lys1-d缺陷型标记介导毕赤酵母超高拷贝质粒整合

Superhigh-copy plasmid integration in Komagataella phaffii mediated by lys1-d auxotrophic marker

传统构建毕赤酵母质粒多拷贝菌株的方法不仅操作复杂、成本高,而且难以获得理想中的高拷贝菌株.近来,一种利用leu2-d缺陷型标记直接筛选毕赤酵母多拷贝克隆的方法显示出很大的便利.本研究发现,使用一个lys1-d缺陷型标记能更加高效地介导毕赤酵母超高拷贝质粒整合.首先构建一个携带lys1-d和EGFP(enhanced green fluorescent protein)报告基因的整合载体,然后将载体转化至敲除了内源lys1基因的毕赤酵母中,最后分别检测了转化子内EGFP含量和质粒拷贝数.结果显示,所有转化子整合的质粒拷贝数均处于19~168之间;并且转化子内质粒拷贝数越高,其胞内表达的EGFP产量也越高.这一系统为构建毕赤酵母超高拷贝质粒整合菌株增添了有效的工具.

In Komagataella phaffii,traditional methods for constructing the strains containing multicopy plasmid are not only complicated and costly,but also difficult to obtain desired high-copy strains.Recently,a method to screen K.phaffii multicopy clones by leu2-d auxotrophic marker exhibited significant advantage.In this study,we found that a lys1-d auxotrophic marker could mediate K.phaffii superhigh-copy plasmid integration more efficiently.Firstly,a vector carrying lys1-d and EGFP(enhanced green fluorescent protein)reporter gene was constructed.Then,the resulting vector was successfully transformed into the lys1-defective K.phaffii.Finally,the EGFP production and plasmid copy number in transformants were measured,respectively.The results exhibited the copy number in all transformants were between 19 and 168.Moreover,the fluorescence intensity was increased with the increase of plasmid copy number.This system provides an efficient tool for constructing K.phaffii superhigh-copy plasmid integrated strains.