New Yarrowia lipolytica strains for the co-expression of steroidogenic mammalian proteins were obtained in this study. For this purpose, a two-step approach for constructing recombinant strains that permits the simple introduction of several expression cassettes encoding heterologous proteins into the yeast genome was successfully applied. This study tested two series of integrative multi-copy expression vectors containing cDNAs for the mature forms of P450scc system components (cytochrome P450scc (CYP11A1), adrenodoxin reductase, adrenodoxin, or fused adrenodoxin-P450scc) or for P45017α (CYP17A1) under the control of the isocitrate lyase promoter pICL1, which were constructed using the basic plasmids p64PT or p67PT (rDNA or the long terminal repeat (LTR) zeta of Ylt1 as integration targeting sequences and ura3d4 as a multi-copy selection marker). This study demonstrated the integration of up to three expression vectors containing different heterologous cDNA via their simultaneous transformation into haploid recipient strains. Additionally, further combinations of the different expression cassettes in one strain were obtained by subsequent diploidisation using selected haploid multi-copy transformants. Thus, recombinant strains containing three to five different expression cassettes were obtained, as demonstrated by Southern blotting. Expression of P450scc system proteins was identified by western blotting. The presented method for recombinant strain construction is a useful tool for the heterologous expression of multi-component enzyme systems in Y. lipolytica.

在该研究中获得了用于共表达类固醇生成哺乳动物蛋白质的新解脂耶氏酵母菌株。为此，成功应用了构建重组菌株的两步方法，该方法允许将几种编码异源蛋白质的表达盒简单地引入酵母基因组。本研究测试了两个系列的整合多拷贝表达载体，其中包含成熟形式的 P450scc 系统成分（细胞色素 P450scc (CYP11A1)、肾上腺素氧还蛋白还原酶、肾上腺素氧还蛋白或融合的肾上腺素氧还蛋白-P450scc）或 P45017α (CYP17A1) 控制下的 cDNA。异柠檬酸裂解酶启动子 pICL1，使用基本质粒 p64PT 或 p67PT（rDNA 或长末端重复序列 (LTR) zetaYlt1 作为整合靶向序列，ura3d4作为多拷贝选择标记）。该研究表明，通过同时转化到单倍体受体菌株中，最多可整合三种含有不同异源 cDNA 的表达载体。此外，通过随后使用选定的单倍体多拷贝转化体进行二倍体化，获得了一种菌株中不同表达盒的进一步组合。因此，如Southern印迹所证明的，获得了含有三到五个不同表达盒的重组菌株。P450scc 系统蛋白的表达通过蛋白质印迹鉴定。所提出的重组菌株构建方法是多组分酶系统异源表达的有用工具解脂耶氏菌。