Isolation and Identification of Non- Yeast Producing 2-Phenylethanol and Study of the Ehrlich Pathway and Shikimate Pathway.

2-phenylethanol (2-PE) has been widely utilized as an aromatic additive in various industries, including cosmetics, beer, olive oil, tea, and coffee, due to its rose-honey-like aroma. However, no reports have investigated the production of 2-PE by . Here, (syn., , and named strain R5) was identified by analysis of morphology, physiology and biochemistry, and 26S rRNA and ITS gene sequence. Then, based on the analysis of whole-genome sequencing and comparison with the KEGG database, it was inferred that strain R5 could synthesize 2-PE from L-phe or glucose through the Ehrlich pathway or shikimate pathway. For further verification of the 2-PE synthesis pathway, strain R5 was cultured in M3 (NH), M3 (NH + Phe), and M3 (Phe) medium. In M3 (Phe) medium, the maximum concentration of 2-PE reached 1.28 g/L, which was 16-fold and 2.29-fold higher than that in M3 (NH) and M3 (Phe + NH) media, respectively. These results indicated that 2-PE could be synthesized by strain R5 through the shikimate pathway or Ehrlich pathway, and the biotransformation from L-phe to 2-PE was more efficient than that from glucose. The qRT-PCR results suggested that compared to M3 (Phe + NH) medium, the mRNA expression levels of were 124-fold and 86-fold higher in M3 (Phe) and M3 (NH) media, respectively, indicating that the transport of L-phe was inhibited when both NH and Phe were present in the medium. In the M3 (Phe) and M3 (Phe + NH) media, the mRNA expression level of was higher than , , , and , and it was 2.6 times higher and 2.48 times higher, respectively, compared to the M3 (NH) medium, revealing that the key gene catalyzing the dehydrogenation of benzaldehyde to 2-PE is . Furthermore, strain R5 exhibits tolerance to high concentrations of 2-PE, reaching 3 g/L, which conferred an ideal tolerance to 2-PE. In summary, the synthesis pathway of 2-PE, mainly for the Ehrlich pathway, was proved for the first time in , which had not been previously explored and provided a basis for non- yeast-producing 2-PE and its applications.