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Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains

Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to...

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Autores principales: Makeeva, Anastasiya, Muzaev, Dmitry, Shubert, Maria, Ianshina, Tatiana, Sidorin, Anton, Sambuk, Elena, Rumyantsev, Andrey, Padkina, Marina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10536657/
https://www.ncbi.nlm.nih.gov/pubmed/37764140
http://dx.doi.org/10.3390/microorganisms11092297
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author Makeeva, Anastasiya
Muzaev, Dmitry
Shubert, Maria
Ianshina, Tatiana
Sidorin, Anton
Sambuk, Elena
Rumyantsev, Andrey
Padkina, Marina
author_facet Makeeva, Anastasiya
Muzaev, Dmitry
Shubert, Maria
Ianshina, Tatiana
Sidorin, Anton
Sambuk, Elena
Rumyantsev, Andrey
Padkina, Marina
author_sort Makeeva, Anastasiya
collection PubMed
description Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate Mut(S) strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii Mut(S) strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes.
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spelling pubmed-105366572023-09-29 Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains Makeeva, Anastasiya Muzaev, Dmitry Shubert, Maria Ianshina, Tatiana Sidorin, Anton Sambuk, Elena Rumyantsev, Andrey Padkina, Marina Microorganisms Article Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate Mut(S) strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii Mut(S) strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes. MDPI 2023-09-12 /pmc/articles/PMC10536657/ /pubmed/37764140 http://dx.doi.org/10.3390/microorganisms11092297 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Makeeva, Anastasiya
Muzaev, Dmitry
Shubert, Maria
Ianshina, Tatiana
Sidorin, Anton
Sambuk, Elena
Rumyantsev, Andrey
Padkina, Marina
Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains
title Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains
title_full Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains
title_fullStr Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains
title_full_unstemmed Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains
title_short Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains
title_sort alternative pcr-based approaches for generation of komagataella phaffii strains
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10536657/
https://www.ncbi.nlm.nih.gov/pubmed/37764140
http://dx.doi.org/10.3390/microorganisms11092297
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