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

ABSTRACT: 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.

SUBMITTER: Yang Q

PROVIDER: S-EPMC10755794 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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

Yang Qilin Q Liu Shuangping S Zhao Yuzong Y Han Xiao X Chang Rui R Mao Jian J

Synthetic and systems biotechnology 20231128 4

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 Aro10 ...[more]

PMID: 38161995

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Project description:Background2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, inhibits growth of the producer strains. However, the limited knowledge regarding 2-PE tolerance mechanisms renders our current knowledge base insufficient to inform rational design.ResultsTo improve the growth phenotype of Saccharomyces cerevisiae under a high 2-PE concentration, adaptive laboratory evolution (ALE) was used to generate an evolved 19-2 strain. Under 2-PE stress, its OD600 and growth rate increased by 86% and 22% than that of the parental strain, respectively. Through whole genome sequencing and reverse engineering, transcription factor Pdr1p mutation (C862R) was revealed as one of the main causes for increased 2-PE tolerance. Under 2-PE stress condition, Pdr1p mutation increased unsaturated fatty acid/saturated fatty acid ratio by 42%, and decreased cell membrane damage by 81%. Using STRING website, we identified Pdr1p interacted with some proteins, which were associated with intracellular ergosterol content, reactive oxygen species (ROS), and the ATP-binding cassette transporter. Also, the results of transcriptional analysis of genes encoded these proteins confirmed that Pdr1p mutation induced the expression of these genes. Compared with those of the reference strain, the ergosterol content of the PDR1_862 strain increased by 72%-101%, and the intracellular ROS concentration decreased by 38% under 2-PE stress. Furthermore, the Pdr1p mutation also increased the production of 2-PE (11% higher).ConclusionsIn the present work, we have demonstrated the use of ALE as a powerful tool to improve yeast tolerance to 2-PE. Based on the reverse engineering, transcriptional and physiological analysis, we concluded that Pdr1p mutation significantly enhanced the 2-PE tolerance of yeast by regulating the fatty acid proportion, intracellular ergosterol and ROS. It provides new insights on Pdr1p mediated 2-PE tolerance, which could help in the design of more robust yeasts for natural 2-PE synthesis.

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

Publications

Enzymatic properties and inhibition tolerance analysis of key enzymes in β-phenylethanol anabolic pathway of <i>Saccharomyces cerevisiae</i> HJ.

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