The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

酵母嗜酸小果蝇（Komagataella phaffii）被广泛用作产生异源蛋白质的微生物宿主。但是，用于该酵母的分子工具基本上仅限于一些整合和复制质粒。最近已经提出四大序列ķ。phaffii着丝粒可用于开发一类新的有丝分裂稳定载体。在这项工作中，我们设计了一种基于颜色的遗传检测方法，以研究质粒在K中的稳定性。phaffii和带K的构造向量。phaffii着丝粒和ADE3标记。这些遗传工具通过转化为有丝分裂稳定性来评估ade2 / ade3营养缺陷型菌株，并通过定量PCR（qPCR）考虑质粒拷贝数。我们的结果证实，由于细胞分裂过程中典型的染色体样分离，着丝粒质粒保持低拷贝数。这些特征与体内组装的可能性相结合，促使这些质粒成为K的新成员。phaffii遗传工具箱。