TY - JOUR

T1 - Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains.

AU - Макеева, Анастасия Станиславовна

AU - Музаев, Дмитрий Михайлович

AU - Шуберт, Мария Андреевна

AU - Яньшина, Татьяна Михайловна

AU - Сидорин, Антон Витальевич

AU - Самбук, Елена Викторовна

AU - Румянцев, Андрей Михайлович

AU - Падкина, Марина Владимировна

PY - 2023/9/12

Y1 - 2023/9/12

N2 - 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 MutS 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 MutS 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.

AB - 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 MutS 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 MutS 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.

KW - Komagataella phaffii

KW - Neo-2/15

KW - Pichia pastoris

KW - heterologous protein production

KW - multicopy strains

KW - split markers

UR - https://www.mendeley.com/catalogue/d2f51c0f-6586-3a2d-8bd7-c68e9c31778d/

U2 - 10.3390/microorganisms11092297

DO - 10.3390/microorganisms11092297

M3 - Article

VL - 11

JO - Microorganisms

JF - Microorganisms

SN - 2076-2607

IS - 9

M1 - 2297

ER -