Isolation And Purification Of Epothilone Synthetase And Preliminary Construction Of Synthetic System In Vitro

Epothilone,a polyketide compound with anti-tumor activity produced by Sorangium cellulosum,is considered to be a newer alternative to the famous anticancer drug paclitaxel.At present,two kinds of epothilone anticancer drugs have been approved for the market for the treatment of advanced breast cancer.However,the biosynthesis of epothilone in vivo is limited by complex regulation of cell metabolism and invisible rate-limiting steps,and the fermentation yield of endogenous producing strains and heterologous expressing strains is still difficult to meet the needs of industrial production after optimization.In vitro cell-free biosynthesis system,the target compound is obtained by reconstructing the in vitro synthesis pathway of the target compound without cell synthesis.Compared to in vivo biosynthesis,the in vitro synthesis system can avoid the complex cell regulation mechanism,and study the biosynthesis of natural products more intuitively.It has the advantages of easy adjustment of reaction components,short cycle,small workload,and 1:1 replication to industrial production.At present,the in vitro synthesis platform has successfully realized the efficient production of farnesene,limonene,astaxanthin,lycopene,and other natural products.Therefore,the separation and purification of epothilone synthetase and the reconstruction of the epothilone synthesis system in vitro are expected to further analyze the synthesis mechanism,and provide theoretical guidance for identifying and breaking through the rate-limiting steps of in vivo synthesis and the synthesis of novel structural analogues of epothilone.However,there are few studies on the synthetic pathway of epothilone reconstructed in vitro.Focusing on the goal of isolating and purifying epothilone synthetase and attempting to build an in vitro synthesis system,this paper attempts to directly extract the epothilone synthetase derived from S.cellulosum and Myxococcus xanthus and expresses and purifies part of the module proteins of epothilone synthetic assembly line in Escherichia coli.The in vitro synthesis system of epothilone was preliminarily established,and the following research results were obtained:1.The epothilone synthetase was extracted by gel filtration chromatography,but no target enzyme complex or proteins after complex depolymerization were obtained,indicating that this method is not suitable for the direct separation and extraction of epothilone synthetase with low intracellular content.2.The epothilone synthetase EPOA was labeled with a protein label,and the epothilone synthetase was extracted by affinity chromatography,but no target protein was obtained.3.The loading modules EPOA-ACP and EPOP proteins of epothilone synthetase,were expressed and purified in E.coli.and the synthesis pathway of 2-methylthiazole,the initial unit of epothilone synthesis,was attempted to reconstruct in vitro.Suspected target products were detected in the reaction system in vitro,and the structure of the products needed to be verified later.In conclusion,the paper successfully expressed and purified the first two module proteins of the epothilone synthesis assembly line in Escherichia coli,and attempted to construct the in vitro synthesis system of 2-methylthiazole,the initial unit of epothilone biosynthesis.This study provides a reference for the expression and purification of other module proteins of epothilone synthetase and lays a foundation for the extension of the subsequent in vitro synthesis pathway of epothilone.