Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity
Abstract Background Previously, we isolated a mutant of Parachlorella kessleri named strain PK4 that accumulated higher concentrations of lipids than the wild-type strain. Resequencing of the PK4 genome identified mutations in three genes which may be associated with the high-lipid phenotype. The fi...
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| Format: | Article |
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BMC
2024-03-01
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| Series: | Biotechnology for Biofuels and Bioproducts |
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| Online Access: | https://doi.org/10.1186/s13068-024-02484-7 |
| _version_ | 1797275470966816768 |
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| author | Yuki Kasai Satsuki Takagi Shuhei Ota Kotaro Ishii Tsuyoshi Takeshita Shigeyuki Kawano Shigeaki Harayama |
| author_facet | Yuki Kasai Satsuki Takagi Shuhei Ota Kotaro Ishii Tsuyoshi Takeshita Shigeyuki Kawano Shigeaki Harayama |
| author_sort | Yuki Kasai |
| collection | DOAJ |
| description | Abstract Background Previously, we isolated a mutant of Parachlorella kessleri named strain PK4 that accumulated higher concentrations of lipids than the wild-type strain. Resequencing of the PK4 genome identified mutations in three genes which may be associated with the high-lipid phenotype. The first gene, named CDMT1, encodes a protein with a calcium-dependent membrane association domain; the second gene, named DMAN1, encodes endo-1,4-β-mannanase, while the third gene, named AATPL1, encodes a plastidic ATP/ADP antiporter-like protein. Results To determine which of these mutant genes are directly responsible for the phenotype of strain PK4, we delivered Cas9-gRNA ribonucleoproteins targeting each of the three genes into the wild-type cells by electroporation and successfully disrupted these three genes separately. The lipid productivity in the disruptants of CDMT1 and DMAN1 was similar to and lower than that in the wild-type strain, while the disruptants of AATPL1 exhibited > 30% higher lipid productivity than the wild-type strain under diurnal conditions. Conclusions We succeeded in improving the lipid productivity of P. kessleri by CRISPR/Cas9-mediated gene disruption of AATPL1. The effective gene-editing method established in this study will be useful to improve Parachlorella strains for industrial applications. |
| first_indexed | 2024-03-07T15:14:51Z |
| format | Article |
| id | doaj.art-3bf49c979150422d910c185ae844af1f |
| institution | Directory Open Access Journal |
| issn | 2731-3654 |
| language | English |
| last_indexed | 2024-03-07T15:14:51Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels and Bioproducts |
| spelling | doaj.art-3bf49c979150422d910c185ae844af1f2024-03-05T17:57:59ZengBMCBiotechnology for Biofuels and Bioproducts2731-36542024-03-0117111710.1186/s13068-024-02484-7Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivityYuki Kasai0Satsuki Takagi1Shuhei Ota2Kotaro Ishii3Tsuyoshi Takeshita4Shigeyuki Kawano5Shigeaki Harayama6Research and Development Initiative, Chuo UniversityResearch and Development Initiative, Chuo UniversityDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of TokyoDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of TokyoDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of TokyoDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of TokyoResearch and Development Initiative, Chuo UniversityAbstract Background Previously, we isolated a mutant of Parachlorella kessleri named strain PK4 that accumulated higher concentrations of lipids than the wild-type strain. Resequencing of the PK4 genome identified mutations in three genes which may be associated with the high-lipid phenotype. The first gene, named CDMT1, encodes a protein with a calcium-dependent membrane association domain; the second gene, named DMAN1, encodes endo-1,4-β-mannanase, while the third gene, named AATPL1, encodes a plastidic ATP/ADP antiporter-like protein. Results To determine which of these mutant genes are directly responsible for the phenotype of strain PK4, we delivered Cas9-gRNA ribonucleoproteins targeting each of the three genes into the wild-type cells by electroporation and successfully disrupted these three genes separately. The lipid productivity in the disruptants of CDMT1 and DMAN1 was similar to and lower than that in the wild-type strain, while the disruptants of AATPL1 exhibited > 30% higher lipid productivity than the wild-type strain under diurnal conditions. Conclusions We succeeded in improving the lipid productivity of P. kessleri by CRISPR/Cas9-mediated gene disruption of AATPL1. The effective gene-editing method established in this study will be useful to improve Parachlorella strains for industrial applications.https://doi.org/10.1186/s13068-024-02484-7Parachlorella kessleriGenetic transformationElectroporationGenome editingCRISPR/Cas9 |
| spellingShingle | Yuki Kasai Satsuki Takagi Shuhei Ota Kotaro Ishii Tsuyoshi Takeshita Shigeyuki Kawano Shigeaki Harayama Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity Biotechnology for Biofuels and Bioproducts Parachlorella kessleri Genetic transformation Electroporation Genome editing CRISPR/Cas9 |
| title | Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity |
| title_full | Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity |
| title_fullStr | Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity |
| title_full_unstemmed | Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity |
| title_short | Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity |
| title_sort | development of a crispr cas9 mediated gene editing method to isolate a mutant of the unicellular green alga parachlorella kessleri strain nies 2152 with improved lipid productivity |
| topic | Parachlorella kessleri Genetic transformation Electroporation Genome editing CRISPR/Cas9 |
| url | https://doi.org/10.1186/s13068-024-02484-7 |
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