Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS

ACS Applied Bio Materials Pub Date : 2024-06-01 Epub Date: 2024-02-28 DOI:10.1007/s10529-024-03466-3

Weixian Wang, Minghai Han, Guofei Zhu, Xiaohui Liu, Tianming Zhao, Xiaoyan Ma, Xun Gong, Cunbin Xu

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Abstract

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 Zeo^R 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.

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通过 Cre/loxP 系统回收可选择标记，构建共同表达多种蛋白质的 Komagataella phaffii 菌株。

目的利用Cre/loxP系统构建可循环选择性标记，构建共表达多种蛋白的Komagataella phaffii菌株：结果：该策略中的质粒由整合了lox71-Sh ble-lox66的pPICZαA产生。首先，质粒插入一个目的蛋白基因，然后转化到 K. phaffii KM71 中。其次，将含有 CRE 重组酶基因的辅助质粒 pPICZαA/cre/his4 进一步染色体插入其中的 Sh ble 基因。最后，通过甲醇诱导产生用于 Cre/loxP 介导重组的 CRE，从而删除 lox71 和 lox66 之间的序列，实现 ZeoR 和 His- 标记的循环。然后，以表达一种目的蛋白的菌株为宿主，用同样的方法插入另一种目的蛋白基因：结论：该方法操作简便，能有效回收可选择标记，从而使两个蛋白质基因在染色体上依次整合，并成功地在酵母中共同表达。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

ACS Applied Bio Materials Chemistry-Chemistry (all)

CiteScore

9.40

自引率

2.10%

发文量

464