RESUMO
Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and immunosuppressive activities. However, the yield of edeines in wild-type (WT) Brevibacillus is extremely low, and chemical synthesis of edeines is a time-consuming process. Genetic engineering has proven to be an effective approach to produce antibiotics with high yield. In this study, the edeine biosynthetic gene cluster (ede BGC), which is involved in edeine production, was identified and characterized in Brevibacillus brevis X23. To improve edeine production in B. brevis X23, the ede BGC promoter was replaced with six different promoters, Pmwp , Pspc , PxylA , Pshuttle-09 , Pgrac or P43 , through double-crossover homologous recombination. The new promoters significantly increased the expression of the ede BGC as well as edeine production by 2.9 ± 0.4 to 20.5 ± 1.2-fold and 3.6 ± 0.1to 8.7 ± 0.7-fold respectively. The highest yield of edeines (83.6 mg l-1 ) was obtained in B. brevis X23 with the Pmwp promoter. This study provides a practical approach for producing high yields of edeines in B. brevis.
Assuntos
Bacillus , Brevibacillus , Antibacterianos/metabolismo , Bacillus/metabolismo , Brevibacillus/genética , Brevibacillus/metabolismo , Edeína/química , Edeína/metabolismoRESUMO
Species that are currently listed under the genus Brevibacillus (formerly, Bacillus brevis cluster) have been a rich source of antimicrobial peptides for many decades. The first known peptide antibiotic, gramicidin, is presumed to be produced by a Brevibacillus sp. Members of the genus are widely spread in nature. They can be found in a variety of environments including intestinal tracts of animals, seawater, and soil. Some Brevibacillus strains have been used commercially as probiotics. Bioactive peptides produced by Brevibacillus spp. include antibacterial, antifungal and anti-invertebrate agents. Brevibacillus antimicrobial peptides are synthesized through ribosomal or nonribosomal pathway; these two groups can be further categorized based on specific structural features such as cyclization and presence of lipid chain. Some of the antimicrobial compounds produced by this genus share structural similarities that were overlooked previously. For example, the structural similarity between BT peptide, brevibacillin, and bogorol was revealed only recently. Here we review and classify Brevibacillus antimicrobial peptides and summarize their bioactivities and potential applications.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacocinética , Brevibacillus/metabolismo , Peptídeos/química , Peptídeos/classificação , Peptídeos/metabolismo , Peptídeos/farmacologia , Animais , Antifúngicos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bacteriocinas/metabolismo , Edeína/metabolismo , Gramicidina/metabolismo , Guanidinas/metabolismo , Lipopeptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Probióticos , Ribossomos/metabolismo , Água do Mar/microbiologia , Microbiologia do Solo , Tirocidina/metabolismoRESUMO
Edeines are atypical cationic peptides produced by Brevibacillus brevis Vm4 with broad-spectrum antimicrobial activity. These linear nonribosomal peptides bind to the 30S ribosomal subunit and block t-RNA binding to the P-site. To identify the mechanism of high-level self-resistance in the producing organism, the B. brevis Vm4 genome was sequenced and the edeine biosynthetic cluster discovered. A potential edeine-modifying enzyme, EdeQ, showed similarity to spermidine N-acetyltransferases. EdeQ was purified and shown to convert edeine to N-acetyledeine, which is inactive against cells in vivo and against cell-free extracts. Unexpectedly, tandem mass spectroscopy and nuclear magnetic resonance demonstrate that N-acylation occurs on the free amine of the internal diaminopropionic acid rather than the N-terminal spermidine polyamine. Acetylation of edeine by EdeQ abolishes its ability to inhibit translation, thus conferring resistance to the antibiotic in the producing organism.
Assuntos
Acetiltransferases/metabolismo , Antibacterianos/metabolismo , Brevibacillus/enzimologia , Edeína/metabolismo , Acetilação , Acetiltransferases/genética , Antibacterianos/química , Brevibacillus/genética , Brevibacillus/metabolismo , Edeína/química , Genes Bacterianos , Modelos Moleculares , Família Multigênica , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/metabolismoAssuntos
Antibacterianos/metabolismo , Bacillus/enzimologia , Edeína/análogos & derivados , Espermidina/análogos & derivados , Ureo-Hidrolases/química , Sequência de Aminoácidos , Sulfato de Amônio/química , Antibacterianos/química , Arginase/química , Arginina/metabolismo , Precipitação Química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Edeína/química , Edeína/metabolismo , Eletroforese em Gel de Poliacrilamida , Hidrólise , Dados de Sequência Molecular , Análise de Sequência , Espermidina/química , Espermidina/metabolismo , Especificidade por Substrato , Ureo-Hidrolases/genética , Ureo-Hidrolases/isolamento & purificaçãoRESUMO
A cell-free protein synthesizing system from a mutant of Saccharomyces cerevisiae translated exogenous mRNA in the presence of 2 microM edeine, while a similar system from wild-type strain was completely inhibited by the drug. The mutant ribosomes showed an affinity for [125I]edeine comparable to the wild-type ribosomes, thereby suggesting that these macromolecules alone were not responsible for the edeine-resistant capacity of the mutant.
