Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina

Gao, Hong; Murugesan, Buvani; Hoßbach, Janina; Evans, Stephanie K.; Stark, W. Marshall; Smith, Margaret C. M.

doi:10.1186/s12896-019-0521-y

Cited by 4 publications

(4 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Plasmids pHG4 [23], pHG5, pHG6 and pHG7 are all integrating vectors encoding the int/attP loci from TG1, SV1, φBT1 and φC31, respectively (). These four plasmids also encode compatible antibiotic resistance loci; apramycin resistance ( aac(3)IV , pHG4), kanamycin resistance ( aphII , pHG5), erythromycin resistance ( ermE , pHG6) and hygromycin resistance ( hygB , pHG7).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Gao

Smith

2021

Access Microbiology

Self Cite

View full text Add to dashboard Cite

Some major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces recipients via conjugal transfer and integration. Pairwise combinations of Escherichia coli donors containing vectors encoding orthogonal serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1 or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into Streptomyces coelicolor and Streptomyces lividans at moderate frequencies after conjugation. This work demonstrated how site-specific recombination based on orthogonal serine integrases can save researchers time in genome engineering experiments in Streptomyces .

show abstract

Section: Methodsmentioning

confidence: 99%

“…Plasmids pHG4 [ 23 ], pHG5, pHG6 and pHG7 are all integrating vectors encoding the int/attP loci from TG1, SV1, φBT1 and φC31, respectively ( Fig. 1 ).…”

Section: Methodsmentioning

confidence: 99%

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Gao

Smith

2021

Access Microbiology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Despite the importance of the genus Amycolatopsis, the development of adapted genetic tools has lagged behind that for other actinobacterial genera such as Streptomyces [8,9]. For some time, the introduction of DNA into Amycolatopsis has been a limiting factor, until this problem could be circumvented by the development of a method of direct transformation of Amycolatopsis mycelia [10] or by using intergeneric conjugation from Escherichia coli to introduce single-stranded DNA into the Amycolatopsis host [11,12].…”

Section: Introductionmentioning

confidence: 99%

Marker-Free Genome Engineering in Amycolatopsis Using the pSAM2 Site-Specific Recombination System

et al. 2022

View full text Add to dashboard Cite

Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis.

show abstract

“…Despite the importance of the genus Amycolatopsis, the development of adapted genetic tools has lagged behind that for other actinobacterial genera such as Streptomyces (8,9).…”

Section: Introductionmentioning

confidence: 99%

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

Santos

Caraty-Philippe

Darbon

et al. 2021

Preprint

View full text Add to dashboard Cite

Actinobacteria belonging to the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as biodegradation or bioconversion. Despite their industrial importance, tools and methods for the genetic manipulation of Amycolatopsis are less developed than in other actinobacteria such as Streptomyces. Moreover, most of the existing methods do not support convenient marker-free genome engineering. Here, we report the use of the pSAM2 site-specific recombination system for the efficient deletion of marker genes or large DNA regions in Amycolatopsis. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the Xis and Int proteins from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the recombination sites attL and attR on each side of a region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei DSM 40773 by obtaining with high efficiency (>95%) a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster.

show abstract

Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina

Cited by 4 publications

References 54 publications

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Marker-Free Genome Engineering in Amycolatopsis Using the pSAM2 Site-Specific Recombination System

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

Contact Info

Product

Resources

About