Abstract
Background: 2-Phenylethanol (2-PE) is an aromatic alcohol with rose fragrance, which is widely used as an additive in food, tobacco and daily chemical industries. Yeast is the main microorganism producing natural 2-PE, but it is limited by low yield and weak tolerance. Nature and fermented products is a resource treasury of yeasts with excellent traits. Screening strains with good phenotypic traits and conducting breeding by cell fusion for genetic pyramiding is an effective way to improve strains. Results: In this study, 25 strains of 2-PE-producing yeasts were isolated from Chinese brewed samples. Three Saccharomyces cerevisiae strains with good traits in tolerance and 2-PE yield were screened out. The strain LSC-1 produces 2-PE of 3.41 g/L with an increase of 9.3% compared to the industrial strain CWY132. The strain NGER shows good tolerance to 2-PE at the concentration of 3.60 g/L in agar plate, and the thermotolerant strain S.C-1 shows growth ability at 41℃. Two rounds of protoplast fusion were performed with these three parent strains for pyramiding of traits. A fusion strain RH2-16 with high 2-PE yield and increased tolerance was obtained. Using 5 g/L L-phenylalanine as the precursor, RH2-16 produced 2-PE of 4.31 g/L through fermentation conversion and the molar conversion rate of L-Phe reached 115% in 36 h. Compared to the yield of the parental strain LSC-1 and the industrial strain CWY132, 2-PE in RH2-16 increased by 26.4% and 38.1%, respectively. Overexpression of the key enzyme genes ARO8, ARO10, and ADH2 in the Ehrlich pathway in RH2-16 did not increase 2-PE production.Conclusion: Diversified S.cerevisiae strains with different traits can be isolated from the brewing related samples. Protoplast fusion technology can effectively pyramid excellent genetic traits and breed yeast strains with significantly improved tolerance and 2-PE yield. Our research provided a breeding strategy for S.cerevisiae and a strain for industrial production of 2-PE. Overexpression of the key enzyme genes in 2-PE synthesis pathway does not necessarily improve increase production.
Saccharomyces Cerevisiae Strains Selected From Nature Significantly Increased the Production of 2-Phenylethanol Through Protoplast Fusion
