cloned using degenerate consensus oligonucleotides to generate specific probes that were in turn used to screen mini-gene libraries. The gene is defined by a 1014 bp ORF predicted to encode a protein 337 amino acids long that shares significant homology with the Mnn9ps of S. cerevisiae, Candida albicans and Hansenula polymorpha, including a putative N-terminal transmembrane domain. Disruption of YlMNN9 leads to phenotypes such as resistance to sodium orthovanadate and sensitivity to hygromycin B, compatible with a glycosylation defect, and hypersensitivity to Calcofluor white, Congo red or zymolyase, characteristic of cell wall defects. Analysis of cell wall proteins present in beta-mercaptoethanol and zymolyase extracts showed significant differences between the parental and the ylmnn9 Delta strain. These results suggest that, as has been the case with the mnn9 strain of S. cerevisiae, the ylmnn9 Delta strain we present in this work, could be used to study the cell wall proteins of Y. lipolytica and how they are organized into the cell wall.
在本研究中，我们描述了酿酒酵母MNN9基因的解脂耶氏酵母同源物的分离，我们将其命名为YlMNN9，并对含有 disrupted YlMNN9等位基因的解脂耶氏酵母菌株进行了表型分析。使用简并共有寡核苷酸克隆YlMNN9，以生成特定探针，这些探针又用于筛选小基因文库。该基因由一个1014 bp的开放阅读框定义，预计编码一个337个氨基酸长的蛋白质，该蛋白质与酿酒酵母、白色念珠菌和多形汉逊酵母的Mnn9ps具有显著同源性，包括一个推定的N端跨膜结构域。YlMNN9的破坏导致对原钒酸钠的抗性和对潮霉素B的敏感性等表型，这与糖基化缺陷一致，并且对荧光增白剂、刚果红或溶菌酶超敏，这是细胞壁缺陷的特征。对存在于β-巯基乙醇和溶菌酶提取物中的细胞壁蛋白的分析表明，亲本菌株和ylmnn9Δ菌株之间存在显著差异。这些结果表明，正如酿酒酵母的mnn9菌株一样，我们在本研究中展示的ylmnn9Δ菌株可用于研究解脂耶氏酵母的细胞壁蛋白以及它们如何组织到细胞壁中。
