Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector
Abstract Background Sustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotric...
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| Format: | Article |
| Language: | English |
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BMC
2023-02-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-023-02033-1 |
| _version_ | 1811165624583323648 |
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| author | Pariya Shaigani Tobias Fuchs Petra Graban Sophia Prem Martina Haack Mahmoud Masri Norbert Mehlmer Thomas Brueck |
| author_facet | Pariya Shaigani Tobias Fuchs Petra Graban Sophia Prem Martina Haack Mahmoud Masri Norbert Mehlmer Thomas Brueck |
| author_sort | Pariya Shaigani |
| collection | DOAJ |
| description | Abstract Background Sustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported. Results The targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions. Conclusion This study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved. |
| first_indexed | 2024-04-10T15:39:41Z |
| format | Article |
| id | doaj.art-40ae7d5d409e4d19a947f201fb973abd |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-10T15:39:41Z |
| publishDate | 2023-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-40ae7d5d409e4d19a947f201fb973abd2023-02-12T12:26:29ZengBMCMicrobial Cell Factories1475-28592023-02-0122111410.1186/s12934-023-02033-1Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sectorPariya Shaigani0Tobias Fuchs1Petra Graban2Sophia Prem3Martina Haack4Mahmoud Masri5Norbert Mehlmer6Thomas Brueck7Department of Chemistry, Werner Siemens-Chair of Synthetic Biotechnology, Technical University of MunichDepartment of Chemistry, Werner Siemens-Chair of Synthetic Biotechnology, Technical University of MunichDepartment of Chemistry, Werner Siemens-Chair of Synthetic Biotechnology, Technical University of MunichDepartment of Chemistry, Werner Siemens-Chair of Synthetic Biotechnology, Technical University of MunichDepartment of Chemistry, Werner Siemens-Chair of Synthetic Biotechnology, Technical University of MunichDepartment of Chemistry, Werner Siemens-Chair of Synthetic Biotechnology, Technical University of MunichDepartment of Chemistry, Werner Siemens-Chair of Synthetic Biotechnology, Technical University of MunichDepartment of Chemistry, Werner Siemens-Chair of Synthetic Biotechnology, Technical University of MunichAbstract Background Sustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported. Results The targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions. Conclusion This study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved.https://doi.org/10.1186/s12934-023-02033-1Oleaginous yeastYeast oilGenome engineeringCRISPR/CasTailored plant oil alternativesCutaneotrichosporon oleaginosus |
| spellingShingle | Pariya Shaigani Tobias Fuchs Petra Graban Sophia Prem Martina Haack Mahmoud Masri Norbert Mehlmer Thomas Brueck Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector Microbial Cell Factories Oleaginous yeast Yeast oil Genome engineering CRISPR/Cas Tailored plant oil alternatives Cutaneotrichosporon oleaginosus |
| title | Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector |
| title_full | Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector |
| title_fullStr | Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector |
| title_full_unstemmed | Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector |
| title_short | Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector |
| title_sort | mastering targeted genome engineering of gc rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector |
| topic | Oleaginous yeast Yeast oil Genome engineering CRISPR/Cas Tailored plant oil alternatives Cutaneotrichosporon oleaginosus |
| url | https://doi.org/10.1186/s12934-023-02033-1 |
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