Abstract
The application of clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas9) technology in the genetic modification of Yarrowia lipolytica is challenged by low efficiency and low throughput. Here, a highly efficient CRISPR-iCas9 (with D147Y and P411T mutants) genetic manipulation tool was established for Y. lipolytica, which was further utilized to integrate carotene synthetic key genes and significantly improve the target product yield. First, CRISPR-iCas9 could shorten the time of genetic modification and enable the rapid knockout of nonsense suppressors. iCas9 can lead to more than 98% knockout efficiency for NHEJ-mediated repair after optimal target disruption of a single gene, 100% knockout efficiency for a single gene-guided version, and more than 80% knockout efficiency for multiple genes simultaneously in Y. lipolytica. Subsequently, this technology allowed for rapid one-step integration of large fragments (up to 9902-bp) of genes into chromosomes. Finally, YL-ABTG and YL-ABTG2Z were further constructed through CRISPR-iCas9 integration of key genes in a one-step process, resulting in a maximum β-carotene and zeaxanthin content of 3.12 mg/g and 2.33 mg/g dry cell weight, respectively. Therefore, CRISPR-iCas9 technology provides a feasible approach to genetic modification for efficient biosynthesis of biological compounds in Y. lipolytica.
Key points
• iCas9 with D147Y and P411T mutants improved the CRISPR efficiency in Y. lipolytica.
• CRISPR-iCas9 achieved efficient gene knockout and integration in Y. lipolytica.
• CRISPR-iCas9 rapidly modified Y. lipolytica for carotenoid bioproduction.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Funding
This research was supported by the Natural Science Foundation of China (NSFC) Project (grant number 32172145) and the 2020 Team Innovation Project from the Fundamental Scientific Research Special Capital Fund of the National Universities, China (GK202001008).
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QHC, YDQ, NYJ, and YHM designed the experiments. YDQ and QHC performed the experiments. QHC, YDQ, and YHM were responsible for data processing and analysis. QHC, YDQ, CYH, and YHM wrote and revised manuscripts.
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Chen, Q.H., Qian, Y.D., Niu, Y.J. et al. Characterization of an efficient CRISPR-iCas9 system in Yarrowia lipolytica for the biosynthesis of carotenoids. Appl Microbiol Biotechnol 107, 6299–6313 (2023). https://doi.org/10.1007/s00253-023-12731-w
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DOI: https://doi.org/10.1007/s00253-023-12731-w