Functional Characterization Of Four NADPH-cytochrome P450 Reductases In Andrographis Paniculata

ObjectiveAndrographis paniculata has the effects of clearing away heat and toxins,anti-inflammatory dampness,etc.It is widely used for the treatment of headache fever,cough and sore throat,dampness and heat diarrhea.Numerous studies have shown that the main active ingredients of A.paniculata include diterpene lactones and flavonoids,in which diterpene lactone content is an important measure of the quality of A.paniculata.The diterpene lactone compound andrographolide is the main effective component of A.paniculata,and it has the anti-virus,cardiovascular protection,immune function enhancement,anti-osteoporosis and anti-malaria activities.Many pharmacological activities show the great potential of andrographolide compounds.Currently the main active ingredient andrographolide mainly obtained from the original plant extract.However,with the increasing demand of A.paniculata,and the influence of pests,diseases and ecological environment,the wild resources have long been unable to meet the needs of the market.However,the lack of selection of cultivated resources and the imprecise planting conditions have made the quality of A.paniculata produced in different places varies greatly,with serious deterioration of quality and low content of active ingredients.This is one of the biggest obstacles to the large-scale entry of A.paniculata into the international market as a high-quality botanical drug.Therefore,there is an urgent need to find an effective way to ease the pressure on the supply of A.paniculata resources.As the technology of synthetic biology to produce high-value medicinal active ingredients has matured,the use of metabolic engineering or synthetic biology to increase the supply of natural products is one of the most promising methods.Therefore,using molecular biology techniques to study the biosynthetic pathway of andrographolide is expected to achieve heterologous and efficient production of andrographolide,thereby alleviating the supply pressure of high quality andrographis paniculata resources.Studies have shown that the diterpene synthase ApCPS2 in A.paniculata can convert the diterpene precursor GGPP into ent-CPP.However,the research on the downstream biosynthesis pathway of andrographolide is still very limited.Based on the structure of andrographolide speculated that the cytochrome P450(CYP450)family of genes catalyzes the biosynthesis downstream of ent-CPP.Cytochrome P450 Reductase(CPR),which provides electrons for the catalytic process of CYP450,is an important part of CYP450 system.The electron transfer reaction between CPR and CYP450 is the rate-limiting step of CYP450 redox reaction,so the study of CPR on the downstream biosynthesis of A.paniculata is of great significance.Therefore,the subject has carried on the following research:(1)Based on the transcriptome information obtained in the earlier stage,the CPR gene of A.paniculata was screened by homology comparison.(2)Functional verification of candidate enzyme genes by both prokaryotic and eukaryotic expression methods.(3)To analyze the tissue specificity and induction mode of CPR gene of A.paniculata.Finally,the function of CPR gene of four andrographis was identified,which laid the foundation for further isolation and identification of CYP450 involved in andrographolide synthesis,which laid the foundation for the downstream biosynthesis pathway of andrographolide.Methods1.The coding sequence of NADPH-cytochrome P450 reductase in A.paniculata was screened by transcriptome sequencing information and homology comparison.Primer Premier 5.0 software was used to design 5’and 3’end-specific primers,and the cDNA of A.paniculata was used as a template to clone cytochrome P450 reductase gene complete coding region(CDS).The basic characteristics of CPR gene of Andrographis paniculata were analyzed by bioinformatics.2.Construction of CPR prokaryotic expression vector of Andrographis paniculata CPR protein.Protein concentration was determined by the Quick Start Bradford Protein Assay kit.The electron transport activity of ApCPRs was measured by the change of optical absorption at 550 nm of reduced cytochrome C(Cyt Cred)and the change of optical absorbance of K3Fe(CN)6 at 424 nm.Absorbance changes were recorded by a Varioskan Flash spectrophotometer.GraphPad Prism 5 software was used to calculate the non-linear regression analysis.Finally,the prokaryotic expression system was used to verify CPR activity of Andrographis paniculata.3.Construct the CPR eukaryotic expression vector of Andrographis paniculata,co-express the CPR protein of A.paniculata and the CYP76AH1 protein of Salvia miltiorrhiza in yeast,and perform the enzymatic reaction with the secondary tanshinone diene.Qualitative detection by GC-MS was performed by quantitative analysis of yeast extract by ultra-high performance liquid chromatography(UPLC)to verify its function.4.The content of andrographolide in different tissues was analyzed by ultra-high performance liquid chromatography(UPLC).The expression of CPR in leaves of Andrographis paniculata after induction by methyl jasmonate was analyzed by RT-qPCR.According to the accumulation pattern of andrographolide,A.paniculata CPR which may be involved in andrographolide biosynthesis was inferred.Results and conclusion1.Based on the PCR technique,the andrographis leaf cDNA was finally obtained using a Takara reverse transcription kit.The complete coding region of ApCPR1,ApCPR2,ApCPR3 and ApCPR4 genes was amplified by specific primers using the cDNA of Andrographis paniculata as a template,and four specific bands of about 2000 bp were obtained and cloned into the pEASY-Blunt Zero vector to obtain recombinant plasmids.Based on the N-terminal sequence of the hydrophobic region,plant CPRs fall into two categories,namely,class Ⅰ and class Ⅱ.The phylogenetic analysis of 31 CPR sequences collected from 21 plants showed that ApCPR1 and AtCPR1 were classified as CPR class Ⅰ,ApCPR2,ApCPR3 and ApCPR4 and AtCPR2 were classified as class Ⅱ.2.ApCPR1,ApCPR2,ApCPR3 and ApCPR4 were cloned into prokaryotic expression vector pET-3 2a by double enzyme digestion and were overexpressed in Escherichia coli Transetta(DE3).Expression was induced by isopropyl b-d-1-thiogalactopyranoside(IPTG)to obtain a recombinant protein.Through in vitro enzymatic reaction,it was demonstrated that ApCPR1,ApCPR2 and ApCPR4 can reduce cytochrome c and ferricyanide in a NADPH-dependent manner,indicating that they have the function of CPR.In addition ApCPR3 is not active.3.The complete coding sequence of CYP76AH 1 was constructed by double enzyme digestion method to obtain the recombinant plasmid pAHl which was expressed in galactose-inducible yeast double expression vector pESC-Leu.Four ApCPR and SmCPRl were constructed by pSAl and pCPA1 respectively.CPR and CYP76AH1 were co-transformed into YJ14 yeast engineered bacteria and analyzed by GC-MS and UPLC after galactose induction.The results showed that ApCPR1,ApCPR2 and ApCPR4 could catalyze the production of rust alcohol by CYP76AH1 in yeast.ApCPR1 protein and CYP76AH1 protein were ligated through different linker peptides and constructed into the yeast double expression vector pESC-His.Yeast extract was analyzed by using transformed yeast engineering bacteria and galactose induced by UPLC.The results show that the content of rust alcohol,which is catalyzed by the fusion protein of ApCPR1 at the C-terminal of CYP76AH1 by the flexible peptide GGGGS or GSG,is greatly increased compared with that of CYP76AH1 and ApCPR1 alone.It shows that under certain conditions,the fusion protein of CPR and P450 is beneficial to increase the catalytic efficiency of the P450 monooxygenase in vivo.4.The content of andrographolide in different tissues of A.paniculata was determined by UPLC and found that the andrographolide contained in leaves was the most.The results of real-time quantitative PCR showed that the expression of ApCPR2 and ApCPR4 increased gradually with the induction time in the methyl jasmonate-treated andrographolide leaves.This pattern of expression is consistent with the trend toward accumulation of andrographolide by methyl jasmonate,suggesting that ApCPR2 and ApCPR4 are associated with the biosynthesis of andrographolide.