Complete Analysis Of The Biosynthetic Pathway Of The Main Flavonoid Components Of Licorice And De Novo Synthesis In Yeas

Glycyrrhizae Radix et Rhizoma,one of the most commonly used rare and endangered medicinal herbs with a long history of application,has shown good and safe preventive and therapeutic effects on various diseases.It is widely used in almost all Chinese medicine prescriptions,medicines and more health care products,even in the food,tobacco and cosmetics industries.Flavonoids are a large group of components with strong biological activity found in Glycyrrhiza uralensis.The main flavonoids of G.uralensis include isoliquiritigenin(Iso-LG),liquiritigenin(LG)and their glycoside derivatives isoliquiritin(Iso-LN)and liquiritin(LN).Because the functional genes in the main flavonoid metabolic pathway of G.uralensis belong to the gene family,especially some downstream key enzymes are super-family genes,with many members and complex functions,which limits the complete analysis of their biosynthetic pathway.The current problems of G.uralensis resource and quality,coupled with the limitations of chemical synthesis,tissue cell culture and other methods,the heterogeneous biosynthesis of the active flavonoids has become an effective strategy.Based on the deep transcriptome combined with multiple analysis methods,this study efficiently screened,cloned and characterized the functional genes of the G.uralensis flavonoids pathway and achieved biosynthesis in Saccharomyces cerevisiae.The specific research contents are as follows:1.Screening,cloning and bioinformatics analysis of candidate genes for all 7 enzymes in the main flavonoids synthesis pathway of G.uralensis.In order to efficiently screen candidate genes for all enzymes genes in the G.uralensis flavonoids pathway,this study performed deep transcriptome sequencing of G.uralensis seedlings after NaCl and MeJA stress,combined with multiple analyses including G.uralensis genome,bioinformatics annotation,known homologous gene cluster,and the major candidate genes were rapidly screened.The transcriptome sequencing results obtained large amount of data,deep sequencing,high accuracy and good quality.A total of 61 potential genes for 7 enzymes(PAL,C4H,4CL,CHS,CHR,CHI,UGT)were screened by transcriptome binding genomic analysis.Then based on the further analysis of genes expression level,and according to the published homologous genes reported in the literatures,the NJ trees were constructed by clustering the transcriptome sequences annotated as chalcone reductase,chalcone isomerase and flavonoid 7-O-glucose transferase to further narrow the range of candidate genes.Finally 7 major candidate genes named as GuPAL1,GuC4H1,Gu4CL1,GuCHS1,GuCHR1,GuCHIl and GuUGT1,as well as 11 other candidate genes were screened based on the above analysis,which would be used for the gene cloning and functional characterization in the next research,in order to rapidly and exactly obtain the 7 enzyme genes with catalytic activities.In order to obtain candidate gene sequences and information,in this study,based on transcriptome gene sequences,all the 18 candidate genes were cloned and the 16 gene sequences were successfully obtained,including GuPAL1,GuPAL2,GuPAL3,GuC4H1,GuC4H2,Gu4CL1,Gu4CL2,Gu4CL3,GuCHS1,GuCHS3,GuCHS4,GuCHR1,GuCHI1,GuUGT1,Gu UGT2,Gu UGT3.Among them,the candidate genes CHS2 and CHI2 failed to be cloned due to the transcriptome sequence splicing problem.Then bioinformatics analy sis of the obtained genes was carried out,which can provide a basis for candidate genes screening validation and subsequent catalytic activity studies.2.Study on catalytic activity of candidate genes based on E.coli expression and yeast expression.In order to characterize the activity of all candidate genes,the E.coli expression system was used to verify the function of PAL,CHS,CHR,CHI and UGT.The expression vectors were constructed by recombination with pET32a(+),transformed into BL21(DE3)and cultured,then induced expression by IPTG,via in vitro enzymatic reaction,and detected products by LC-QTOF-MS/MS.The results showed that the candidate genes PAL1 and PAL2 could catalyze the production of the target product cinnamic acid;the candidate genes CHS1 and CHS3 could catalyze the production of the target product naringenin chalcone;the candidate gene CHR1 could catalyze the production of the target product Iso-LG;the candidate gene CHI1 could catalyze the target product LG;the candidate genes UGT1,UGT2 and UGT3 were capable of catalyzing the production of the target products Iso-LN and LN.The functions of C4H and 4CL were verified by the yeast expression system.The results showed that the candidate genes C4H1 and C4H2 had the function of synthesizing coumaric acid;the candidate genes 4CL1 and 4CL2 had the corresponding catalytic activities.In summary,all 7 enzymes in the pathway with their respective catalytic activities have been successfully obtained.3.Preliminary study on the potential catalytic mechanism of the important enzyme flavonoid UGT in G.uralensis.In order to further study the potential catalytic mechanism of important key enzyme UGT,this study researched the catalytic parameters of GuUGT1,GuUGT2 and GuUGT3,and preliminarily studied its catalytic mechanism through homologous modeling,molecular docking and amino acid scanning of GuUGT1.The results of enzyme kinetic parameter analysis showed that UGT 1 had a Km of 68.17 μM,a Vmax of0.87 μmol·(min·mg)-1,and a Vmax/Km of 12.49,indicating that UGT1 had better activity and higher catalytic efficiency,which also verified the correctness of EUGT1 as a major candidate gene.Through the homology modeling and molecular docking simulation,the binding modes and binding affinity of the receptor UGT1 with UDPG,LG and other compounds were predicted.The ligand LG could produce hydrogen bonds with the side chains of Ser363 and Thr143 respectively,which were potential important binding sites.The ligand UDPG could form hydrogen bonds with the skeletons of Asn362,Gly14,Ser363,Ser13,Met17,Gly277,Ala341 and the side chains of His358,respectively.Amino acid scanning predicted that Gly-14 and Gly-277 were key residues affecting the gly cosylation of LG by UGT 1 protein.4.De novo synthesis of the main active flavonoids of G.uralensis in S.cerevisiae.In order to achieve heterogenous biosynthesis of the main flavonoid components of G.uralensis,one or more of the major candidate genes with known catalytic functions were inserted into pESC series vectors,and a total of 7 different recombinant plasmids were obtained.The recombinant plasmids obtained above were then combined into different synthesis modules and transformed into S.cerevisiae WAT11,and a total of 9 different recombinant yeast strains were obtained.Based on the upstream substrate and intermediate supply mechanism of S.cerevisiae WAT11,the obtained recombinant yeast WM1 produced p-coumaric acid,and the yield after 36 hours was 7.59 μmol·L-1;A small amount of Iso-LG was detected in WM2-1 fermentation;WM3-1 and WM4-1 produced LG and LN respectively;Most of the Iso-LG in WM4-1 was glycosylated before isomerization,and the yield of Iso-LN was 4.7 times higher than that of LN;WM3-2 and WM4-2 promoted the conversion of Iso-LG to LG by overexpressing GuCHI,and the yield of LG or LN increased by 1.3 times;In WM2-2,WM3-3 and WM4-3,GuCHS1::GuCHR1 was overexpressed,so the production of Iso-LG was 18.2 times higher than that of WM2-1,and the yield of LG was 5.3 times that of WM3-1,and LN accumulation was 3.1 times higher than WM4-2 and 6.4 times higher than WM4-1.To sum up,in this study,18 candidate genes of all 7 enzymes responsible for biosynthesis of main flavonoids in G.uralensis were efficiently screened out by combination of deep transcriptome sequencing and multiple analysis methods,then cloning,bioinformatics analysis and functional characterization of these genes were performed,finally 13 candidate genes with corresponding catalytic functions were obtained,which complete analyzed metabolic pathway of main active flavonoids in G.uralensis for the first time.Afterwards,7 major candidate genes with catalytic activity were reconstructed their metabolic pathways in S.cerevisiae,which realized de novo biosynthesis of Iso-LG,LG and their glycoside derivatives Iso-LN and LN in S.cerevisiae for the first time,providing a new model for the production of G.uralensis main flavonoids and sustainable utilization of G.uralensis resources.