A nonribosomal peptide synthetase involved in the biosynthesis of ampullosporins in Sepedonium ampullosporum.
Recently, the saprophytic ascomycete Sepedonium ampullosporum strain HKI-0053 was isolated from a basidiomycete on account of its premature induction of pigment formation in Phoma destructiva, a process often related to the neuroleptic activity of the inducing compound. The active substance was identified as the 15-membered peptaibol type peptide Ampullosporin. Although to date more than 300 peptaibols have been discovered, their biosynthetic machinery has not been characterized yet. By improving the culture conditions it was possible to grow S. ampullosporum in a submerged culture and to increase Ampullosporin production by more than three times to 33 mg/l at reduced fermentation times. The appearance of two high molecular weight proteins, HMWP1 (1.5 MDa) and HMWP2 (350 kDa) was closely related to the production of Ampullosporin during the course of fermentation. Both proteins showed a cross-reaction with antibodies against a core fragment of nonribosomal peptide synthetases (NRPSs). Biochemical characterization of the partially purified enzymes exhibited selectivity for the substrate amino acid alpha-aminoisobutyric acid (Aib). substantiating their involvement in Ampullosporin biosynthesis. Our data suggest that Ampullosporin synthetase has been isolated, and provides the basis for the characterization of the entire biosynthetic gene cluster. Furthermore, this knowledge will enable the manipulation of its NRPS template, in order to engineer mutant strains of Sepedonium ampullosporum which could produce more potent analogues of Ampullosporin.
Reiber
Kathrin
K
Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstrasse 11a, D-07745 Jena, Germany.
Neuhof
Torsten
T
Ozegowski
Jörg H
JH
von Döhrend
Hans
H
Schwecke
Torsten
T
D000595:Amino Acid Sequence; D000621:Aminoisobutyric Acids / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000138:chemical synthesis; D001203:Ascomycota / Q000201:enzymology*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000138:chemical synthesis; D005810:Multigene Family; D054848:Peptaibols; D010453:Peptide Synthases / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D013379:Substrate Specificity
D000621:Aminoisobutyric Acids; D000900:Anti-Bacterial Agents; D005656:Fungal Proteins; D054848:Peptaibols; D010455:Peptides; C109004:ampullosporin; D010453:Peptide Synthases