A novel Streptomyces spp. integration vector derived from the S. venezuelaephage, SV1

Fayed, Bahgat; Younger, Ellen; Taylor, Gabrielle; Smith, Margaret C. M.

doi:10.1186/1472-6750-14-51

Cited by 32 publications

(25 citation statements)

References 24 publications

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“…The sequences of attB sites recognised by a variety of integrases (ϕC31(34), ϕJoe(35), Bxbl(36), R4(32), SPBc(37), SV1(38), TG1(29) and TP901(39)) were used in BLAST searches of the genome sequence of Amycolatopsis marina DSM45569 (NCBI Genome Database NZ_FOKG00000000) (Table 2). The most significant hits for R4 and TG1 attB sites had the highest identities and lowest E-value.…”

Section: Resultsmentioning

confidence: 99%

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

Gao

Murugesan

Hoßbach

et al. 2018

Preprint

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Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify integrating vectors, derived using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from E. coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. The presence of homologous TG1 and R4 attB sites in other species of this genus indicates that vectors based on TG1 and R4 integrases could be widely applicable.ImportanceRare Actinomycetes have the same potential of natural product discovery as Streptomyces, but the potential has not been fully explored due to the lack of efficient molecular biology tools. In this study, we identified two serine integrases, TG1 and R4, which could be used in the rare Actinomycetes species, Amycolatopsis marina, as tools for genome integration. The high level of conservation between the attB sites for TG1 and R4 in a number of Amycolatopsis species suggested that plasmids with the integration systems from these phages should be widely useful in this genus.

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Section: Resultsmentioning

confidence: 99%

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

Gao

Murugesan

Hoßbach

et al. 2018

Preprint

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“…45 Innate attB sites are less conserved in rare actinomycetes but can be introduced prior to integration. 45 In addition, there are similar TG1, 46 R4, 47 and SV1 48 systems involving large serine recombinases that catalyze recombination between attP with bacterial attB sites. Tyrosine integrases such as the cre/ loxP and Flp/ FRT systems have also been reconstituted in actinomycetes.…”

Section: Advances In Genetic Engineering Of Actinomycetesmentioning

confidence: 99%

Engineering microbial hosts for production of bacterial natural products

et al. 2016

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Microbial fermentation provides as an attractive alternative to chemical synthesis for the production of structurally complex natural products. In most cases, however, production titers are low and need to be improved for compound characterization and/or commercial production. Owing to advances in functional genomics and genetic engineering technologies, microbial hosts can be engineered to overproduce a desired natural product, greatly accelerating the traditionally time-consuming strain improvement process. This review covers recent developments and challenges in the engineering of native and heterologous microbial hosts for production of bacterial natural products, focusing on the genetic tools and strategies for strain improvement. Special emphasis is placed on bioactive secondary metabolites from actinomycetes. The considerations for the choice of host systems will also be discussed in this review.

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“…The alternatives to the ΦC31 integrative site in Streptomyces are the ΦBT1 (Gregory et al 2003), TG1 (Morita et al 2009) and SV1 (Fayed et al 2014) integrative sites. The ΦBT1 derived vectors integrate into the attP site localized into the SCO4848 gene (S. coelicolor genome) (Gregory et al 2003), while the TG1 plasmids integrate at SCO3658 (Fayed et al 2014) and SV1 plasmids at SCO4383 (Fayed et al 2014).…”

Section: Introductionmentioning

confidence: 99%

“…The ΦBT1 derived vectors integrate into the attP site localized into the SCO4848 gene (S. coelicolor genome) (Gregory et al 2003), while the TG1 plasmids integrate at SCO3658 (Fayed et al 2014) and SV1 plasmids at SCO4383 (Fayed et al 2014). To Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7271-0) contains supplementary material, which is available to authorized users.…”

Section: Introductionmentioning

confidence: 99%

New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces

Gonzalez-Quiñonez

López-García

Yagüe

et al. 2016

Appl Microbiol Biotechnol

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Integrative plasmids are one of the best options to introduce genes in low copy and in a stable form into bacteria. The ΦC31-derived plasmids constitute the most common integrative vectors used in Streptomyces. They integrate at different positions (attB and pseudo-attB sites) generating different mutations. The less common ΦBT1-derived vectors integrate at the unique attB site localized in the SCO4848 gene (S. coelicolor genome) or their orthologues in other streptomycetes. This work demonstrates that disruption of SCO4848 generates a delay in spore germination. SCO4848 is cotranscribed with SCO4849, and the spore germination phenotype is complemented by SCO4849. Plasmids pNG1-4 were created by modifying the ΦBT1 integrative vector pMS82 by introducing a copy of SCO4849 under the control of the promoter region of SCO4848. pNG2 and pNG4 also included a copy of the P ermE * in order to facilitate gene overexpression. pNG3 and pNG4 harboured a copy of the bla gene (ampicillin resistance) to facilitate selection in E. coli. pNG1-4 are the only integrative vectors designed to produce a neutral phenotype when they are integrated into the Streptomyces genome. The experimental approach developed in this work can be applied to create phenotypically neutral integrative plasmids in other bacteria.

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A novel Streptomyces spp. integration vector derived from the S. venezuelaephage, SV1

Cited by 32 publications

References 24 publications

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

Engineering microbial hosts for production of bacterial natural products

New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces

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