Shibui Tatsuro, Hara Hiroyoshi
Food Biotechnology Laboratory, School of Food Sciences, Nippon Veterinary and Life Science University, 1-7-1 Kyounamcho, Musashinoshi, Tokyo, 180-8602, Japan.
Biotechnol Prog. 2017 Sep;33(5):1201-1208. doi: 10.1002/btpr.2541. Epub 2017 Sep 4.
Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017.
巴斯德毕赤酵母已被用于生产多种重组蛋白，并且已构建了许多有用的突变菌株。然而，通过基因靶向分离突变体的效率通常较低，对于那些缺乏酵母遗传学经验的人来说，该过程也很困难。为了克服这些问题，我们开发了一种新的基因破坏系统，该系统使用可诱导的Cre/突变型loxP系统和一个拯救基因。该系统的基因破坏盒仅具有短同源区域，就能用一个补偿性拯救基因取代其含有突变的靶基因位点。由于该盒含有AOX1启动子驱动的Cre基因，当靶向菌株在含有甲醇的培养基上生长时，盒中突变型loxP序列之间的DNA片段，即标记基因、拯救基因和Cre基因，会被切除，基因组中仅留下剩余的突变型loxP序列，因此可以分离出靶基因被破坏的突变体。该系统最初在ADE2基因破坏实验中得到验证，在该实验中，通过菌落颜色变化可以很容易地检测到基因破坏。然后，该系统被应用于敲除URA3和OCH1基因，据报道，传统的基因靶向方法很难实现这一点。所有三个带有拯救基因的基因破坏盒都取代了它们的靶基因，并且Cre/突变型loxP系统运行良好，成功分离出了它们的敲除突变体。本研究确定了一种新的基因破坏系统，该系统可用于有效且策略性地敲除感兴趣的基因，特别是那些缺失对生长有害的基因，而无需在巴斯德毕赤酵母中使用特殊菌株，例如缺乏非同源末端连接的菌株。© 2017美国化学工程师学会 生物技术进展，33:1201 - 1208，2017。
