Construction and characterization of centromeric plasmids for Komagataella phaffii using a color-based plasmid stability assay.

Luiza Cesca Piva,Lidia Maria Pepe de Moraes,Janice Lisboa De Marco,Fernando Araripe Gonçalves Torres,Viviane Castelo Branco Reis,Cecile Fairhead

doi:10.1371/journal.pone.0235532

Luiza Cesca Piva, Lidia Maria Pepe de Moraes + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0235532

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Journal: PloS one	Publication Date: Jul 2, 2020
Citations: 5	License type: CC BY 4.0

Affiliation: Universidade de Brasília

Abstract

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.

Highlights

Komagataella phaffii is a methylotrophic yeast of great industrial importance that has been used for more than 30 years as a heterologous protein production platform [1]
In order to expand the molecular toolbox used in K. phaffii genetic manipulation, in this study we developed a genetic system based on an ade2/ade3 auxotrophic strain and a replicative vector carrying the wild-type ADE3 gene
The adenine synthesis pathway is used as a tool for auxotrophic selection, gene copy number indication, and for plasmid stability analyses [32]

Summary

Introduction

Komagataella phaffii is a methylotrophic yeast of great industrial importance that has been used for more than 30 years as a heterologous protein production platform [1]. Vectors carrying K. phaffii centromeres were constructed and transformed to assess plasmid copy number and mitotic stability. The maps for pPICH-ADE3 and all centromeric plasmids constructed in this work are present on S1 Fig. pPICH-ADE3 was digested with BamHI for the cloning of all four K. phaffii centromeres.

Results

Conclusion