Screening And Functional Characterization Of The Genes Related To Camptothecin Biosynthesis

Camptothecin(CPT)is an important class of anti-cancer drugs,belonging to monoterpene indole alkaloids(MIA)compounds.Camptothecin and its derivatives exert its anti-cancer effect by targeting topoisomerase I(Topo I)to inhibit DNA synthesis.At present,multiple camptothecin derivatives such as topotecan,irinotecan and belotican have been approved for clinical treatment of cancer.Camptotheca acuminata and Nothapodytes nimmoniana are the main source plants of the anticancer drug camptothecin.The production of camptothecin-type anticancer drugs mainly relies on the extraction and modification of natural camptothecin.However,the low content of camptothecin in plants has become the main bottleneck in the production of camptothecin-type drugs.The research on the biosynthesis of camptothecin will lay the foundation for the synthesis of camptothecin through metabolic engineering or synthetic biology technology,which is of great significance for the production and research and development of camptothecinderived drugs.This study accurately quantified the expression of more than 30,000 genes in ten C.acuminata tissues(lateral root,stem,young leaf,mature leaf,flower of mature plants,cotyledon,seedling taproot,seedling hypocotyl,seedling epicotyl and leaf of seedings)by absolute quantitative transcriptome sequencing with unique molecular identifiers(UMIs).A total of 7,854 proteins from five tissues(root,stem,young leaf,mature leaf,and flower)were quantified by label free quantitative proteomics.The integrated analysis of transcriptome and proteome data found that the correlation between mRNA and protein abundance of genes related to camptothecin was very low.This phenomenon may suggest the complex post-transcriptional and post-translational modifications in this species.Based on phylogenetic analysis and coexpression analysis,a total of five CacCYP450s and three CacOMTs were selected as candidate enzymes involved in the biosynthesis of CPT and its derivatives.With WGCNA analysis and qRT-PCR validation,fifteen TFs possibly regulating camptothecin synthesis were screened out.The full-length transcriptome of five tissues(roots,stems,young leaves,mature leaves and flowers)of C.acuminata was sequenced using ONT(Oxford Nanopole Technologies)sequencing platform,and 5,692 alternative splice events(AS)were found in 4,746 genes.In all AS events,IR is the most frequent AS type.The AS events of six genes in five tissues were validated using RT-PCR,and the results were consistent with sequencing data.In addition,four identified pathway genes(GES,10HGO,STR1,and 10OMT)in CPT biosynthesis were found to undergo AS events,indicating that AS may regulate the tissue-specific expression of genes related to camptothecin synthesis.Strictosidine synthase(STR)can catalyze the condensation of secologanin and tryptamine to produce strictosidine,which is a common precursor of all MIAs.This study measured the content of camptothecin in five tissues of N.nimmoniana and found that the content of camptothecin was the highest in the roots.This result provides a basis for screening STR candidate genes through co-expression in the future.Based on the annotated information of the N.nimmoniana genome,we obtained 30 STR-like genes.Two candidate genes NniSTR2 and NniSTR19 that may be involved in camptothecin synthesis were screened through phylogenetic analysis and co expression analysis.In the subsequent experiments,the CroSTR of Catharanthus roseus was used as the positive control for enzyme activity detection,along with the CacSTRl and CacSTR2 in C.acuminata obtained by our group in the early stage,for functional identification of the STR genes.First,the above five genes were cloned and constructed into the expression vector pET28a,and then transformed into Escherichia coli BL21(DE3)for heterologous expression.After induction by IPTG,crude protein was extracted and detected using SDS-PAGE and Western blot,respectively.The target proteins were purified using Ni2+ affinity chromatography column,and CroSTR,NniSTR2 and NniSTR19 proteins were successfully purified.After in vitro catalysis,the products were detected using HPLC and LC-MS.It was found that CroSTR can catalyze the synthesis of strictosidine from tryptamine and secologanin.However,it cannot catalyze the formation of strictosidinic acid from tryptamine and secologanin acid,which is consistent with the previous report.Strictosidine and strictosidinic acid were detected in the catalytic products of NniSTR2 and NniSTR19,suggesting that they may be bifunctional enzymes.Due to the formation of inclusion bodies,the CacSTRl and CacSTR2 could not be purified.In the later stage,it is necessary to optimize the prokaryotic expression system of STR or explore the plant transient expression system to further investigate their function.In this study,we generated large-scale transcriptomic and proteomic data from multiple C.acuminata tissues,which provided valuable resources for the further study of functional genomics of C.acuminata.The integrated analysis of multiple omics data revealed the biosynthesis of camptothecin and its multi-level regulatory processes in different tissues.Candidate enzymes/transcription factors involved in the biosynthesis of camptothecin and its derivatives were screened.Two STR enzymes from N.nimmoniana were preliminarily identified by heterologously expressing them in E.coli and detecting their in vitro enzymatic activities.This work laid important foundation for further elucidating the biosynthesis pathway of camptothecin and its derivatives.