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J. Biol. Chem., Vol. 276, Issue 37, 34824-34831, September 14, 2001
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From the Department of Chemistry, Philipps University,
D-35032 Marburg, Germany
Nonribosomal peptides are processed on
multifunctional enzymes called nonribosomal peptide synthetases
(NRPSs), whose modular multidomain arrangement allowed the rational
design of new peptide products. However, the lack of natural competence
and efficient transformation methods for most of nonribosomal peptide
producer strains prevented the in vivo manipulation of
these biosynthetic gene clusters. In this study, we present methods for
the construction of a genetically engineered Bacillus
subtilis surrogate host for the integration and heterologous
expression of foreign NRPS genes. In the B. subtilis
surrogate host, we deleted the resident 26-kilobase srfA
gene cluster encoding the surfactin synthetases and subsequently used
the same chromosomal location for integration of the entire 49-kilobase
bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method.
Synthesis of the branched cyclic peptide antibiotic bacitracin in the
engineered B. subtilis strain was achieved at high level,
indicating a functional production and proper posttranslational
modification of the bacitracin synthetases BacABC, as well as the
expression of the associated bacitracin self-resistance
genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new
bacitracin derivatives.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF007865.
Engineered Biosynthesis of the Peptide Antibiotic Bacitracin
in the Surrogate Host Bacillus subtilis*
*
This work was supported by the Deutsche
Forschungsgemeinschaft and Fonds der Chemischen Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-6421-282-5722; Fax: 49-6421-282-2191; E-mail:
marahiel@chemie.uni-marburg.de.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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