Rational Discovery Of Pentaketide Ansamycins In Three Actinobacterial Strains

Ansamycins is a special family of polyketide antibiotics,which has AHBA(3-amino-5-hydroxybenzoic acid)as a starter unit.18 skeletons and nearly 300 compounds have been discovered by now.Ansamycins usually have significant biological activity.One of the representative compounds is rifamycin,derivatives of which are still first-line antituberculous drugs.The derivatives of maytansine were approved for HER2-positive metastatic breast cancer treatment in 2013.Geldanamycin is famous for its activity as an inhibitor of HSP 90.From here we see that ansamycin has a high potential for patent medicine.Thus,finding new anamomycin compounds is favored by natural product chemists.In our early work,about 80 AHBA synthase gene positive strains were gained utilizing PCR screening,further sequence analysis revealed dozens of gene clusters probably encode ansamycins with novel skeletons and two unknown natural product(containing AHBA but not ansamycin)biosynthetic gene clusters.Follow up research shows a silent situation of these gene clusters.In this thesis,rational discovery was carried out around four new AHBA positive bacteria(Streptomyces sp.S033,Streptomyces sp.S035,Streptomyces sp.S051 and Actinomadura sp.S046)To activate the silent gene cluster using transcriptional activation strategy,useful genetic operation systems need to be established.Firstly,we found out the optimum conditions for sporing,and the sensitivity of the four strains to different antibiotics was tested.On this foundation,the condition of spore conjugation(medium,concentration of Mg2+,germination condition)was optimized using an integrative plasmid pMELK(containing melanin report gene).Conjugation efficiency of S035 can be 2×10-4;other three strains have lower conjugation efficiency from 1×10-7 to 1×10-8.Three potential novel pentaketide ansamycin biosynthetic gene clusters were predicted in S033,S035 and S046.Gene prediction showed that all three gene clusters contained SARP(Streptomyces Antibiotic Regulatory Protein)and LAL(Large ATP-binding regulators of the LuxR family)family genes,which are conserved in benzenoid ansamycin gene clusters and make a positive regulation in the biosynthesis of ansamycin.Therefore,overexpression and co-overexpression of these two regulator genes were carried out.As for S035,metabolic profilings of all three mutant strains have changed,Master of Feifei Wei(Shandong University School of Pharmacy)isolated and identificated aminoansamycin A-G from S035-SL,isolated L2 from S035-LAL,skeleton of which areexact match with bioinformatics prediction.It is worth mentioning that the metabolic profiling of S035-LAL is totally different with its counterpart of S035-SL,we speculate there're more novel derivative compounds of aminoansamycin,isolation and identification is in progress.Different from knowns,aminoansamycins have good cytotoxic activity,aminoansamycin D/E's IC50 to HeLa cell is 0.9 ?M,IC50 to HCT116 cell of aminoansamycin D is 0.21?M.Master of Feifei Wei isolated and identificated an open-loop pentaketide ansamycin with novel skeleton from S033-SARP.Its ring-opening is similar with microansamycin I reported by our Lab before.Because of the low conjugation efficiency of S046,only one mutant strain S046-LuxR was gained,there was no different in their metabolic profiling through HPLC detection.The AHBA gene cluster in S051 does not contain pks gene and amide synthase gene,implying that the gene cluster does not encoded an ansamycin.This gene cluster contains 6 atypical nrps genes.13 genes containing AHBA synthetic genes are similar to mitomycin's biosynthetic genes,but there's only one nrps gene for mitomycin.Hence we predict that the AHBA gene cluster in S051 probably encodes a very new AHBA skeleton.There's a SARP family gene earby the gene cluster,an overexpression was carried out and a known compound naphthgeranine A was isolated and identificated.RT-PCR of type ? pks gene nph C and prenyltransferase gene nph B showed that the nphS051 didn't express in wild type,but well-expressed in the mutant,which proved a specific positive regulation of SARP to the nph gene cluster.To get the complete sequence of aminoansamycin gene cluster(aas),we sequenced the whole genome of S035.Genome of S035 was devided into two parts:a 7.6 Mb linear chromosome and a 1.4 Mb big linear plasmid.AntiSMASH 4.0 helped predict 29 and 10 secondary metabolic pathway,many of which have potential to encode structure-new natural product.According to the transcriptional condition of these genes,5 gene clusters were chosen and overexpressions of possible positive regulator genes were done.4 of 6 mutant strains had their metabolic profile changed,which needed to be further analyzed.In summary,in this thesis,we successfully activated two cryptic pentaketide ansamycin biosynthetic gene clusters in Streptomyces sp.S033 and Streptomyces sp.S035,two novel pentaketide ansamycins with new skeletons were isolated and identified,one of them,aminoansamycins,are the first kind of pentaketide ansamycin which shows cytotoxicity.