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CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria


It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products’ roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

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Fig. 1: Chromosomal integration of BGCs mediated through CRAGE.
Fig. 2: Design and architecture of BGC constructs.
Fig. 3: Expression of BGC4 in a panel of phylogenetically diverse chassis strains.
Fig. 4: Expression of BGC7 in a panel of phylogenetically diverse chassis strains.
Fig. 5: Relationship between secondary metabolite production and genetic identity for each representative chassis strain and P. luminescens subsp. laumondii TTO1.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.


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The work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under contract no. DE-AC02-05CH11231. The work performed by the Environmental Molecular Sciences Laboratory, a National Scientific User Facility sponsored by DOE’s Office of Biological and Environmental Research and located at PNNL, is operated by Battelle for the DOE under contract no. DE-AC05-76RL01830. Work in the Bode laboratory was supported by the DFG within the priority programme SPP1617 and the LOEWE Center for Translational Biodiversity Genomics (LOEWE TBG). We thank A. Wahler for professional editing support and P. Jensen for reading and commenting on our manuscript. We thank A. Deutschbauer for providing the pKMW2 plasmid and W.W. Metcalf for providing the E. coli BW29427 strain.

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G.W., Z. Zhao, D.R., R.E., S.D., J.-F.C. and Y.Y. designed and built all CRAGE and BGC constructs. G.W., Z. Zhao, Y.M. and K.C. performed QA/QC of all CRAGE transformation and analysed lux expression. G.W. and J.K. performed and analysed BGC expression and metabolite production in multiple chassis. K.L., S.K., M.D.R., L.S. and T.N. performed LC-HRMS analyses. G.W., J.K., Z. Zhang, Y.E., Y.-M.S., B.B. and L.S. performed both targeted and untargeted metabolite analyses. Y.E., Y.-M.S. and H.B.B. performed structural characterization of metabolites from BGC7. Y.E., Y.-M.S., K.B., D.W.H., N.M.W., C.F., A. Luhrs, A. Lubbe and H.B.B. performed structural characterization of metabolites from BGC5. G.W., Z. Zhao and B.W. extended the utility of CRAGE to α- and β-Proteobacteria and Actinobacteria. B.W. and H.O. tried to extend the utility of CRAGE to Streptomyces sp. G.W., J.K., Y.-M.S., E.M.R., N.J.M., A.V., H.B.B. and Y.Y. wrote the manuscript. H.B.B. and Y.Y. supervised the study.

Corresponding authors

Correspondence to Helge B. Bode or Yasuo Yoshikuni.

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Competing interests

Lawrence Berkeley National Laboratory filed a United States patent application for CRAGE technology (US patent 20190048354). The patent is currently pending. The application lists Y.Y., G.W., Z. Zhao., J.F.C. and D.R. as inventors.

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Supplementary Information

Supplementary Figs. 1–32, Supplementary Tables 8–10, Supplementary Results, Supplementary Dataset legends and Supplementary References.

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Supplementary Tables

Supplementary Tables 1–7 and Supplementary Tables 11–14.

Supplementary Dataset 1

DNA sequence of the pW5Y-Apr plasmid.

Supplementary Dataset 2

DNA sequence of the pW17 plasmid.

Supplementary Dataset 3

DNA sequence of the pW34 plasmid.

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Wang, G., Zhao, Z., Ke, J. et al. CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria. Nat Microbiol 4, 2498–2510 (2019).

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