Cloning and characterization of the peptide synthetase gene cluster involved in the nonribosomal biosynthesis of fusaricidin-type antifungal antibiotics in Paenibacillus polymyxa PKB1.

摘要

Paenibacillus polymyxa (formerly Bacillus polymyxa) PKB1 produces fusaricidin-type antifungal antibiotics that inhibit the growth of Leptosphaeria maculans, a plant pathogenic 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.;To confirm the involvement of fusA in fusaricidin production, a modified PCR-targeting mutagenesis protocol was developed to create a fusA mutation on the chromosome of PKB1. A DNA fragment internal to fusA was replaced by a gene disruption cassette containing two antibiotic resistance markers for independent selection of apramycin resistance in E. coli and chloramphenicol resistance in P. polymyxa . The inclusion of an oriT site in the disruption cassette allowed efficient transfer of the inactivated fusA allele into P. polymyxa by intergeneric conjugation from E. coli. Targeted disruption of fusA led to the complete loss of the antifungal activity against L. maculans, suggesting that fusA plays an essential role in the nonribosomal synthesis of fusaricidin. The boundaries of the fus gene cluster were determined using the same mutagenesis strategy.;The results presented in this thesis provide the basis for genetic manipulation of fusaricidin production in P. polymyxa PKB1, and furthermore, for construction of novel antibiotics by combinatorial biosynthesis.;Using a reverse genetic approach based on the conserved motifs of nonribosomal peptide synthetases, the entire fusaricidin biosynthetic gene cluster ( fus) was cloned and sequenced, and spans 32.4 kb including an open reading frame (fusA) encoding a six-module peptide synthetase. The second, fourth and fifth modules of fusaricidin synthetase (FusA) each contain an epimerization domain, consistent with the incorporation of D-amino acids in these positions of fusaricidin. However, the sixth module, corresponding to D-Ala, lacks an epimerization domain. The sixth adenylation domain of FusA was produced at high levels in Escherichia coli and shown to activate D-Ala specifically, providing evidence for direct selection and activation of a D-amino acid by a typical prokaryotic peptide synthetase. The fus cluster also includes genes presumably involved in biosynthesis, modification, and activation of the lipid moiety of fusaricidin. However, no genes for regulation, resistance, or transport functions were encountered.