School of Bioscience, University of Sheffield, Sheffield, UK.
Future address: Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia.
Yeast. 2023 Nov;40(11):550-564. doi: 10.1002/yea.3902. Epub 2023 Oct 23.
Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.
汉逊德巴利酵母是一种具有相当生物技术潜力的酵母，它是一种耐渗透压、耐应激的油脂微生物。然而，靶向基因组修饰工具是有限的，需要带有营养缺陷型标记的菌株。同源重组的基因靶向已被报道效率不高，但在这里我们描述了一组试剂和一种方法，可在野生型分离物中高效进行基因靶向。它使用简单的聚合酶链反应（PCR）扩增，该扩增将完全异源的选择性标记物延伸 50bp 侧翼序列，与基因组中的靶位点完全相同。转化体通过同源重组高频（>75%）整合 PCR 产物。我们通过高效地破坏基因和从安全的染色体栖位表达异源蛋白来说明该方法的潜力。这些方法应该刺激和促进对汉逊德巴利酵母菌株的进一步分析，并为生物技术工程菌株开辟道路。
