Research On The Discovery And Biosynthesis Of Microbial Natural Products Driven By Multi-omics

Microbial natural products are the main source of new drug development,and play an important role in many fields such as medicine,food,health,and agriculture.Especially with the widespread sequencing of microbial genomes,it has been found that there are a large number of silent gene clusters in microorganisms that have not been exploited,and the potential for exploiting novel natural products is enormous.However,traditional methods of microbial natural products,such as systematic separation based on "take things all of a lump" or targeted separation based on biological activity,currently face problems such as "high blindness,low innovation rate,and repeated discovery".Therefore,it is necessary to innovate mining methods.Fortunately,with the emergence of genomics,metabolomics,and various emerging gene editing methods,the mining of natural products has entered a new stage.Based on a multi-omics mining strategy combining genomics and metabolomics,this article focused on two strains of myxobacteria and one strain of entomophytic actinomycetes.A total of 8 compounds,including 5 new compounds,were efficiently isolated and identified from them.Multiomics methods significantly improve the directional mining efficiency of novel natural products "dark matter",and conduct biological activity testing and preliminary exploration of biosynthesis of the obtained novel compounds.Based on the large myxobacteria resource library of the research group,using onedimensional nuclear magnetic metabolomics characterization and chemometrics methods,the Archangium violaceum SDU8 with specific nuclear magnetic signals was selected,and further integrated genome analysis,bioinformatics structure prediction and two-dimensional nuclear magnetic metabolomics characterization to guide targeted separation,a class of novel nineteen membered macrolide compounds archangiumide A-C(1-3)was obtained.Among them,archeniumide A is the first discovered novel 19 membered macrolide skeleton with a rare diene structure fragment,which is the only case in the macrolide superfamily.Through sequence conservation analysis and isotope labeling experiments,we have deduced the biosynthetic pathway of archangimides-an assembly line that conforms to trans AT type I PKS.We have also conducted preliminary biochemical experiments in vitro to explore the key enzymatic mechanism for the formation of the malodiene group in archangimides A.In addition,in the biological activity test,it was found that archangimide A has significant anti vesicular stomatitis virus(VSV)activity.Further,based on 1D NMR metabonomics characterization,chemometrics methods and MyxoDB database of myxobacteria natural products,another myxobacteria strain Corallococcus exercis SDU142 was selected from the highly complex crude extracts of the myxobacteria resource database of the research group.Known compounds were quickly removed from highly complex crude extracts through nuclear magnetic resonance characterization.After that,we further integrated genomics and nuclear magnetic metabolomics analysis,especially through two-dimensional nuclear magnetic metabolomics to characterize the structural fragments corresponding to the "probe" nuclear magnetic signal,assisted by MyxoDB database retrieval and mass spectrometry metabolomics characterization,to predict structural novelty,thereby targeting the discovery of a new compound,pyxipyrrolone C(4),and deriving its biosynthetic pathway.After testing the cytotoxic activity of pyxipyrrolone C,it was found that it had moderate antitumor activity against human acute monocytic leukemia THP-1(IC50 was 26.92 ± 0.33 μM)。Apply the established multi-group natural product mining method was used to the actinomycetes from insect intestines,excavate the first naturally sourced glycoside substitution product of cycloheximide family α-L-rhamnosyl-actiphenol(5)and three known cycloheximide compounds(6—8)from Streptomyces sp.ML6,which crude extract has anti-Candida albicans activity.Analyze its biosynthetic pathway and find the production of α-L-rhamnosyl-actiphenol is the result of the joint action of two nonadjacent gene clusters in the genome of Streptomyces sp.ML6.Subsequently,the product 7-O-β-D-glucoside-cycloheximide(9)was obtained by enzymatic glycosylation in vitro.The antifungal and cytotoxic activities of the compounds were determined,and the activity of the two glycosylation products was significantly lower than that of cycloheximide.Thus,we speculate that glycosylation of compounds in strain ML6 may have potential ecological significance for self resistance of parasitic bacteria and hosts.In short,this paper successfully bridged the gap between "genotype" and"chemical type" in microbial natural products through the combination of genomics,NMR-based metabolomics.MS-based metabolomics,MyxoDB database retrieval,and activity screening.Isolated the lead compounds with novel skeleton and prominent activity from both myxobacteria and actinomycetes in special habitats,which fully proved that the combination of multiple groups can make up for the defects of a single method.It has outstanding advantages in improving the mining efficiency of natural products.This study provides a good methodological basis for the subsequent exploration of microbial natural products and the derivation of biosynthetic pathways.