CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria

Ke, Jing; Robinson, David; Wu, Zong-Yen; Kuftin, Andrea; Louie, Katherine; Kosina, Suzanne; Northen, Trent; Cheng, Jan-Fang; Yoshikuni, Yasuo

doi:10.1016/j.chembiol.2021.08.009

Title: CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria

Journal Article · Fri Sep 10 00:00:00 EDT 2021 · Cell Chemical Biology

DOI:https://doi.org/10.1016/j.chembiol.2021.08.009· OSTI ID:1881880

Ke, Jing ^[1]; Robinson, David ^[1]; Wu, Zong-Yen ^[1];

^[1]; Louie, Katherine ^[1]; Kosina, Suzanne ^[1]; Northen, Trent ^[1]; Cheng, Jan-Fang ^[1];

^[2]

Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Hokkaido University (Japan)

With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1881880

Journal Information:: Cell Chemical Biology, Vol. 29, Issue 4; ISSN 2451-9456

Publisher:: Cell PressCopyright Statement

Country of Publication:: United States

Language:: English

References (45)

Entomopathogenic bacteria as a source of secondary metabolites Bode, Helge B. Current Opinion in Chemical Biology, Vol. 13, Issue 2 https://doi.org/10.1016/j.cbpa.2009.02.037	journal	April 2009
Structure Elucidation and Activity of Kolossin A, the D -/ L -Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase Bode, Helge B.; Brachmann, Alexander O.; Jadhav, Kirtikumar B. Angewandte Chemie International Edition, Vol. 54, Issue 35 https://doi.org/10.1002/anie.201502835	journal	June 2015
Entomopathogenic bacteriaPhotorhabdus luminescensas drug source againstLeishmania amazonensis Antonello, Ana Maria; Sartori, Thaís; Folmer Correa, Ana Paula Parasitology, Vol. 145, Issue 8 https://doi.org/10.1017/S0031182017002001	journal	November 2017
Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation Jinek, M.; Jiang, F.; Taylor, D. W. Science, Vol. 343, Issue 6176 https://doi.org/10.1126/science.1247997	journal	February 2014
CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus Peng, Ran; Wang, Ye; Feng, Wan-wan Microbial Cell Factories, Vol. 17, Issue 1 https://doi.org/10.1186/s12934-018-0867-1	journal	January 2018
Distribution of siderophore gene systems on a Vibrionaceae phylogeny: Database searches, phylogenetic analyses and evolutionary perspectives Thode, Sunniva Katharina; Rojek, Ewelina; Kozlowski, Mikolaj PLOS ONE, Vol. 13, Issue 2 https://doi.org/10.1371/journal.pone.0191860	journal	February 2018
Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria Dong, Chen; Fontana, Jason; Patel, Anika Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-04901-6	journal	June 2018
Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting Fu, Jun; Bian, Xiaoying; Hu, Shengbaio Nature Biotechnology, Vol. 30, Issue 5 https://doi.org/10.1038/nbt.2183	journal	April 2012
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA Nishimasu, Hiroshi; Ran, F. Ann; Hsu, Patrick D. Cell, Vol. 156, Issue 5 https://doi.org/10.1016/j.cell.2014.02.001	journal	February 2014
Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9 Mougiakos, Ioannis; Bosma, Elleke F.; Weenink, Koen ACS Synthetic Biology, Vol. 6, Issue 5 https://doi.org/10.1021/acssynbio.6b00339	journal	February 2017
Heterogeneity in Bacterial Specialized Metabolism Tobias, Nicholas J.; Bode, Helge B. Journal of Molecular Biology, Vol. 431, Issue 23 https://doi.org/10.1016/j.jmb.2019.04.042	journal	November 2019
CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes Gilbert, Luke A.; Larson, Matthew H.; Morsut, Leonardo Cell, Vol. 154, Issue 2, p. 442-451 https://doi.org/10.1016/j.cell.2013.06.044	journal	July 2013
Multi-functional genome-wide CRISPR system for high throughput genotype–phenotype mapping Lian, Jiazhang; Schultz, Carl; Cao, Mingfeng Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-13621-4	journal	December 2019
Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems Liu, Hualan; Robinson, David S.; Wu, Zong-Yen PLOS ONE, Vol. 15, Issue 11 https://doi.org/10.1371/journal.pone.0241867	journal	November 2020
DNA Repair: Common Approaches to Fixing Double-Strand Breaks Hiom, Kevin Current Biology, Vol. 19, Issue 13 https://doi.org/10.1016/j.cub.2009.06.009	journal	July 2009
A specialized metabolic network selectively modulates Arabidopsis root microbiota Huang, Ancheng C.; Jiang, Ting; Liu, Yong-Xin Science, Vol. 364, Issue 6440 https://doi.org/10.1126/science.aau6389	journal	May 2019
CRISPR/Cas9 in Genome Editing and Beyond Wang, Haifeng; La Russa, Marie; Qi, Lei S. Annual Review of Biochemistry, Vol. 85, Issue 1 https://doi.org/10.1146/annurev-biochem-060815-014607	journal	June 2016
Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds Zalatan, Jesse G.; Lee, Michael E.; Almeida, Ricardo Cell, Vol. 160, Issue 1-2 https://doi.org/10.1016/j.cell.2014.11.052	journal	January 2015
Tuning genetic control through promoter engineering Alper, H.; Fischer, C.; Nevoigt, E. Proceedings of the National Academy of Sciences, Vol. 102, Issue 36, p. 12678-12683 https://doi.org/10.1073/pnas.0504604102	journal	August 2005
One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor Stein, M. L.; Beck, P.; Kaiser, M. Proceedings of the National Academy of Sciences, Vol. 109, Issue 45 https://doi.org/10.1073/pnas.1211423109	journal	October 2012
The regulation of pathogenicity and mutualism in Photorhabdus Joyce, Susan A.; Watson, Robert J.; Clarke, David J. Current Opinion in Microbiology, Vol. 9, Issue 2 https://doi.org/10.1016/j.mib.2006.01.004	journal	April 2006
Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen Ffrench-Constant, Richard; Waterfield, Nicholas; Daborn, Phillip FEMS Microbiology Reviews, Vol. 26, Issue 5 https://doi.org/10.1111/j.1574-6976.2003.tb00625.x	journal	January 2003
Glidobactins A, B and C, new antitumor antibiotics. I. Production, isolation, chemical properties and biological activity. Oka, Masahisa; Nishiyama, Yuji; Ohta, Shinichi The Journal of Antibiotics, Vol. 41, Issue 10 https://doi.org/10.7164/antibiotics.41.1331	journal	January 1988
Accessing Bioactive Natural Products from the Human Microbiome Milshteyn, Aleksandr; Colosimo, Dominic A.; Brady, Sean F. Cell Host & Microbe, Vol. 23, Issue 6 https://doi.org/10.1016/j.chom.2018.05.013	journal	June 2018
MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data Pluskal, Tomáš; Castillo, Sandra; Villar-Briones, Alejandro BMC Bioinformatics, Vol. 11, Issue 1 https://doi.org/10.1186/1471-2105-11-395	journal	July 2010
Multiplexed and tunable transcriptional activation by promoter insertion using nuclease-assisted vector integration Brown, Alexander; Winter, Jackson; Gapinske, Michael Nucleic Acids Research, Vol. 47, Issue 12 https://doi.org/10.1093/nar/gkz210	journal	April 2019
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation Gilbert, Luke A.; Horlbeck, Max A.; Adamson, Britt Cell, Vol. 159, Issue 3 https://doi.org/10.1016/j.cell.2014.09.029	journal	October 2014
Refining the Natural Product Repertoire in Entomopathogenic Bacteria Tobias, Nicholas Jay; Shi, Yi-Ming; Bode, Helge B. Trends in Microbiology, Vol. 26, Issue 10 https://doi.org/10.1016/j.tim.2018.04.007	journal	October 2018
Discovery of microbial natural products by activation of silent biosynthetic gene clusters Rutledge, Peter J.; Challis, Gregory L. Nature Reviews Microbiology, Vol. 13, Issue 8 https://doi.org/10.1038/nrmicro3496	journal	June 2015
Computational approaches to natural product discovery Medema, Marnix H.; Fischbach, Michael A. Nature Chemical Biology, Vol. 11, Issue 9 https://doi.org/10.1038/nchembio.1884	journal	August 2015
Insect-Specific Production of New GameXPeptides in Photorhabdus luminescens TTO1, Widespread Natural Products in Entomopathogenic Bacteria Nollmann, Friederike I.; Dauth, Christina; Mulley, Geraldine ChemBioChem, Vol. 16, Issue 2 https://doi.org/10.1002/cbic.201402603	journal	November 2014
Merging chemical ecology with bacterial genome mining for secondary metabolite discovery Vizcaino, Maria I.; Guo, Xun; Crawford, Jason M. Journal of Industrial Microbiology and Biotechnology, Vol. 41, Issue 2 https://doi.org/10.1007/s10295-013-1356-5	journal	February 2014
CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria Wang, Gaoyan; Zhao, Zhiying; Ke, Jing Nature Microbiology, Vol. 4, Issue 12 https://doi.org/10.1038/s41564-019-0573-8	journal	October 2019
Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements Fontana, Jason; Dong, Chen; Kiattisewee, Cholpisit Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-15454-y	journal	April 2020
Production of Cytotoxic Glidobactins/Luminmycins by Photorhabdus asymbiotica in Liquid Media and Live Crickets Theodore, Christine M.; King, Jarrod B.; You, Jianlan Journal of Natural Products, Vol. 75, Issue 11 https://doi.org/10.1021/np300623x	journal	October 2012
Secondary Metabolites Produced by Heterorhabditis Symbionts and Their Application in Agriculture: What We Know and What to Do Next Stock, S. Patricia; Kusakabe, Ayako; Orozco, Rousel A. Journal of Nematology, Vol. 49, Issue 4 https://doi.org/10.21307/jofnem-2017-084	journal	January 2018
The New State of the Art: Cas9 for Gene Activation and Repression La Russa, Marie F.; Qi, Lei S. Molecular and Cellular Biology, Vol. 35, Issue 22 https://doi.org/10.1128/MCB.00512-15	journal	September 2015
Simple “On-Demand” Production of Bioactive Natural Products Bode, Edna; Brachmann, Alexander O.; Kegler, Carsten ChemBioChem, Vol. 16, Issue 7 https://doi.org/10.1002/cbic.201500094	journal	March 2015
The Fish Pathogen Vibrio ordalii Under Iron Deprivation Produces the Siderophore Piscibactin Ruiz, Pamela; Balado, Miguel; Fuentes-Monteverde, Juan Carlos Microorganisms, Vol. 7, Issue 9 https://doi.org/10.3390/microorganisms7090313	journal	September 2019
Enabling Graded and Large-Scale Multiplex of Desired Genes Using a Dual-Mode dCas9 Activator in Saccharomyces cerevisiae Deaner, Matthew; Mejia, Julio; Alper, Hal S. ACS Synthetic Biology, Vol. 6, Issue 10 https://doi.org/10.1021/acssynbio.7b00163	journal	June 2017
Structures of cepafungins I, II and III. Terui, Yoshihiro; Nishikawa, Junko; Hinoo, Hiroshi The Journal of Antibiotics, Vol. 43, Issue 7 https://doi.org/10.7164/antibiotics.43.788	journal	January 1990
CRAGE-Duet Facilitates Modular Assembly of Biological Systems for Studying Plant–Microbe Interactions Wang, Bing; Zhao, Zhiying; Jabusch, Lauren K. ACS Synthetic Biology, Vol. 9, Issue 9 https://doi.org/10.1021/acssynbio.0c00280	journal	July 2020
CRISPR interference (CRISPRi) for sequence-specific control of gene expression Larson, Matthew H.; Gilbert, Luke A.; Wang, Xiaowo Nature Protocols, Vol. 8, Issue 11 https://doi.org/10.1038/nprot.2013.132	journal	October 2013
Regulating Alternative Lifestyles in Entomopathogenic Bacteria Crawford, Jason M.; Kontnik, Renee; Clardy, Jon Current Biology, Vol. 20, Issue 1 https://doi.org/10.1016/j.cub.2009.10.059	journal	January 2010
Systematic testing of enzyme perturbation sensitivities via graded dCas9 modulation in Saccharomyces cerevisiae Deaner, Matthew; Alper, Hal S. Metabolic Engineering, Vol. 40 https://doi.org/10.1016/j.ymben.2017.01.012	journal	March 2017

Similar Records

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria

Journal Article · Mon Oct 14 00:00:00 EDT 2019 · Nature Microbiology · OSTI ID:1881880

Wang, Gaoyan; Zhao, Zhiying; Ke, Jing; +28 more

CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria

Journal Article · Mon Dec 02 00:00:00 EST 2019 · Nature Microbiology · OSTI ID:1881880

Wang, Gaoyan; Zhao, Zhiying (Jean); Ke, Jing; +28 more

Development of platforms for functional characterization and production of phenazines using a multi-chassis approach via CRAGE

Journal Article · Wed Dec 08 00:00:00 EST 2021 · Metabolic Engineering · OSTI ID:1881880

Ke, Jing; Zhao, Zhiying; Coates, Cameron R.; +8 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CRISPR-Cas9
CRAGE
secondary metabolites
BGC activation
multiple sgRNA sites
BGC deletion
BGC-to-compound characterization

Title: CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria

Citation Formats

References (45)

Similar Records

Related Subjects