Mining Of Novel Natural Products From Two Strains Of Bacteria

Microorganisms as an important source of new antibiotics and antitumor drugs can produce many natural products with diverse structures and activities.The continuous emergence of drug-resistant bacteria poses a great threat to human public health,so it is particularly urgent to discover natural products with new structures or new mode of action from microorganisms.Burkholderiales are ecologically and metabolically diverse gram-negative bacteria.Because of their large number of natural products with novel structures and good biological activities,they have gradually acted as important natural product producers and target bacteria for natural products mining.In addition,the marine environment is also an important source of new natural products.In order to adapt to the complex and changeable marine environment,many marine microorganisms produce a large number of secondary metabolites with unique structures.So they are also important producers of new natural products.This study aims to discover new natural products from Chitinimonas koreensis DSM 1 7726,which belongs to Burkholderiaceae family,by genome sequence analysis and heterologous expression of gene clusters.At the same time,OSMAC(One Strain Many Compounds)strategy was used to the isolation and identification of natural products from a potential noval strain,Marinoscillum sp.YZ09,isolated from marine sediments in Aoshan Bay,Qingdao.The main research contents and results are as follows:(1)Targeted mining of non-ribosomal peptide from Chitinimonas koreensis based on genome sequenceGenome sequence analysis revealed that Chitinimonas koreesis DSM 17726 has four unknown non-ribosomal peptide synthetase(NRPS)biosynthetic gene clusters—BGC1,BGC3,BGC10,BGC11,all of which show low similarity to known NRPS.It is speculated that new lipopeptide or cyclic peptide compounds may be produced.The size of BGC1 is 54 kb.containing 12 NRPS modules and the initiation module has a starter condensation domain.It is speculated that its product is a dodecapeptide compound with a lipid chain.A series of peaks were detected after direct cloning of gene clusters,insertion of transposable elements and constitutive promoter Papra,and heterologous expression in the heterologous host Schlegelalla brevitalea DSM 7029Δglb.Two new lipopeptides.chitinipeptins A and B,were isolated and identified by HRMS(High resolution mass spectrometry),NMR(Nuclear magnetic resonance)and Marfey’s hydrolysis.The N-terminus of these compounds contain a N-tetradecanoyl group and polypeptides backbone consisting of unnatural amino acids-Dhb(dehydrobutyrine),D-Dab(2,4-diaminobutyric acid),D-Ser,D-Lys,D-Glu,D-Asp.The further fragments were analyzed by MS/MS and the structures of four other compounds-chitinipeptins C,D,E,F were deduced.The results of biological activity test show that the compounds have weak cytotoxicity and strong surfactant activity,which can promote the swarming of bacteria on the surface of solid substrate.The size of BGC3 is 61 kb,and it contains permease and dehydrogenase genes related to Fe3+,so its products may be siderophore compounds.The gene cluster was captured by direct cloning and inserted into transposable element and promoters.The product of BGC3 was detected by HPLC-MS(chromatography-mass spectrometry)and siderophore activity test.After MS/MS analysis,it was found that this product was same to the siderophore-vanchrobactin,produced by Vibrio anguillarum serotype O2 strains,but there are some differences in the composition of gene clusters,and there may be evolutionary differences.Siderophore can capture Fe3+ in the external environment for bacteria’s metabolism.At the same time,the direct cloning and heterologous expression of BGC10 and BGC11 have been successfully finished,but the products have not been detected from the fermentation broth,and further optimization of conditions and methods are required.(2)Isolation of potential novel bacteria strain in marine sediments and isolation,identification of the natural products9 strains of bacteria were isolated from sea mud,marine animals and marine plant samples from Aoshan Bay,Qingdao.Through the alignment of 16s rDNA,it was found that the YZ09 strain was 94.95%similar to the known strain Marinoscillum luteum strain A03A-221 of the genus Marinoscillum,which may be a potential novel strain and was named Marinoscillum sp.YZ09.The fermentation detection and chemical screening of YZ09 strain were carried out by OSMAC method,and a novel pyrazinone alkaloid compound marinopyrazinone C was isolated and identified.The compound has a pyrazinone ring as the basic nucleus and is substituted by 6-methyloctyl and two other methyl groups.The biosynthetic mechanism of this compound was preliminarily speculated by genome sequence analysis and isotopic amino acid feeding experiments.In conclusion,in this study,a series of novel lipopeptide natural products,chitinipeptins,were discovered from C.koreensis DSM 17726 with good surfactant activity via using new natural product mining technology based on bacterial genome sequence.Besides,utilizing the classic OSMAC mining strategy based on novel strain resources,a novle pyrazinone alkaloid-marinopyrazinone C was obtained from a potential novel strain,Marinoscillum sp.YZ09.This study not only discovered and identified new natural products,but also proved that genome mining and screening of potential novel strain resources guided by genome sequences are important methods for novel natural produts discovery.It shows the importance and feasibility of this method for natural product mining.