This study was conducted to screen the microorganisms producing novel natural antimicrobial substances.The target strains were screened by co-culture method;the strains were identified by 16S rDNA,physiological and biochemical properties,and whole genome sequencing;and the stability of the antimicrobial substances,the prediction of secondary metabolites in the genome of the strains,and the minimum inhibitory concentration (MIC) were utilized to study the biological properties of the antimicrobial substances.The results showed that a strain named Paenibacillus elgii CL-1 was screened from the soil,which produced antimicrobial substances with broad-spectrum antimicrobial properties and was resistant to catalase,pepsin,trypsin,proteinase K,and acid/base,and unstable at high temperatures.The antiSMASH analysis was used to find out that Paenibacillus elgii CL-1 contains 17 secondary metabolite gene clusters,including penisin and octapeptin C4. Further analysis revealed that the antimicrobial substances isolated and purified from the fermentation broth of CL-1 by high-performance liquid chromatography had a broad antimicrobial spectrum and good antimicrobial activity,with a minimum inhibitory concentration value of up to 1 μg/mL.Through mass spectrometry analysis,it was found that the Bacillus-like bacterium Paenibacillus elgii CL-1 was able to produce pelgipeptin B,which has a broad antimicrobial spectrum,good stability,and strong antimicrobial activity.The present study provides a chassis strain for the exploration of the natural antimicrobial drug pelgipeptin B and lays a foundation for its research and development as well as its application.