Semi-rational Design Of DBAT Towards Synthesis Of Baccatin ? Based On Low-cost Acyl Donor

Paclitaxel(taxol?)is a widely used chemotherapeutic drug.Baccatin III is an important precursor of taxol in its biosynthesis pathway.The decrease in the production cost of baccatin III is expected to decline the price of taxol and meet its market demand.For the problem of the high cost of the natural acyl donor(acetyl-CoA)to produce baccatin III in the biosynthetic pathway,this study sought to replace it with an inexpensive acyl donor Vinyl acetate(VA).VA is a commonly used activated acyl donor which has an acetyl group.Compared to the natural substrate acetyl-CoA,VA is relatively inexpensive.In addition,the reaction by-product using VA as acyl donor is vinyl alcohol,which is unstable and can tautomerize to form volatilizable acetaldehyde immediately,thereby,driving the reaction towards product.Its irreversibility leads to a quick acylation.In order to find economic and efficient acyl donor,this work explored whether VA could replace acetyl-CoA as an alternative acyl donor with 10-DAB as co-substrate to generate baccatin III under the catalysis of DABT.In addition,DBAT was modified to improve its catalytic efficiency via semi-rational design.The main findings of this paper are as follows:(1)An expression vector for the DBAT enzyme was constructed.Using pET32a(+)plasmid as framework,Sac I and BamH I as restriction sites and dbat as target gene to construct pET32a-dbat vector for heterologous protein expression.The results showed that the new constructed vector could be used for heterologous DBAT expression.(2)The novel acyl donor VA,which can replace the natural acyl donor acetyl-CoA was determined.In the catalytic reaction of replacing acetyl-CoA as an acyl donor with VA In vitro,it was speculated that the His-162 of DBAT acts as a catalytic base,first deprotonating the C10 hydroxyl group of the acyl acceptor 10-DAB,thereby carrying out the carbonyl carbon of VA.The nucleophilic attack forms a tetrahedral intermediate,which is then rearranged to produce the paclitaxel precursor baccatin III and the by-product vinyl alcohol.Vinyl alcohol immediately experiences tautomerism to form acetaldehyde and volatilizes out of the system.(3)Build a 3D model of DBAT by homology modeling method.Since the crystal structure data of DBAT has not been reported yet,this study used homology modeling method to predict the three-dimensional structure of DBAT.The rationality of the constructed model was evaluated by PROCHECK.The results showed that the model was reasonable.(4)In order to improve the catalytic efficiency of DBAT for the reaction system with VA as acyl donor,a semi-rational design strategy combining rational design and directed evolution was used to engineer DBAT.Based on the knowledge that the enhancing of hydrogen bonding interaction between substrates and enzyme can stabilize the charge between them,and can enhance the orientation interaction of the substrate,so that the substrate and the enzyme can be combined rapidly and the reaction state of the transition state can be stabilized,the virtual saturated mutation of all amino acids(10)in the substrate binding pocket of DBAT was performed(all of them were mutated to other 19 amino acids)to produce 190 single mutants.Analysis of the resulting 190 DBAT mutants revealed that additional hydrogen bonds were introduced only between I43 S,I43T,D390 H and D390 R and VA.(5)The above four single mutants were constructed by PCR site-directed mutagenesis and their kinetic parameters were determined.The results showed that for the enzymatic reaction of DBAT in vitro catalyzing the formation of baccatin III from 10-DAB and VA,catalytic efficiency of I43 S,I43T and D390 R were 2.12,2.09 and 2.29 times higher than that of wild type DBAT,respectively.A double mutant I43 S / D390 R was constructed by combining the positive effects of the single mutants.The catalytic efficiency of I43 S / D390 R was increased by about 3.30-fold compared with wild type DBAT with 10-DAB and VA as substrates,while about 2.99 times more efficient in reaction with wild type DBAT as enzyme and 10-DAB and acetyl-CoA as substrates.This study can provide economical and environmental friendly methods for paclitaxel production and provide a reference for the exploration of new acyl donors to modify natural products pathways.