Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens

Raynal, Alain; Friedmann, Annick; Tuphile, Karine; Guérineau, Michel; Pernodet, Jean‐Luc

doi:10.1099/00221287-148-1-61

Cited by 13 publications

(11 citation statements)

References 30 publications

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“…In order to clarify the roles of thgH, thgI, thgJ and thgK in the biosynthesis of trehangelin, the four genes were PCR amplified from genomic DNA of P. rubra K07‐0510. The fragment (3.5 kb) containing the four genes was cloned downstream of the ermE* promoter of vector pOSV556t ( oriT, pSAM2‐derived int gene, attP site) . The resulting plasmid pOSV556‐ thgHIJK was introduced into Streptomyces albus J1074 by conjugation, whereupon it integrated at the chromosomal attB site.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of Trehangelin in Polymorphospora rubra K07–0510: Identification of Metabolic Pathway to Angelyl‐CoA

et al. 2016

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Trehangelins are trehalose angelates discovered from endophytic actinomycete Polymorphospora rubra K07-0510. We identified the trehangelin biosynthetic gene cluster, including genes that encode a glycoside hydrolase-like protein (thgC), α-amylase (thgD), 3-ketoacyl-ACP synthase III (thgI), 3-ketoacyl-ACP reductase (thgK), enoyl-CoA hydratase (thgH) and acyl transferase (thgJ). Heterologous expression of thgH, thgI, thgJ and thgK confirmed the importance of these genes in the biosynthesis of trehangelin A. Enzymatic activity studies showed that ThgI catalyses the condensation of acetyl-CoA and methylmalonyl-CoA to 2-methylacetoacetyl-CoA (MAA-CoA), ThgK catalyses NADPH-dependent reduction of MAA-CoA to 3-hydroxy-2-methylbutyryl-CoA (HMB-CoA) and ThgH catalyses the dehydration of HMB-CoA to angelyl-CoA (AN-CoA). This is the first report on the elucidation of the enzymatic formation of AN-CoA.

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Section: Resultsmentioning

confidence: 99%

Biosynthesis of Trehangelin in Polymorphospora rubra K07–0510: Identification of Metabolic Pathway to Angelyl‐CoA

et al. 2016

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“…It has also been shown that a 26-bp att sequence (attB26) centered on the region encoding the anticodon stemloop of the tRNA and not completely included in the identity segment retains the entire functionality of attB (18). The minimal attP site has also been defined (17). Therefore, after site-specific intermolecular recombination between attP and attB26, it is possible to obtain what may be considered the minimal attL and attR sites.…”

Section: Resultsmentioning

confidence: 99%

“…The expression of both int and xis leads to excision via intramolecular recombination between attL and attR. The att sequences required for site-specific recombination have been studied in detail and precisely defined (17,18). Our work aimed to produce an attL-antibiotic resistance geneattR cassette that carries blunt end restriction sites at both extremities.…”

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confidence: 99%

Excisable Cassettes: New Tools for Functional Analysis of Streptomyces Genomes

Raynal

Karray

Tuphile

et al. 2006

Appl Environ Microbiol

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The functional analysis of microbial genomes often requires gene inactivation. We constructed a set of cassettes consisting of single antibiotic resistance genes flanked by the attL and attR sites resulting from site-specific integration of the Streptomyces pSAM2 element. These cassettes can easily be used to inactivate genes by in-frame deletion in Streptomyces by a three-step strategy. In the first step, in Escherichia coli, the cassette is inserted into a cloned copy of the gene to be inactivated. In the second step, the gene is replaced by homologous recombination in Streptomyces, allowing substitution of the wild-type target gene with its inactivated counterpart. In the third step, the cassette can be removed by expression of the pSAM2 genes xis and int. The resulting strains are marker-free and contain an "attB-like" sequence of 33, 34, or 35 bp with no stop codon if the cassette is correctly chosen. Thus, a gene can be disrupted by creating an in-frame deletion, avoiding polar effects if downstream genes are cotranscribed with the target gene. A set of cassettes was constructed to contain a hygromycin or gentamicin resistance gene flanked by the attL and attR sites. The initial constructions carrying convenient cloning sites allow the insertion of any other marker gene. We tested insertion and excision by inserting a cassette into orf3, the third gene of an operon involved in spiramycin biosynthesis. We verified that the cassette exerted a polar effect on the transcription of downstream genes but that, after excision, complementation with orf3 alone restored spiramycin production.

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“…In addition, it can integrate into the Streptomyces chromosome through site-specific recombination between the pSAM2 site within the vector and the corresponding site of the chromosome. This recombination reaction is mediated by the pSAM2 integrase encoded within pOSV556t (Raynal, Friedmann, Tuphile, Guerineau, & Pernodet, 2002). In some Streptomyces strains, DNA inserted into such phage attachment sites is stable and no antibiotic selection is therefore needed to maintain a construct once it is integrated into the chromosome, whereas in other strains, DNA inserted into these sites is not stable and constant antibiotic selection is required to maintain the insert.…”

Section: Preparation Of Gene Expression Constructsmentioning

confidence: 99%