A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production

Frontiers in Microbiology
Jan-Philipp SchwarzhansKarl Friehs

Abstract

Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an autonomously replicating sequence (ARS). Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named "pMito" consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displaye...Continue Reading

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Citations

Nov 19, 2017·Scientific Reports·Kate E Royle, Karen Polizzi
May 29, 2018·Applied and Environmental Microbiology·Yasuyuki NakamuraAkihiko Kondo
Jan 29, 2021·Frontiers in Microbiology·Lukas BernauerAnita Emmerstorfer-Augustin

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Datasets Mentioned

BETA
CBS7435
FR839632
LT724168

Methods Mentioned

BETA
fluorescence microscopy
PCR
confocal microscopy
electrophoresis
Fluorescence

Software Mentioned

Find Individual Motif Occurrences ( FIMO )
BLASTn
MEME
CONSED
GS De Novo Assembler
pMito
softWoRx
FIMO

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