Enzymatic properties and inhibition tolerance analysis of key enzymes in β-phenylethanol anabolic pathway of Saccharomyces cerevisiae HJ

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, Aro9pHJ (−24.05 kJ/mol) and Aro10pHJ (−14.33 kJ/mol) exhibited lower binding free energies compared to Aro9pS288C (−21.93 kJ/mol) and Aro10pS288C (−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 Vmax(Aro9pHJ: 23.89 μmol·(min∙g)−1) > Aro9pS288C: 21.3 μmol·(min∙g)−1) and inhibition constant Ki values (Aro9pHJ: 0.28 mol∙L−1>Aro9pS288C 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), Aro10pHJ exhibited a stronger affinity than Aro10pS288C. Furthermore, Aro9pHJ and Aro10pHJ 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 Aro9pHJ and Aro10pHJ 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.