Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The data that support the findings of this study are available from the corresponding author upon request.
References
Donia, M. S. & Fischbach, M. A. Human microbiota. Small molecules from the human microbiota. Science 349, 1254766 (2015).
Keller, N. P. Translating biosynthetic gene clusters into fungal armor and weaponry. Nat. Chem. Biol. 11, 671–677 (2015).
Vorholt, J. A. Microbial life in the phyllosphere. Nat. Rev. Microbiol. 10, 828–840 (2012).
Waters, C. M. & Bassler, B. L. Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346 (2005).
Blacher, E., Levy, M., Tatirovsky, E. & Elinav, E. Microbiome-modulated metabolites at the interface of host immunity. J. Immunol. 198, 572–580 (2017).
Pusztahelyi, T., Holb, I. J. & Pocsi, I. Secondary metabolites in fungus–plant interactions. Front. Plant Sci. 6, 573 (2015).
Kim, E., Moore, B. S. & Yoon, Y. J. Reinvigorating natural product combinatorial biosynthesis with synthetic biology. Nat. Chem. Biol. 11, 649–659 (2015).
Smanski, M. J. et al. Synthetic biology to access and expand nature’s chemical diversity. Nat. Rev. Microbiol. 14, 135–149 (2016).
Weber, T. et al. antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 43, W237–W243 (2015).
Skinnider, M. A. et al. Genomic charting of ribosomally synthesized natural product chemical space facilitates targeted mining. Proc. Natl Acad. Sci. USA 113, E6343–E6351 (2016).
Mukherjee, S. et al. 1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life. Nat. Biotechnol. 35, 676–683 (2017).
Hadjithomas, M. et al. IMG-ABC: a knowledge base to fuel discovery of biosynthetic gene clusters and novel secondary metabolites. mBio 6, e00932 (2015).
Rutledge, P. J. & Challis, G. L. Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nat. Rev. Microbiol. 13, 509–523 (2015).
Cohen, L. J. et al. Commensal bacteria make GPCR ligands that mimic human signalling molecules. Nature 549, 48–53 (2017).
Guo, C. J. et al. Discovery of reactive microbiota-derived metabolites that inhibit host proteases. Cell 168, 517–526 (2017).
Shao, Z. et al. Refactoring the silent spectinabilin gene cluster using a plug-and-play scaffold. ACS Synth. Biol. 2, 662–669 (2013).
Wenzel, S. C. & Muller, R. Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways. Curr. Opin. Biotechnol. 16, 594–606 (2005).
Fu, J. et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat. Biotechnol. 30, 440–446 (2012).
Yoshikuni, Y., Ferrin, T. E. & Keasling, J. D. Designed divergent evolution of enzyme function. Nature 440, 1078–1082 (2006).
Yoshikuni, Y., Dietrich, J. A., Nowroozi, F. F., Babbitt, P. C. & Keasling, J. D. Redesigning enzymes based on adaptive evolution for optimal function in synthetic metabolic pathways. Chem. Biol. 15, 607–618 (2008).
Ziemert, N. et al. Diversity and evolution of secondary metabolism in the marine actinomycete genus Salinispora. Proc. Natl Acad. Sci. USA 111, E1130–E1139 (2014).
Jensen, P. R. Natural products and the gene cluster revolution. Trends Microbiol. 24, 968–977 (2016).
Soucy, S. M., Huang, J. & Gogarten, J. P. Horizontal gene transfer: building the web of life. Nat. Rev. Genet. 16, 472–482 (2015).
Williams, K. P. et al. Phylogeny of Gammaproteobacteria. J. Bacteriol. 192, 2305–2314 (2010).
McDonald, B. R. & Currie, C. R. Lateral gene transfer dynamics in the ancient bacterial genus Streptomyces. mBio 8, e00644–12 (2017).
Williams, D., Gogarten, J. P. & Papke, R. T. Quantifying homologous replacement of loci between Haloarchaeal species. Genome Biol. Evol. 4, 1223–1244 (2012).
Engel, Y., Windhorst, C., Lu, X., Goodrich-Blair, H. & Bode, H. B. The global regulators Lrp, LeuO and HexA control secondary metabolism in entomopathogenic bacteria. Front. Microbiol. 8, 209 (2017).
Iqbal, H. A., Low-Beinart, L., Obiajulu, J. U. & Brady, S. F. Natural product discovery through improved functional metagenomics in Streptomyces. J. Am. Chem. Soc. 138, 9341–9344 (2016).
Nah, H. J., Pyeon, H. R., Kang, S. H., Choi, S. S. & Kim, E. S. Cloning and heterologous expression of a large-sized natural product biosynthetic gene cluster in Streptomyces species. Front. Microbiol. 8, 394 (2017).
Bierman, M. et al. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116, 43–49 (1992).
Gregory, M. A., Till, R. & Smith, M. C. Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors. J. Bacteriol. 185, 5320–5323 (2003).
Brophy, J. A. N. et al. Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria. Nat. Microbiol. 3, 1043–1053 (2018).
Wozniak, R. A. & Waldor, M. K. Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow. Nat. Rev. Microbiol. 8, 552–563 (2010).
Santos, C. N., Regitsky, D. D. & Yoshikuni, Y. Implementation of stable and complex biological systems through recombinase-assisted genome engineering. Nat. Commun. 4, 2503 (2013).
Santos, C. N. & Yoshikuni, Y. Engineering complex biological systems in bacteria through recombinase-assisted genome engineering. Nat. Protoc. 9, 1320–1336 (2014).
Lampe, D. J., Akerley, B. J., Rubin, E. J., Mekalanos, J. J. & Robertson, H. M. Hyperactive transposase mutants of the Himar1 mariner transposon. Proc. Natl Acad. Sci. USA 96, 11428–11433 (1999).
Hickman, A. B., Chandler, M. & Dyda, F. Integrating prokaryotes and eukaryotes: DNA transposases in light of structure. Crit. Rev. Biochem. Mol. Biol. 45, 50–69 (2010).
Dubendorff, J. W. & Studier, F. W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45–59 (1991).
Bilyk, B., Horbal, L. & Luzhetskyy, A. Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074. Micro. Cell Fact. 16, 5 (2017).
Englaender, J. A. et al. Effect of genomic integration location on heterologous protein expression and metabolic engineering in E. coli. ACS Synth. Biol. 6, 710–720 (2017).
Sousa, C., de Lorenzo, V. & Cebolla, A. Modulation of gene expression through chromosomal positioning in Escherichia coli. Microbiology 143, 2071–2078 (1997).
Moriguchi, K., Yamamoto, S., Ohmine, Y. & Suzuki, K. A fast and practical yeast transformation method mediated by Escherichia coli based on a trans-kingdom conjugal transfer system: just mix two cultures and wait one hour. PLoS ONE 11, e0148989 (2016).
Frost, L. S. in Encyclopedia of Microbiology 3rd edn (Ed. Baldauf, S. L. et al.) 517–531 (2009).
Trieu-Cuot, P., Carlier, C., Martin, P. & Courvalin, P. Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria. FEMS Microbiol. Lett. 48, 289–294 (1987).
Forst, S., Dowds, B., Boemare, N. & Stackebrandt, E. Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51, 47–72 (1997).
Somvanshi, V. S. et al. A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states. Science 337, 88–93 (2012).
Stock, S. P., Kusakabe, A. & Orozco, R. A. Secondary metabolites produced by Heterorhabditis symbionts and their application in agriculture: what we know and what to do next. J. Nematol. 49, 373–383 (2017).
Bode, H. B. Entomopathogenic bacteria as a source of secondary metabolites. Curr. Opin. Chem. Biol. 13, 224–230 (2009).
Tobias, N. J. et al. Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus. Nat. Microbiol. 2, 1676–1685 (2017).
Cai, X. et al. Entomopathogenic bacteria use multiple mechanisms for bioactive peptide library design. Nat. Chem. 9, 379–386 (2017).
Duchaud, E. et al. The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens. Nat. Biotechnol. 21, 1307–1313 (2003).
Dudnik, A., Bigler, L. & Dudler, R. Heterologous expression of a Photorhabdus luminescens syrbactin-like gene cluster results in production of the potent proteasome inhibitor glidobactin A. Microbiol. Res. 168, 73–76 (2013).
Bian, X., Plaza, A., Zhang, Y. & Muller, R. Luminmycins A–C, cryptic natural products from Photorhabdus luminescens identified by heterologous expression in Escherichia coli. J. Nat. Prod. 75, 1652–1655 (2012).
Schimming, O., Fleischhacker, F., Nollmann, F. I. & Bode, H. B. Yeast homologous recombination cloning leading to the novel peptides ambactin and xenolindicin. Chembiochem 15, 1290–1294 (2014).
Nollmann, F. I. et al. Insect-specific production of new GameXPeptides in Photorhabdus luminescens TTO1, widespread natural products in entomopathogenic bacteria. Chembiochem 16, 205–208 (2015).
Bian, X., Plaza, A., Yan, F., Zhang, Y. & Muller, R. Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo. Biotechnol. Bioeng. 112, 1343–1353 (2015).
Bode, E. et al. Simple ‘on-demand’ production of bioactive natural products. Chembiochem 16, 1115–1119 (2015).
Bode, H. B. et al. Structure elucidation and activity of kolossin A, the d-/l-pentadecapeptide product of a giant nonribosomal peptide synthetase. Angew. Chem. Int. Ed. 54, 10352–10355 (2015).
Lambalot, R. H. et al. A new enzyme superfamily—the phosphopantetheinyl transferases. Chem. Biol. 3, 923–936 (1996).
Winson, M. K. et al. Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs. FEMS Microbiol. Lett. 163, 193–202 (1998).
Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E. & Khosla, C. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291, 1790–1792 (2001).
Stein, M. L. et al. One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor. Proc. Natl Acad. Sci. USA 109, 18367–18371 (2012).
Theodore, C. M., King, J. B., You, J. & Cichewicz, R. H. Production of cytotoxic glidobactins/luminmycins by Photorhabdus asymbiotica in liquid media and live crickets. J. Nat. Prod. 75, 2007–2011 (2012).
DeFelice, B. C. et al. Mass spectral feature list optimizer (MS-FLO): a tool to minimize false positive peak reports in untargeted liquid chromatography-mass spectroscopy (LC-MS) data processing. Anal. Chem. 89, 3250–3255 (2017).
Myers, O. D., Sumner, S. J., Li, S., Barnes, S. & Du, X. One step forward for reducing false positive and false negative compound identifications from mass spectrometry metabolomics data: new algorithms for constructing extracted ion chromatograms and detecting chromatographic peaks. Anal. Chem. 89, 8696–8703 (2017).
Clevenger, K. D. et al. A scalable platform to identify fungal secondary metabolites and their gene clusters. Nat. Chem. Biol. 13, 895–901 (2017).
Zhao, L., Cai, X., Kaiser, M. & Bode, H. B. Methionine-containing rhabdopeptide/xenortide-like peptides from heterologous expression of the biosynthetic gene cluster kj12ABC in Escherichia coli. J. Nat. Prod. 81, 2292–2295 (2018).
Harding, C. R., Schroeder, G. N., Collins, J. W. & Frankel, G. Use of Galleria mellonella as a model organism to study Legionella pneumophila infection. J. Vis. Exp. 2013, e50964 (2013).
Louwerse, J. D. et al. Stable recombinase-mediated cassette exchange in arabidopsis using Agrobacterium tumefaciens. Plant Physiol. 145, 1282–1293 (2007).
Glaser, S., Anastassiadis, K. & Stewart, A. F. Current issues in mouse genome engineering. Nat. Genet. 37, 1187–1193 (2005).
Xu, Z. et al. Large-scale transposition mutagenesis of Streptomyces coelicolor identifies hundreds of genes influencing antibiotic biosynthesi. Appl. Environ. Microbiol. 83, e02889-16 (2017).
Suzuki, H., Takahashi, S., Osada, H. & Yoshida, K. Improvement of transformation efficiency by strategic circumvention of restriction barriers in Streptomyces griseus. J. Microbiol. Biotechnol. 21, 675–678 (2011).
Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E. & Khosla, C. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291, 1790–1792 (2001).
Wetmore, K. M. et al. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio 6, e00306–e00315 (2015).
Anastassiadis, K. et al. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis. Model. Mech. 2, 508–515 (2009).
Salis, H. M. The ribosome binding site calculator. Method Enzym. 498, 19–42 (2011).
Kouprina, N. & Larionov, V. Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology. Chromosoma 125, 621–632 (2016).
Wargacki, A. J. et al. An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335, 308–313 (2012).
Clasquin, M. F., Melamud, E. & Rabinowitz, J. D. LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine. Curr. Protoc. Bioinformatics 14, 14.11.1–14.11.23 (2012).
Pluskal, T., Castillo, S., Villar-Briones, A. & Oresic, M. MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics 11, 395 (2010).
Wesche, F. et al. Combined approach of backbone amide linking and on-resin N-methylation for the synthesis of bioactive and metabolically stable peptides. J. Med. Chem. 61, 3930–3938 (2018).
Nollmann, F. I. et al. Synthesis of szentiamide, a depsipeptide from entomopathogenic Xenorhabdus szentirmaii with activity against Plasmodium falciparum. Beilstein J. Org. Chem. 8, 528–533 (2012).
Bodenhausen, G. & Ruben, D. J. Natural abundance N-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett. 69, 185–189 (1980).
Bax, A. & Summers, M. F. 1H and 13C assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc. 108, 2093–2094 (1986).
Braunschweiler, L. & Ernst, R. R. Coherence transfer by isotropic mixing—application to proton correlation spectroscopy. J. Magn. Reson. 53, 521–528 (1983).
Lerner, L. & Bax, A. Sensitivity-enhanced two-dimensional heteronuclear relayed coherence transfer NMR-spectroscopy. J. Magn. Reson. 69, 375–380 (1986).
Rance, M. et al. Improved spectral resolution in Cosy H-1-NMR spectra of proteins via double quantum filtering. Biochem. Biophys. Res. Commun. 117, 479–485 (1983).
Delaglio, F. et al. NMRpipe—a multidimensional spectral processing system based on unix pipes. J. Biomol. NMR 6, 277–293 (1995).
Acknowledgements
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.
Author information
Authors and Affiliations
Contributions
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
Ethics declarations
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.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Information
Supplementary Figs. 1–32, Supplementary Tables 8–10, Supplementary Results, Supplementary Dataset legends and Supplementary References.
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.
Rights and permissions
About this article
Cite this article
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). https://doi.org/10.1038/s41564-019-0573-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41564-019-0573-8
This article is cited by
-
Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling
Microbial Cell Factories (2024)
-
Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts
Nature Communications (2024)
-
Exploring a general multi-pronged activation strategy for natural product discovery in Actinomycetes
Communications Biology (2024)
-
Genome mining reveals novel biosynthetic gene clusters in entomopathogenic bacteria
Scientific Reports (2023)
-
Microbial engineering strategies to utilize waste feedstock for sustainable bioproduction
Nature Reviews Bioengineering (2023)