Identification And Functional Analysis Of Genes To Biosynthetic Of Sanguinarine And Chelerythrine In Macleaya Cordata

Macleaya cordata(Willd.)R.Br.is a perennial herb that belongs to the Papaveraceae family and is typically prescribed as a traditional antibacterial medicine,whose effect and usage were well documented in Ben-Cao-Shi-Yi,a Chinese encyclopedia of botany and medicine from the early Tang dynasty.More importantly,a high level of anti-inflammatory,regulating intestinal microbiota and promoting animal growth benzylisoquinoline alkaloids(BIAs),such as sanguinarine(SAN)and chelerythrine(CHE)are produced in M.cordata,whereas addictive BIAs,such as morphine and codeine,are completely absent.Therefore,M.cordata has become an alternative to antibiotic feed additive and widely used in zootechny in many countries around the world.As more and more countries limit and ban antibiotic feed additive,the market demand for M.cordata will fall exponentially,resulting in a sharp decline in the wild resource.It is very important to improve the SAN and CHE content in M.cordata by molecular breeding and using the heterogeneous production of SAN and CHE in industrial microbes,but the prerequisite is to find the synthetic gene of SAN and CHE.SAN and CHE belong to BIAs according to its chemical structure.This is a complex biosynthetic pathway involved many genes.In this paper,the whole genome and transcriptome of M.cordata were sequenced,and combination of genomic,transcriptomic and metabolism profile investigations to predict and screen the candidate genes involved in the synthesis of SAN and CHE.Finally,we using heterologous expression and precursor feeding in Saccharomyces cerevisiae to test and verify the function of the candidate gene.The major findings are as follows1.This study completed the whole genome sequencing of M.cordata,which is the first species to complete genome-wide sequencing in the Papaveraceae.The 19-kmer analysis of these sequences suggested a genome size of 540.5 Mb and a heterozygous rate of 0.92%,a total of 22,328 protein-coding genes were predicted.Compare the genome-wide data of M.cordata within biosynthetic genes of SAN and CHE in Papaver bracteatum,Coptisjaponica,Eschscholtzia californica and Papaver somniferum for homologous gene alignment,39 candidate gene sequences were obtained.Then,based on the transcriptome and metabolic profile data of different organs in M.cordata,it is concluded that the synthesis of SAN and CHE in M.cordata may have tissue-organ specificity,the protopine(PRO)and allocryptopine(ALL)which is the precursors of SAN and CHE synthesized in roots,while SAN and CHE are synthesized in capsules.According to this hypotheses,16 genes were screened from 39 candidate genes.It includes 6 methyltransferase genes,4 flavoprotein oxidase genes,6 cytochrome P450 enzyme genes and 1 cytochrome P450 reductase gene.All candidate genes were transferred into Saccharomyces cerevisiae for heterologous expression verification,and finally,the genes which participated in 14 steps synthetic pathway from norcoclaurine to SAN and CHE were identified.The Mco2833 gene is responsible for catalyzing the first step from norcoclaurine to SAN and CHE synthesis,methylating norcoclaurine to form coclaurine;2 methyltransferase genes Mco8567 and Mco769 catalyze the second step;The cytochrome P450 oxidase gene Mco2661 catalyzes the 3'-hydroxylation ofN-methylcoclaurine to form 3'-hydroxy-N-methylcoclaurine in step 3;The Mco2833 gene can also transfer a methyl group to the 4'-hydroxyl group of 3'-hydroxy-N-methylcoclaurine in step 4 to form a reticuline;Step 5 is characterized by the flavoprotein oxidase gene Mco20113 catalyzing the formation of scoulerine;The first step to the SAN synthesis branch is completed by the cytochrome P450 oxidase gene Mco217;the second step catalyzes the scoulerine-generated cheilanthifoline gene failed to find;the third step is catalyzed by two methyltransferase genes Mco830 and Mco830 to convert stylopine to N-m ethyl sty lopine;The gene that catalyzes the production of protopine by N-methylstylopine in the fourth step reaction could not be found;Two cytochrome P450 oxidase genes,Mco11229 and Mco11218,can be completed step 5 catalyzes the reaction of PRO to produce 6'-hydroxyprotopine and undergoes spontaneous reaction to form dihydrogensanguinarine(DHSAN);Finally,two flavoprotein oxidase genes,Mco6407 and Mco6408,can catalyze the formation of SAN by DHSAN;The first step from scoulerine to CHE synthesis is completed by the methyltransferase gene Mco9487;the second step catalyzes the cytochrome P450 oxidase gene of tetrahydrocolumbamine to produce canadine is Mco9485;the third step is also composed of two methyltransferase genes.Mco830 and Mco830 are completed;The gene for the reaction in step 4 was not found;the reaction in step 5 was also completed by both Mco11229 and Mco11218,which catalyzed the formation of 6'-hydroxy allocryptopine by allocryptopine and spontaneously reacted to form dihydrochelerythrine(DHCHE);in the last step,there are also Mco6407 and Mco64082 genes that can catalyze DHCHE to generate CHE2.This study demonstrates the synthesis pathway of reticuline which is the important synthetic intermediates of SAN and CHE proposed by Battersby for the first time.Mco2833 catalyzes the production of 6'-O-methylnorlaudanosoline by norlaudanosoline to catalyze the formation of norreticuline,and then both genes Mco769 and Mco8567 can catalyze the production of reticuline by norreticuline.This pathway has been confirmed to provide a new explanation for the biosynthesis of reticuline in higher plants,and provides a new route for the re-editing of SAN and CHE synthetic pathways.3.This study found that the methyltransferase Mco2833 in M.cordata participated in the 4 steps reaction in the SAN and CHE synthesis pathways.The four different substrates of norlaudanosoline,6'-O-methylnorlaudanosoline,norcoclaurine and 3'-hydroxy-N-methylcoclaurine are catalyzed to form 6'-O-methylnorlaudanosoline,norreticuline,coclaurine and reticuline,respectively.This result provides genes to build an efficient synthetic SAN and CHE pathways in industrial microbiology.4.This study explored the possibleN-methylstylopine synthesis bypass in M.cordata.Although both Mco830 and Mco833 genes can be used to form N-methylscoulerin and N-methylcheilanthifoline,respectively,as scoulerine and cheilanthifoline as methylation substrates,Mco217 cannot catalyze the formation of N-methylcheilanthifoline by N-methylscoulerin.The PsSPS gene in poppy does not catalyze the production of N-methylstylopine by N-methylcheilanthifoline.This work identified new catalytic substrates for Mco830 and Mco833 in M.cordata and provided new ideas and evidence for the synthetic pathways of some intermediate compounds in SAN and CHE.