Tags

Type your tag names separated by a space and hit enter

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria.

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.

Authors+Show Affiliations

US Department of Energy Joint Genome Institute, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.Molecular Biotechnology, Department of Biosciences and Buchmann Institute for Molecular Life Sciences, Goethe Universität Frankfurt, Frankfurt am Main, Germany.Molecular Biotechnology, Department of Biosciences and Buchmann Institute for Molecular Life Sciences, Goethe Universität Frankfurt, Frankfurt am Main, Germany.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.Emery Pharma, Alameda, CA, USA.Emery Pharma, Alameda, CA, USA.Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA.Emery Pharma, Alameda, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. h.bode@bio.uni-frankfurt.de. LOEWE Centre for Translational Biodiversity Genomics, Frankfurt, Germany. h.bode@bio.uni-frankfurt.de.US Department of Energy Joint Genome Institute, Berkeley, CA, USA. yyoshikuni@lbl.gov. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. yyoshikuni@lbl.gov. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. yyoshikuni@lbl.gov. Center for Advanced Bioenergy and Bioproducts Innovation, Urbana, IL, USA. yyoshikuni@lbl.gov. Global Institution for Collaborative Research and Education, Hokkaido University, Hokkaido, Japan. yyoshikuni@lbl.gov.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31611640

Citation

Wang, Gaoyan, et al. "CRAGE Enables Rapid Activation of Biosynthetic Gene Clusters in Undomesticated Bacteria." Nature Microbiology, 2019.
Wang G, Zhao Z, Ke J, et al. CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria. Nat Microbiol. 2019.
Wang, G., Zhao, Z., Ke, J., Engel, Y., Shi, Y. M., Robinson, D., ... Yoshikuni, Y. (2019). CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria. Nature Microbiology, doi:10.1038/s41564-019-0573-8.
Wang G, et al. CRAGE Enables Rapid Activation of Biosynthetic Gene Clusters in Undomesticated Bacteria. Nat Microbiol. 2019 Oct 14; PubMed PMID: 31611640.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria. AU - Wang,Gaoyan, AU - Zhao,Zhiying, AU - Ke,Jing, AU - Engel,Yvonne, AU - Shi,Yi-Ming, AU - Robinson,David, AU - Bingol,Kerem, AU - Zhang,Zheyun, AU - Bowen,Benjamin, AU - Louie,Katherine, AU - Wang,Bing, AU - Evans,Robert, AU - Miyamoto,Yu, AU - Cheng,Kelly, AU - Kosina,Suzanne, AU - De Raad,Markus, AU - Silva,Leslie, AU - Luhrs,Alicia, AU - Lubbe,Andrea, AU - Hoyt,David W, AU - Francavilla,Charles, AU - Otani,Hiroshi, AU - Deutsch,Samuel, AU - Washton,Nancy M, AU - Rubin,Edward M, AU - Mouncey,Nigel J, AU - Visel,Axel, AU - Northen,Trent, AU - Cheng,Jan-Fang, AU - Bode,Helge B, AU - Yoshikuni,Yasuo, Y1 - 2019/10/14/ PY - 2019/01/08/received PY - 2019/08/27/accepted PY - 2019/10/16/entrez PY - 2019/10/16/pubmed PY - 2019/10/16/medline JF - Nature microbiology JO - Nat Microbiol N2 - 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. SN - 2058-5276 UR - https://www.unboundmedicine.com/medline/citation/31611640/CRAGE_enables_rapid_activation_of_biosynthetic_gene_clusters_in_undomesticated_bacteria L2 - http://dx.doi.org/10.1038/s41564-019-0573-8 DB - PRIME DP - Unbound Medicine ER -