Heterologous Expression And Combinatorial Biosynthesis Of The Anticancer Drug Romidepsin (FK228)

Cancer is a disease with extremely high morbidity and mortality in the world,which is a serious threat to human health.Epigenetic disorders are closely and importantly related to the occurrence of cancer.The acetylation and deacetylation of histones is one form of epigenetic inheritance.Histone acetyltransferases(HAT)and histone deacetylases(HDAC)regulate chromatin transcription by controlling the acetylation or deacetylation of histone lysine residues,and thus regulate the expression of genes related to tumorigenesis.In recent years,deacetylase has become a target for effective treatment of cancer,and Histone deacrtylase inhibitors(HDACi)exerts antitumor activity by inhibiting the HDAC activity,improve the level of acetylation in cells,promote the expression of tumor suppressor gene transcription to inhibit the growth of cancer cells,inducing cancer cell differentiation,guiding the direction of the cancer cells to normal cells or directly inducing the apoptosis of cancer cells,and it also makes the reserach of HDACi becoming a hot spot.Romidepsin(FK228)is the first natural HDACi to play antitumor activity.It is a highly effective and selective HDACi approved by the FDA for the clinical treatment of cutaneous T-cell lymphoma.In view of the limitation of the production strain of FK228 in China and the difficulty of its chemical synthesis,in this study,the biosynthetic gene cluster tdp of Thailandepsins,a structural analog of FK228,was used as the skeleton.By embedding the coding sequence of the non-ribosomal peptide synthase module 4-7 of the FK228 gene cluster,the coding sequence of the nonribosomal peptide synthase module 4-7 of the tdp gene cluster was replaced,the substrate selection was changed from Val-Cys-Thr-Val to Met-Cys-Val/Ile and malonyl coenzyme A for biosynthesis.FK228 was successfully synthesized in Burkholderia thailandensis E264 by the fusion gene cluster tdp-dep.Subsequently,FK228 production was increased to be 94 mg/L by expression of the path-specific transcriptional regulator tdpR.Meanwhile,in this study,by replacing the non-ribosomal peptide synthase module 4 of the tdp gene cluster,its substrate selection was converted from Met to Val for biosynthesis,and a new Thailandepsins derivative Thailandepsin G was obtained.The main research results of this paper are summarized as follows:1、The complete biosynthetic gene cluster tdp of Thailandepsins was obtained by RecET direct cloning.The expression vector of Thailandepsins was constructed and expressed in DSM7029.2、The fusion biosynthetic gene cluster tdp-dep was constructed by Red/ET recombineering combined with DNA fragment assembly.FK228 was successfully expressed in E264.Gene expression level affects biosynthesis efficiency,and gene transcription level is related to transcription regulation genes.The positive regulation gene tdpR was introduced to increase the yield of FK228 to be 94 mg/L.3、The non-ribosomal peptide synthase module 4,6 and 7 of Thailandepsins biosynthetic gene cluster tdp was replaced,respectively.A new compound was obtained after the replacement of module 4,which was identified as a new derivative of Thailandepsins.The structure of this new derivative was identified and named as Thailandepsin G.The inhibitory effect of Thailandepsin G on MIA-Paca-2 and Kasumi was similar to that of Thailandepsin A.In this study,Thailandepsins biosynthetic gene cluster tdp was reconstructed to realize the heterologous expression of FK228 by the recombinant gene cluster.At the same time,new structural derivatives were obtained by replacing single module of nonribosomal peptide synthase,which provided a theoretical basis for the construction of new anabolic pathways and the production of drugs with application value,as well as for the synthesis of new compounds derivant offer new ideas.