Genome Mining Drives The Study Of Antibacterial Secondary Metabolites Of Streptomyces Sulphureus DSM 40104

The rapid emergence and widespread distribution of antimicrobial resistance（AMR）is considered to be one of the most serious challenges to human health worldwide.Due to the widespread occurrence of drug-resistant bacteria and the severe contamination of antibiotic-resistance genes,novel antibiotics are urgently needed to cope with increasing drug-resistant pathogens.Actinomyces were once known as the"synthesis factory"of natural drugs,and it was reported that they could produce a large number of anticancer,antifungal,and anti-infective drugs.In addition,the establishment of high throughput gene sequencing technology has directly promoted the development of bioinformatics,which assisted the development of genome mining technology.This experiment conducted genome mining research through bioinformatics analysis,guided by polyketidesynthase（PKS）,nonribsomal peptide synthetase（NRPS）,and drug-resistance genes gyr B,and obtained an actinomycete Streptomyces sulphureus DSM 40104with the potential to produce antibiotics.In this study,the one strain-many compounds（OSMAC）strategy was used to explore and optimize the fermentation conditions of strain DSM 40104,and 22 compounds were isolated and purified from the fermentation products of the strain DSM 40104 by comprehensive use of various chromatographic techniques,including medium-pressure liquid chromatography,Sephadex LH-20 column,normal phase chromatography at atmospheric pressure,high-performance liquid chromatography,combined with molecular networking.We used various spectroscopic techniques such as ultraviolet,nuclear magnetic resonance,X-ray single crystal diffraction,and mass spectrometry,and identified 14 compounds,including six Pyridinopyrones compounds with four novel compounds,Pyridinopyrone E（1）Pyridinopyrone F（2）,Pyridinopyrone G（3）and Pyridinopyrone H（4）,and two known compounds Pyridinopyrone C（5）and Pyridinopyrone D（6）.There were eight other known compounds been identified including a polyaromatic cyclic polyketone Resistomycin（7）,a tanshinone compound Cryptotanshinone（8）,two cyclic pentapeptide compounds Malformin E（10）and Malformin C（11）,three cyclic dipeptide compounds Cyclo-（Leu-Val）（12）,Cyclo-（Leu-Phe）（13）and Cyclo-（Ile-Phe）（14）,and one fatty acid compound palmitic acid（17）.Antibacterial activity tests of some compounds showed that compound 7 had good anti-methicillin-resistant staphylococcus aureus（MRSA）activity,with a minimal inhibitory concentration（MIC）value of 0.781μM.Meanwhile,the anti-inflammatory activity of compounds 1,2,and 3 was tested on BV-2 cells and compounds 1,2,and 3 all showed inhibitory effects on the production of NO in Lipopolysaccharide（LPS）-induced BV-2 cells,with IC₅₀ of 8.384μM,7.739μM,10.280μM respectively.Finally,based on the genomic information of the strain and the chemical structure of Pyridinopyrones,we have proposed the biosynthesis pathway of Pyridnopyrones.Compounds 1～6 have a rare class of structures with pyridine ring andα-pyranone ring linked by a polyene chain,and only four compounds with above-mentioned structure have been published up to now.And,our study demonstrates the diversity of polyene pyrone compounds regarding their chemical structure and bioactivity while providing new insights into their biosynthesis pathway.Compound 7 has significant anti-MRSA activity,and it is easily isolated from the metabolites of DSM 40104,which provides a basis for its simple industrial production.Compound 8 is a compound with a wide range of biological activities,most of which were previously isolated from Salvia miltiorrhiza.However,this is the first time that compound 8 has been isolated from microorganisms,which provides convenience for its future industrial production.