Screening of High-Yield 2-Phenylethanol Producing Strain from Wild-Type Saccharomyces cerevisiae and Optimization of Fermentation Parameters

Abstract

2-Phenylethanol (2-PE), an aromatic alcohol with a rose-like fragrance, is widely used in the food, pharmaceutical, and high-end cosmetic industries. In this study, a high-yield 2-PE-producing strain was isolated and identified as Saccharomyces cerevisiae based on morphological characterization and taxonomic identification. Fermentation medium components (carbon and nitrogen sources) were optimized through single-factor experiments in shaking flasks, and fermentation medium with 40 g/L glucose, 5 g/L malt extract, 1.75 g/L corn steep liquor, 2.5 g/L yeast extract, 5 g/L malt extract, 1.75 g/L corn steep liquor was considered suitable for 2-PE production. RT-qPCR results indicated that corn steep liquor activates expression of genes related to the shikimate pathway and Ehrlich pathway (pha2, aro4, aro8, and aro9), thereby promoting the synthesis of 2-PE through these pathways. Excess yeast extract inhibited the expression of aro8 and aro9, while enhancing the expression of tdh3 and adh2, thus promoting the de novo synthesis of 2-PE. Furthermore, fermentation in a 5 L bioreactor was applied to investigate the effects of feeding strategies, inoculum proportion, and pH on 2-PE production. With a pH of 5.5 and10% inoculum proportion, the supplementation of the substrate L-Phe led to a 2-PE production of 4.81 g/L after 24 h of fermentation. Finally, in situ product recovery (ISPR) techniques was applied to alleviate 2-PE cytotoxicity, achieving a production of 6.41 g/L. This process offers a promising strategy for producing 2-PE efficiently and naturally, paving the way for further industrial applications in food, pharmaceutical, and cosmetic sectors.

Keywords: 2-phenylethanol; Saccharomyces cerevisiae; fermentation condition optimization; strain screening.

Figure A1

The effect of different 2-PE concentrations on cell growth of S. cerevisiae D-22.

Figure 1

Metvbabolic pathway of 2-phenylethanol in microorganisms. GLC glucose, G6P glucose-6-phosphate, E4P erythose-4-phosphate, PEP phosphoenolpyruvate, DAHP 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, SHIK shikimate, CHO chorismate, PREP prephenate, PPY phenylpyruvate, PHE phenylalanine, PAC phenylacetaldehyde, 2PE 2-phenylethonal.

Figure 2

Isolation of the 2-PE-producing strains. (A) Comparison of 2-PE production of different strains in YPD medium; (B) Scanning electron microscope images of S. cerevisiae D-22; (C) Neighbor-joining phylogenetic tree based on 18S rRNA gene sequences of S. cerevisiae D-22.

Figure 3

Influence of initial glucose and nitrogen sources concentration on 2-PE production. S. cerevisiae D-22 was cultured in FM medium with different concentrations of (A) initial glucose, (B) corn steep liquor, (C) yeast extract and (D) Malt extract for 72 h. The concentrations of both L-Phe and 2-PE were analyzed using HPLC. Error bars represent the standard deviation of three independent assays. Different letters indicated the significant difference based on one-way analysis of variance (ANOVA) (p < 0.05).

Figure 4

Influence of different nitrogen source conditions on 2-PE biosynthesis genes. (A) RT-qPCR assay to confirm the effect of the transcript level related to key 2-PE biosynthesis genes under different nitrogen source conditions. The transcript level of pha2 in the control group (CK) was set to 1 for normalization. Error bars represent the standard deviation of three independent assays. Asterisks indicated the significant difference based on unpaired Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001); (B) GLC glucose, G6P glucose-6-phosphate, E4P erythose-4-phosphate, PEP phosphoenolpyruvate, DAHP 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, SHIK shikimate, CHO chorismate, PREP prephenate, TYR L-tyrosine, PPY phenylpyruvate, PHE L-phenylalanine, PAC phenylacetaldehyde, PAA phenylacetate, 2PE 2-phenylethonal.

Figure 5

Influence of pH and inoculum proportion on 2-PE production. Cell growth, glucose and L-Phe consumption, and 2-PE production with (A) pH = 5; (B) pH = 5.5; (C) pH = 6; (D) pH = 7; (E) 20% inoculum proportion; (F) 10% inoculum proportion.

Figure 6

Fed-batch fermentation of S. cerevisiae D-22 in a 5 L bioreactor. Cell growth, glucose and L-Phe consumption, and 2-PE production. (A) following the addition of 2 g/L L-Phe at 12 h; (B) following the addition of 1 L oleci acid at 10 h. Discussion.

Figure 7

The upregulation of adh2 and tdh3 sustains NAD⁺/NADH homeostasis and enhances the de novo synthesis of 2-PE. GLC glucose, PEP phosphoenolpyruvate, PAC phenylacetaldehyde, 2PE 2-phenylethonal, TDH3 glyceraldehyde-3-phosphate dehydrogenase, ADH2 alcohol dehydrogenase 2.