Study On Tyrosol Production From L-tyrosine Using A Muti-enzyme Cascade

Tyrosol is a phenethyl alcohol derivative with pharmacological activity,which is a precursor of functional compounds such as hydroxytyrosol and salidroside.It is widely used in pharmaceutical,cosmetic and food industries.Conventional plant extraction and chemical synthesis have the disadvantages of low extraction yield,harsh reaction conditions and complicated purification process.The enzymatic conversion method has the advantages of mild reaction,high conversions and little environmental pollution,which is a potential tyrosol production method.In this study,a cascade pathway was designed by mimicking the Ehrlich pathway of Saccharomyces cerevisiae for whole-cell catalytic synthesis of tyrosol from L-tyrosine.After identifying the rate-limiting enzyme in the multi-enzyme reaction,the expression levels of the cascade pathway were optimized by modular assembly and gene duplication of the rate-limiting enzyme pyruvate decarboxylase(PDC).The synergism of L-amino acid deaminase(LAAD),pyruvate decarboxylase,alcohol dehydrogenase and glucose dehydrogenase was obtained.Then the optimized recombinant strain was used to further improve the tyrosol production by in situ product removal.The main contents were described as follows.(1)Design and construct a cascade pathway for tyrosol biosynthesis.First,based on the three-step reaction of transamination,decarboxylation and reduction of yeast Ehrlich pathway,deamination-decarboxylation-reduction was elicited and a cofactor regeneration system was constructed for the reduction reaction.Second,L-amino acid deaminase from Proteus mirabilis(Pm LAAD),pyruvate decarboxylase from Candida tropicails(Ct PDC),alcohol dehydrogenase from Saccharomyces cerevisiae(Sc ADH6)and glucose dehydrogenase from Bacillus megaterium(Bm GDH)were screened as pathway enzymes for the cascade reaction.Then,the formation of tyrosol was detected by cascade catalytic reaction in vitro,which verified the feasibility of this cascade pathway.Finally,PDC was identified as the rate-limiting enzyme of the cascade reaction via measuring the kinetic parameters of the four enzymes.(2)Assembly and optimization of multi-enzyme cascade pathway.Co-expression strains of Pm LAAD,Ct PDC,Sc ADH6 and Bm GDH were reconstructed to verify the feasibility of the cascade reaction in vivo by whole-cell conversions.A series of co-expression strains were constructed by co-expressing these four enzymes in two plasmids,and four plasmids with different copy numbers were used to regulate the relative activities of the four enzymes and balance the reaction rate of each enzyme.Comparing the tyrosol-producing ability of the 12 constructed strains(E.coli 01-12),the best tyrosol-synthesizing strain E.coli 07 showed accumulation of the intermediate product p-hydroxyphenylpyruvate(4-HPP)when increasing the concentration of substrate,and the enzyme activity ratio of LAAD to PDC in whole cells was detected as 1:0.8.In vitro conversions for different activity ratios of LAAD to PDC suggested that ratio of 1:1.5 was the optimal.Based on the results of in vitro optimization,gene duplication strategy was used to improve the expression level of PDC and obtained E.coli 07-2 strain with the ratio of enzyme activity increased to 1:1.5.The conditions for catalysts preparation and conversion system were optimized with E.coli 07-2 strain,and the conversion reached 95.5%with 30g·L^-1L-tyrosine as substrate.In addition,it was found that the limiting factor affecting the further increase of yield was product inhibition.The product concentration of 22 g·L^-1could completely inhibit the conversion of whole cells.(3)In situ product separation method to improve the yield of tyrosol.The resin XAD4was selected by comparing the adsorption capacity of tyrosol and L-tyrosine.Secondly,the addition of resin was optimized,in which 10%(dry,w/v)XAD4 was able to adsorb 17.5g·L^-1of tyrosol;Under the condition of high concentration of ethanol,the tyrosol on the resin could be desorbed effectively.In addition,the desorption time also had an effect on the yield of tyrosol.The yield of tyrosol was 97.8%after two batches of 3 times volume of anhydrous ethanol were desorbed for 8 h.Then,the effect of different substrate concentrations on the adsorption conversion were investigated.Finally,the concentration of tyrosol prepared on a scale using E.coli 07-2 binding resin XAD4 reached 35.7 g·L^-1for 32 h,with 93.6% conversion.