A Novel Vector for Construction of Markerless Multicopy Overexpression Transformants in Pichia pastoris.
A Novel Vector for Construction of Markerless Multicopy Overexpression Transformants in Pichia pastoris.
复制标题
构建毕赤酵母无标记多拷贝过表达转化子的新载体
DOI：
10.3389/fmicb.2017.01698
复制
发表时间：
2017
影响因子：
5.2
通讯作者：
Cui Z
中科院分区：
文献类型：
--
作者：
Li D;Zhang B;Li S;Zhou J;Cao H;Huang Y;Cui Z
毕赤酵母因其高容量生产力而被广泛用作异源蛋白表达的平台。靶基因的多拷贝整合通常用于提高靶蛋白的生产。Cre/lox重组系统是一种强大的工具，可以在一个选择标记的多个集成过程中拯救标记。本文报道了一种基于Cre/lox重组系统的新型表达载体，用于靶基因的多次整合，构建了多拷贝表达菌株。将PAOX1启动子与cre融合，构建甲醇诱导的cre重组酶。通过引入操作基因lacO，阻断了Cre重组酶在大肠杆菌中的漏表达。所设计的表达载体pMCO-AOXα在大肠杆菌中稳定，能有效地挽救Zeocin耐药基因，为下一轮在帕斯德酵母中的整合提供依据。选择大肠杆菌植酸酶AppA作为报告基因。用一种抗生素构建具有2-16个appA拷贝的转化子。当整合<12拷贝时，appA的表达与基因剂量有关。与单拷贝整合相比，整合12个拷贝的appA蛋白产量提高了4.45倍。结果表明，pMCO-AOXα可有效地合理构建pastoris的多拷贝转化。
Pichia pastoris is widely used as a platform for heterologous protein expression because of its high volumetric productivity. Multicopy integration of the target gene is commonly used to improve the production of the target protein. Cre/lox recombination system is a powerful tool for the marker rescue during multiple integrations with one selection marker. Here we reported a novel expression vector based on the Cre/lox recombination system for multiple integrations of target gene to construct multicopy expression strain of P. pastoris. PAOX1 promoter was fused to cre to construct a methanol inducible Cre recombinase. The leakage expression of Cre recombinase in Escherichia coli was blocked by introducing the operator gene lacO. The expression vector designed pMCO-AOXα was stable in E. coli and could effectively rescue the Zeocin resistance gene for next round of integration in P. pastoris. Phytase AppA from E. coli was chosen as a reporter gene. Transformants with 2–16 copies of appA were constructed by using a single antibiotic. Expression of appA was gene dosage dependent when <12 copies were integrated. The protein yield increased 4.45-folds when 12 copies of appA were integrated comparing with the single copy integration. Our results showed that pMCO-AOXα was highly effective for rational construction of multicopy transformat in P. pastoris.