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PiggyBac transposon-mediated mutagenesis and application in yeast Komagataella phaffii

  • Jinxiang Zhu
  • Qiaoyun Zhu
  • Ruiqing Gong
  • Qin Xu
  • Menghao Cai
  • Tianyi Jiang
  • Xiangshan Zhou
  • Mian Zhou
  • Yuanxing Zhang
Original Research Paper

Abstract

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

Keywords

Mutagenesis Komagataella phaffii piggyBac Transposon 

Notes

Acknowledgements

We gratefully acknowledge Prof. Li-Lin Du (National Institute of Biological Sciences, Beijing, China) for helpful suggestions. We thank Prof. Tian Xu (Institute of Developmental Biology and Molecular Medicine School of Life Sciences Fudan University, Shanghai, China) and the Sanger Institute Archives (http://www.sanger.ac.uk/technology/clonerequests/) for providing piggyBac plasmids (pCMV-PBase and PB[SV40-neo]) and hyPBase expression vector (pCMV-hyPBase), respectively. We also thank Prof. James M. Cregg (Keck Graduate Institute of Applied Life Science) for providing JC303 strain.

Supporting information

Supplementary Table 1—Oligonucleotide primers used in this study.

Funding

This work was sponsored by National Natural Science Foundation of China (31600056); the Fundamental Research Funds for the central Universities (222201714021); Shanghai Chenguang Program (15CG27).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10529_2018_2592_MOESM1_ESM.docx (22 kb)
Supplementary material 1 (DOCX 22 kb)

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiPeople’s Republic of China
  2. 2.Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB)ShanghaiPeople’s Republic of China
  3. 3.Roche R&D Center (China) Ltd.ShanghaiPeople’s Republic of China

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