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  - 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/2019/01/10/433417.short
4100  - http://biorxiv.org/content/early/2019/01/10/433417.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 each centromere 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.Author summary The methylotrophic yeast Komagataella phaffii is considered as one of the most important platforms for the production of proteins and metabolites. We sought in this study to develop a color-based genetic system widely used in other yeasts to assess mitotically stability of vectors carrying the proposed K. phaffii centromeres. First, we constructed a K. phaffii strain (LA3) mutant for ADE2 and ADE3; this resulted in a strain that forms white colonies and when transformed with a vector (pPICH-ADE3) carrying ADE3 turns red. Next, the four K. phaffii centromeres were cloned into pPICH-ADE3 and tested in LA3 for copy number and plasmid stability. Centromeres are responsible for proper chromosome segregation during cell division, hence guaranteeing that both daughter cells receive one copy of the duplicated DNA. Our results show that three K. phaffii centromeres behaved as expected conferring extra stability to the replicative plasmids and maintaining them at low copy number. Once characterized, centromeres can be used as parts in the construction of advanced genetic manipulation tools, thus allowing the construction of strains capable of expressing large metabolic pathways for the production of complex biochemicals.