Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector.
CRISPR/Cas
Cocoa butter
Cutaneotrichosporon oleaginosus
Fatty acid biosynthesis
Genome engineering
High-oleic sunflower oil
Oleaginous yeast
Tailored plant oil alternatives
Yeast oil
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
08 Feb 2023
08 Feb 2023
Historique:
received:
16
12
2022
accepted:
31
01
2023
entrez:
9
2
2023
pubmed:
10
2
2023
medline:
11
2
2023
Statut:
epublish
Résumé
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. 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. 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.
Sections du résumé
BACKGROUND
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
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
CONCLUSIONS
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.
Identifiants
pubmed: 36755261
doi: 10.1186/s12934-023-02033-1
pii: 10.1186/s12934-023-02033-1
pmc: PMC9906925
doi:
Substances chimiques
Plant Oils
0
Fatty Acids
0
RNA, Messenger
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
25Subventions
Organisme : Bundesministerium für Bildung und Forschung
ID : 033RC012B
Organisme : Bundesministerium für Bildung und Forschung
ID : 031B0662B
Organisme : Bundesministerium für Bildung und Forschung
ID : 03SF0577A
Organisme : Bundesministerium für Bildung und Forschung
ID : 031B0853A
Informations de copyright
© 2023. The Author(s).
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