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CRISPR–Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters

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Abstract

Here we report an efficient CRISPR–Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

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Figure 1: CRISPR–Cas9-based promoter knock-in strategy to activate silent biosynthetic gene clusters in streptomycetes.
Figure 2: Activation of biosynthetic gene clusters in multiple streptomycetes.
Figure 3: Activation of type II PKS biosynthetic gene cluster in S. viridochromogenes yields a novel pigmented compound.

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Acknowledgements

We gratefully acknowledge financial support from the National Institutes of Health (GM077596) (H.Z.), the A*STAR Visiting Investigator Program (H.Z.), and the National Research Foundation, Singapore (NRF2013-THE001-094) (M.M.Z., F.T.W., Y.H.L., E.L.A. and H.Z.). NMR data collection at the UIUC IGB Core was funded by NIH (S10-RR028833). Conjugative donor strains of E. coli were gifted by Prof. William Metcalf at UIUC. We thank members of the Zhao laboratory in UIUC, co-workers in MEL and MERL in A*STAR for constructive comments, Dr. Xudong Guan and Dr. Lingyang Zhu from UIUC for assisting with NMR data acquisition, and Dr. Ying Swan Ho from BTI, A*STAR for HRMS data acquisition.

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Author notes

  1. Mingzi M Zhang and Fong Tian Wong: These authors contributed equally to this work.

Authors and Affiliations

  1. Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology, and Research (A*STAR), Singapore

    Mingzi M Zhang, Wan Lin Yeo, Ee Lui Ang & Huimin Zhao

  2. Molecular Engineering Lab, Biomedical Science Institutes, A*STAR, Singapore

    Fong Tian Wong & Elena Heng

  3. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Yajie Wang, Shangwen Luo, Ryan E Cobb, Behnam Enghiad & Huimin Zhao

  4. Organic Chemistry, Institute of Chemical and Engineering Sciences, A*STAR, Singapore

    Yee Hwee Lim

Authors
  1. Mingzi M Zhang
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  2. Fong Tian Wong
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  3. Yajie Wang
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  4. Shangwen Luo
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  5. Yee Hwee Lim
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  6. Elena Heng
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  7. Wan Lin Yeo
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  8. Ryan E Cobb
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  10. Ee Lui Ang
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  11. Huimin Zhao
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Contributions

M.M.Z., F.T.W., and H.Z. conceived and designed the research. M.M.Z., F.T.W., Y.W., E.H., W.L.Y., R.E.C., and B.E. performed the molecular biology, conjugation and fermentation experiments. S.L. and Y.H.L. performed structure elucidation of compounds. M.M.Z., F.T.W., E.L.A., and H.Z. analyzed the data. M.M.Z., F.T.W., and H.Z. wrote the manuscript.

Corresponding author

Correspondence to Huimin Zhao.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Tables 1–8, Supplementary Figure 1–17 and Supplementary Note (PDF 3726 kb)

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Zhang, M., Wong, F., Wang, Y. et al. CRISPR–Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters. Nat Chem Biol 13, 607–609 (2017). https://doi.org/10.1038/nchembio.2341

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