The Construction of Heterothallic Strains of <i>Komagataella kurtzmanii</i> Using the I-SceI Meganuclease
The methylotrophic yeast <i>Komagataella kurtzmanii</i> belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typi...
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2025-01-01
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| author | Daria D. Sokolova Philipp I. Akentyev Kristina O. Petrova Lyudmila V. Lyutova Aleksei A. Korzhenkov Irek I. Gubaidullin Stepan V. Toshchakov Dmitry G. Kozlov |
| author_facet | Daria D. Sokolova Philipp I. Akentyev Kristina O. Petrova Lyudmila V. Lyutova Aleksei A. Korzhenkov Irek I. Gubaidullin Stepan V. Toshchakov Dmitry G. Kozlov |
| author_sort | Daria D. Sokolova |
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| description | The methylotrophic yeast <i>Komagataella kurtzmanii</i> belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typically contain a mixture of sexually dimorphic cells that are prone to self-diploidisation and spore formation accompanied by genetic rearrangements. These characteristics pose a significant challenge to the development of genetically stable producers for industrial use. In the present study, we constructed heterothallic strains of <i>K. kurtzmanii</i>, ensuring a constant mating type by unifying the genetic sequences in the active and silent MAT loci. To obtain such strains, we performed site-directed inactivation of one of the two yeast MAT loci, replacing its sequence with a selective HIS4 gene surrounded by I-SceI meganuclease recognition sites. We then used transient expression of the SCE1 gene, encoding a recombinant I-SceI meganuclease, to induce site-specific cleavage of HIS4, followed by damage repair by homologous recombination in mutant cells. As a result, heterothallic strains designated ‘Y-727-2(alpha)’ and ‘Y-727-9(a)’, which correspond to the α and <b>a</b> mating type, respectively, were obtained. The strains demonstrated a loss of the ability to self-diploidize. The results of PCR and whole genome analysis confirmed the identity of the contents of the MAT loci. Analysis of the genomes of the final strains, however, revealed a fusion of chromosome 3 and chromosome 4 in strain Y-727-2(alpha)-1. This finding was subsequently confirmed by pulsed-field gel electrophoresis of yeast chromosomes. However, the ability of the Y-727-2(alpha)-derived producers to efficiently secrete recombinant β-galactosidase was unaffected by this genomic rearrangement. |
| format | Article |
| id | doaj-art-c415d28112744f8a91fbc2059d4ceabe |
| institution | Kabale University |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-01-01 |
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| spelling | doaj-art-c415d28112744f8a91fbc2059d4ceabe2025-01-24T13:25:10ZengMDPI AGBiomolecules2218-273X2025-01-011519710.3390/biom15010097The Construction of Heterothallic Strains of <i>Komagataella kurtzmanii</i> Using the I-SceI MeganucleaseDaria D. Sokolova0Philipp I. Akentyev1Kristina O. Petrova2Lyudmila V. Lyutova3Aleksei A. Korzhenkov4Irek I. Gubaidullin5Stepan V. Toshchakov6Dmitry G. Kozlov7National Research Center “Kurchatov Institute”, 123182 Moscow, RussiaNational Research Center “Kurchatov Institute”, 123182 Moscow, RussiaNational Research Center “Kurchatov Institute”, 123182 Moscow, RussiaNational Research Center “Kurchatov Institute”, 123182 Moscow, RussiaNational Research Center “Kurchatov Institute”, 123182 Moscow, RussiaNational Research Center “Kurchatov Institute”, 123182 Moscow, RussiaNational Research Center “Kurchatov Institute”, 123182 Moscow, RussiaNational Research Center “Kurchatov Institute”, 123182 Moscow, RussiaThe methylotrophic yeast <i>Komagataella kurtzmanii</i> belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typically contain a mixture of sexually dimorphic cells that are prone to self-diploidisation and spore formation accompanied by genetic rearrangements. These characteristics pose a significant challenge to the development of genetically stable producers for industrial use. In the present study, we constructed heterothallic strains of <i>K. kurtzmanii</i>, ensuring a constant mating type by unifying the genetic sequences in the active and silent MAT loci. To obtain such strains, we performed site-directed inactivation of one of the two yeast MAT loci, replacing its sequence with a selective HIS4 gene surrounded by I-SceI meganuclease recognition sites. We then used transient expression of the SCE1 gene, encoding a recombinant I-SceI meganuclease, to induce site-specific cleavage of HIS4, followed by damage repair by homologous recombination in mutant cells. As a result, heterothallic strains designated ‘Y-727-2(alpha)’ and ‘Y-727-9(a)’, which correspond to the α and <b>a</b> mating type, respectively, were obtained. The strains demonstrated a loss of the ability to self-diploidize. The results of PCR and whole genome analysis confirmed the identity of the contents of the MAT loci. Analysis of the genomes of the final strains, however, revealed a fusion of chromosome 3 and chromosome 4 in strain Y-727-2(alpha)-1. This finding was subsequently confirmed by pulsed-field gel electrophoresis of yeast chromosomes. However, the ability of the Y-727-2(alpha)-derived producers to efficiently secrete recombinant β-galactosidase was unaffected by this genomic rearrangement.https://www.mdpi.com/2218-273X/15/1/97<i>Komagataella kurtzmanii</i><i>Komagataella phaffii</i>heterothallic yeastcell mating typeI-SceI meganucleaserepair |
| spellingShingle | Daria D. Sokolova Philipp I. Akentyev Kristina O. Petrova Lyudmila V. Lyutova Aleksei A. Korzhenkov Irek I. Gubaidullin Stepan V. Toshchakov Dmitry G. Kozlov The Construction of Heterothallic Strains of <i>Komagataella kurtzmanii</i> Using the I-SceI Meganuclease Biomolecules <i>Komagataella kurtzmanii</i> <i>Komagataella phaffii</i> heterothallic yeast cell mating type I-SceI meganuclease repair |
| title | The Construction of Heterothallic Strains of <i>Komagataella kurtzmanii</i> Using the I-SceI Meganuclease |
| title_full | The Construction of Heterothallic Strains of <i>Komagataella kurtzmanii</i> Using the I-SceI Meganuclease |
| title_fullStr | The Construction of Heterothallic Strains of <i>Komagataella kurtzmanii</i> Using the I-SceI Meganuclease |
| title_full_unstemmed | The Construction of Heterothallic Strains of <i>Komagataella kurtzmanii</i> Using the I-SceI Meganuclease |
| title_short | The Construction of Heterothallic Strains of <i>Komagataella kurtzmanii</i> Using the I-SceI Meganuclease |
| title_sort | construction of heterothallic strains of i komagataella kurtzmanii i using the i scei meganuclease |
| topic | <i>Komagataella kurtzmanii</i> <i>Komagataella phaffii</i> heterothallic yeast cell mating type I-SceI meganuclease repair |
| url | https://www.mdpi.com/2218-273X/15/1/97 |
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