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

作　　者：Qilin Yang Shuangping Liu Yuzong Zhao Xiao Han Rui Chang Jian Mao 

机构地区：[1]National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing,State Key Laboratory of Food Science and Technology,School of Food Science and Technology,Jiangnan University,Wuxi,Jiangsu,214122,China [2]Shaoxing Key Laboratory of Traditional Fermentation Food and Human Health,Jiangnan University(Shaoxing)Industrial Technology Research Institute,Shaoxing,Zhejiang,312000,China [3]National Engineering Research Center of Huangjiu,Zhejiang Guyuelongshan Shaoxing Wine Co.,Ltd.,Shaoxing,Zhejiang,312000,China [4]Jiangsu Provincial Engineering Research Center for Bioactive Product Processing Technology,Jiangnan University,Wuxi,Jiangsu,214122,China

出　　处：《Synthetic and Systems Biotechnology》2023年第4期772-783,共12页合成和系统生物技术（英文）

基　　金：supported by the National Natural Science Foundation of China(32072205,22138004);Shaoxing Science and Technology Plan Project(2022B43001).

摘　　要：Huangjiu is known for its unique aroma,primarily attributed to its high concentration ofβ-phenylethanol(ranging from 40 to 130 mg/L).Phenylalanine aminotransferase Aro9p and phenylpyruvate decarboxylase Aro10p are key enzymes in theβ-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 Vmax(Aro9p^(HJ):23.89μmol⋅(min·g)^(-1)>Aro9p^(S288C):21.3μmol⋅(min·g)^(-1))and inhibition constant Ki 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 metabolitesβ-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 increasedβ-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 theβ-phenylethanol metabolic pathway.

关 键 词：Ehrlich pathway Phenylalanine aminotransferase and phenylpyruvate decarboxylase Saccharomyces cerevisiae Metabolic engineering Escherichia coli 

分 类 号：Q814[生物学—生物工程]