Investigations into PoyH, a promiscuous protease from polytheonamide biosynthesis.

Polytheonamides are the most extensively modified ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) currently known. In RiPP biosynthesis, the processed peptide is usually released from a larger precursor by proteolytic cleavage to generate the bioactive terminal product of the pathway. For polytheonamides, which are members of a new RiPP family termed proteusins, we have recently shown that such cleavage is catalyzed by the cysteine protease PoyH acting on the precursor PoyA, both encoded in the polytheonamide biosynthetic gene cluster. We now report activity for PoyH under a variety of reaction conditions for different maturation states of PoyA and demonstrate a potential use of PoyH as a promiscuous protease to liberate and characterize RiPPs from other pathways. As a proof of concept, the identified recognition motif was introduced into precursors of the thiopeptide thiocillin and the lanthipeptide lichenicidin VK1, allowing for their site-specific cleavage with PoyH. Additionally, we show that PoyH cleavage is inhibited by PoyG, a previously uncharacterized chagasin-like protease inhibitor encoded in the polytheonamide gene cluster.

Exploring the chemistry of uncultivated bacterial symbionts: antitumor polyketides of the pederin family.

Symbiotic bacteria have long been proposed as being responsible for the production of numerous natural products isolated from invertebrate animals. However, systematic studies of invertebrate-symbiont associations are usually associated with serious technical challenges, such as the general resistance of symbionts to culturing attempts and the complexity of many microbial consortia. Herein an overview is provided on the culture-independent, metagenomic strategies recently employed by our group to contribute to a better understanding of natural product symbiosis. Using terrestrial Paederus spp. beetles and the marine sponge Theonella swinhoei as model animals, the putative genes responsible for the production of pederin-type antitumor polyketides have been isolated. In Paederus fuscipes, which uses pederin for chemical defense, these genes belong to an as-yet unculturable symbiont closely related to Pseudomonas aeruginosa. To study the extremely complex association of T. swinhoei and its multispecies bacterial consortium, we used a phylogenetic approach that allowed the isolation of onnamide/theopederin polyketide synthase genes from an uncultured sponge symbiont. Analysis of the biosynthesis genes provided unexpected insights into a possible evolution of pederin-type pathways. Besides revealing new facets of invertebrate chemical ecology, these first gene clusters from uncultivated symbiotic producers suggest possible biotechnological strategies to solve the supply problem associated with the development of most marine drug candidates.

Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei.

Bacterial symbionts have long been suspected to be the true producers of many drug candidates isolated from marine invertebrates. Sponges, the most important marine source of biologically active natural products, have been frequently hypothesized to contain compounds of bacterial origin. This symbiont hypothesis, however, remained unproven because of a general inability to cultivate the suspected producers. However, we have recently identified an uncultured Pseudomonas sp. symbiont as the most likely producer of the defensive antitumor polyketide pederin in Paederus fuscipes beetles by cloning the putative biosynthesis genes. Here we report closely related genes isolated from the highly complex metagenome of the marine sponge Theonella swinhoei, which is the source of the onnamides and theopederins, a group of polyketides that structurally resemble pederin. Sequence features of the isolated genes clearly indicate that it belongs to a prokaryotic genome and should be responsible for the biosynthesis of almost the entire portion of the polyketide structure that is correlated with antitumor activity. Besides providing further proof for the role of the related beetle symbiont-derived genes, these findings raise intriguing ecological and evolutionary questions and have important general implications for the sustainable production of otherwise inaccessible marine drugs by using biotechnological strategies.