Cloning And Prokaryotic Expression Of Genes Involved In The Biosynthetic Pathways Of Podophyllotoxin And Its Precursor Coniferol From Sinopodophyllum Hexandrum

Podophyllotoxin and its derivatives,as a class of widely effective antitumor drugs,are inadequate to meet their high market demand by current production methods.With the rapid development of synthetic biology and the successive elucidation of podophyllotoxin biosynthesis pathway,the heterologous biosynthesis of podophyllotoxin has already been performed in tobacco,Escherichia coli and yeast in recent years.As judged from the available results,there are still considerable space,necessity and advantages for deepening the exploration based on microbial systems.Therefore,this study will focus on an attempt in this aspect using E.coli as a chassis.Podophyllotoxin is a lignan compound,which is directly derived form its central precursor coniferol.The synthesis of coniferol belongs to phenylpropanoid pathway.In Sinopodophyllum hexandrum,10 enzymes related to coniferol biosynthesis include Sh PAL,Sh C4 H,Sh C3 H,Sh4CL,Sh HCT,Sh CSE,Sh COMT,Sh CCo A-OMT,Sh CCR and Sh CAD.The podophyllotoxin biosynthesis pathway started from coniferol contains 14 related enzymes: Sh DIR,Sh Lac1,Sh Lac2,Sh PLR,Sh SDH,Sh CPR,Sh CYP719A23,Sh OMT3,Sh CYP71CU1,Sh OMT1,Sh2 ODD,Sh CYP71BE54,Sh CYP82D61 and Sh UGT78D2-like.In the beginning of this work,the blastn or tblastn program was used to search the best matches against the SRA nucleic acid data package SRX4112555 of S.hexandrum deposited on NCBI website,for designing two pairs of primers specific to the full-length gene of each enzyme mentioned above.Then,a procedure composed of a fist round of RT-PCR and subsequent second round of nested PCR was applied to amplify the CDS of each gene from the root of S.hexandrum plant sourced from Aba district,Sichuan Province.After purification,the PCR fragments were inserted into plasmid p ET32a（+）by enzymatic digestion ＆ ligation or recombinant cloning to construct their corresponding p ET-serial prokaryotic expression vectors which were further verified by sequencing.The results showed that most of the cloned genes had no significant differences in DNA and amino acid sequences,as compared with the reference items reported by abroad research groups.Moreover,through backtracking comparison with SRA data package SRX4112555,it was found for most genes the matched hints could be assembled into a full contig,implying the serial genes cloned in this work are authentically originated from the main ecotype of S.hexandrum plant.Additionally,bioinformatics analyses such as conserved domain search,protein homologue alignment and homologous modeling of high-order structure were accomplished for the deduced proteins of many cloned genes.All these genes have been indexed in public nucleic acid databases（e.g.Gen Bank）with accession numbers MW531732–MW531752,MW625919–MW625921.Finally,all these cloned genes were subjected to a test of recombinant expression in E.coli.Their p ET-serial expression vectors were transformed into E.coli strain BL21（DE3）,followed by IPTG induction and SDS-PAGE analysis.The results showed that 15 enzyme genes among them could efficiently express,yielding recombinant proteins with a size close to the theoretical molecular weight,while most of the other 9 enzymes with a failure in recombinant expression belongs to the family of cytochrome P450 monooxygenases（CYPs）.Eukaryotic CYPs are generally endoplasmic reticulum membrane-associated proteins,which are often difficult to express in E.coli lacking endomembrane system.Therefore,the next step of this work will concentrate to solve this bottleneck by exploiting different prokaryotic expression strategies for membrane proteins.In conclusion,the work of this paper has completed the cloning and prokaryotic expression analysis of all enzyme genes（24 in total）involved in the biosynthetic pathways of podophyllotoxin and its precursor coniferol.This has made a concrete foundation for constructing a fully self-sufficient engineering strain for heterologous biosynthesis of podophyllotoxin in E.coli,and also gives it a certain amount of cues for evaluating the feasibility.Prospectively,this study could contribute to the synthetic biology of podophyllotoxin,and further advance its production and application.