Rapamycin enhanced the production of 2-phenylethanol during whole-cell bioconversion by yeast.

2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, has been widely utilized in food, perfume, and beverages. Saccharomyces cerevisiae is one of the most promising microorganisms for the biosynthesis of natural 2-PE. However, the growth of S. cerevisiae is generally inhibited by 2-PE, which makes its production in yeast cell factories challenging. Here, the whole-cell bioconversion was used to avert growth inhibition, leading to an increase in the concentration and productivity of 2-PE. Moreover, rapamycin (Rap) addition further improved the efficiency of 2-PE synthesis. The concentration of 2-PE (2.20 g/L) was 1.68-fold higher than that in the absence of Rap during the whole-cell bioconversion by S. cerevisiae BY4741. RT-qPCR results showed that Rap addition increased the transcription of ARO9, ARO10, ADH2, GAP1, ARO80, GLN3, and GDH2. When the GLN3 was knocked out, the transcriptional levels of the genes were dramatically decreased, and the concentration of 2-PE significantly decreased to 0.21 g/L. The results indicated that Rap enhanced the flux of the Ehrlich pathway, and Gln3 exerted a central role in the regulation of Rap. Furthermore, commercial yeast (S. cerevisiae FY202001) was selected to verify the applicability of Rap. In the presence of Rap, 3.67 g/L 2-PE was obtained by whole-cell bioconversion in flask, which was increased by 9% than that in the absence of Rap. Finally, the 2-PE titer reached 4.93 g/L by whole-cell bioconversion in a 5 L bioreactor, with a yield of 84 mol% from L-phenylalanine and a productivity of 0.103 g/L h, which was far higher than that of the currently reported in S. cerevisiae. These findings provided a new idea for the efficient synthesis of 2-PE. KEY POINTS: • Whole-cell bioconversion was used to produce 2-PE. • The regulation of the Ehrlich pathway by Rap provides a theoretical basis for developing an effective yeast cell factory to produce 2-PE. • The 2-PE productivity of 0.103 g/L h is far higher than that of the currently reported in S. cerevisiae .