Huangjiu is known for its unique aroma, primarily attributed to its high concentration of β-phenylethanol (ranging from 40 to 130 mg/L). Phenylalanine aminotransferase Aro9p and phenylpyruvate decarboxylase Aro10p are key enzymes in the β-phenylethanol synthetic pathway of Saccharomyces cerevisiae HJ. This study examined the enzymatic properties of these two enzymes derived from S. cerevisiae HJ and S288C. After substrate docking, Aro9p^HJ (-24.05 kJ/mol) and Aro10p^HJ (-14.33 kJ/mol) exhibited lower binding free energies compared to Aro9p^S288C (-21.93 kJ/mol) and Aro10p^S288C (-12.84 kJ/mol). ARO9 and ARO10 genes were heterologously expressed in E. coli BL21. Aro9p, which was purified via affinity chromatography, showed inhibition by l-phenylalanine (L-PHE), but the reaction rate V_max(Aro9p^HJ: 23.89 μmol·(min∙g)^-1) > Aro9p^S288C: 21.3 μmol·(min∙g)^-1) and inhibition constant K_i values (Aro9p^HJ: 0.28 mol L^-1>Aro9p^S288C 0.26 mol L^-1) indicated that Aro9p from S. cerevisiae HJ was more tolerant to substrate stress during Huangjiu fermentation. In the presence of the same substrate phenylpyruvate (PPY), Aro10p^HJ exhibited a stronger affinity than Aro10p^S288C. Furthermore, Aro9p^HJ and Aro10p^HJ were slightly more tolerant to the final metabolites β-phenylethanol and ethanol, respectively, compared to those from S288C. The study suggests that the mutations in Aro9p^HJ and Aro10p^HJ may contribute to the increased β-phenylethanol concentration in Huangjiu. This is the first study investigating enzyme tolerance mechanisms in terms of substrate and product, providing a theoretical basis for the regulation of the β-phenylethanol metabolic pathway.

• PMID: 38161995
• DOI full text
• PMC full text
• PubMed
• PubTator

Reference Type

Journal Article

Authors

Yang Q, Liu S, Zhao Y, Han X, Chang R, Mao J

Primary Lit For

Additional Lit For

Review For