Use of an inducible promoter for antibiotic production in a heterologous host

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this op...

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Published inApplied microbiology and biotechnology Vol. 87; no. 1; pp. 261 - 269
Main Authors Dangel, Volker, Westrich, Lucia, Smith, Margaret C. M, Heide, Lutz, Gust, Bertolt
Format Journal Article
LanguageEnglish
Published Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.06.2010
Springer-Verlag
Springer
Springer Nature B.V
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Abstract The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2 mg L⁻¹ of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25-2.0 mg L⁻¹). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.
AbstractList The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2 mg L^sup -1^ of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25-2.0 mg L^sup -1^). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.[PUBLICATION ABSTRACT]
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2 mg L⁻¹ of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25-2.0 mg L⁻¹). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2mgL super(-1) of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25-2.0mgL super(-1)). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2 mg L −1 of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25–2.0 mg L −1 ). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2 mg L(-1) of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25-2.0 mg L(-1)). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.
Author Gust, Bertolt
Westrich, Lucia
Heide, Lutz
Dangel, Volker
Smith, Margaret C. M
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IsPeerReviewed true
IsScholarly true
Issue 1
Keywords Streptomyces
Genetic engineering
Novobiocin
Inducible promoter
Actinomycetales
Antibiotic
Streptomycetaceae
Production
Bacteria
Actinomycetes
Language English
License CC BY 4.0
http://www.springer.com/tdm
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PublicationTitle Applied microbiology and biotechnology
PublicationTitleAbbrev Appl Microbiol Biotechnol
PublicationTitleAlternate Appl Microbiol Biotechnol
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  year: 2003
  ident: 2435_CR23
  publication-title: Current Topics in Medicinal Chemistry
  doi: 10.2174/1568026033452500
  contributor:
    fullname: A Maxwell
– volume: 151
  start-page: 1949
  year: 2005
  ident: 2435_CR11
  publication-title: Microbiology
  doi: 10.1099/mic.0.27669-0
  contributor:
    fullname: AS Eustáquio
– volume: 33
  start-page: e87
  year: 2005
  ident: 2435_CR26
  publication-title: Nucleic Acids Res
  doi: 10.1093/nar/gni086
  contributor:
    fullname: A Rodriguez-Garcia
SSID ssj0012866
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Snippet The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for...
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SubjectTerms Anti-Bacterial Agents - biosynthesis
Antibiotics
Applied Genetics and Molecular Biotechnology
Binding sites
Biological and medical sciences
Biomedical and Life Sciences
Biosynthesis
Biotechnology
Chemical reactions
E coli
Fundamental and applied biological sciences. Psychology
Genes
Genetic Engineering
Inducible promoter
Life Sciences
Medical research
Microbial Genetics and Genomics
Microbiology
novobiocin
Novobiocin - biosynthesis
Plasmids
Promoter Regions, Genetic
Ribonucleic acid
RNA
Streptococcus infections
Streptomyces
Streptomyces coelicolor
Streptomyces coelicolor - genetics
Streptomyces coelicolor - metabolism
Studies
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Title Use of an inducible promoter for antibiotic production in a heterologous host
URI https://link.springer.com/article/10.1007/s00253-009-2435-4
https://www.ncbi.nlm.nih.gov/pubmed/20127238
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