Dataset Information

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

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 displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector transformation in Saccharomyces cerevisiae, widening the potential for biotechnological application. pMito displayed strong potential to reduce clonal variability in experiments targeting recombinant protein production. These findings also showcase the as of yet largely untapped potential of mitochondrial ARS from different yeasts for biotechnological applications.

SUBMITTER: Schwarzhans JP

PROVIDER: S-EPMC5411459 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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A Mitochondrial Autonomously Replicating Sequence from <i>Pichia pastoris</i> for Uniform High Level Recombinant Protein Production.

Schwarzhans Jan-Philipp JP Luttermann Tobias T Wibberg Daniel D Winkler Anika A Hübner Wolfgang W Huser Thomas T Kalinowski Jörn J Friehs Karl K

Frontiers in microbiology 20170502

<i>Pichia pastoris</i> 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 ...[more]

PMID: 28512458

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Project description:The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability.IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.

Project description:Yeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the 13C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism.The physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking.This systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion.

Dataset Information

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

Publications

A Mitochondrial Autonomously Replicating Sequence from <i>Pichia pastoris</i> for Uniform High Level Recombinant Protein Production.

Similar Datasets

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