Design Of Biosynthesis Pathway Of Paclitaxel Side Chain And Cloning And Functional Verification Of Key Enzymes

Paclitaxel is a highly effective and broad-spectrum anticancer drug,which is in short supply.At present,chemical semi-synthesis is the main source method,but the process of chemical synthesis of side chain involves the protection and de-protection of some sites,the synthesis process is complicated and there are many byproducts.The biosynthetic pathway to create paclitaxel side chain instead of chemical synthesis is an important development direction of green production of paclitaxel.The synthesis pathway of taxol side chain in Taxus chinense has not been solved yet.The development of synthetic biology technology provides a new way to analyze the heterosynthesis pathway of taxol side chain in Taxus chinense.In this paper,based on the similarity principle of enzyme reaction of substrates,a possible biosynthetic pathway of paclitaxel side chain was designed,and the related enzymes in the design pathway were cloned,and their functions were verified,which laid a foundation for heterologically achieving the biosynthesis of paclitaxel side chain.The main results are as follows:(1)Design of synthetic pathways for side chains of non-natural paclitaxel.Based on the search of metabolic pathway database,two highly feasible biosynthesis pathways of paclitaxel side chain were systematically designed by using molecular docking simulation technology to reflect the structure similarity principle of substrates.Both of the two feasible pathways involve 4 steps of enzyme catalysis,which provides a more convenient idea of side chain synthesis.(2)Construction of a genetically engineered strain that can produce phenylalanine,a precursor of paclitaxel side chain synthesis.In this study,we optimized aro G and phe A,the key ring-limiting enzymes in the main pathway of phenylalanine synthesis in Escherichia coli K12 MG1655 ?rec A ?end A(DE3),and integrated them into the substrate cells by CRISPR/Cas9 gene editing technology.Two branching pathways of phenylalanine synthesis were knocked out,and the key gene of tyrosine synthesis,Tyr A,and the key gene of tryptophan synthesis,Trp E,were knocked out to weaken the shunt of substances.Finally,a genetically engineered strain GZ-5 with high ?-Phe yield was obtained.The fermentation yield of the strain reached 1.831 g/L,which was more than 3 times of the original strain.(3)Cloning and physicochemical characterization of candidate genes for paclitaxel side chain synthesis pathway.We cloned the Bad A gene from Rhodopseudomonas palustris and the PAM,C2'OH,CPR,DBTNBT genes from Taxus chinensis.The rare codon of each protein was analyzed and optimized,and the protein containing signal peptide was truncated to enhance its expression in the chassis strain.The potential functions of candidate proteins were predicted through the analysis of protein conserved domains,which provided theoretical support for the verification of protein functions in vitro.(4)In vitro reaction verification of candidate protein and synthesis of N-benzoyl-?-phenylalanine,an important precursor of paclitaxel side chain.It was confirmed by in vitro reaction that PAM protein,Bad A protein and DBTNBT protein expressed in Escherichia coli K12 MG1655 ?rec A ?end A(DE3)could exert the catalytic activity of substrates.In this study,the reaction products were verified by HPLC and LC-MS,and ?-phenylalanine,sulphoester benzoate and N-benzoyl-?-phenylalanine were successfully synthesized in vitro.Among them,N-benzoyl-?-phenylalanine is a new non-natural product synthesized for the first time in this study,which is only one step away from the synthesis of the final paclitaxel side chain.In vitro function verification experiments of DBTNBT enzyme also confirmed that the acyltransferases have a wide range of substrate catalytic activity.In this study,?-phenylalanine,thiolipid benzoate and N-benzoyl-?-phenylalanine were successfully synthesized by in vitro reaction.N-benzoyl-?-phenylalanine was synthesized for the first time in this study.At the same time,N-benzoyl-?-phenylalanine was used as an important precursor for the synthesis of paclitaxel side chain,and only one step hydroxylation was needed to the final synthesis of paclitaxel.The synthesis of N-benzoyl-?-phenylalanine proves that the pathway designed in this study is feasible to a certain degree,which provides a more convenient and short biosynthetic pathway for the synthesis of paclitaxel side chain,and provides a new idea to solve the contradiction between supply and demand of paclitaxel drug market.