Heterologous Coexpression Of The Doxorubicin Biosynthesis Genes In E.Coli

Doxorubicin is a broad-spectrum antitumor drug.Owing to its remarkable biological activities,it has been widely recruited for clinical treatment of gastric cancer,lung cancer,ovarian cancer and other diseases.Currently,doxorubicin is of high cost and thus expensive due to the backwardness of production process and long production cycle.Using advanced biotechnology approaches,Streptomyces ceucetius ATCC 27952 was shown to harbor doxorubicin synthesis pathways and the related genes have been identified.In this study,the doxorubicin synthesis genes were rearranged and heterologously expressed in E.coli BL21 to further elucidate the roles of the key doxorubicin synthesis modules.In addition,this study aimed to address the problems arising from microbial fermentation production of doxorubicin.The research contents consist of the following aspects:1.First,doxorubicin related synthase genes were individually heterologously expressed in E.coli.It has been shown that biosynthesis of doxorubicin involves 24 synthase genes and 1 main resistance genes.In this study,a total of 18 expression vectors were constructed and SDS-PAGE was employed to determine whether E.coli BL21 is appropriate for expressing doxorubicin synthesis enzyme genes.2.Second,the genes in type ? polyketone synthase modules were assemblied,integrated and heterologous expressed in E.coli.To do so,the PKS ? metabolism pathway was subcloned into two plasmids and transformed into Escherichia coli.The construction of type ? polyketone synthase module was to synchronize the expression of multiple genes.SDS-PAGE analysis showed that the genes in plasmids were successfully expressed in E.coli.3.Finally,the double-plasmid recombinant strain was cultivated in shake-flasks containing M9 medium.Cell growth and free fatty acid were measured to determine metabolic flux allocation.Specially,the 36 h broth was analyzed by mass spectrometry.Results showed that no desired chemical was observed.The reason behind deserves in-depth exploration.4.To elevate substrate concentration in the cell,the genes in E.coli were deleted.For biosynthesis of doxorubicin,propionyl-CoA and malonyl-CoA are two major substrates.Substrate concentration is crucial for biosynthesis of doxorubicin precursor.In view of previous studies,genes were knocked out from the E.coli genome to improve Malonyl-CoA concentration.