Biosynthetic Pathway Elucidation And Microbial Cell Factory Construction Of Active Diterpenoids Of Andrographis Paniculata

The main active ingredients of Andrographis paniculata are lactone ring-containing diterpenoids andrographolide（AD）and neoandrographolide（NAD）,which are widely used in the treatment of various infections and acute lung injury.However,its unique lactone ring has strong species-specificity,which is a difficulty in pathway elucidation.With the rapid development of synthetic biology,the microbial"cell factories"based on Escherichia coli and Saccharomyces cerevisiae provide high throughput screening platforms for the rapid identification of catalytic enzymes,and the heterologous synthesis of important and complex natural products can also be achieved.Meanwhile,multi-omics integration analysis of genome,transcriptome and metabolome can more accurately understand the characteristics of active compounds biosynthesis and metabolism,and get more accurate range of candidate genes.This study focused on the mining of multi-omics data of A.paniculata and the construction of two microbial cell factories,E.coli and S.cerevicae,together with the functional identification of a class of kaurene synthase-like（KSL）and cytochrome P450 monooxygenase（CYP）,which were selected by multi-omics integration analysis based on these two microbial cell factories.Based on the special mechanism of plant diterpene biosynthesis and the specificity of A.paniculata lactone ring,four conjectural mechanisms of diterpenoid biosynthesis of A.paniculata were proposed,namely,KSL pathway,CYP pathway,pyrophosphatase pathway（PPase）and 2-oxoglutarate-dependent dioxygenase（2-ODD）pathway,among which the KSL pathway and CYP pathway were mainly considered.In addition,15 kinds of diterpenoids of A.paniculata were supplemented by the network pharmacology database.According to the structure variation of diterpenoids,the biosynthesis steps of active diterpene lactones in A.paniculata were reasonably deduced,which provided reliable basis for exploring the key enzyme genes of A.paniculata.Through multi-omics integration analysis and homology comparisons among functional genes of other species by phylogenetic tree,a total of 6 target Ap KSL genes were obtained,and 4 Ap KSL genes were focused after excluding 2 Ap KSL genes which had been reported by the cooperator.Further,2 Ap KSL genes were finally identified as candidate key genes by gene differential expression analysis.A total of 369 Ap CYP genes were obtained after screening,among which 59 were CYP71 and CYP76 families.While 28 members were highly expressed in leaves and stems,and 23 members of them could be induced by Me JA.At last,16 full-length Ap CYP genes were obtained by cloning.By means of metabolic engineering,the vector p GG-Ap CPS2 containing At GGPPS gene from Arabidopsis thaliana and Class I diterpene synthase gene Ap CPS2 were transformed into E.coli chassis cells for expression,and the E.coli cell factory capable for producing the universal diterpene precursor ent-CPP of A.paniculata was successfully constructed.Based on this chassis cell,we verified the function of two candidate Ap KSL,and found that Ap KSL4 could catalyze the formation of ent-sandaracopimiradiene,ent-isopimiradiene and a new unknown product.The structure of the new product was identified as a new diterpenoid by NMR,named（4a S,4b R,8S,8a S,10a R）-1,1,4a-trimethyl-7-methylene-8-vinyltetradecahydrophenanthrene,abbreviated TMV,formula for C₂₀H₃₂,and the molecular weight is 272.48.Meanwhile,the contents of TMV in different organs of A.paniculata were detected.The content of TMV was 0.852±0.017μg/g in the leaf and0.472±0.152μg/g in the stem of A.paniculata.The target gene expression frame was assembled using CRISPR/Cas9 technology and fusion PCR method,and the Ap CPS2 and Arabidopsis cytochrome P450 reductase gene At CPR1 were homologous recombination at the neutral site of the genome for expression.The cell factory of S.cerevicae producing ent-CPP was successfully constructed.Based on the cell factory,the expression frame containing Ap KSL4 gene was introduced,and the cell factory of S.cerevicae capable of producing TMV was preliminarily obtained through genome reconstruction.By the idea of cell metabolic engineering,the central metabolism was enhanced by overexpression of ERG10,HMG2 and HMGr,and the biosynthetic pathway of terpenoid precursors was enhanced by fusion expression of GGPPS.By constructing Ap CPS2 and Ap KSL4 fusion expression boxes,the metabolic flow was guided into the biosynthesis direction of the product TMV.And the strategy of enhancing the regulatory factor module by overexpressing Upc2,a transcription factor that positively regulates the metabolism of S.cerevicae,optimized the cell factory for producing TMV.The TMV yield was increased from 34.62μg/L to 386.42μg/L by 11.16 folds.CCK-8 method was used to detect the effect of TMV on RAW264.7 cell viability.The effects of TMV on the secretion of inflammatory cytokines and the regulation of genes and proteins expression related to inflammation were detected by ELISA,Western Blot and q PCR to systematically evaluate the pharmacological activity of TMV.It was found that TMV had a certain inflammatory inhibition effect,and the anti-inflammatory effect was enhanced with the increase of its concentration,which was a potential pharmacodynamic substance.Based on the two microbial factories of S.cerevicae and E.coli,the functions of 16Ap CYPs were tested in batches.The results showed that Ap CYP14 could catalyze ent-CPP to produce an unknown product,while the other 15 Ap CYPs were no obvious catalytic reactions nor new products were generated.The mass spectrogram of Ap CYP14 catalytic product was retrieved in NIST and other databases,but no matching information was found,so that further identification of the unknown product by NMR is needed,and this result will provide clues and possibilities for the elucidation of the biosynthetic pathway of the active diterpene lactone in A.paniculata.In conclusion,this study successfully constructed the microbial factories of E.coli and S.cerevicae capable for producing ent-CPP,aiming at solving the problems of unclear biosynthetic pathway of active diterpene lactones in A.paniculata and difficulties in elucidation.The active diterpene biosynthetic pathway of A.paniculata was explored and a new diterpene compound was heterologous synthesized and yield optimized by the biosynthetic pathway prediction and derivation,multi-omics integration analysis to screen genes and microbial cell factories verification.In addition,the anti-inflammatory effect of the new obtained diterpenoid was also evaluated.This study laid a foundation for the analysis,cell factory construction and modification of the biosynthesis of active components AD and NAD in A.paniculata,which is of great significance for the quality control of the medicinal materials,the cultivation of improved varieties and the hetero-synthesis of active components for A.paniculata.