RESUMO
Labyrinthopeptins are class III lantibiotics produced by the actinomycete Actinomadura namibiensis. The most characteristic structural feature is the posttranslationally installed triamino triacid labionin with a quaternary α-carbon. In addition to the unique structure, labyrinthopeptin A2 possess remarkable antiviral and antiallodynic biological activities. To harness the substrate tolerance of the biosynthetic machinery, we developed an efficient system for the generation of labyrinthopeptin analogs. Streptomyces lividans was used as a heterologous host since the natural producer Actinomadura namibiensis remained genetically intractable. Generation of a library of 39 mutants allowed identification of variable and invariable regions in the labyrinthopeptin structures. Additional data on the flexibility of the biosynthetic machinery were provided by in vitro experiments. This study is detailed investigation on the potential to generate analogs of class III lantibiotics by genetic engineering.
Assuntos
Actinobacteria/genética , Bacteriocinas/genética , Mutagênese Sítio-Dirigida , Actinobacteria/química , Actinobacteria/metabolismo , Sequência de Aminoácidos , Bacteriocinas/química , Bacteriocinas/metabolismo , Clonagem Molecular , Genes Bacterianos , Genes Sintéticos , Hidrólise , Dados de Sequência Molecular , Família Multigênica , Mutação , Plasmídeos/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismoRESUMO
Lantibiotics are a large group of ribosomally synthesized peptides post-translationally modified to incorporate the amino acid lanthionine. They are classified, according to their biosynthetic pathway and bioactivity, into three major subtypes. Of Actinomycetes type III lantibiotics, only four peptides (SapB, SapT, LabA1, and LabA2) have been described and structurally characterized, although homologous gene clusters are abundant in other Actinomycetes. All these gene clusters share a similar architecture with a characteristic Ser/Ser/Cys motif in precursor peptides, which has previously been suggested to act as a precursor for lanthionine (SapB) and labionin (LabA2) rings. Mass spectrometry screening led to the discovery and characterization of three new representatives of type III lantibiotics: Avermipeptin (Avi), Erythreapeptin (Ery), and Griseopeptin (Gri) from Streptomyces avermitilis DSM 46492, Saccharopolyspora erythraea NRRL 2338, and Streptomyces griseus DSM 40236, respectively. Apart from the assignment of these peptides to their corresponding gene clusters, additional investigations on Avi, Ery and Gri peptides indicate stepwise leader processing by putative aminopeptidase-like protease(s), thus yielding mixtures of differently N-terminal-processed lantibiotic peptides. Similar peptide processing was observed for a heterologously expressed eryth biosynthetic gene cluster expressed in a Streptomyces host system. Remarkably, all isolates of the new type III lantibiotics contain both the amino acids lanthionine and labionin, thus implying dual-mode cyclase activity of the processing lyase-kinase-cyclase enzymes. These findings have implications for the structures and maturation of other type III lantibiotics from Actinomycetes.
Assuntos
Alanina/análogos & derivados , Antibacterianos/química , Bacteriocinas/química , Saccharopolyspora/química , Streptomyces griseus/química , Streptomyces/química , Sulfetos/química , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Antibacterianos/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Genes Bacterianos , Espectrometria de Massas , Dados de Sequência Molecular , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , Sulfetos/metabolismoAssuntos
Antibacterianos/biossíntese , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Macrolídeos , Micromonosporaceae , Família Multigênica/genética , Ácido 4-Aminobenzoico/antagonistas & inibidores , Antibacterianos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Macrolídeos/farmacologia , Micromonosporaceae/genética , Micromonosporaceae/metabolismoRESUMO
This study demonstrated, for the first time, that immunity genes licFGEHI are not essential for self-protection and production of the two-component lantibiotic lichenicidin in the Gram-negative heterologous host Escherichia coli BLic5. Additionally, it was experimentally demonstrated that lichenicidin lantibiotics are active against the E. coli imp4213 strain, a mutant strain possessing a permeable outer membrane.
Assuntos
Proteínas de Bactérias/biossíntese , Bacteriocinas/biossíntese , Escherichia coli/metabolismo , Bacteriocinas/genética , Bacteriocinas/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Engenharia Genética , Mutação , Fases de Leitura AbertaRESUMO
Lichenicidin is a class II two-component lantibiotic produced by Bacillus licheniformis. It is composed of the two peptides Bliα and Bliß, which act synergistically against various Gram-positive bacteria. The lichenicidin gene cluster was successfully expressed in Escherichia coli, thus constituting the first report to our knowledge of a full reconstitution of a lantibiotic biosynthetic pathway in vivo by a Gram-negative host. This system was further exploited to characterize and assign the function of proteins encoded in the biosynthetic gene cluster in the maturation of lichenicidin peptides. Moreover, a trans complementation system was developed for expression of Bliα and Bliß variants in vivo. This contribution will spur future studies in the heterologous expression and engineering of lantibiotics.
