Structural characterization of the siderophore rhodochelin from Rhodococcus jostii RHA1 and elucidation of its biosynthetic machinery

Rhodococci represent an important genus of industrial interest, both because of their role in bioremediation and biocatalysis, as well as for their potential as producers of natural products. In this context, the genome sequencing of the biphenyl-degrading soil bacterium Rhodococcus jostii RHA1 r...

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Bibliographic Details
Main Author: Bosello, Mattia
Contributors: Marahiel, Mohamed A. (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2012
Chemie
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Summary:Rhodococci represent an important genus of industrial interest, both because of their role in bioremediation and biocatalysis, as well as for their potential as producers of natural products. In this context, the genome sequencing of the biphenyl-degrading soil bacterium Rhodococcus jostii RHA1 represents the first attempt to harness the biosynthetic metabolic potential of the genus Rhodococcus, by enabling the systematic exploration of its natural product-producing capabilities. The genome of R. jostii RHA1 contains 23 secondary metabolite gene clusters, all considered to be orphan with respect to their product, including two clusters putatively involved in siderophore biosynthesis. In this study, the isolation, structural characterization and genetic analysis of the biosynthetic origin of rhodochelin, a unique mixed-type catecholate-hydroxamate siderophore isolated from R. jostii RHA1, which represents the first characterized NRPS-derived natural product of the strain, is reported. Structure elucidation of rhodochelin was accomplished via MSn- and NMR-analysis and revealed the tetrapeptide to contain an unusual ester bond between an L-δ-N-formyl-δ-N-hydroxyornithine (L-fhOrn)moiety and the side chain of a threonine residue. Bioinformatic analysis of the R. jostii RHA1 genome revealed the enzymes responsible for siderophore biosynthesis to be encoded in three distant NRPS gene clusters. Single gene deletions within the three putative biosynthetic gene clusters abolished rhodochelin production, proving that the ORFs responsible for rhodochelin biosynthesis are located in different chromosomal loci. Biochemical characterization of the monooxygenase Rmo and the formyltransferase Rft established a route for the biosynthesis of the nonproteinogenic amino acid L-fhOrn, prior to its incorporation into the peptide scaffold by the NRPS-assembly line. The insights gained from the structural and functional characterization of rhodochelin, together with the genetic and biochemical characterization of the respective biosynthetic gene clusters, allowed the proposal of a biosynthetic model for rhodochelin assembly. Finally, the efficient and, in this work, first reported cross-talk between three distantly located secondary metabolite gene clusters provides deep insights into natural product biosynthesis that may facilitate future attempts to isolate new natural products.
DOI:https://doi.org/10.17192/z2012.0767