Cloning And Functional Analysis Of Terpene Synthase And Cytochrome P450 Genes Involved In Triptolide Biosynthesis

Triptolide,as one of the main bioactive ingredients in Tripterygium wilfordii,has been demonstrated to possess remarkable anti-inflammatory,immunosuppressive,and antitumour activities and to exhibit potential medicinal value for central nervous system diseases(e.g.,Parkinson's and Alzheimer's diseases).It has become one of the leading compounds of the next "blockbuster" drug.Moreover,several derivatives of triptolide have undergone clinical trials.Currently,triptolide is mainly derived from:(1)chemical synthesis,(2)extraction and separation from Tripterygium wilfordii.Due to its complex structure and multiple chiral centers,the chemical synthesis process is cumbersome,resulting in an extremely low yield,Also the content in Tripterygium wilfordii plants is extremely low and the plants grow lowly,leading to a low production.The two methods mentioned above are difficult to meet the needs of commercialization,especially industrialization needs after triptolide and its derivatives becoming the drugs.Therefore,it is urgent to develop new strategies and technologies to obtain natural active products such as triptolide.In recent years,synthetic biology technique has been applied successfully in the research and production of paclitaxel,artemisinin and tanshinone,suggesting that it will become a potential new method by designing and engineering microbial strains to produce natural bioactive products.The bioactive ingredients of Traditional Chinese Medicine(TCM)are the pharmacodynamic substance basis of Chinese Materia Medica Resource,and its formation is regulated by unique functional gene groups involved in the secondary metabolic pathway in plants.Hence the purpose of this study is analyzing the key genes involved in triptolide biosynthesis and clarifying its biosynthetic pathway,then designing and engineering microbial strains to produce triptolide and its derivatives efficiently based on the synthetic biology techniques.Previously,our research group established a stable and controllable suspension cell system of T.wilfordii using tissue culture technology,and completed the deep sequencing analysis of T.wilfordii transcriptome,and clarified the upstream biosynthetic pathway of triptolide,and identified four diterpene synthases(TwCPS1,TwCPS2,TwCPS3 and TwKS),confirming that TwCPS1 catalyzes GGPP to(+)-CPP,TwCPS2 has no biological activity,TwCPS3 catalyzes GGPP to ent-CPP,TwKS catalyzes ent-CPP to 16a-hydroxy-ent-kaurane and ent-kaurene.However,the structure of the diterpene intermediate of triptolide has not been determined,and the key enzyme gene that catalyzes(+)-CPP to abietane-type diterpene intermediate has not been identified.Therefore,this study urgently need to solve two difficulties in the process of promoting the elucidation of triptolide biosynthetic pathway:(1)to clarify the structure of triptolide diterpene intermediate,(2)to clarify key terpene synthase responsible for the formation of diterpene intermediate via(+)-CPP.To this end,UPLC/Q-TOF MS was used to characterize the metabolites between the suspension cells,culture medium and different tissues of the plant,and the abietane-type miltiradiene was detected by GC-MS in T.wilfordii suspension cells.Then we confirmed that miltiradiene was the diterpene intermediate compound of triptolide through feeding experiments.Based on this,the terpene synthase gene TwMS was further screened out,and its encoded protein catalyzed(+)-CPP to miltiradiene,successfully revealing the biological process of triptolide from linear GGPP to the cyclized abietane-type diterpene intermediate miltiradiene.To further investigate the modification processes catalyzed by CYP450 involved in the downstream biosynthetic pathway,we first cloned and characterized four NADPH-cytochrome P450 reductase genes in T.wilfordii.Then we constructed a "CYP450 gene-metabolite" regulatory network map through integration analysis of the genomics,metabolomics and transcriptomics,and thirty-two candidate CYP450 genes which were positively correlated with triptolide accumulation were screened out.Combined with transcriptome data of different tissues,ten CYP450 genes highly expressed in T.wilfordii roots were screened out.Then RNAi was employed with suspension cell cultures to provide more direct evidence of a role in triptolide biosynthesis for the candidate genes.The results showed that the gene expression level and triptolide accumulation were significantly down-regulated in CYP728B70,TW011445.1,TW012149.1,TW006625.1-RNAi suspension cells.And we successfully identified the first CYP450 gene(CYP728B70)involved in triptolide biosynthetic pathway through the engineering yeast experiments.In vitro enzymatic studies confirmed that it could catalyze two consecutive oxidation reactions from miltiradiene or abietatriene(miltiradiene auto-oxidation product)to the corresponding carboxylic acid products.Furthermore,a large number of fermentation-enriched products were prepared and purified through the engineering yeast,and identified by the high-resolution mass spectrometry and1H-NMR and 13C-NMR.The two-step modification process involved in triptolide biosynthetic pathway was successfully clarified,laying the basis for further investigation of triptolide biosynthetic pathway and its production based on synthetic biology.The main results are as follows:1.Identifying the diterpene intermediate of triptolideUPLC/Q-TOF MS was used to detect and compare the metabolite difference between the suspension cells,culture medium and different tissues of the plant,indicated that the suspension cells selectively enriched triptolide and celastrol which further confirming its use in investigating triptolide biosynthetic pathway.GC-MS analysis revealed that T.wilfordii suspension cells contain abietane-type diterpene intermediate miltiradiene.The triptolide accumulation increased significantly when feeding miltiradiene,confirming that miltiradiene is the diterpene intermediate of triptolide.2.Screening out the key candidate enzyme genes that regulate the formation of triptolide diterpene intermediateThe full-length sequences of diterpene synthase TwCPS4 and TwKSL1 were screened out based on T.wilfordii transcriptome data.The monoterpene synthase gene TwMS was obtained by analyzing T.wilfordii transcriptome data using the SmMS amino acid sequence of Salvia miltiorrhiza as the query sequence.Informatics analysis showed that TwCPS4 has a 'DxDD'functional domain at the N-terminus and clustered with TwCPS1,speculating that it has the function of catalyzing GGPP protonation and cyclization into(+)-CPP;TwKSL1 has a 'DDxxD' functional domain at the C-terminus and clustered with TwKS,presumeing that it has the function of catalyzing ent-CPP dephosphorylation and cyclization to diterpene compounds;TwMS and TwTPS27 clustered together,presuming that it has the function of catalyzing(+)-CPP phosphorylation and cyclization into miltiradiene.3.Revealing the biological process of triptolide from linear GGPP to the cyclized abietane-type diterpene intermediate miltiradieneTwo terpene synthase TwCPS4 and TwMS,which may involve in the synthesis of miltiradiene,and one diterpene synthase TwKSL1,which may regulate the synthesis of unknown diterpene,were cloned.The heterologous expression and in vitro functional characterization confirmed that TwCPS4 catalyzed GGPP to(+)-CPP,TwKSL1 catalyzed the formation of ent-pimara-8(14).15-diene,8a-hydroxy-ent-pimar-15-ene,ent-kaurene via ent-CPP,TwMS catalyzed(+)-CPP to miltiradiene.Then we successfully revealed the biological process of triptolide from linear GGPP to the cyclized abietane-type diterpene intermediate miltiradiene.through further characterization the biological functions of TwCPS1,TwCPS4 and TwMS in vivo and in vitro.This study promoted the process of triptolide biosynthetic pathway,and laid the foundation for the investigation of CYP450 involved in the downstream biosynthetic pathway.4.Cloning and functional analysis of NADPH-cytochrome P450 reductase genes from T.wilfordiiFour NADPH-cytochrome P450 reductase genes were screened out and cloned based on T.wilfordii transcriptome data,and ligated into the prokaryotic expression vector HIS-MBP-pET28a.Then the soluble protein was induced.In vitro enzymatic studies showed that four CPRs were displayed the similar enzyme activity(ie.,electron donor capacity).Furthermore,each CPR was transformed into the engineering yeast with the identified ent-kaurene oxidase(TwKO),and it is found that the yields of ent-kaurenoic acid and 16a-hydroxy-ent-kaurenoic acid were high when TwKO is combined with CPR3,suggesting that CPR3 has a more efficient electron donating ability.We speculated that CPR3 is more suitable for the functional study of other CYP450 monooxygenases in T.wilfordii.5.Identifying the first CYP450 gene involved in triptolide biosynthetic pathwayWe constructed a "CYP450 gene-metabolite" regulatory network map through integration analysis of the genomics,metabolomics and transcriptomics,and screened out thirty-two candidate CYP450 genes which were positively correlated with triptolide accumulation.Combined with transcriptome data of different tissues,ten CYP450 genes highly expressed in T.wilfordii roots were screened out.Then RNAi was employed with suspension cell cultures to provide more direct evidence of a role in triptolide biosynthesis for the candidate genes.The results showed that the gene expression level and triptolide accumulation were significantly down-regulated in CYP728B70,TW011445.1,TW012149.1,TW006625.1-RNAi suspension cells.Combined with the functional annotation information of CYP728B70,it was speculated that it might be involved in the hydroxylation reaction of diterpene intermediate miltiradiene,and the engineering yeast experiments confirmed that CYP728B70 could catalyze the formation of new products from miltiradiene.Furthermore,a large number of fermentation-enriched products were prepared and purified through the engineering yeast,and the chemical structures were identified as 8,12-abietadienol?dehydroabietinol?8,12-abietadienoic acid?dehydroabietic acid by the high-resolution mass spectrometry and 1H-NMR and 13C-NMR.Feeding experiments clarified the catalytic process of CYP728B70.Overexpression of CYP728B70 in suspension cells using a biolistic gene gun and the results confirmed that CYP728B70 regulated the triptolide biosynthesis.In summary,this study used the feeding substrate,gene cloning and heterologous expression,in vitro enzymatic reaction,engineering yeast,separation and purification of the target products,RNAi and overexpression of gene via a biolistic gene gun,to clarify triptolide biosynthetic pathway in T.wilfordii.Here we successfully revealed the biological process of triptolide from linear GGPP to the cyclized abietane-type diterpene intermediate miltiradiene,and further identified the first CYP450(CYP728B70)which could catalyze two consecutive oxidation reactions from miltiradiene or abietatriene(miltiradiene auto-oxidation product)to the corresponding carboxylic acid products,and successfully clarified the downstream two-step modification process catalyzed by CYP450 involved in triptolide biosynthetic pathway.These results laid the basis for further investigation of triptolide biosynthetic pathway and its production based on synthetic biology.