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Identification and Characterization of the Lysobactin Biosynthetic GeneCluster and Its Unusual Termination Module
Nonribosomal peptides (NRPs) constitute a class of structurally and functionally diverse natural products, which are synthesized by nonribosomal peptide synthetases (NRPSs). NRPs exhibit a wide range of bioactivities, including antimicrobial, antifungal, antiviral, immunosuppressive and antitumor...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2012
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| Zusammenfassung: | Nonribosomal peptides (NRPs) constitute a class of structurally and functionally diverse
natural products, which are synthesized by nonribosomal peptide synthetases (NRPSs).
NRPs exhibit a wide range of bioactivities, including antimicrobial, antifungal, antiviral,
immunosuppressive and antitumor properties. Numerous of these compounds have
been discovered via screening of microbial extracts. In recent years, increasing
knowledge of the biosynthesis of natural products and development of new sequencing
techniques lead to the identification of gene clusters, which are putatively involved in
the biosynthesis of nonribosomal peptides.
Based on the sequencing result of the genome of Lysobacter sp. ATCC 53042 and the
former work from Bernhard et al.[1] on the gene fragment involved in the biosynthesis of
lysobactin, the entire biosynthetic gene cluster of lysobactin was identified and
characterized. The cluster encodes two multimodular nonribosomal peptide synthetases
(LybA and LybB). Due to the correlation of the number of modules found within the
lysobactin gene cluster and the primary sequence of lysobactin, the biosynthesis of
lysobactin follows the colinearity principle. Investigation of the adenylation domain
substrate specificities confirmed the direct association between the synthetases and
lysobactin biosynthesis.
Furthermore, an unusual tandem thioesterase domain architecture (PCP-TE1-TE2) of the
LybB termination module was identified. Biochemical characterization of the individual
thioesterases in vitro proved that the first thioesterase is responsible for the cyclization
and the release of the final product, while the second thioesterase showed a type II TE
activity, which is responsible for the regeneration of the mis-primed peptide carrier
protein during the biosynthesis of lysobactin. Together with the observation of the
proteolytic degradation during the heterologous production of LybB-PCP-TE1-TE2 giving
rise of LybB-PCP-TE1, we have proposed that the LybB is also cleaved to generate
lone-standing LybB-TE2 prior to lysobactin synthesis in the native strain. The resulting
lone-standing TE2 serves as external type II TE to regenerate mis-primed peptide carrier
protein via hydrolytic cleavage of the PCP-bound noncognate substrates.
Additionally, the sequence of the genome of Lysobacter sp. ATCC 53042 was
bioinformatically analyzed. The analysis result delivered further potential NRPS and
PKS-NRPS hybrid gene clusters. Based on the proposed substrate specificities of the
adenylation domains, the chemical structures of the products were proposed. However,
further experiments are needed to confirm the production of these compounds. |
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| DOI: | 10.17192/z2012.0484 |