Elucidation And Functional Study Of Key Enzymes In Salvianolic Acids Biosynthesis Pathway In Salvia Miltiorrhiza

A variety of complex natural products from medicinal plants are formed by certain laws and biosynthesis pathway research of these products remains in frontier field.The critical study to elucidate biosynthesis pathway is functional evaluation of key catalytic enzymes.Only by fully understanding the functions of key enzymes,can they be used as the basic elements for heterologous synthesizing natural products in synthetic biology;as the potential targets for regulating in metabolic engineering;as the important candidates for protein engineering in structural biology to realize directed evolution.After nearly 20 years of exploration by researchers,the biosynthesis pathway of the salvianolic acids in Salvia miltiorrhiza has been basically elucidated.The function of most of key enzymes in this pathway have been verified.The 4-coumarolyl-Co A from phenylalanine pathway and 4-hydroxyphenyllactic acid or 3,4-dihydroxyphenyllactic acid(Salvianic acid)was catalyzed by Rosmarinic acid Synthase(Sm RAS)to form precursor of rosmarinic acid,and then oxidized by Cytochromes P450 monooxygenase 98A(Sm CYP98A)family genes to generate rosmarinic acid.However,the biosynthesis mechanism of products in downstream of rosmarinic acid(RA),such as salvianolic acid B(SAB),salvianolic acid E(SAE)as well as other natural active ingredients is still unclear.It is indirectly suggested that the Laccase family(Sm LAC)may play a role in SAB biosynthesis based on some in vivo experiments.For these key enzymes,their functions and catalytic mechanism s still need to be further studied.Therefore,this project focuses on the three key enzyme families in the salvianolic acids biosynthesis pathway,which are Sm RAS,Sm CYP98 A and Sm LAC.The research strategy as following: Screening out the target enzyme by integratinge omics data and bioinformatics analysis;Obtaining the recombinant enzyme protein through heterologous expression;Studying the enzyme catalytic function and mechanism by in vitro enzyme catalysis experiment combined with molecular docking;Investigating the in vivo function by gene knocked out and overexpressed technology;Profiling the gene expression through expression profiling analysis experiment,to further uncover function of three key enzymes in the biosynthetic pathway of salvianolic acid.Eleven Sm RASs were mined from the S.miltiorrhiza genome and Sm RAS10 was selected as target through transcriptome and phylogentic analysis on account of higher expression level in roots and closely related to rosmarinic acid synthase from other Lamiaceae plants.Sm RAS10 recombinant protein was obtained through Escherichia coli heterologous expression system.In vitro catalysis experiments suggested that Sm RAS10 can catalyze different types of acyl donors and acyl acceptors.Kinetic parameters of relevant reaction were aslso measured.Through molecular docking,key catalytic sites of Sm RAS10 were predicted.After Sm RAS10 was knocked out or overexpressed using CRISPR/Cas9 and overexpression methods,the acculmation of RA and SAB in S.miltiorrhiza were significantly changed.That indicated Sm RAS10 plays an important role in the biosynthesis of salvianolic acids.It can be used as a potential target in metabolic engineering strategy for increasing the content of salvianolic acid in transgenic hairy roots.Three Sm CYP98 A family enzyme genes were mined from the genome of S.miltiorrhiza,namely Sm CYP98A75,Sm CYP98A76 and Sm CYP98A14.Through transcriptome and phylogentic analysis,expression profile in different organs of three genes and their relationship with CYP98 A genes in other species were studied.Based on the heterologous expression system of Saccharomyces cerevisiae,microsomal recombined proteins containing Sm CYP98A75,Sm CYP98A76 and Sm CYP98A14 were obtained.According to in vitro catalysis reaction,Sm CYP98A75 and Sm CYP98A14 can oxidize various rosmarinic acid precursors to generate rosmarinic acid.Furthermore,Sm CYP98A75 can also catalyze the production of salvianic acid from 4-hydroxyphenyllactic acid in the case of 4-hydroxyphenyllactic acid and Sm RAS existence.It is suggested that Sm CYP98A75 plays an equally key role as Sm CYP98A14 in the biosynthesis of salvianolic acid.The kinetic parameters of Sm CYP98A75 and Sm CYP98A14 were determined respectively,and the key amino acid sites for the catalytic action of two enzymes were also predicted.The singletarget and dual-targets knockout vectors for Sm CYP98A14 and Sm CYP98A75 were constructed via CRISPR/Cas9 system.Compared with single-target knockout hairy roots,the decrease in phenolic acid content in dual-targets knockout hairy roots is more significant,indicating that Sm CYP98A75 and Sm CYP98A14 are essential to the biosynthesis of salvianolic acid components.After Sm CYP98A75 or Sm CYP98A14 was overexpressed,the content of RA and SAB in the transgenic hairy roots were significantly increased,suggested these two enzymes can be used as potential targets in metabolic engineering strategy for increasing the content of salvianolic acid in transgenic hairy roots.On the basis of previous study of Sm LACs by our group,Sm LACs overexpression or silencing resulted in changes of salvianolic acid content.Due to lacking of verification by in vitro catalysis experiment,the function of Sm LAC needs to be further confirmed.Using the Pichia pastoris heterologous expression system,recombined proteins of Sm LAC7,Sm LAC20 and Sm LAC28 were obtained.In vitro enzyme catalysis reaction showed that none of the three enzymes could directly oxidize RA to generate SAB.However,the Sm LAC7 can catalyze some stilbene compounds with stilbene nucleus,such as oxidized resveratrol and cedar alcohol to form the dimer containing furan ring.Because of the numerous of members and redundant function in plant laccase family,conserved domains editing via CRISPR/Cas9 system to knock out most of Sm LACs is a good strategy to study the function of them.Single-target and dual-targets knockout vector for two copper ion conserved domains of Sm LACs were constructed,and transgenic hairy roots were obtained.It was observed that the growth as well as development of Sm LACs-knockout hairy roots were significantly inhibited,and the accumulation of lignin as well as phenolic acids decreased significantly.Through microscopic observation,it was found that the root structure was abnormal,the xylem and phloem areas appeared irregular arrangement with blurred boundary after Sm LACs were knocked out.Studies on Sm LAC showed they plays a key role in the growth as well as development of S.miltiorrhiza roots and they are closely related to the biosynthesis of lignin and phenolic acid.Three Sm LACs fail to catalyze RA to form SAB does not mean that the family of Sm LAC didn’t participate in SAB generation.Due to the large number of members of the Sm LAC family,other members may still play a catalytic role and need further researches.Moreover,the dimerization of stilbene compounds with stilbene nucleus by Sm LAC7 also offers a reference for the study biosynthesis pathway of medicinal plants such as Polygonum cuspidatum,whose stilbene glycosides usually as the main natural products.In conclusion,this study conducted a more detailed study on the key enzymes of the three families in salvianolic acid biosynthesis pathways and elucidated their biological functions.This research remaps the biosynthesis pathway of salvianolic acids,which will provide more reliable elements for future study on the salvianolic acid synthetic biology,provide potential targets for metabolic engineering strategies based on key enzymes and enrich theoretical basis of rosmarinic acid biosynthesis pathway in Lamiaceae plants.