• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.399-405
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• 1998

From the sequence alignment of various non-ribosomal peptide synthetases, several motifs of highly conserved sequences have been identified within each domain of peptide synthetases. We designed PCR primers based on the highly conserved nucleotide sequences to amplify and isolate a ∼7.2-kb DNA fragment of the Bacillus subtilis 713 which was isolated and reported to produce an antifungal peptide compound. Nucleotide sequence analysis of 4.8 kb of the predicted amino acids revealed significant homology to various peptide synthetases over the whole sequence and also revealed two amino acid-activating domains with highly conserved Core 1 to Core 6 and spacer motif. This suggests that the isolated DNA fragment is part of a peptide synthetase gene for antifungal peptide.

• Korean Journal of Microbiology
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• v.55 no.3
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• pp.303-305
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• 2019

An actinobacterial strain designated Gordonia sp. MMS17-SY073 (=KCTC 49257) was isolated from a coastal soil of an island, and its complete genome was analyzed. A single contig consisting of 5,962,176 bp with the G + C content of 67.4% was obtained, and the annotation resulted in 5,201 protein-coding genes, 6 rRNA genes and 45 tRNA genes. Strain MMS17-SY073 was closest to the type strain of Gordonia soli based on the 16S rRNA gene sequence comparison, sharing 98.5% sequence similarity. A number of biosynthetic gene clusters for secondary metabolites, non-ribosomal peptide synthetase types in particular, could be identified from the genome.

• KSBB Journal
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• v.22 no.5
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• pp.362-365
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• 2007

Cyanobacteria have been identified as one of the most promising group producing novel biochemically active natural products. Cyanobacteria are a very old group of prokaryotic organisms that produce very diverse secondary metabolites, especially non-ribosomal peptide and polyketide structures. Large multienzyme complexes which are responsible for the non-ribosomal biosynthesis of peptides are modular for the addition of a single amino acid. An activation of amino acid substrates results in an amino adenylate occuring via an adenylation domain (A-domain). A-domains are responsible for the recognition of amino acids as substrates within NP synthesis. The A-domain contains ten conserved motifs, A1 to A10. In this study, ten conserved motifs from A1 to A10 were checked regarding their amino acid sequence of the NRPS-module of Microcystis aeruginosa PCC 7806. The part of the amino acid sequence chosen was that which contained as many conserved motives as possible, and then these amino sequence were compared between other cyanobacteria to design a new degenerate primer. A new degenerate primer (A3/A7 primer) was designed to detect any putative NP synthetase region in unkwon cyanobacteria by a reverse translation of the conserved amino acid sequence and a search for cyanobacterial DNA bank.

• 한국균학회소식:학술대회논문집
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• 2014.05a
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• pp.27-28
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• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.689-699
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• 2020

Brevibacillus brevis GZDF3 is a gram-positive, plant growth-promoting rhizosphere bacterium (PGPR) isolated from the rhizosphere soil of Pinellia ternata (an important herb in traditional Chinese medicine). The GZDF3 strain produces certain active compounds, such as siderophores, which are the final metabolite products of non-ribosomal peptide synthetase (NRPS) and independent non-ribosomal peptide synthetase (NIS) activity. With the present study, we attempted to investigate the siderophore production characteristics and conditions of Bacillus sp. GZDF3. The antibacterial activity of the siderophores on pathogenic fungi was also investigated. Optimal conditions for the synthesis of siderophores were determined by single factor method, using sucrose 15 g/l, asparagine 2 g/l, 32℃, and 48 h. The optimized sucrose asparagine medium significantly increased the production of siderophores, from 27.09% to 54.99%. Moreover, the effects of different kinds of metal ions on siderophore production were explored here. We found that Fe³⁺ and Cu²⁺ significantly inhibited the synthesis of siderophores. The preliminary separation and purification of siderophores by immobilized-metal affinity chromatography (IMAC) provides strong antibacterial activity against Candida albicans. The synergistic effect of siderophores and amphotericin B was also demonstrated. Our results have shown that the GZDF3 strain could produce a large amount of siderophores with strong antagonistic activity, which is helpful in the development of new biological control agents.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.448-452
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• 2018

The full genome sequence of Bacillus safensis KCTC 12796BP which had been isolated from the marine sponge in the seawater of Jeju Island, was determined by Pac-Bio next-generation sequencing system. A circular chromosome in the length of 3,935,874 bp was obtained in addition to a circular form of plasmid having 36,690 bp. The G + C content of chromosome was 41.4%, and that of plasmid was 37.3%. The number of deduced CDSs in the chromosome was 3,980, whereas 36 CDS regions were determined in a plasmid. Among the deduced CDSs in chromosome, 81 tRNA genes and 24 rRNA genes in addition to one tmRNA were allocated. More than 30 CDSs for sporulation, 16 CDSs for spore coat, and 20 CDSs for germination were also assigned in the chromosome. Several genes for capsular polysaccharide biosynthesis and for flagella biosynthesis and chemotaxis in addition to genes for osmotic tolerance through glycine-choline betaine pathway were also identified. Above all, the biosynthetic gene cluster for anti-allergic compounds seongsanamides were found among two non-ribosomal peptide synthetase (NRPS) gene clusters for secondary metabolites.