Sonication-Dependent Electroporation of the Erythromycin-Producing Bacterium
            <i>Saccharopolyspora erythraea</i>

Fitzgerald, Nancy B.; English, Robert S.; Lampel, J S; Boom, Thomas J. Vanden

doi:10.1128/aem.64.4.1580-1583.1998

Cited by 22 publications

(4 citation statements)

References 11 publications

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“…While Nonomuraea is highly sensitive to glycine (600 μg ml −1 is sufficient to retard growth in RARE3 medium), several attempts to prepare protoplasts were unsuccessful (S. Azimonti, unpublished results). Similar lack of success was encountered in the attempts to adapt to Nonomuraea the direct transformation [4–6] or electroporation [1,8–9] systems described for other actinomycetes.…”

Section: Resultsmentioning

confidence: 69%

“…However, conditions for protoplast formation and regeneration have to be empirically determined and this approach has met with limited success with non‐ Streptomyces strains. Alternative systems have been developed for other actinomycete genera, and gene transfer systems based on direct transformation or electroporation are now available for Amycolatopsis , Actinomadura and Rhodococcus [4–9]. Intergeneric transfer of plasmid DNA from Escherichia coli to Streptomyces spp.…”

Section: Introductionmentioning

confidence: 99%

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A gene transfer system for the glycopeptide producerNonomuraeasp. ATCC39727

Stinchi¹,

Azimonti²,

Donadio³

et al. 2003

FEMS Microbiology Letters

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The filamentous actinomycete Nonomuraea sp. ATCC39727 produces the industrially important glycopeptide antibiotic A40926. We developed a gene transfer system based on intergeneric conjugation from Escherichia coli. Analysis of the ex-conjugants revealed that the incoming plasmid pSET152 had integrated at two sites in the Nonomuraea genome. One of these was characterized and found to be highly related to other PhiC31 attB sites described in Streptomyces spp., including the core TTS sequence, where crossover occurs. Surprisingly, pSET152 was also found in episomic form in the Nonomuraea ex-conjugants.

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Section: Resultsmentioning

confidence: 69%

Section: Introductionmentioning

confidence: 99%

A gene transfer system for the glycopeptide producerNonomuraeasp. ATCC39727

Stinchi¹,

Azimonti²,

Donadio³

et al. 2003

FEMS Microbiology Letters

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“…Actinoplanes garbadinensis ATCC31049 was maintained by growing on medium ABB13 (Fitzgerald et al, 1998) at 30°C. S. lividans 1326, Escherichia coli BW2113/pIJ790 and E. coli ET12567/pUZ8002 were kindly supplied by Professor Mervyn Bibb (John Innes Centre, Norwich, UK) and manipulated as previously described (Kieser et al, 2000;Gust et al, 2004).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Organization of the genes encoding the biosynthesis of actagardine and engineering of a variant generation system

Boakes¹,

Cortés

Appleyard

et al. 2009

Molecular Microbiology

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SummaryThe biosynthetic pathway of the type B lantibiotic actagardine (formerly gardimycin), produced by Actinoplanes garbadinensis ATCC31049, has been cloned, sequenced and annotated. The gene cluster contains the gene garA that encodes the actagardine prepropeptide, a modification gene garM, involved in the dehydration and cyclization of the prepeptide, several putative transporter and regulatory genes as well as a novel luciferase-like monooxygenase gene designated garO. Expression of these genes in Streptomyces lividans resulted in the production of ala(0)-actagardine while deletion of the garA gene from A. garbadinensis generated a strain incapable of producing actagardine. Actagardine production was successfully restored however, by the delivery of the plasmid pAGvarX. This plasmid contains an engineered cassette of the actagardine encoding gene garA and offers an alternative route to generating extensive libraries of actagardine variants. Using this plasmid, an alanine scanning library has been constructed and the mutants analysed. Further modifications include the removal of the novel garO gene from A. garbadinensis. Deletion of this gene resulted in the production of deoxy variants of actagardine, demonstrating that the formation of the sulfoxide group is enzyme catalysed and not a spontaneous chemical modification as previously believed.

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“…Ultrasound pre-treatment prior to electrotransformation was used previously e.g., for Saccharopolyspora erythraea [ 38 ] or Streptomyces spp. [ 39 ].…”

Section: Discussionmentioning

confidence: 99%

Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation

Kolek

Sedlář

Provazník

et al. 2016

Biotechnol Biofuels

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BackgroundButanol is currently one of the most discussed biofuels. Its use provides many benefits in comparison to bio-ethanol, but the price of its fermentative production is still high. Genetic improvements could help solve many problems associated with butanol production during ABE fermentation, such as its toxicity, low concentration achievable in the cultivation medium, the need for a relatively expensive substrate, and many more. Clostridium pasteurianum NRRL B-598 is non-type strain producing butanol, acetone, and a negligible amount of ethanol. Its main benefits are high oxygen tolerance, utilization of a wide range of carbon and nitrogen sources, and the availability of its whole genome sequence. However, there is no established method for the transfer of foreign DNA into this strain; this is the next step necessary for progress in its use for butanol production.ResultsWe have described functional protocols for conjugation and transformation of the bio-butanol producer C. pasteurianum NRRL B-598 by foreign plasmid DNA. We show that the use of unmethylated plasmid DNA is necessary for efficient transformation or successful conjugation. Genes encoding DNA methylation and those for restriction-modification systems and antibiotic resistance were searched for in the whole genome sequence and their homologies with other clostridial bacteria were determined. Furthermore, activity of described novel type I restriction system was proved experimentally. The described electrotransformation protocol achieved an efficiency 1.2 × 102 cfu/μg DNA after step-by-step optimization and an efficiency of 1.6 × 102 cfu/μg DNA was achieved by the sonoporation technique using a standard laboratory ultrasound bath. The highest transformation efficiency was achieved using a combination of these approaches; sono/electroporation led to an increase in transformation efficiency, to 5.3 × 102 cfu/μg DNA.ConclusionsBoth Dam and Dcm methylations are detrimental for transformation of C. pasteurianum NRRL B-598. Methods for conjugation, electroporation, sonoporation, and a combined method for sono/electroporation were established for this strain. The methods described could be used for genetic improvement of this strain, which is suitable for bio-butanol production.

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Sonication-Dependent Electroporation of the Erythromycin-Producing Bacterium Saccharopolyspora erythraea

Cited by 22 publications

References 11 publications

A gene transfer system for the glycopeptide producerNonomuraeasp. ATCC39727

A gene transfer system for the glycopeptide producerNonomuraeasp. ATCC39727

Organization of the genes encoding the biosynthesis of actagardine and engineering of a variant generation system

Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation

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