Biotechnology letters · 2024
Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.
СтатьяНазвание и аннотация: Перевод в очереди
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Wang Weixian, Han Minghai, Zhu Guofei, Liu Xiaohui, Zhao Tianming, Ma Xiaoyan, Gong Xun, Xu Cunbin
Аннотация
OBJECTIVE: A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.
RESULTS: A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recycling of ZeoR and His- markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures.
CONCLUSIONS: With easy manipulation, the method was effective in recycling of the selectable markers, and consequently two protein genes were sequential integrated chromosomally and successfully co-expressed in the yeast.
RESULTS: A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recycling of ZeoR and His- markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures.
CONCLUSIONS: With easy manipulation, the method was effective in recycling of the selectable markers, and consequently two protein genes were sequential integrated chromosomally and successfully co-expressed in the yeast.