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An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA

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Abstract

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

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Acknowledgments

We are grateful to Jacob Bauer for critically reading the manusrtipt and correcting the English. We are also grateful to Dr. Bertold Gust (John Innes Centre, Norwich, UK) for the pIJ773 plasmid.

Funding

This work was supported by the Slovak Research and Development Agency under contract No. APVV-15-0410 and by VEGA grant 2/0002/16 from the Slovak Academy of Sciences. The research leading to these results has received funding from the European Commission’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement STREPSYNTH (project No. 613877). This work was co-funded by the Slovak Research and Development Agency under contract No. DO7RP-0037-12.

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Authors and Affiliations

  1. Institute of Molecular Biology, v.v.i, Slovak Academy of Sciences, Dubravska cesta 21, 845 51, Bratislava, Slovak Republic

    Bronislava Rezuchova, Dagmar Homerova, Beatrica Sevcikova, Renata Novakova, Lubomira Feckova & Jan Kormanec

  2. c/Colegio Santo Domingo de Guzmán, EntreChem SL, Vivero Ciencias de la Salud, 33011, Oviedo, Spain

    Luz Elena Núñez & Jesús Cortés

  3. Institute of Virology Biomedical Research Center, v.v.i, Slovak Academy of Sciences, 845 05, Bratislava, Slovak Republic

    Ludovit Skultety

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  1. Bronislava Rezuchova
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  2. Dagmar Homerova
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  3. Beatrica Sevcikova
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  4. Luz Elena Núñez
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  5. Renata Novakova
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  8. Jesús Cortés
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  9. Jan Kormanec
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Correspondence to Jan Kormanec.

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Rezuchova, B., Homerova, D., Sevcikova, B. et al. An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA. Appl Microbiol Biotechnol 102, 10231–10244 (2018). https://doi.org/10.1007/s00253-018-9393-7

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  • DOI: https://doi.org/10.1007/s00253-018-9393-7

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