Monday, July 30, 2007
P116

Nonribosomal biosynthesis of fusaricidin in Paenibacillus polymyxa PKB1 involves direct incorporation of a D-amino acid

Jingru Li and Susan E. Jensen. Biological Sciences, University of Alberta, CW-405 Biological Sciences Building, Edmonton, AB T6G2E9, Canada

Paenibacillus polymyxa PKB1 produces fusaricidin-type antifungal metabolites that inhibit the growth of Leptosphaeria maculans, a fungus causing blackleg disease of canola. Fusaricidin consists of a guanidino-modified beta-hydroxy fatty acid linked to a cyclic hexapeptide with four residues present in the D-configuration. The entire fusaricidin biosynthetic gene cluster spanning 32.4 kb has been cloned and sequenced. A large open reading frame, designated fusA, encodes a single nonribosomal peptide synthetase (NRPS) containing six functional modules. The organization of fusaricidin synthetase (FusA) follows the collinearity rule of NRPSs, bearing condensation, adenylation, and thiolation domains in each module as well as epimerization domains responsible for the transformation of amino acid residues from L to D form, in the second, fourth and fifth modules. However, the sixth module, corresponding to D-Ala, lacks an epimerization domain. The substrate specificity of the sixth adenylation domain of FusA (expressed in Escherichia coli) was determined to be D-Ala only, which represents the first biochemical evidence for the direct selection and activation of a D-amino acid by an isolated adenylation domain of a peptide synthetase. Other genes coding for the components of fatty acid synthases were identified at the N-terminal end of FusA, suggesting their role in modification and activation of the lipid moiety of fusaricidin. A PCR-targeting mutagenesis protocol was developed to disrupt fusA, which abolished the antifungal activity of PKB1 against L. maculans. Together, these findings provide further evidence of the biosynthetic versatility of NRPSs that may be exploited for production of novel antibiotics by combinatorial biosynthesis.

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