Isolation and Identification of Non- Saccharomyces 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 Starmerella bacillaris . Here, S. bacillaris (syn., Candida zemplinina , 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 4 + ), M3 (NH 4 + + 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 4 + ) and M3 (Phe + NH 4 + ) 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 4 + ) medium, the mRNA expression levels of YAT were 124-fold and 86-fold higher in M3 (Phe) and M3 (NH 4 + ) media, respectively, indicating that the transport of L-phe was inhibited when both NH 4 + and Phe were present in the medium. In the M3 (Phe) and M3 (Phe + NH 4 + ) media, the mRNA expression level of ADH5 was higher than PDC , hisC , GOT1 , and YAT , and it was 2.6 times higher and 2.48 times higher, respectively, compared to the M3 (NH 4 + ) medium, revealing that the key gene catalyzing the dehydrogenation of benzaldehyde to 2-PE is ADH5 . 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 S. bacillaris , which had not been previously explored and provided a basis for non- Saccharomyces yeast-producing 2-PE and its applications.