Identification and cloning of C. albicans SC5314 genes encoding L-methionine biosynthetic pathway enzymes.

Enzymes of fungal L-methionine biosynthetic pathway: homoserine O-acetyltransferase (Met2p), O-acetylhomoserine sulfhydrylase (Met15p) and cystathionine-γ-synthase (Str2p) could be exploited as molecular targets for antifungal chemotherapy. The goal of the study was to identify and clone genes encoding mentioned above enzymes. MET2, MET15 and STR2 genes were identified with the use of Candida Genome Database (http://www.candidagenome.org/). Amplifications of genes were carried out on C. albicans SC5314 genomic DNA with the use of polymerase chain reaction (PCR) and appropriate designed primers. PCR products that give satisfying molecular weight were selected and then cloned in a vector for its maintenance to produce the target protein. The constructed expression plasmids were designed in three different versions: encoding a native protein or recombinants with oligo-His tags added either at the C- or N-end. Introduction of fusion domains would facilitate future isolation of the gene products via metal-affinity chromatography. The additional oligo-His domains were introduced by PCR with the help of appropriate designed primers. A direct mutagenesis was carried out in a case of the plasmid containing MET2 gene because it contains a codon CTG that is translated differently in bacteria and yeast cells. In C. albicans it encodes L-Serine, whereas in E. coli cells L-Leucine.This dataset contains the description of methodology, used primers sequences and three image files with the results of agarose gel electrophoresis of obtained PCR products, agarose gel electrophoresis of isolated plasmids DNA and restriction analysis.The studies were financially supported from the project OPUS 20 financed by the National Science Centre (No. UMO-2020/39/B/NZ7/01519).

真菌L-甲硫氨酸生物合成途径中的三类酶可作为抗真菌化疗的分子靶点，分别为高丝氨酸O-乙酰基转移酶（homoserine O-acetyltransferase，Met2p）、O-乙酰高丝氨酸巯基解酶（O-acetylhomoserine sulfhydrylase，Met15p）以及胱硫醚-γ-合酶（cystathionine-γ-synthase，Str2p）。本研究的目标为鉴定并克隆编码上述酶的基因。研究人员借助念珠菌基因组数据库（Candida Genome Database，http://www.candidagenome.org/）成功鉴定出MET2、MET15及STR2基因。以白色念珠菌SC5314的基因组DNA为模板，通过聚合酶链式反应（polymerase chain reaction，PCR）及定制引物完成基因扩增。选取分子量符合预期的PCR产物，将其克隆至载体中以维持序列并表达目标蛋白。本研究构建的表达质粒分为三种不同版本：分别编码天然蛋白，或在蛋白的C端、N端添加寡聚组氨酸（oligo-His）标签的重组蛋白。引入融合结构域可便于后续通过金属亲和色谱法分离基因表达产物。额外的寡聚组氨酸结构域通过定制引物介导的PCR反应引入。针对携带MET2基因的质粒需进行定点诱变，因该基因含有CTG密码子，该密码子在细菌与酵母细胞中的翻译方式存在差异：在白色念珠菌中该密码子编码L-丝氨酸，而在大肠杆菌（E. coli）中则编码L-亮氨酸。本数据集包含实验方法说明、所用引物序列，以及三张图像文件，分别为所得PCR产物的琼脂糖凝胶电泳结果、分离得到的质粒DNA的琼脂糖凝胶电泳结果，以及限制性酶切分析结果。本研究由国家科学中心资助的OPUS 20项目（编号UMO-2020/39/B/NZ7/01519）提供经费支持。