Targeted Discovery Of Novel Lipopeptides From Lysobacter And Investigation Of Potential The Relationships Between WBP-29479A1 And The Biosynthesis Of Phenazines And Phenoxazines

The discovery of antibiotics with a novel structure and mode of action is a pressing need due to the constant emergence of drug-resistant pathogens.Cyclic lipopeptides provide a distinct group of lead compounds in the development of antibiotics due to their diverse structures,broad biological activities and unique mode of action.Therefore,it has attracted extensive attention to develop novel potent cyclic lipopeptides against resistant bacteria.Bioactive natural products produced by microbes have been a primary source of lead compounds.The exploitation of new microbes is a profitable approach to discover novel bioactive natural products Previous studies indicated that the largely unexplored Lysobacter strains contain abundant unknown non-ribosomal peptide synthesis pathways,which provide new resources for the discovery of novel cyclic lipopeptides.In this thesis,we performed the targeted discovery of novel cyclic lipopeptides from Lysobacter antibioticus ATCC 29479 and initiated an investigation into the relationships between the cyclic lipopeptide WBP-29479A1 and the biosynthesis of phenazines and phenoxazines1.Targeted discovery of novel cyclic lipopeptides from Lysobacter antibioticus ATCC 29479.We analyzed the genome sequence of strain 29479 and found it contains two novel NRPS gene clusters,wbp and GC5.A novel cyclic lipopeptide WBP-29479A1 containing the 11-amino acid residues and a 3-hydroxy fatty acid was isolated from the metabolites of strain 29479.The gene cluster responsible for WBP-29479A1 biosynthesis was confirmed by gene deletion,and the biosynthetic pathway of WBP-29479A1 was proposed.The work laid a foundation for further structure modification and yield optimization.In addition,WBP-29479A1 exhibited activity against MRSA in a menaquinone-dependent manner.At present,the identification of the products of GC5 gene cluster is still in progress.2.Investigation of the relationships between WBP-29479A1 and the biosynthesis of phenazines and phenoxazines.We found that WBP-29479A1 along with other two classes of compounds disappeared in strain 29479 ? WBP compared with the wild type.The analysis of the UV spectra and LC-MS data suggested that one class of the disappeared compounds might be phenazines.Based on the metabolite profile of the deletion mutant of ubiquinone oxidase genes in the wbp gene cluster,we proposed that WBP-29479A1 might promote the biosynthesis of phenazines through the inhibition of ubiquinone oxidase activity,but the specific mechanism remains to be further clarified.Furthermore,the analysis of the transcriptome showed that the transcription levels of phenazines related genes in mutant 29479 ? WBP were comparable to those in the wild type.Therefore,we speculated that the elimination of WBP-29479A1 might influence the translation levels of phenazines related enzymes,protein modifications or the supply of biosynthetic precursors.We found the other class of disappeared compounds belong to phenoxazines.One of them was identified as 2-aminophenoxazine,which is biosynthesized using 2-aminophenol as a precursor.The precursor feeding experiment indicated that the insufficiency of 2-aminophenol might be the reason for the elimination of phenoxazines in mutant 29479?WBP.Futher studies are needed to understand the relationship between WBP-29479A1 and the biosynthesis of phenoxazines,paticularly the biosynthesis of the 2-aminophenol precursor in strain 29479.In conclusion,this research identified a novel anti-MRSA cyclic lipopeptide WBP-29479A1,which provides a good example for genome mining of novel natural products from Lysobacter strains.Moreover,the research revealed a potentially new way for regulation of the secondary metabolites in microorganisms,in which a cyclic lipopeptide plays an intriguing role in the production of phenazines and phenoxazines.