A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material
Abstract Background Komagataella phaffii (Pichia pastoris) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being ex...
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| Format: | Članak |
| Jezik: | English |
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BMC
2023-12-01
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| Serija: | Microbial Cell Factories |
| Teme: | |
| Online pristup: | https://doi.org/10.1186/s12934-023-02262-4 |
| _version_ | 1827581320292728832 |
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| author | Dariusz Abramczyk Maria del Carmen Sanchez Olmos Adan Andres Ramirez Rojas Daniel Schindler Daniel Robertson Stephen McColm Adele L. Marston Paul N. Barlow |
| author_facet | Dariusz Abramczyk Maria del Carmen Sanchez Olmos Adan Andres Ramirez Rojas Daniel Schindler Daniel Robertson Stephen McColm Adele L. Marston Paul N. Barlow |
| author_sort | Dariusz Abramczyk |
| collection | DOAJ |
| description | Abstract Background Komagataella phaffii (Pichia pastoris) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers. Results Here, we describe a linear “nano”chromosome (of 15–25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of P AOX1 and demonstrated that the secreted protein was active as a regulator of the complement system. Conclusions We have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain. |
| first_indexed | 2024-03-08T22:34:42Z |
| format | Article |
| id | doaj.art-d96f7342d724424bbcf9697fae49fd08 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-03-08T22:34:42Z |
| publishDate | 2023-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-d96f7342d724424bbcf9697fae49fd082023-12-17T12:34:14ZengBMCMicrobial Cell Factories1475-28592023-12-0122111910.1186/s12934-023-02262-4A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic materialDariusz Abramczyk0Maria del Carmen Sanchez Olmos1Adan Andres Ramirez Rojas2Daniel Schindler3Daniel Robertson4Stephen McColm5Adele L. Marston6Paul N. Barlow7School of Chemistry, University of EdinburghMax Planck Institute for Terrestrial MicrobiologyMax Planck Institute for Terrestrial MicrobiologyMax Planck Institute for Terrestrial MicrobiologySchool of Biological Sciences, University of EdinburghIngenza Ltd ScotlandThe Wellcome Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of EdinburghSchool of Chemistry, University of EdinburghAbstract Background Komagataella phaffii (Pichia pastoris) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers. Results Here, we describe a linear “nano”chromosome (of 15–25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of P AOX1 and demonstrated that the secreted protein was active as a regulator of the complement system. Conclusions We have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain.https://doi.org/10.1186/s12934-023-02262-4Pichia pastorisYeastBiomanufactureArtificial chromosomeTherapeutic proteinsComplement factor H |
| spellingShingle | Dariusz Abramczyk Maria del Carmen Sanchez Olmos Adan Andres Ramirez Rojas Daniel Schindler Daniel Robertson Stephen McColm Adele L. Marston Paul N. Barlow A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material Microbial Cell Factories Pichia pastoris Yeast Biomanufacture Artificial chromosome Therapeutic proteins Complement factor H |
| title | A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material |
| title_full | A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material |
| title_fullStr | A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material |
| title_full_unstemmed | A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material |
| title_short | A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material |
| title_sort | supernumerary synthetic chromosome in komagataella phaffii as a repository for extraneous genetic material |
| topic | Pichia pastoris Yeast Biomanufacture Artificial chromosome Therapeutic proteins Complement factor H |
| url | https://doi.org/10.1186/s12934-023-02262-4 |
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