Studies On The Biosynthesis Of Phenylacrylic Acid Derivatives By Enzymatic Cascades

Phenacrylic acids are important plant secondary metabolites widely used in medicine and cosmetics.Currently,plant extraction,chemical synthesis,and microbial conversion are the main methods for phenacrylic acid production.Microbial convertion has some advantages such as,mild reaction conditions,simple procedures,environment-friendly,and broad application prospects.In this study,we tried to develop enzymatic cascades to synthesize phenacrylic acids from phenol and its derivatives.The main results are as follows.(1)Construction and analysis of enzymatic cascades.Enzymatic cascades were constructed by combining tyrosine phenol-lyase(TPL)from Erwinia herbicola,Citrobacter freundii,Rhodobacter capsulatus,and tyrosine ammonia-lyase(TAL)from Rhodotorula glutinis and Trichosporon cutaneum.In Escherichia coli BL21(DE3),the enzymatic cascade was fermented with catechol,sodium pyruvate and ammonium chloride.The intermediate L-DOPA and end-product caffeic acid were detected by HPLC to verify the catalytic activity of the enzymatic cascade.The results showed that the enzymatic cascade consisting of tyrosine phenol-lyase(Eh TPL)from E.herbicola and tyrosine ammonia-lyase(Rg TAL)from R.glutinis was more efficient.(2)Enzymatic cascade substrate profiling.The potential substrates phenol,catechol,resorcinol,and guaiacol were assayed.The corresponding products(p-coumaric acid,caffeic acid,2,4-dihydroxycinnamic acid,and ferulic acid)were detected by HPLC to verify the scope of substrate profiles for the enzyme cascade reaction.The results showed that the substrates of enzymatic cascade were phenol,catechol,and resorcinol.Moreover,phenol was a better substrate than catechol and resorcinol.(3)Optimization of the enzyme cascade reaction.After fermentation with 4 g/L glycerol as carbon source,yeast powder and peptone as nitrogen source,seed culture time of 10 hours,IPTG concentration of 0.1 m M,induction temperature of 20°C,induction onset time of 2 hours,and induction for 10 hours,p H was adjusted to 9.0,and then substrates(phenol,catechol,resorcinol)were added in batch replenishment mode for 8 hours at 35°C.Under the optimum conditions,p-coumaric acid yield reached 936 mg/L(8.13-fold increased);caffeic acid yield reached 154 mg/L(11.5-fold increased);and 2,4-dihydroxycinnamic acid yield reached 84 mg/L(7.4-fold increased).