Research On Heterologous Expression Of Taxadiene Precursor Of Anti-Cancer Drug Taxol In Tumor-Targeting Bacteria

Tumor-targeting bacteria can specifically proliferate at tumor sites,some of them are currently applied to clinical trials.As an anti-cancer drug delivery vehicle,tumor-targeting bacteria have significant advantages over traditional chemotherapy Chemotherapy acts by intravenous injection and relies on blood circulation to reach the tumor sites,which is harmful to other tissues and cells besides tumor.In contrast,tumor-targeting bacteria can actively target to the tumor site and make a continuous release of drugs as their reproduction,so that the drugs can accumulate to a higher concentration locally in the tumor and have a good anti-tumor effect.E.coli Nissle1917(EcN),a kind of tumor-targeting bacteria with strong tumor-targeting and targeting-multiplication ability,has been used as a probiotic in the past for the treatment of dysentery and intestinal care with a good safety profile and has been on the market for more than 100 years.EcN has been used as a carrier to express a variety of anti-inflammatory and anti-bacteria drugs,and is an excellent carrier for targeted delivery of anti-cancer drugs.Taxol is a natural terpenoid anti-cancer drug with good therapeutic effects on breast cancer and lung cancer and so on.Exploring its biosynthesis in tumor-targeting bacteria is of great significance for the development of novel anti-cancer drug delivery systems.Since the biosynthetic pathway of taxol has not been elucidated totally,the present study was conducted to the taxol precursor taxadiene,which has been elucidated in the biosynthetic pathway,the aim is to successfully construct taxadiene efficient biosynthetic system in EcN Firstly,a taxadiene expression vector containing two exogenous genes which encode geranylgeranyl diphosphate synthase(GGPPS)and taxadiene synthase(TS)respectively,was successfully constructed by Gibson Assembly and realized the heterologous expression of taxadiene in EcN.Later,by optimizing the metabolic pathway of taxadiene,the four endogenous genes which encode 1-deoxy-D-xylulose-5-phosphate synthase(DXS),isopentenyl-diphosphate delta-isomerase(IDI),2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase(IspD),and 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase(IspF)together with two exogenous genes were combined in the taxadiene expression vector to increase the supply of precursors during taxdiene synthesis,aiming to increase the relative yield of taxadiene.On this basis,the relationship between gene copy number,promoter strength,fermentation temperature,fermentation time and the efficiency of taxadiene biosynthesis was established in the heterologous expression strain through systematic testing and optimization,realizing the efficient biosynthesis of taxadiene In addition,the short peptide tags RIAD and RIDD was added to the taxadiene expression strain,to create a scaffold enzyme assembly and to explore the effect of scaffold proteins on taxadiene synthesis.After optimization,the relative yield of taxadiene was increased to 111.2-folds of the original yield.This study was the first to achieve heterologous expression of taxadiene in the tumortargeting bacteria EcN,which laid the foundation for the development of taxadiene targeting delivery system and provided a reference for the heterologous expression of other terpenoids in EcN.