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 UTC 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.

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Research Organization:: Lawrence Berkeley National Laboratory (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, Journal Name: Cell Chemical Biology Journal Issue: 4 Vol. 29; ISSN 2451-9456

Publisher:: Cell PressCopyright Statement

Country of Publication:: United States

Language:: English

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