Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains.

Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2-electron reduction of the enzyme-bound thioester releasing the generated peptides as C-terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde-generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.

Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus.

Xenorhabdus and Photorhabdus species dedicate a large amount of resources to the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria undergo symbiosis with nematodes, which is followed by an insect pathogenic phase. So far, the molecular basis of this tripartite relationship and the exact roles that individual metabolites and metabolic pathways play have not been well understood. To close this gap, we have significantly expanded the database for comparative genomics studies in these bacteria. Clustering the genes encoded in the individual genomes into hierarchical orthologous groups reveals a high-resolution picture of functional evolution in this clade. It identifies groups of genes-many of which are involved in secondary metabolite production-that may account for the niche specificity of these bacteria. Photorhabdus and Xenorhabdus appear very similar at the DNA sequence level, which indicates their close evolutionary relationship. Yet, high-resolution mass spectrometry analyses reveal a huge chemical diversity in the two taxa. Molecular network reconstruction identified a large number of previously unidentified metabolite classes, including the xefoampeptides and tilivalline. Here, we apply genomic and metabolomic methods in a complementary manner to identify and elucidate additional classes of natural products. We also highlight the ability to rapidly and simultaneously identify potentially interesting bioactive products from NRPSs and PKSs, thereby augmenting the contribution of molecular biology techniques to the acceleration of natural product discovery.

Bioactive derivatives of isopropylstilbene from mutasynthesis and chemical synthesis.

Isopropylstilbene is a natural product from Photorhabdus luminescens TT01, with multiple biological activities. A mutant deficient in the production of both anthraquinones and cinnamic acid was constructed, thus giving a clean background according to UV detection. This anthraquinone and stilbene deficient (ASD) mutant was used in mutasynthesis experiments to obtain new stilbene derivatives, which were detected by GC-MS. The structures of the new derivatives were confirmed by detailed MS analysis and then chemically synthesised; all of the natural and synthetic compounds were tested against protozoa that cause tropical diseases. Two compounds obtained by mutasynthesis showed the highest activity against Trypanosoma cruzi, the causative agent of Chagas disease, and Leishmania donovani, which causes leishmaniasis.

Characterisation of taxlllaids A-G; natural products from Xenorhabdus indica.

Six new lipodepsipeptides and an additional linear derivative named taxlllaids A-G (1-7) have been identified in the entomopathogenic bacterium Xenorhabdus indica. The structures of the main compounds have been solved by detailed NMR spectroscopic analysis and the structures of minor derivatives were elucidated by a combination of labelling experiments and detailed MS experiments. The absolute configuration of the taxlllaids was deduced by using the advanced Marfey method and analysis of the biosynthesis gene cluster showing the presence of epimerisation domains, which was subsequently proved to be correct by solid-phase peptide synthesis of all taxlllaids. The exchange of a single amino acid in the adenylation domain was shown to be responsible for substrate promiscuity of the third A domain, resulting in the incorporation of leucine, phenylalanine or tyrosine. Bioactivity testing revealed the taxlllaids to be weakly active against Plasmodium falciparum and against a number of eukaryotic cell lines.

Biosynthesis of the natural fluorophore legioliulin from legionella.

Let it shine: The biosynthesis of the UV fluorophore legioliulin (1) from Legionella spp. was elucidated and the phenylalanine ammonium lyase LglD responsible for the formation of the starter unit cinnamic acid was biochemically characterized. Additionally, two novel derivatives differing in the starter unit have been identified by mutasynthesis experiments.