| Plant-derived natural products are one of the important ways to create new pesticides.Phryma leptostachya is a perennial herb.As a traditional insecticidal plant,it has been used for pest control in East Asia for a long time.The plant contains a variety of lignans with high insecticidal activity,especially(+)-haedoxan A has extremely high insecticidal activity against diptera and lepidoptera pests.Previous studies have also shown that(+)-haedoxan A has significant selectivity and low cytotoxicity,and is relatively safe for humans and higher animals.Therefore,the development of botanical insecticides with(+)-haedoxan A as the main insecticidal active ingredient has broad prospects.However,just like the problems encountered in the development of most plant-derived natural products,the shortage of resources is the bottleneck restricting the industrialization of(+)-haedoxan A.The traditional way of obtaining(+)-haedoxan A is to directly extract and separate from the root of P.leptostachya,but(+)-haedoxan A is only found in P.leptostachya,and the content is very low.Consequently,it is not feasible to rely on this method to produce pesticides.Although(+)-haedoxan A has been fully synthesized by chemical methods,its harsh reaction conditions and more than 20 synthetic reaction steps make it impossible to industrialize.With the rise of synthetic biology,the “bottom-up” strategy to achieve the purpose of “building to apply” is one of the most effective ways to solve the shortage of plant resources,and has achieved great success in solving the supply of artemisinin and paclitaxel resources.The analysis of biosynthetic pathways is the premise and foundation of synthetic biology research.Currently,there are very few studies on the biosynthesis of(+)-haedoxan A,only one cytochrome P450(Pl CYP81Q38),which may be involved in its biosynthesis,has been reported,and no genomic and transcriptomic data of P.leptostachya in the public databases.In this study,based on the transcriptome sequencing of different tissues of P.leptostachya,bioinformatics analysis was used to predict the candidate genes that may be involved in the biosynthesis of(+)-haedoxan A,focusing on the functional studies of cytochrome P450(P450)and O-methyltransferase(OMT)involved in post-modification reactions in the biosynthesis pathway of(+)-haedoxan A.The main results are as follows:1.Using HPLC,LC-MS and other methods,it was confirmed that the root tissue was the main site of accumulation of lignans metabolized by P.leptostachya.Based on this,the transcriptome sequencing of different tissues of P.leptostachya was completed by using the strategy of comparative transcriptomics and combined with the second-generation and third-generation high-throughput sequencing technologies.A total of 72,796 full-length transcripts were obtained from the third-generation Pac Bio sequencing results,which were used as the reference transcriptome database of P.leptostachya without reference genome.The second-generation BGISEQ-500 platform was employed to sequence the roots,stems and leaves of P.leptostachya,and a total of 68,536 transcripts were finally detected,with an average alignment rate of 77.48% with the Pac Bio reference gene set.By analyzing the upstream genes of(+)-haedoxan A biosynthesis,it was found that 22 of the 37 differentially expressed genes related to coniferyl alcohol biosynthesis were up-regulated in roots,and Pl CYP81Q38 was also specifically expressed in roots.The expression pattern of these genes was basically consistent with the accumulation pattern of lignans.Thus,the tissue expression pattern of Pl CYP81Q38 was used to screen downstream genes,and 18 P450 s and 15 OMTs were obtained as candidate genes for further research.2.(+)-Sesamin was identified as the key precursor for the biosynthesis of lignans in P.leptostachya by using the substrate feeding experiment of P.leptostachya seedlings.Metabolic profiling analysis showed that two products could be generated after feeding(+)-sesamin,and LC-HRMS preliminarily speculated that both products were first hydroxylated and then methylated products of(+)-sesamin.Then,the 14 P450 s screened previously were successfully expressed in Saccharomyces cerevisiae.Combined with whole-cell catalysis and in vitro biochemical tests,it was confirmed that a new P450 enzyme Pl CYP706AA1 catalyzed the next reaction of(+)-sesamin in P.leptostachya.After separation,purification and structural identification of the reaction products,it was determined that Pl CYP706AA1 catalyzed the reaction of(+)-sesamin to generate two products,namely(+)-sesaminol and(+)-sesamin-2-ol,in which(+)-sesaminol was the main product.3.Three endogenous NADPH-cytochrome P450 reductases(Pl CPRs)were found in P.leptostachya,and it was preliminarily confirmed that the 3 Pl CPRs have electron transfer function by prokaryotic expression and in vitro enzymatic tests.Subsequently,it was determined by the whole-cell catalytic system of S.cerevisiae that all 3 Pl CPRs could improve the catalytic efficiency of CYP706AA1,among which Pl CPR2 showed the highest matching degree with CYP706AA1.Pl CPR2 can increase the total yield of the products(+)-sesaminol and(+)-sesamin-2-ol by more than 3 times,and the total yield of products in shake flasks was increased from 3.00 mg/L to a maximum of 9.40 mg/L.In addition,the universal test showed that Pl CPRs could also enhance the catalytic activity of Pl CYP81Q38 and Si CYP92B14.4.Eight OMT genes of P.leptostachya(PlOMTs)were cloned,and 7 soluble recombinant Pl OMTs proteins were successfully expressed in E.coli.In vitro enzymatic test showed that Pl OMT2 had the highest catalytic activity for the two substrates(+)-sesaminol and(+)-sesamin-2-ol.After separation,purification and structural identification of the reaction products,it was determined that Pl OMT2 methylated the above two substrates to generate(+)-sesangolin and 1,3-benzodioxole,5-[4-(1,3-benzodioxol-5-yl)tetrahydro-1H,3H-furo[3,4-c]furan-1-yl]-4-methoxy-,[1S-(1α,3aα,4α,6aα)]-(9CI),respectively.At the same time,the presumption of LC-HRMS in the early stage was also proved,that is,the two methylated products generated by the P1OMT2 reaction were the same as those two products generated by feeding(+)-sesamin to the seedlings of P.leptostachya.5.RT-qPCR analysis showed that the newly discovered genes Pl CYP706AA1,Pl OMT2 and Pl CPRs exhibited highly similar spatiotemporal expression pattern with the reported Pl CYP81Q38 gene,that is,they were highly expressed in the roots after the six-leaf stage of P.leptostachya.Considering that the root tissue was the main accumulation site of lignans,this indicated that the synthesis of lignans may be consistent with the accumulation site,that is,it mainly occurred in the root tissue of P.leptostachya.In conclusion,based on the concept of synthetic biology,this study carried out the analysis of the biosynthetic pathway of(+)-haedoxan A in P.leptostachya by means of natural product chemistry,transcriptomics,molecular biology,biochemistry,etc.It was determined that the root tissue was the main accumulation site of lignans metabolized by P.leptostachya,and the first transcriptome sequencing of P.leptostachya was completed.Two new enzymes Pl CYP706AA1 and Pl OMT2 involved in the biosynthesis of lignans were identified from P.leptostachya,and three Pl CPRs that can improve the catalytic efficiency of Pl CYP706AA1 were obtained.Based on the above results,we successfully revealed the two-step reaction of lignans biosynthetic pathway in P.leptostachya,that is,using(+)-sesamin as the substrate to promote the two-step main reaction to produce(+)-sesangolin.This lays a foundation for the next step to comprehensively analyze the biosynthetic pathway of(+)-haedoxan A and the production of(+)-haedoxan A by microorganisms in the future to break the bottleneck of resource shortage in the development of botanical pesticides and realize its industrialization. |
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