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Sci Rep. 2015 Mar 24;5:9383. doi: 10.1038/srep09383.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis.

Author information

1
1] KAUST Global Collaborative Research, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong [2] Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093, United States.
2
KAUST Global Collaborative Research, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
3
1] Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093, United States [2] JNC Corporation, Yokohama Research Center, 5-1 Okawa, Kanazawa-ku, Yokohama, Kanagawa 2368605, Japan.
4
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, United States.
5
1] Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093, United States [2] Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093, United States.

Abstract

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning "plug-and-play" approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

PMID:
25807046
PMCID:
PMC4894447
DOI:
10.1038/srep09383
[Indexed for MEDLINE]
Free PMC Article

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