Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
Key Laboratory of Medical Microbiology and Parasitology of Education Department of Guizhou, Guizhou Medical University, Guiyang, China.
Yeast. 2020 Sep;37(9-10):505-513. doi: 10.1002/yea.3467. Epub 2020 Apr 28.
Nonconventional yeast Candida famata and Ogataea polymorpha are interesting organisms for basic and applied studies. O. polymorpha is methylotrophic thermotolerant yeast capable of xylose alcoholic fermentation whereas C. famata is capable of riboflavin overproduction. Still, the new tools for molecular research of these species are needed. The aim of this study was to develop the new dominant selective markers for C. famata and O. polymorpha usable in metabolic engineering experiments. In this work, the BSD gene from Aspergillus terreus coding for blasticidin S deaminase, O. polymorpha AUR1 gene required for sphingolipid synthesis and IMH3 gene, which encodes IMP dehydrogenase, were tested as the new dominant selective marker genes. Our results showed that AUR1 and IMH3 genes could be used as dominant selective markers for O. polymorpha with frequencies of transformation of 40 and 20 transformants per microgram of DNA, respectively. The IMH3 gene was successfully used as the marker for construction of O. polymorpha strains with increased ethanol production from xylose due to overexpression of TAL1, TKL1 and AOX1 genes. The BSD gene from A. terreus, conferring resistance to blasticidin, was found to be efficient for selection of C. famata transformants.
非传统酵母 Candida famata 和 Ogataea polymorpha 是基础和应用研究的有趣生物体。O. polymorpha 是一种甲醇营养型耐热酵母，能够进行木糖酒精发酵，而 C. famata 能够过量生产核黄素。尽管如此，仍需要新的工具来进行这些物种的分子研究。本研究旨在为 C. famata 和 O. polymorpha 开发新的优势选择标记，可用于代谢工程实验。在这项工作中，我们测试了来自 Aspergillus terreus 的 BSD 基因（编码博来霉素 S 脱氨酶）、O. polymorpha 中用于合成鞘脂的 AUR1 基因和编码 IMP 脱氢酶的 IMH3 基因作为新的优势选择标记基因。结果表明，AUR1 和 IMH3 基因可分别作为 O. polymorpha 的优势选择标记基因，转化频率分别为每微克 DNA 40 和 20 个转化体。IMH3 基因成功地用于构建 O. polymorpha 菌株，通过过量表达 TAL1、TKL1 和 AOX1 基因，从木糖生产提高乙醇产量。来自 A. terreus 的 BSD 基因赋予博来霉素抗性，被发现可有效地选择 C. famata 转化体。
