Abstract
Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.
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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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The authors gratefully acknowledge all the members for their kind cooperation in completion of this work.
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This work was supported by the National key research and development program of China (2021YFC2104300) and Natural Science Foundation of Jiangsu Province (BK20210573).
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All authors contributed to the study conception and design. STQ designed the experiments. SQ and LYW performed the experiments. LYW, YF, WJ, JJ, YWX, HDC and SP analyzed the data. LYW wrote the manuscript. STQ revised the manuscript. All authors read and approved the final manuscript.
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Shen, Q., Yan, F., Li, YW. et al. Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology. Biotechnol Lett 46, 37–46 (2024). https://doi.org/10.1007/s10529-023-03444-1
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DOI: https://doi.org/10.1007/s10529-023-03444-1