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Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli


Nonribosomal peptides (NRPs) are a class of microbial secondary metabolites that have a wide variety of medicinally important biological activities, such as antibiotic (vancomycin), immunosuppressive (cyclosporin A), antiviral (luzopeptin A) and antitumor (echinomycin and triostin A) activities1,2. However, many microbes are not amenable to cultivation and require time-consuming empirical optimization of incubation conditions for mass production of desired secondary metabolites for clinical and commercial use3. Therefore, a fast, simple system for heterologous production of natural products is much desired. Here we show the first example of the de novo total biosynthesis of biologically active forms of heterologous NRPs in Escherichia coli. Our system can serve not only as an effective and flexible platform for large-scale preparation of natural products from simple carbon and nitrogen sources, but also as a general tool for detailed characterizations and rapid engineering of biosynthetic pathways for microbial syntheses of novel compounds and their analogs.

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Figure 1: The echinomycin biosynthetic cluster from S. lasaliensis.
Figure 2: LC-MS analyses of Ecm18-catalyzed thioacetal formation.
Figure 3: Chemical characteristic spectra of compound 1 produced by the engineered E. coli strain.
Figure 4: Chemical characteristic spectra of compound 2 produced by the engineered E. coli strain.

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We thank M.J. Waring and M. Searcey for kindly providing the authentic reference samples of triostin A and TANDEM, respectively, and H. Kinashi (Hiroshima University) for advice in handling the streptomycete linear plasmid. This work was financially supported by Grant-in-Aids for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (A) 17208010 (H.O.), by Uehara Memorial Foundation PK220920 (H.O.), by National Institute of General Medical Sciences grant GM 075857-01 (C.C.C.W.) and by American Cancer Society grant RSG-06-010-01-CDD (C.C.C.W.). K.W. is a recipient of fellowships from the Japan Antibiotics Research Association, Pfizer Infectious Disease Foundation and the Agricultural Chemical Research Foundation. A.M. is a recipient of JSPS predoctoral fellowship 177009104.

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Correspondence to Kenji Watanabe or Hideaki Oikawa.

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Supplementary information

Supplementary Fig. 1

SDS-PAGE analysis of the purified E. coli-produced echinomycin biosynthetic proteins. (PDF 2970 kb)

Supplementary Fig. 2

Maps of the plasmid-borne echinomycin biosynthetic gene cluster. (PDF 84 kb)

Supplementary Fig. 3

Assays for checking the maintenance and stability of the plasmids carrying the echinomycin biosynthetic cluster in E. coli DH5α. (PDF 2878 kb)

Supplementary Fig. 4

Chemical characteristic spectra of the authentic reference for echinomycin. (PDF 161 kb)

Supplementary Fig. 5

Chemical characteristic spectra of the authentic reference for triostin A. (PDF 167 kb)

Supplementary Methods

Methods used for echinomycin biosynthetic cluster isolation and a table of the sequences of the primers used in this manuscript. (PDF 147 kb)

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Watanabe, K., Hotta, K., Praseuth, A. et al. Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli. Nat Chem Biol 2, 423–428 (2006).

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