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An efficient system for stable markerless integration of large biosynthetic gene clusters into Streptomyces chromosomes

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Abstract

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage–based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage–based integration plasmid pMTMF. The system was also more stable.

Key points

• Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes

• Efficient heterologous production of MTM in the stable engineered S. lividans strain

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Acknowledgements

We are grateful to Andriy Luzhetskyy (Universitat des Saarlandes, Saarbrucken, Germany) for the cosmid H2-26 and the plasmid pUWLoriT, to Justin Nodwell (McMaster University, Ontario, Canada) for the plasmid pMU1s.

Funding

This work was supported by the Slovak Research and Development Agency under contract nos. APVV-15-0410 and APVV-19-0009, and by VEGA grant 2/0026/20 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, Slovak Academy of Sciences, Dubravska cesta 21, 845 51, Bratislava, Slovak Republic

    Dominika Csolleiova, Renata Knirschova, Bronislava Rezuchova, Dagmar Homerova, Beatrica Sevcikova, Renata Novakova, Lubomira Feckova, Rachel Javorova & Jan Kormanec

  2. Institute of Chemistry, Slovak Academy of Sciences, 845 38, Bratislava, Slovak Republic

    Maria Matulova

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

    Luz Elena Núñez & Jesús Cortés

Authors
  1. Dominika Csolleiova
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  2. Renata Knirschova
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  3. Bronislava Rezuchova
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  4. Dagmar Homerova
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  5. Beatrica Sevcikova
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  6. Maria Matulova
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  7. Luz Elena Núñez
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  8. Renata Novakova
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  9. Lubomira Feckova
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  10. Rachel Javorova
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  11. Jesús Cortés
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  12. Jan Kormanec
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Contributions

JK conceived and designed research. DC, RN, BR, DH, BS, MM, LN, RN, LF, and RJ conducted experiments. LN, JC, JK, MM, DC and BR analyzed data. DC and JK wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Jan Kormanec.

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Csolleiova, D., Knirschova, R., Rezuchova, B. et al. An efficient system for stable markerless integration of large biosynthetic gene clusters into Streptomyces chromosomes. Appl Microbiol Biotechnol 105, 2123–2137 (2021). https://doi.org/10.1007/s00253-021-11161-w

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