Efficient synthesis of tyrosol from L-tyrosine via heterologous Ehrlich pathway in Escherichia coli

For the efficient conversion of L-tyrosine(L-Tyr)to tyrosol,which is an aromatic compound widely used in the pharmaceutical and chemical industries,a novel four-enzyme cascade pathway based on the Ehrlich pathway of Saccharomyces cerevisiae was designed and reconstructed in Escherichia coli.Then,the expression levels of the relevant enzymes were coordinated using a modular approach and gene duplication after the identification of the pyruvate decarboxylase from Candida tropicalis(CtPDC)as the rate-limiting enzymatic step.In situ product removal(ISPR)strategy with XAD4 resins was explored to avoid product inhibition and further improve tyrosol yield.As a result,the titer and conversion rate of tyrosol obtained were 35.7 g·L^(-1) and 93.6%,respectively,in a 3-L bioreactor.Results presented here provide a potential enzymatic process for industrial production of tyrosol from cheap amino acids.

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

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.