RESUMEN
The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera Streptomyces, Micromonospora, and Saccharothrix. Two different systems based on Cre/loxP and Dre/rox have been utilized for numerous applications. The activity of the Cre recombinase on the heterospecific loxLE and loxRE sites was similar to its activity on wild-type loxP sites. Moreover, an apramycin resistance marker flanked by the loxLERE sites was eliminated from the Streptomyces coelicolor M145 genome at a surprisingly high frequency (80%) compared to other bacteria. A synthetic gene encoding the Dre recombinase was constructed and successfully expressed in actinomycetes. We developed a marker-free expression method based on the combination of phage integration systems and site-specific recombinases. The Cre recombinase has been used in the deletion of huge genomic regions, including the phenalinolactone, monensin, and lipomycin biosynthetic gene clusters from Streptomyces sp. strain Tü6071, Streptomyces cinnamonensis A519, and Streptomyces aureofaciens Tü117, respectively. Finally, we also demonstrated the site-specific integration of plasmid and cosmid DNA into the chromosome of actinomycetes catalyzed by the Cre recombinase. We anticipate that the strategies presented here will be used extensively to study the genetics of actinomycetes.
Asunto(s)
Actinomycetales/genética , Ingeniería Genética/métodos , Genética Microbiana/métodos , Genoma Bacteriano , Biología Molecular/métodos , Mutagénesis Insercional , Plásmidos , Recombinasas/genética , Recombinasas/metabolismo , Recombinación Genética , Eliminación de SecuenciaRESUMEN
Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the ß-glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter.
Asunto(s)
Actinobacteria/enzimología , Genes Reporteros/genética , Glucuronidasa/genética , Actinobacteria/genética , Codón Iniciador/genética , Diterpenos , Vectores Genéticos , Glicósidos/biosíntesis , Glicósidos/genética , Lisina/análogos & derivados , Nebramicina/análogos & derivados , Nebramicina/farmacología , Regiones Promotoras Genéticas , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Actinomycetes are Gram-positive bacteria with a complex life cycle. They produce many pharmaceutically relevant secondary metabolites, including antibiotics and anticancer drugs. However, there is a limited number of biotechnological applications available as opposed to genetic model organisms like Bacillus subtilis or Escherichia coli. We report here a system for the functional expression of a synthetic gene encoding the I-SceI homing endonuclease in several streptomycetes. Using the synthetic sce(a) gene, we were able to create controlled genomic DNA double-strand breaks. A mutagenesis system, based on the homing endonuclease I-SceI, has been developed to construct targeted, non-polar, unmarked gene mutations in Streptomyces sp. Tü6071. In addition, we have shown that homologous recombination is a major pathway in streptomycetes to repair an I-SceI-generated DNA double-strand break. This novel I-SceI-based tool will be useful in fundamental studies on the repair mechanism of DNA double-strand breaks and for a variety of biotechnological applications.
Asunto(s)
Proteínas Bacterianas/metabolismo , Reparación del ADN , Desoxirribonucleasa I/metabolismo , Técnicas Genéticas , Streptomyces/genética , Proteínas Bacterianas/genética , Roturas del ADN de Doble Cadena , Desoxirribonucleasa I/genética , Genes Sintéticos , Mutagénesis , Recombinación Genética , Streptomyces/metabolismoRESUMEN
Analysis of the alpha-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to the identification of five putative regulatory genes, which are congregated into a subcluster. Analysis of the lipReg1-4 and lipX1 showed that they encode components of two-component signal transduction systems (LipReg1 and LipReg2), multiple antibiotics resistance-type regulator (LipReg3), large ATP-binding regulators of the LuxR family-type regulator (LipReg4), and small ribonuclease (LipRegX1), respectively. A combination of targeted gene disruptions, complementation experiments, lipomycin production studies, and gene expression analysis via RT-PCR suggests that all regulatory lip genes are involved in alpha-lipomycin production. On the basis of the obtained data, we propose that LipReg2 controls the activity of LipReg1, which in its turn govern the expression of the alpha-lipomycin pathway-specific regulatory gene lipReg4. The ribonuclease gene lipX1 and the transporter regulator lipReg3 appear to work independently of genes lipReg1, lipReg2, and lipReg4.
Asunto(s)
Antibacterianos/biosíntesis , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Glicósidos/genética , Streptomyces aureofaciens/genética , Antibacterianos/química , Antibacterianos/metabolismo , Mapeo Cromosómico , Clonación Molecular , Escherichia coli/genética , Genes Reguladores , Prueba de Complementación Genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Familia de Multigenes , Mutagénesis , Polienos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Streptomyces aureofaciens/metabolismoRESUMEN
We report here a system for the functional expression of the Flp recombinase in several actinomycetes: Streptomyces coelicolor, S. lividans, and Saccharotrix espanaensis. We have constructed a synthetic gene encoding the Flp recombinase with a GC content of 60.6% optimized for expression in high-GC bacteria. Using the synthetic flp(a) gene, we have removed an apramycin resistance gene flanked by FRT sites from the chromosome of actinomycetes with an efficiency of 40%. Sequencing the region of chromosome showed that excision of the apramycin cassette by Flp recombinase was specific.
Asunto(s)
Actinomycetales/genética , ADN Nucleotidiltransferasas/metabolismo , Farmacorresistencia Microbiana/genética , Ingeniería Genética/métodos , Cromosomas Fúngicos , ADN Nucleotidiltransferasas/genética , Expresión Génica , Genes Sintéticos , Marcadores Genéticos , Genoma Fúngico , Nebramicina/análogos & derivados , Nebramicina/farmacología , Recombinación Genética , Streptomyces coelicolor/genética , Streptomyces lividans/genéticaRESUMEN
Site-specific recombinases revolutionized "in vivo" genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. We have constructed a synthetic gene encoding Cre recombinase with the GC content 67.7% optimized for expression in high-GC bacteria and demonstrated this gene to be functional in Streptomyces lividans. Using the synthetic cre(a) gene, we have removed an apramycin resistance gene flanked by loxP sites from the chromosome of S. lividans with 100% efficiency. Sequencing of the chromosomal DNA part showed that excision of the apramycin cassette by Cre recombinase was specific.
Asunto(s)
Expresión Génica , Ingeniería Genética/métodos , Integrasas/genética , Streptomyces lividans/genética , Clonación Molecular , Integrasas/metabolismo , Plásmidos/genética , Streptomyces lividans/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The soil-borne and marine gram-positive Actinomycetes are a particularly rich source of carbohydrate-containing metabolites. With the advent of molecular tools and recombinant methods applicable to Actinomycetes, it has become feasible to investigate the biosynthesis of glycosylated compounds at genetic and biochemical levels, which has finally set the basis for engineering novel natural product derivatives. Glycosyltransferases (GT) are key enzymes for the biosynthesis of many valuable natural products that contain sugar moieties and they are most important for drug engineering. So far, the direct cloning of unknown glycosyltransferase genes by polymerase chain reaction (PCR) has not been described because glycosyltransferases do not share strongly conserved amino acid regions. In this study, we report a method for cloning of novel so far unidentified glycosyltransferase genes from different Actinomycetes strain. This was achieved by designing primers after a strategy named consensus-degenerate hybrid oligonucleotide primer (CODEHOP). Using this approach, 22 novel glycosyltransferase encoding genes putatively involved in the decoration of polyketides were cloned from the genomes of 10 Actinomycetes. In addition, a phylogenetic analysis of glycosyltransferases from Actinomycetes is shown in this paper.