In this study, YlACL2 was inactivated by two methods: traditional approach based on homologous recombination and uracil marker and markerless system using CRISPR/Cas9. The efficiency of YlACL2 inactivation using traditional approach was 4% (one ΔYlacl2 strain out of 24 tested transformants) whereas knockout efficiency using CRISPR/Cas9 system was 75% (18 ΔYlacl2 strains out of 24 tested transformants). YlACL2 null mutant strains were not able to utilize citrate as a single carbon source. Growth kinetics was investigated in the media with glucose and acetate as a single carbon source. The fact that ΔYlacl2 is able to grow in the minimal medium with glucose as a single carbon source provides evidence that there is an alternative source of acetyl-CoA on carbohydrate substrates in Y. lipolytica.
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The work was carried out using the equipment of the Unique Scientific Facility of the All-Russia Collection of Industrial Microorganisms National Bioresource Center of the Kurchatov Institute National Resource Center (GOSNIIgenetika).
The work was financially supported by the Russian Federation (State Task no. 595-00003-19 PR) and partially funded by grant no. MK-2241.2019.7.
The authors declare that they have no conflicts of interest.
This article does not contain any studies involving animals performed by any of the authors.
This article does not contain any studies involving human participants performed by any of the authors.
Translated by I. Gordon
Abbreviations: ACL—ATP citrate lyase; CRISPR—clustered regularly interspaced short palindromic repeats; OD600—optical density at a wavelength of 600 nm; PAM—protospacer adjacent motif; sgRNA—small guide RNA.
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Yuzbasheva, E.Y., Yuzbashev, T.V., Vinogradova, E.B. et al. Inactivation of Yarrowia lipolytica YlACL2 gene Coding Subunit of ATP Citrate Lyase Using CRISPR/Cas9 System. Appl Biochem Microbiol 56, 885–892 (2020). https://doi.org/10.1134/S0003683820090112
- Yarrowia lipolytica
- CRISPR/Cas9 system
- ATP citrate lyase