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Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Its Subsequent Fermentation for Optimized Production

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Metagenomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2555))

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Abstract

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40% of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60%, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC Gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 25 years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article, an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans as a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Stable and efficient marker-less integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR23A-based system will be explained. Finally, a basic protocol for bench-top bioreactor experiments which can form the start in the production process optimization and up-scaling will be provided.

Authors Yuriy Rebets and Jan Kormanec are contributed equally to this work.

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Acknowledgements

The research leading to these results has received funding from the European Commission’s Seventh Framework Program (FP7/2007–2013) under the grant agreement STREPSYNTH (Project No. 613877). In addition, Jan Kormanec was also supported by the Slovak Research and Development Agency under the contracts APVV-19-0009 and DO7RP-0037-12, and by the VEGA grant 2/0026/20 from Slovak Academy of Sciences.

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Authors and Affiliations

  1. Department of Microbiology, Immunology and Transplantation, lab. Molecular Bacteriology, Rega Institute, KU Leuven, Leuven, Belgium

    Jozef Anné

  2. Department of Chemical Engineering, Chemical and Biochemical Reactor Engineering and Safety Division, KU Leuven, Leuven, Belgium

    Kristel Bernaerts

  3. Department of Pharmaceutical Biotechnology, University of Saarland, Saarbrücken, Germany

    Andriy Lutzhetskyy

  4. Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), University of Saarland, Saarbrücken, Germany

    Andriy Lutzhetskyy

  5. Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic

    Jan Kormanec

  6. Explogen LLC, Lviv, Ukraine

    Yuriy Rebets

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Correspondence to Jozef Anné .

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Editors and Affiliations

  1. Department of Microbiology and Biotechnology, University of Hamburg, Hamburg, Germany

    Wolfgang R. Streit

  2. Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Göttingen, Germany

    Rolf Daniel

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Rebets, Y., Kormanec, J., Lutzhetskyy, A., Bernaerts, K., Anné, J. (2023). Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Its Subsequent Fermentation for Optimized Production. In: Streit, W.R., Daniel, R. (eds) Metagenomics. Methods in Molecular Biology, vol 2555. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2795-2_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2795-2_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2794-5

  • Online ISBN: 978-1-0716-2795-2

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