{"title":"开发 CRISPR/Cas9 介导的基因编辑方法,以分离出具有更高脂质生产率的单细胞绿色藻类 Parachlorella kessleri 菌株 NIES-2152 突变体","authors":"Yuki Kasai, Satsuki Takagi, Shuhei Ota, Kotaro Ishii, Tsuyoshi Takeshita, Shigeyuki Kawano, Shigeaki Harayama","doi":"10.1186/s13068-024-02484-7","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>Previously, we isolated a mutant of <i>Parachlorella kessleri</i> 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 <i>CDMT1</i>, encodes a protein with a calcium-dependent membrane association domain; the second gene, named <i>DMAN1</i>, encodes endo-1,4-β-mannanase, while the third gene, named <i>AATPL1</i>, encodes a plastidic ATP/ADP antiporter-like protein.</p><h3>Results</h3><p>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 <i>CDMT1</i> and <i>DMAN1</i> was similar to and lower than that in the wild-type strain, while the disruptants of <i>AATPL1</i> exhibited > 30% higher lipid productivity than the wild-type strain under diurnal conditions.</p><h3>Conclusions</h3><p>We succeeded in improving the lipid productivity of <i>P. kessleri</i> by CRISPR/Cas9-mediated gene disruption of <i>AATPL1</i>. The effective gene-editing method established in this study will be useful to improve <i>Parachlorella</i> strains for industrial applications.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.1000,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02484-7","citationCount":"0","resultStr":"{\"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\",\"authors\":\"Yuki Kasai, Satsuki Takagi, Shuhei Ota, Kotaro Ishii, Tsuyoshi Takeshita, Shigeyuki Kawano, Shigeaki Harayama\",\"doi\":\"10.1186/s13068-024-02484-7\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><p>Previously, we isolated a mutant of <i>Parachlorella kessleri</i> 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 <i>CDMT1</i>, encodes a protein with a calcium-dependent membrane association domain; the second gene, named <i>DMAN1</i>, encodes endo-1,4-β-mannanase, while the third gene, named <i>AATPL1</i>, encodes a plastidic ATP/ADP antiporter-like protein.</p><h3>Results</h3><p>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 <i>CDMT1</i> and <i>DMAN1</i> was similar to and lower than that in the wild-type strain, while the disruptants of <i>AATPL1</i> exhibited > 30% higher lipid productivity than the wild-type strain under diurnal conditions.</p><h3>Conclusions</h3><p>We succeeded in improving the lipid productivity of <i>P. kessleri</i> by CRISPR/Cas9-mediated gene disruption of <i>AATPL1</i>. The effective gene-editing method established in this study will be useful to improve <i>Parachlorella</i> strains for industrial applications.</p></div>\",\"PeriodicalId\":494,\"journal\":{\"name\":\"Biotechnology for Biofuels\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":6.1000,\"publicationDate\":\"2024-03-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02484-7\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biotechnology for Biofuels\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://link.springer.com/article/10.1186/s13068-024-02484-7\",\"RegionNum\":1,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology for Biofuels","FirstCategoryId":"5","ListUrlMain":"https://link.springer.com/article/10.1186/s13068-024-02484-7","RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
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
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.
期刊介绍:
Biotechnology for Biofuels is an open access peer-reviewed journal featuring high-quality studies describing technological and operational advances in the production of biofuels, chemicals and other bioproducts. The journal emphasizes understanding and advancing the application of biotechnology and synergistic operations to improve plants and biological conversion systems for the biological production of these products from biomass, intermediates derived from biomass, or CO2, as well as upstream or downstream operations that are integral to biological conversion of biomass.
Biotechnology for Biofuels focuses on the following areas:
• Development of terrestrial plant feedstocks
• Development of algal feedstocks
• Biomass pretreatment, fractionation and extraction for biological conversion
• Enzyme engineering, production and analysis
• Bacterial genetics, physiology and metabolic engineering
• Fungal/yeast genetics, physiology and metabolic engineering
• Fermentation, biocatalytic conversion and reaction dynamics
• Biological production of chemicals and bioproducts from biomass
• Anaerobic digestion, biohydrogen and bioelectricity
• Bioprocess integration, techno-economic analysis, modelling and policy
• Life cycle assessment and environmental impact analysis