| Natural products produced by actinomycetes are the main source of small molecule drugs,especially clinical antibiotics.However,due to the abuse of antibiotics,the spread of drugresistant bacteria and multidrug resistant strains has become an urgent problem to public Health.Therefore,searching for novel natural products with new mechanisms of action and/or new structure is long-term demand in the field of natural product chemistry.With the development of the technology,the paradigm of natural products discovery has gradually shifted from the traditional activity-oriented method to the genome-oriented method.However,there are still some challenges in the discovery of actinobacterial natural products,such as repeated discovery of known compounds,high labor intensity,and low efficiency.So new approaches are still needed to improve the discovery efficiency of dark matter of natural products.In this thesis,we carried out rational mining of natural products and exploration of genome mining approaches in 3 actinomycete strains.Verrucosispora sp.NS0172 is a 3-amino-5-hydroxybenzoic acid(AHBA)synthase genepositive strain isolated from intertidal soil collected in Xiamen.Bioinformatic analysis of NS0172 genome identified a typical pentaketide ansamycin gene cluster vas.According to the substrate specificity of polyketide synthase,the product of vas was predicted to be novel,with the backbone of AHBA-C3-C3-C2-C2.By constitutively co-overexpression of the conserved SARP and LAL family positive regulatory genes vasRl and vasR2,we successfully activated silent vas gene cluster.The yield of the compounds was further increased by feeding the precursor 3,5-AHBA,and four new compounds(1-4)were obtained by fermentation on 30 L YMG agar plate(containing 200 mg/L AHBA)and HPLC-guided isolation.The crystal of compound 1 was obtained by solvent natural volatilization method.The structure of compounds 1-4,named as verransamycins A-D,was determined by NMR and X-ray single crystal diffraction.Verransamycins is dimerized ansamycins.In addition to the macrolactam ring,verransamycins contains dihydroindole or benzodihydropyran ring on one side,and a unique 5/5/5/6/6 fused ring system on the other side,indicating that it has undergone complex postPKS tailoring.The bioactivity assay showed that verransamycin B(2)had weak cytotoxicity in the colon cancer cell line HCT116 with IC50 values of 18.7 μM.In addition,we also obtained the crystal of another dimerized ansamycin juan limy cin C(5)by liquid phase diffusion method,and its precise structure was also determined by X-ray single crystal diffraction.Juanlimycin C also has the benzodihydropyran ring on one side,but the fused ring system on the other side was different,which may reflect the difference of post-PKS modification.Streptomyces sp.LZ35 is also an AHBA synthase-positive strain isolated from the rhizosphere soil of Kandelia candel collected in the intertidal zone of Jimei,Xiamen.It has more than 40 secondary metabolic gene clusters.Previously,more than 80 natural products of 17 types have been identified through systematic separation or genome mining.We isolated and identified two glycinocin family cyclic lipopeptides 6 and 7 from the SG liquid fermentation products of LZ35 knockout mutant SR111.Among them,6 was analyzed as a new compound,and its aliphatic chain is one CH2 less than glycinocin C.Compounds 6 and 7 have good antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis,and Ca2+can enhance its activity,which is consistent with the characteristics of calcium-dependent antibiotic(CDA)family.In addition,in order to reduce the interference of known compounds on the subsequent genome mining of LZ35,we sequentially knocked out BGC26.5(quinolamines),BGC8(hexacosalactones)and BGC28(haoxinamides)in SR112,in which 9 known biosynthetic gene clusters were deleted,and constructed a mutant strain LZ35Δ12 with cleaner background.In the last chapter,we tried to modify the melanin reporter gene-guided media screening method in Streptomyces sp.S001.The blue pigment reporter gene idgS was introduced after melC1/C2,and 1 0 "silent" gene clusters were subjected to media screening.Although many blue pigment-producing media were identified,but no significant differential peaks or spots were found in HPLC and TLC profiles of metabolites between knockout and wild-type strains,indicating that these conditions may be "false positive".We speculate the possible reason is that the sensitivity of the idgS is much higher than that of melC1/C2.Although the target gene cluster is expressed in the "false positive" media,but the expression level is still low,and the amount of target compound is under the detection limit or covered by other metabolites.In summary,a new class of dimeric pentaketide ansamycins verransamycins(1-4)was discovered and identified from marine actinomycete Verrucosispora sp.NS0172 by rational genome mining,and the crystal structures of verransamycin A(1)and juanlimycin C(5)were determined by X-ray single crystal diffraction.Two glycinocins(6-7)were identified from Streptomyces sp.LZ35 by media screening,of which 6 was analyzed as a new compound.The targeted discovery method based on melC/1C2 and idgS reporter genes was explored in Streptomyces sp.S001,which may helpful for similar researches. |
