Isolation and genome characterization of Paenibacillus polymyxa 188, a potential biocontrol agent against fungi.

AIMS: In this work, we aimed to isolate marine bacteria that produce metabolites with antifungal properties. METHODS AND RESULTS: Paenibacillus polymyxa 188 was isolated from a marine sediment sample, and it showed excellent antifungal activity against many fungi pathogenic to plants (Fusarium tricinctum, Pestalotiopsis clavispora, Fusarium oxysporum, F. oxysporum f. sp. Cubense (Foc), Curvularia plantarum, and Talaromyces pinophilus) and to humans (Aspergillus terreus, Penicillium oxalicum, and Microsphaeropsis arundinis). The antifungal compounds produced by P. polymyxa 188 were extracted and analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The complete genome sequence and biosynthetic gene clusters of P. polymyxa 188 were characterized and compared with those of other strains. A total of 238 carbohydrate-active enzymes (CAZymes) were identified in P. polymyxa 188. Two antibiotic gene clusters, fusaricidin and tridecaptin, exist in P. polymyxa 188, which is different from other strains that typically have multiple antibiotic gene clusters. CONCLUSIONS: Paenibacilluspolymyxa 188 was identified with numerous biosynthetic gene clusters, and its antifungal ability against pathogenic fungi was verified.

Editing Aspergillus terreus using the CRISPR-Cas9 system.

CRISPR-Cas9 technology has been utilized in different organisms for targeted mutagenesis, offering a fast, precise and cheap approach to speed up molecular breeding and study of gene function. Until now, many researchers have established the demonstration of applying the CRISPR/Cas9 system to various fungal model species. However, there are very few guidelines available for CRISPR/Cas9 genome editing in Aspergillus terreus. In this study, we present CRISPR/Cas9 genome editing in A. terreus. To optimize the guide ribonucleic acid (gRNA) expression, we constructed a modified single-guide ribonucleic acid (sgRNA)/Cas9 expression plasmid. By co-transforming an sgRNA/Cas9 expression plasmid along with maker-free donor deoxyribonucleic acid (DNA), we precisely disrupted the lovB and lovR genes, respectively, and created targeted gene insertion (lovF gene) and iterative gene editing in A. terreus (lovF and lovR genes). Furthermore, co-delivering two sgRNA/Cas9 expression plasmids resulted in precise gene deletion (with donor DNA) in the ku70 and pyrG genes, respectively, and efficient removal of the DNA between the two gRNA targeting sites (no donor DNA) in the pyrG gene. Our results showed that the CRISPR/Cas9 system is a powerful tool for precise genome editing in A. terreus, and our approach provides a great potential for manipulating targeted genes and contributions to gene functional study of A. terreus.