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.

A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris

“…Moreover, the cost analysis does not take into account reduced labour costs and those associated with reagent expiry to which restriction cloning and ligation are more susceptible. We hope this research will complement recent efforts to bring a synthetic approach to biopharmaceutical production with Pichia pastoris , such as recently reported work on the restriction site-free cloning vector system for testing different affinity tags and fusion proteins 8 and the generation of episomal expression vectors 10 , 29 .…”

Identification and Characterization of the Mitochondrial Replication Origin for Stable and Episomal Expression in Yarrowia lipolytica

“…Due to the unclear replication mechanism of p-mtORI plasmid, it is possible that copies of p-mtORI were present in the mitochondria. In P. pastoris , the episomal vectors containing a mitochondrial ARS had been proven to present in nucleus through the fluorescence localization analysis . Thus, green fluorescent protein hrGFP gene was expressed in Y. lipolytica by using mtORI and CEN/ARS plasmids, and the intracellular location of recombinant protein was analyzed via fluorescence microscope.…”

“…Mitochondrial origins of ori2, ori3, and ori5 were amplified from the total DNA of S. cerevisiae BY4741. 1443-bp mitochondrial DNA was amplified from the total DNA of P. pastoris GS115 . These mtDNA fragments were assembled with Not I digested pKi-hrGFP to obtain circular plasmids (p-Sce-mtORI2, p-Sce-mtORI3, p-Sce-mtORI5, and p-Ppa-mtORI) containing mtORI sequences from different yeast species.…”

Komagataella phaffii as Emerging Model Organism in Fundamental Research

“…Similar to the S. cerevisiae -specific ARS, PARS1 enabled the high-efficiency transformation of K. phaffii with circular plasmids ( Weninger et al, 2016 ; Pan et al, 2018 ). Recently, a 452 bp ARS element (panARS) was identified in K. lactis and shown to facilitate transformation in a wide range of yeast species, including K. phaffii ( Liachko and Dunham, 2014 ; Camattari et al, 2016 ), and a mitochondrial DNA (mtDNA) fragment was discovered to function as a novel ARS in K. phaffii ( Schwarzhans et al, 2017 ). All plasmids bearing these ARS sequences were found to be poorly stable for replication and segregation in K. phaffii , which restricted their use in many applications.…”