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In the last couple of years the research on natural products concerning ecological questions has gained more and more interest. Especially natural products play an important role for the maintenance of symbiotic relationships.
Here we present the application of the “overlap extension PCR-yeast homologous recombination“(ExRec) to simplify the availability of natural products. We successfully cloned a 45 kb gene cluster and characterized two new peptides ambactin and xenolindicin from Xenorhabdus – the latter derived from a silent gene cluster. ExRec is a very efficient cloning technique and resembles a powerful method regarding the assembly of large gene clusters as well as the cloning from metagenomic libraries or RNA pools.
In addition, we discovered bacterial pyrrolizidine alkaloids from Xenorhabdus, referred to as pyrrolizixenamides. The gene cluster consisted of a NRPS and a hydroxylase encoding gene. Surprisingly, this gene cluster and its variations (type A to D) can be found throughout the bacterial kingdom which might indicate an essential function. While these substances are mainly known to play a role in the defense mechanism of plants, the function of the identified pyrrolizixenamides from Xenorhabdus yet remains unsolved.
Moreover, we firstly identified a phosphopantetheinyl transferase (PPTase) from the lichenized fungus of Evernia prunastri. The gene eppA encoding a Sfp-type PPTase was heterologously expressed in Escherichia coli and Saccharomyces cerevisiae and functional characterized by indigoidine production and complementation of lys5, respectively. All represented results contribute to the elucidation of natural products and thereby to their role in nature with special regard to symbiotic associations.
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.