ABSTRACT
In the last decades, natural products from lichens have gained more interest for pharmaceutical application due to the broad range of their biological activity. However, isolation of the compounds of interest directly from the lichen is neither feasible nor sustainable due to slow growth of many lichens. In order to develop a pipeline for heterologous expression of lichen biosynthesis gene clusters and thus the sustainable production of their bioactive compounds we have identified and characterized the phosphopantheteinyl transferase (PPTase) EppA from the lichen Evernia prunastri. The Sfp-type PPTase EppA was functionally characterized through heterologous expression in E. coli using the production of the blue pigment indigoidine as readout and by complementation of a lys5 deletion in S. cerevisiae.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Lichens/enzymology , Lichens/genetics , Transferases (Other Substituted Phosphate Groups)/genetics , Transferases (Other Substituted Phosphate Groups)/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Genetic Complementation Test , Lichens/classification , Molecular Sequence Data , Piperidones/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Alignment , Transferases (Other Substituted Phosphate Groups)/chemistryABSTRACT
Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram-negative and Gram-positive bacteria indicates an important biological function in bacteria.
Subject(s)
Bacteria/metabolism , Pyrrolizidine Alkaloids/chemistry , Pyrrolizidine Alkaloids/metabolism , Bacteria/chemistry , Bacteria/enzymology , Bacterial Proteins/metabolism , Biosynthetic Pathways , Magnetic Resonance Spectroscopy , Oxidation-Reduction , Peptide Synthases/metabolism , Xenorhabdus/chemistry , Xenorhabdus/enzymology , Xenorhabdus/metabolismABSTRACT
Heterologous production of GameXPeptide A (1), as well as of the novel peptide natural products ambactin (2) and xenolindicins A-C (3 a-c), was achieved by using the "overlap extension PCR-yeast homologous recombination" (ExRec) method. ExRec cloning is based on the ability of yeast to assemble overlapping DNA fragments into functional plasmids. Here we used this technique to clone a total of 15 biosynthesis gene clusters from Photorhabdus and Xenorhabdus with sizes of up to 45 kb. The structures of the novel compounds 2 and 3 a, which were produced in Escherichia coli, were elucidated by detailed MS and bioinformatics analysis, and additionally confirmed by their chemical synthesis.