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

Abstract

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 (NH₄⁺), M3 (NH₄⁺ + 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 (NH₄⁺) and M3 (Phe + NH₄⁺) 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 + NH₄⁺) medium, the mRNA expression levels of YAT were 124-fold and 86-fold higher in M3 (Phe) and M3 (NH₄⁺) media, respectively, indicating that the transport of L-phe was inhibited when both NH₄⁺ and Phe were present in the medium. In the M3 (Phe) and M3 (Phe + NH₄⁺) 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 (NH₄⁺) 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.