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Triple-CRISPRi-mediated down-regulation of the shikimate pathway branch genes for enhancing 2-PE biosynthesis in Saccharomyces cerevisiae

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Abstract

The 2-phenylethanol biosynthesis in Saccharomyces cerevisiae is limited by multiple-branch metabolism in the shikimate pathway. In this research, a total of 4 × 4 (sites × genes) guide sequences from four branch genes (TYR1, ARO8, AAT2 and ALD3) were designed. A single-gene down-regulation library of 4 × 4 Saccharomyces cerevisiae strains was constructed. By the assessment of gene expression level and 2-phenylethanol production, the optimal guide sequences of TYR1/AAT2/ALD3 were identified. On these bases, we first developed a high-yielding 2-phenylethanol strain carrying triple-CRISPRi system for simultaneous three branch repression. The INVScI-TYR1.AAT2.ALD3 successfully achieved the desired transcriptional repression effect and led to a 1.89-fold increase in 2-phenylethanol production compared to the starting strain. Triple-CRISPRi-mediated down-regulation of the shikimate pathway branch genes provided a convenient and efficient solution for the development of 2-phenylethanol high-yield Saccharomyces cerevisiae engineering strain.

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Data availability

The data that support the findings of this study are available from corresponding author, [Huazhong Agricultural University], upon reasonable request.

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Authors and Affiliations

  1. College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Shuaihu Fang, ** Huang & **aobao Qi

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  1. Shuaihu Fang
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  2. **anghao Fan
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  3. **lan Li
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  4. Zi**g Zhu
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  5. **** Huang
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Correspondence to **aobao Qi.

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Fang, S., Fan, X., Li, J. et al. Triple-CRISPRi-mediated down-regulation of the shikimate pathway branch genes for enhancing 2-PE biosynthesis in Saccharomyces cerevisiae. Eur Food Res Technol (2024). https://doi.org/10.1007/s00217-023-04461-0

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