Prokaryotic Expression And Functional Characterization Of Epoxide Hydrolase Genes From Camptotheca Acuminata

Camptothecin（CPT）is a natural topoisomeraseⅠinhibitor disaplaying anti-tumor effect.The comprehensive elucidation of the CPT biosynthesis pathway is a prerequisite for realizing CPT biomanufacturing,and currently there is no progress in the functional analysis of unknown genes involved in the downstream pathway of CPT biosynthesis.During the research of the CPT biosynthesis pathway,our laboratory found that the downstream synthesis pathway of CPT involves the ring opening step of C-2 and C-7epoxy structure of the strictosamide epoxide,which is one of the key downstream steps of CPT biosynthesis.Through mining the gene resource database of Camptotheca acuminata（SAU-Genometa Resource）,several candidate sequences of epoxide hydrolase EHs were observed.Therefore,screening the sequence through bioinformatics analysis,phylogenetic analysis and expression analysis was initated.Their epoxide substrates were futher determinded through in vitro functional characterization.To screen the enzyme that can catalyze the ring-opening reaction of strictosamide epoxide epoxy structure,the main research results are as follows:（1）In this study,four complete epoxide hydrolase candidate genes were mined and screened from the Camptotheca acuminata gene database,and their encoded proteins were analyzed by bioinformatics analysis,phylogenetic analysis and expression analysis.The results showed that the physicochemical properties,secondary structure and tertiary structure of these candidate sequences were similar to that of the characterized EHs sequences.The four candidate sequences all belong to the s EH subfamily.（2）The results of gene quantitative analysis showed that the expression levels of EH-Cac＿g013200 and EH-Cac＿g006313 candidate genes rised to the highest in leaves at60 d,and the expression levels of both EH-Cac＿g006314 and EH-Cac＿g011673 candidate genes rised to the highest in leaves at 75 d.（3）All the EH recombinant proteins are inclusion proteins.They were denatured using solubilized buffer containing 8 mol/L urea,purified using Ni NTA affinity chromatography,and refolded using dilution gradient dialysis method,Soluble EH proteins were finally obtained after vigourous trials.（4）Three substrates,including（S）-Styrene oxide,（R）-Styrene oxide and trans-stilbene oxide were used for functional analysis of four EH recombinant proteins.The results showed that the recombinant protein EH-Cac＿g013200,EH-Cac＿g006313,EH-Cac＿g006314,EH-Cac＿g011673 can transform（S）-styrenee oxide,（S）-styrenee oxide and trans-stilbene oxide to（S）-styrenee glycol,（R）-styrenee glycol and meso-1,2-diphenyl-1,2-ethanediol.（5）The kinetic characterization results of four EHs recombinant proteins showed that the（S）-styrene oxide was the most suitable substrate for EH-Cac＿g013200,EH-Cac＿g006313,and EH-Cac＿g011673.The corresponding K_mvalues are 73.65,61.82,and 67.95μmol/L.（R）-styrene oxide was the most suitable substrate for EH-Cac＿g006314with a K_mvalue is 63.32μmol/L.For the substrate trans stilbene oxide,EH-Cac＿G006314has the strongest affinity,with a K_mof 86.58μmol/L,EH-Cac＿G013200 has the highest catalytic efficiency,with a K_cat/K_mof 0.9μM^-1S^-1。（6）To verify whether it participates in the downstream pathway of CPT biosynthesis,a two-step tandem reaction was performed using strictosamide as the starting substrate for CYP71BE206 microsomal protein and four EHs recombinant proteins,respectively.The MS/MS spectrums showed that the four recombinant EHs proteins could hydrolyze strictosamide epoxide to generate the downstram biosynthetic precursor for CPT,strictosamide 3,7-diol.