Isolation Of Actinomycetes From Rhizospheric Soils Of Trewia Nudiflora&Maytanus Hookeri And Biosynthetic Study On Compound RF-3192C

Microorganism is an important source of small molecule drugs.Among them,actinomycetes have always attracted the attention of natural product researchers due to their impressive natural product biosynthetic potential.In the field of basic research on microbial natural products,the discovery of novel actinomycete resources and novel natural products,and the elucidation of new biosynthetic pathways are still the constant themes at this stage and in the foreseeable future.This thesis has carried out research on these three aspects respectively.One of the themes of this thesis is the isolation and screening of actinomycetes from the rhizospheric soils of maytansinoid-producing plants.In order to search for direct evidence to support the hypothesis that "maytansinoids isolated from plants are actually produced by their endophytic microorganisms",we performed selective isolation of actinomycetes from rhizospheric soil samples of Trewia nudiflora and Maytanus hookeri,and finally obtained 225 isolates of actinomycetes.Through PCR screening of 3-amino-5-hydroxybenzoic acid(AHBA)synthase gene,which is highly conserved in ansamycin biosynthesis,we obtained 16 AHBA synthase gene-positive strains.In addition,we performed complete genome sequencing of 5 positive strains with novel AHBA synthase gene.So far,we have obtained the genome sequences of 3 strains(LR1-1,LR4-17 and LR5-3).The predictive products of ansamycin biosynthetic gene cluster of these 3 strains are all novel pentaketide ansamycins,which needs to be further explored.Although the expected potential plant-type maytansinoid-producing strains could not be obtained,more than 200 actinomycetes(including 38 rare actinomycetes)and 5 novel ansamycin potential-producing strains were obtained in this study,which lays the foundation for further searching for new ansamycin and other natural products through genome mining.Biosynthetic study of compound RF-3192C in Streptomyces sp.S001.We revised the structure of SRT-A1,product of a type Ⅲ PKS gene cluster in Streptomyces sp.S001,by single-crystal X-ray diffraction.It is identified as a known polyketide dimer compound RF-3192C(1).RF-3192C was previously reported as a fungal product,and this is the first time to identified this compound in Streptomyces sp.We determined that the type Ⅲ PKS(RppAS001)and P450 enzyme(P-450melS001)(and their redox partners)genes in the sm8 gene cluster are sufficient to biosynthsize RF-3192C through gene knockout,complementation and heterologous expression experiments.In the P-450melS001 gene knockout mutant,the putative precursor 1,3,6,8-Tetrahydroxynaphthalene(THN)spontaneously oxidized to the shunt product flaviolin(2).We speculate that THN,product of RppAS001,is catalyzed by P-450melS001 to generate RF-3192C through free radical mediated dimerization.However,in the in vitro assay,the widely used spinach FdR/Fdx could not reconstitute the catalytic activity of this P-450melS001.Then,the native FdR/Fdx from Streptomyces sp.S001 was co-expressed with RppAS001 and P-450melS001 genes in surrogate host SR113.The result showed that multiple pairs of native FdR/Fdx could significantly increase the yield of RF-3192C in heterologous expression host.The in vitro enzymatic activity and catalytic mechanism of P-450melS001 remain need to be further determined.In addition,we also isolated and identified the metabolites of Streptomyces sp.SR111.The results of HPLC showed that a series of previously unidentified peaks appeared in the fermentation extract of Streptomyces sp.SR111 in M27 medium.We traced these peaks and identified 5 known compounds:tryptophan(3),cyclo(L-Pro-L-Tyr)(4),(2E)-3methylthioacrylic acid(5),3-methylthiopropionic acid(6)and phenylacetic acid(7).Among them,compound 4 has significant inhibitory activity against food-borne pathogens and plant pathogenic fungi.To our surprise,no NRPS(nonribosomal peptide synthetase)and CDPS(cyclodipeptide synthase)gene clusters responsible for cyclic dipeptide biosynthesis were identified from Streptomyces sp.SR111 genome by bioinformatic analysis.Therefore,the biosynthetic gene of compound 4 in SR111 remains to be further identified.