Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris
| Autor: | Xinying Zhang, Xueyun Zheng, Qingyan Yuan, Ying Lin, Xihao Liao, Nuo Pang, Cheng Li, Yuanyuan Huang, Shuli Liang |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Genetic Markers Genetic Vectors Gene Dosage Heterologous Gene Expression lcsh:Medicine Pichia Article Pichia pastoris 03 medical and health sciences Plasmid Gene Order Vector (molecular biology) Lipase lcsh:Science Selectable marker Genetics Recombination Genetic 6-Phytase Multidisciplinary biology lcsh:R biology.organism_classification 030104 developmental biology Xylosidases Biochemistry Genetic marker biology.protein Xylanase lcsh:Q Plasmids |
| Zdroj: | Scientific Reports, Vol 7, Iss 1, Pp 1-10 (2017) Scientific Reports |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-017-11494-5 |
| Popis: | Pichia pastoris is a widely used heterologous protein production workhorse. However, with its multiple genetic modifications to solve bottlenecks for heterologous protein productivity, P. pastoris lacks selectable markers. Existing selectable marker recycling plasmids have drawbacks (e.g., slow growth and conditional lethality). Here, zeocin-resistance marker recycling vectors were constructed using the Cre/loxP recombination system. The vectors were used to (i) knock in heterologous phytase, xylanase and lipase expression cassettes, (ii) increase the phytase, xylanase and lipase gene copy number to 13, 5, and 5, respectively, with vector introduction and (iii) engineer the secretion pathway by co-overexpressing secretion helper factors (Sly1p and Sec1p) without introducing selectable markers, giving a phytase field of 0.833 g/L. The vectors allow selectable marker recycling and would be a useful tool to engineer P. pastoris for high heterologous protein productivity. |
| Databáze: | OpenAIRE |
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