文献类型：期刊文献

英文题名：Genome-Wide Determination of Gene Essentiality by Transposon Insertion Sequencing in Yeast Pichia pastoris

作者：Zhu, Jinxiang[1];Gong, Ruiqing[1];Zhu, Qiaoyun[1];He, Qiulin[1];Xu, Ning[1];Xu, Yichun[1];Cai, Menghao[1];Zhou, Xiangshan[1];Zhang, Yuanxing[1,2];Zhou, Mian[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]SCICB, Shanghai 200237, Peoples R China

年份：2018

卷号：8

外文期刊名：SCIENTIFIC REPORTS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000437414500009)】；

基金：We gratefully acknowledge Prof. Li-Lin Du (National Institute of Biological Sciences, Beijing, China), Prof. Henry L. Levin (Eunice Kennedy Shriver National Institute of Child Health and Human Development) and Prof. Matthew K. Waldor (Department of Microbiology, Harvard Medical School, Boston, Massachusetts, United States of America), for helpful advices. We also thank Prof. Nancy L. Craig (Johns Hopkins University School of Medicine, Baltimore, MD), Zsuzsanna Izsvak (Max Delbruck Center for Molecular Medicine, Berlin, Germany) and Thomas Vogl (Institute for Molecular Biotechnology, Graz University of Technology) for providing TcBuster plasmids, Sleeping Beauty plasmids, a CRISPR/Cas9 plasmid, respectively. This work was sponsored by National Natural Science Foundation of China [31600056]; the Fundamental Research Funds for the central Universities [222201714021] and Shanghai Chenguang Program [15CG27].

语种：英文

摘要：In many prokaryotes but limited eukaryotic species, the combination of transposon mutagenesis and high-throughput sequencing has greatly accelerated the identification of essential genes. Here we successfully applied this technique to the methylotrophic yeast Pichia pastoris and classified its conditionally essential/non-essential gene sets. Firstly, we showed that two DNA transposons, TcBuster and Sleeping beauty, had high transposition activities in P. pastoris. By merging their insertion libraries and performing Tn-seq, we identified a total of 202,858 unique insertions under glucose supported growth condition. We then developed a machine learning method to classify the 5,040 annotated genes into putatively essential, putatively non-essential, ambig1 and ambig2 groups, and validated the accuracy of this classification model. Besides, Tn-seq was also performed under methanol supported growth condition and methanol specific essential genes were identified. The comparison of conditionally essential genes between glucose and methanol supported growth conditions helped to reveal potential novel targets involved in methanol metabolism and signaling. Our findings suggest that transposon mutagenesis and Tn-seq could be applied in the methylotrophic yeast Pichia pastoris to classify conditionally essential/non-essential gene sets. Our work also shows that determining gene essentiality under different culture conditions could help to screen for novel functional components specifically involved in methanol metabolism.