PiggyBac transposon-mediated mutagenesis and application in yeast Komagataella phaffii
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.
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.
Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.
KeywordsMutagenesis Komagataella phaffii piggyBac Transposon
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.
Supplementary Table 1—Oligonucleotide primers used in this study.
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.
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