文献类型：期刊文献

英文题名：A modified method of gene disruption in Komagataella phaffii with Cre/ loxP system

作者：Han, Minghai Wang, Weixian Gong, Xun Zhu, Guofei Liu, Xiaohui Yu, Zhihai Zhou, Jianli Ma, Chao Ma, Xiaoyan

第一作者：Han, Minghai

通信作者：Han, MH[1]|[144402eaf094eaf27b6d2]韩铭海;

机构：[1]Guizhou Inst Technol, Coll Food & Pharmaceut Engn, Caiguan Rd 1, Guiyang 550000, Peoples R China

第一机构：贵州理工学院食品药品制造工程学院

通信机构：corresponding author), Guizhou Inst Technol, Coll Food & Pharmaceut Engn, Caiguan Rd 1, Guiyang 550000, Peoples R China.|贵州理工学院食品药品制造工程学院;贵州理工学院;

年份：2022

卷号：347

起止页码：40-48

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：20220811703099);Scopus(收录号：2-s2.0-85125018512);WOS:【SCI-EXPANDED(收录号:WOS:000766104600005)】；

基金：This study was funded by the National Natural Science Foundation of China (Grant No. 31760022) and by Guizhou Provincial Science and Technology Foundation (Grant No. [2020] 1Y148) .

语种：英文

外文关键词：Komagataella phaffii; Pichia pastoris; Gene disruption; Marker rescue; Cre; loxP

摘要：To increase protein production, technologies of gene manipulation for engineering the yeast Komagataella phaffii are extensively exploited. In this study, we developed a convenient gene disruption method in the yeast via Cre/ loxP system. First, the simple gene disruption cassette [upstream homologous region (UP)-lox71-Sh ble-lox66downstream homologous region (DW)] was constructed and transformed into the yeast to replace target gene. Second, the Sh ble gene of the cassette integrated in the chromosome was inserted with the auxiliary plasmid pPICZ alpha A/cre/his4, resulting in an expanded cassette of UP-lox71-Sh ble-pPICZ alpha A/cre/his4-lox66-DW. The auxiliary plasmid was generated via sequential insertion of cre and his4 genes into pPICZ alpha A, and linearized with SmaI before its transformation. Finally, for deletion of the sequence between lox71 and lox66 sites in the expanded cassette, CRE protein responsible for Cre/loxP-mediated recombination was produced by methanol induction. Consequently, the corresponding sequence was eliminated permanently, only leaving a scar of lox72 site in the disrupted genes. This strategy was verified by disrupting two genes in the yeast. As the markers were recycled, it was also suitable for multiple gene disruption.