Mass Spectrometry And Genome-Guided Discovery Of Novel Non-ribosomal Peptides

Non-ribosomal peptide（NRP）natural products are widely used in pharmaceutical,agriculture,and food industries due to their complex structure and excellent biological activities.Conventional natural product discovery approaches lack the ablity of precise identification of polypeptide substances,and thus are not suitable for targeted mining of novel NRPs.The current study employed multiple modern mass spectrometry techniques,in combination with hierarchical clustering and molecular networking analyses,to accomplish high-throughput screening and dereplication of fungal NRPs.As a result,a novel class of antimalarial cyclodepsipeptide named xylomyrocins,and a new group of antibacterial lipopeptide named halovirs were discovered,and their essential biosynthetic genes were identified.Discovery of addtional xylomyrocin-like NRPs were achieved by implementing targeted mining on the basis of the correlation information of structure,mass spectroscopic feature,and biosynthetic genes.Detailed descriptions of the results are as follows:1.Rapid detection and dereplication of NPRs from 1748 soil-derived filamentous fungi were achieved by exploiting the high-throughput screening and data analyzing capability of MALDI-TOF mass spectrometry（MS）and hierarchical clustering.A total of 61 representative fungal strains that produce characteristic NRPs were identified.Next,a molecular network was constructed using the UHPLC-HRESI-q TOF MS/MS data of these characteristic NRPs,and 19 out of the 70 NRPs clades generated thereof were predicted to contain novel cyclopeptides.Performing large-scale fermentation and MS-guided isolation on Paramyrothecium sp.XJ0827,the producing fungus of one of the cyclopeptide families,afforded three single compounds named xylomyrocins A–C（1–3）.Structure characterization of 1–3 using multiple spectroscopic approaches,such as nuclear magnetic resonance spectrometry,HRESI-MS,and X-ray single crystal diffraction,revealed that these compounds are10-mer cyclodepsipeptides featuring a rare D-xylonic acid and three uncanonical N-methylated amino acid building blocks.2.Bioinformatics analyses of the sequenced genome of Paramyrothecium sp.XJ0827 enabled the annotation of 98 natural product biosynthetic gene clusters（BGCs）,including the putative xylomyrocin BGC pxxmc.Next,targeted gene disruption experiments confirmed that the NAD-depedent dehydrogenase encoding pxxmc E and non-ribosomal peptide synthetase（NRPS）encoding pxxmc G were the essential genes for xylomyrocin biosynthesis.Last,the biosynthetic origin of the D-xylonic acid unit and the biosynthetic mechanism of xylomyrocin were deduced based on the results of precursor supplementation and stable isotope labeling experiments.3.Searches of homologous genes of Pxxm E and pxxmc G in the published fungal genomes revealed that Paramyrothecium and Colletotrichum spp.harbor pxxmc-like BGCs that are likely to biosynthesize xylomyrocin-type NRPs.MALDI-TOF MS analyses were carried out after collecting 50 different fungal strains of the genus Paramyrothecium or Colletotrichum,and culturing them under different conditions.The results showed that seven fungal isolates,including Paramyrothecium roridum NRRL 2183,P.roridum MN113194,and Colletotrichum sp.XJ1040 were able to produce 10 different groups of NRPs.Further comparison of the MS/MS fragmentation patterns of these NRPs with those of xylomyrocins confirmed that three out of the 10 groups of NRPs are indeed xylomyrocin analogs.Subsequent large-scale fermentation and MS-guided isolation of the corresponding fungi yielded xylomyrocins D–L（4–12）.Antimalarial activity assay showed that several xylomyrocins significantly inhibited the growth of Plasmodium falciparum 3D7,while 1 and 11 were the most active compounds having IC₅₀ values of13.5 and 10.0μM,respectively.4.Apart from xylomyrocins,another group of NRPs was discovered from P.roridum NRRL 2183during MALDI-TOF MS-based screening.MS-guided isolation and structural characterization of these NPRs revealed that they are linear 6-mer lipopeptaibols,named halovirs I–K（29–31）,featuring N-terminal lauroyl substitution and non-proteinogenic 2-aminoisobutyric acid（Aib）building block.Compounds 29 and 30 strongly inhibited the growth of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium by showing MIC values of 20μg/m L.Moreover,29 and 30also exhibited significant anti-proliferative activity against the human lung cancer A549 cells,human cervical cancer He La cells,and human breast cancer MCF-7 cells with IC₅₀values ranging from 1.3 to3.3μM.Last,the halovir BGC prhal was identified after the comprehensive bioinformatics analyses of P.roridum NRRL 2183 genomic data,and the halovir synthetase Pr Hal A was shown to adopt an unconventional in trans substrate loading mechanism during the assembly of halovirs.To sum up,the present study developed a modern MS technique-based fungal NRPs mining platform,and discovered two novel classes of antimalarial and antibacterial fungal NRPs named xylomyrocins and halovirs.Upon elucidation of the biosynthetic mechanisms of xylomyrocins and halovirs,the correlation information of peptide structure,mass spectroscopic feature,and biosynthetic genes were used for targeted mining of additional xylomyrocin analogs,which vastly expanded the chemical diversity of this novle class of NRP.The results of this study not only provided an efficient approach for the discovery of bioactive fungal NRPs but also offered novel lead compounds for the development of antimalarial and antibacterial drugs.