Cloning And Enzymatic Synthesis Of A Novel BAHD Acyltransferase CtAT5 From Cistanche Tubulos

Cistanche tubulosa(Schrenk)Wightis a precious Chinese herbal medicine,which has been used to reinforce the vital function of kidney and induce laxation.The main active components isolated from C.tubulosa are phenylethanoides(PhGs).Modern pharmacologicalstudies have shown that PhGs exhibit a wide range of significant pharmacological activities such as anti-oxidation,neuro-protection,anti-inflammatory,enhancing immunity,improving the repairment of memory,treating AD,anti-aging,and anti-fatigue,which have attracted extensive attentions from scholars.Besides,the phGs were ususally substituted by diverse types of acyl moieties with different numbers at various positions.The common acyl substitution types were caffeoylandferuloyl moiety which attached at the 4-hydroxylgroup or the 6-hydroxylgroup of the central glucose,as well as the acetyl moiety substituted at the 2-hydroxylgroup of the central glucose group,which indicating that there must be a number of novel acyltransferases to be explored in the genome of C.tubulosa.Given that the acyltransferases in C.tubulosa have not been cloned and identified,this study aimed at digging out the potential novel acyltransferases for further characterising and enzymatic synthesis application through molecular biology techniques accompanying with the in vitro enzymatic catalysis identification.It was expected to obtain the acyltransferases invovled in the biosynthesis pathways of phGs on one hand.The obtained novel BAHD acyltransferase could also be used in the enzymatic aclytion of diverse kinds of natural products on the other hand,to be furture applied in the construction of the "non-natural small molecule acylated compounds library" to provide candidates for leading compoundsscreening in drug discovery.In this study,6 novel acyltransferases’ recombinant proteins CtAT1,CtAT2,CtAT3,CtAT4,CtAT5,and CtAT6 were cloned and expressed from C.tubulosa by the RT-PCR,RACE and PCR.The in vitro enzymatic catalysis reactions were performed and detected by the HPLC-HR-ESI-MS,and the results demonstrated that the CtAT5 was a typical malonyltransferase with significant malonylation activity and haboring a wide range of acceptor tolerance.It could catalyzed the malonylation of glycosidic compounds substituted by diverse glycosyl group types at different positions such as phGs,stilbene glycosides,naphthyl glycosides,anthraquinone glycosides,flavonoid glycosides,coumarin glycosides,iridoid glycosides,lignans glycosides,long chain unsaturated fatty acid glycosides and cyanophoric glycosides,and so on.As the active acyl donor of CtAT5,malonyl-CoA was expensive in commecial and unaccessible by chemical methods,which limited the further investigation and application of CtAT5 to a large extent.In order to solve the source problem of malonyl-CoA,a malonyl-CoA synthetase gene MatB was cloned and expressed fromArabidopsis thaliana,which successfully realized the in vitro enzymatic synthesis of malonyl-CoA,using malonic acid and coenzyme A as the raw materials.To make the utmost of MatB and CtAT5 to efficiently catalysis the in vitro enzymatic malonylation,the one pot biosynthesis system of MatB and CtAT5 was constructed.The results showed that all of the positive substrates also could be catalysed to obtain the same malonylated products with using the CtAT5 and standard malonyl-CoA alone,and it was worthy to mention that mostly of the tested substrates exhibited a dramatically increased conversion rate in comparision with the single enzymatic reaction,part of which even close to 100%.The establishment of the biosynthesis system eliminated the addition of malonyl-CoA,substituted for directly adding the cheap and readily available compounds such as malonic acid,and with all reactants in a reactor,shorten the number of steps required overall,aviod a lenghthy seperation process and purification of the intermediate compounds while remarkably reduce the possiblity of the degradation of the malonyl-CoA,being more economical,efficient,simple,time-saving.Based on the dramatically increased conversion rate in the one-pot biosynthesis system,it was applied to large scale preparation of the enzymatic product to further reveal the specific malonylation catalytic characteristics of CtAT5.The salidroside and icariin,both with broad and significant pharmacological effects and being structural specific,were picked out as the representative compounds to obtain its corresponding malonyl products by the in vitro enzymatic synthesis,and combining the semi-preparative liquid chromatography for the separation and purification of the products,and the NMR technique for the further structural analysis.The results showed that the malonylation of the two substrates were both present at the 6-hydroxyl group of the glucose chain,suggesting that CtAT5 may have a function of specifically catalyzing the malonylation of the 6-hydroxyl group of the glucose chain.Finally,the correlation between CtAT5 and the active components of phGs in C.tubulosa was also discussed.Using the real-time fluorescence quantitative PCR technique,the acyl transferase CtAT5 relative expression amount and the content distribution of phGs index components echinacoside and verbascoside at different time points induced by methyl jasmonate for the suspension cell of C.tubulosa was took into comparison.The results manifested that the relative trend of the phGs content changes were consistent with the CtAT5 relative expression amount,indicating that the CtAT5 was correlated with the biosynthesis of phGs.To sum up,in this study,the novel plant BAHD acyltransferase CtAT5 was successfully cloned from the genome of C.tubulosa.The functional identification showed that the recombinant protein could catalyze the malonylation of diverse kinds of natural products,and not be restricted by the glycosyl types,positions,numbers and the glycoside bond types.Meanwhile,to solve the source problem of the acyl donor malonyl-CoA,enzymatic charaterization of the malonyl-CoA synthatase MatB was performed,and the one pot biosynthesis system of MatB and CtAT5 was constructed,which greatly simplifing the malonylation enzymatic synthesis steps,posessing a strong practical value and economic value;Selecting the representative compounds salidroside and icariin for the preparative in vitro enzymatic synthesis,two structural novel non-natural malonylated products were obtained,and structure identification results showed that the malonylation of the two substrates were both occured at the 6-hydroxyl group of the glucose chain;A positive correlation between CtAT5 expression level and phGs contents in C.tubulosa was also discussed by real time PCR test.All of the above results revealed that the CtAT5 is promising to be used as a universal tool for the malonylation modification of structural diverse natural glycoside compounds for active leading compounds discovery.Our study also provided a reference for the further study of acyltransferase in C.tubulosa,with both theoretical and practical significance.