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

The 2-phenylethanol biosynthesis in Saccharomyces cerevisiae is limited by multiple-branch metabolism in the shikimate pathway. In this research, a total of 4 x 4 (sites x genes) guide sequences from four branch genes (TYR1, ARO8, AAT2 and ALD3) were designed. A single-gene down-regulation library of 4 x 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.