The Mining Of Natural Products From Marine Gliding Bacteria,and Heterologous Expression And Tumor-targeting Delivery Of Disorazoles

Cancer is one of the major public health issues worldwide which still lacks effective treatments presently.Chemotherapy is the major strategy to treat cancer.As a number of chemotherapeutic agents originated from natural products(NPs)or derivatives thereof,and many novel NPs have been used to search new antitumor targets,therefore,searching novel NPs is an important part of fighting cancers.However,due to the problem of re-discovery,it is becoming more and more difficult to discover novel valuable compounds from redundant natural products,which may result from too many interests are focused on the traditional natural product-producing taxa.According to the results of bioinformatics analysis,apart from these traditional natural product producers,such as actinomycetes and bacillus,marine gliding bacteria also possess the huge potential of natural product biosynthesis.In this study,we focused on marine gliding bacteria for natural product mining.Apart from searching novel NPs,utilizing existing compounds reasonable is also an important part of fighting cancers.Disorazoles are a series of macrocyclic compounds obtained from Sorangium cellulosum,the cytotoxicity of which is 100-fold higher than epothilone.Due to the limited tumor-targeting ability,disorazoles may cause severe side effects,which restricts their application.With the development of tumor-targeting biotechnologies,especially the development of tumor-targeting bacteria,it is possible to convey these compounds to tumor tissues directly with a suitable vehicle.With these strategies,the compounds with high toxicity can kill cancer cells without affecting normal tissues,which is an promising research direction.Our research is based on the problem of cancer,and focused on the mining of NPs and tumor-targeting delivery of disorazoles.The main research contents and results are as follows:(1)The isolation of novel marine gliding bacteria and evaluation of biosynthetic potential.We isolated more than 100 strains from marine samples using the so-called baiting method,from which we prioritized 11 potential novel species belonging to marine gliding bacteria.The isolation of these bacteria allowed us to explore their secondary metabolites.These isolated strains were fermented with multi conditions,and then the fermentation products were evaluated with bioactivity and LC-MS spectra.Complete genome sequencing was performed on one of the strains designated W222 which demonstrates antifungal activity.A number of novel biosynthetic gene clusters of secondary metabolites are predicted based on the bioinformatic analysis of the genomic sequence,which indicated that strain W222 possesses some biosynthetic potential.(2)The mining of novel siderophore fulvivirgamide and the verification of its biosynthetic pathway.There is a putative desferrioxamine-like biosynthesis gene cluster within the predicted clusters of strain W222.Compared with the known gene cluster of DFOB,it contains two unusual genes fulE and fulF which are conserved in many Bacteroidetes species.Bioinformatic analysis suggested these two genes with unknown function may possess important biological functions,which indicated that strain W222 may be capable of producing novel desferrioxamines(DFOs).Combination of CAS assay and inhibition of siderophores production by adding Fe(?),we observed possible absorption peaks and MWs of siderophores in the fermentation products of strain W222 detected by LC-MS.According to the accurate MWs determined by HRMS and possible molecular formulas,these siderophores were speculated as derivatives of DFOG1 with its primary amine acylated,which were named fulvivirgamide(FVM).To identify their structures,we purified four compounds(1-4)with high yield using chromatographic separation,and the structures of them are elucidated by NMR and HRMS,combined with MS/MS.Compound 1-4 are the derivatives of desferrioxamine G1 with the primary amine acylated with phenylacetyl,acetyl,propionyl,and isobutyryl.The elucidation of structures of compounds 1-4 allowed us to speculate the putative structures of other FVMs with HRMS and MS/MS spectra data.The results demonstrated that all of them are the derivatives of DFOG1 with its primary amine acylated with various fatty acids or aromatic fatty acids.To validate the biosynthetic route of FVM,we transformed the co-expression plasmids harboring the genes from the FVM gene cluster into E.coli BL21(DE3).The fermentation products of different transformants suggested that the FulF is an acyltransferase responsible for the acylation of DFOG1 to produce FVMs.To determine the optimal substrate of FulF,we detected the fermentation products of BL21(DE3)::fulF in the presence of DFOGi and myristic acid and found the presence of FVM14,which indicated that FulF could catalyze the primary amine acylation of DFOG1.The siderophore can chelate iron ion and inhibit the rapid proliferation of cells,hence most of them show some cytotoxic effect.We test the cytotoxicity of compounds 1-3 and all of them showed antiproliferative activity to three cancer lines from the human with IC50 values ranging from 9-19 ?M.(3)Heterologous expression of disorazoles.Heterologous expression can not only facilitate the research of disorazoles but also be the basic premise of tumor-targeting delivery.Burkholderia thailandensis E264 is a Gram-negative strain isolated from a rice field in Thailand,compared with other tumor-trageting bacteria,strain E264 can produce various NPs,which could provide various precursors or substrates for heterologous expression and indicate it could be an excellent host.But strain E264 is resistant to most common antibiotic markers,which restrains the anti-resistant screening for transformants.In this study,we obtained a mutant E264?BAC sensitive to most common selection-markers by knockout the multidrug efflux pumps of B.thailandensis E264 using a traditional seamless recombineering mediated by two separated single crossovers combined with counter-selection.In addition to the advantage of drug resistance,we also tested the effects on growth after knockout pumps and found that the growth rate of the mutant is similar to wild type.After that,we introduced the biosynthetic gene cluster of disorazole A1 to the mutant E264ABAC by conjugation mediated by E.coli WM3064 to obtain the transformants E264?BAC::disA,and three specific UV absorption peaks in the fermentation products of M8 medium were observed.The NMR results verified the peak with retention time at 16.8 min is disorazole F2,the derivative of disorazole A1.Comparing the structures of disorazole A1 and F2,we reckoned this may result from the absence of methyltransferase and cytochrome P450,which are presumably locate out of the BGC in the genome of the original producer.The result of heterologous expression of disorazole Ai facilitated the analysis of its biosynthetic route by excluding the interference of modified enzymes which locate out of the BGC.We encountered similar situations when we tried to express the BGC of disorazole Z in E264ABAC,we detected a specific molecular weight which we think is an analog of disorazole Z lack of two ester groups.According to the biosynthesis pathways,we speculate the ester groups may be catalyzed by a cytochrome P450 and a carboxylesterase which may also locate out of the BGC of disorazole Z.(4)The tumor-targeting characterization of B.thailandensis E264 and related attenuated strains.The success of the expression of disorazoles provided the basis for conveying these high cytotoxic compounds to tumor tissue directly due to the property of strain E264 of tumor-targeting.However,strain E264 is toxic to hosts,to reduce its toxicity and increase safety,we knock the genes wanN or relA and spoT to construct attenuated strains E264AwaaN and E264ArelAAspoT,which had been verified to be successful in strains of Salmonella.We evaluated the toxicity of strain E264 and two attenuated strains and found that the mutant E264AwaaN is low toxicity while low concentration wild type E264 is also safe to host.Then,we tested the ability of tumor colonization of strains E264 and E264AwaaN and found that the efficiency of attenuated strain was lower than wild type strain,and the low concentration E264 could guarantee the safety and colonization efficiency.Given consideration to the safety and colonization efficiency,we utilized low concentration E264 as the vehicle to convey the disorazoles into the tumor tissue to achieve a more significant antitumor effect.But the results suggested that the antitumor effect of E264::disA was not significantly improved than strain E264,which may result from the rigorous tumor microenvironment which inhibits the growth of colonization strains and expression of heterologous BGCsIn conclusion,we discovered a series of novel DFO siderophores from non-traditional natural product producers,marine gliding bacteria;we constructed a new heterologous expression platform using E264ABAC as host,and expressed the high potent cytotoxic disorazoles successfully;finally,we explored the possibilities and limitations of conveying high cytotoxic compounds to tumor tissue with the vehicle B thailandensis E264.