PT  - JOURNAL ARTICLE
AU  - Luiza Cesca Piva
AU  - Janice Lisboa De Marco
AU  - Lidia Maria Pepe de Moraes
AU  - Viviane Castelo Branco Reis
AU  - Fernando Araripe Gonçalves Torres
TI  - Cloning and functional characterization of <em>Komagataella phaffii</em> centromeres by a color-based plasmid stability assay
AID  - 10.1101/433417
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 433417
4099  - http://biorxiv.org/content/early/2019/10/10/433417.short
4100  - http://biorxiv.org/content/early/2019/10/10/433417.full
AB  - 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 genetic stability in K. phaffii. Plasmids bearing K. phaffii centromeres and the ADE3 marker were evaluated in terms of mitotic stability in an ade2/ade3 auxotrophic strain which allows plasmid screening through colony color. Plasmid copy number was verified through qPCR. Our results confirmed that the centromeric plasmids were maintained at low copy number as a result of typical chromosome-like segregation during cell division. These features, combined with high transformation efficiency and in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.