A microbiome-dependent gut-brain pathway regulates motivation for exercise.

Exercise exerts a wide range of beneficial effects for healthy physiology1. However, the mechanisms regulating an individual's motivation to engage in physical activity remain incompletely understood. An important factor stimulating the engagement in both competitive and recreational exercise is the motivating pleasure derived from prolonged physical activity, which is triggered by exercise-induced neurochemical changes in the brain. Here, we report on the discovery of a gut-brain connection in mice that enhances exercise performance by augmenting dopamine signalling during physical activity. We find that microbiome-dependent production of endocannabinoid metabolites in the gut stimulates the activity of TRPV1-expressing sensory neurons and thereby elevates dopamine levels in the ventral striatum during exercise. Stimulation of this pathway improves running performance, whereas microbiome depletion, peripheral endocannabinoid receptor inhibition, ablation of spinal afferent neurons or dopamine blockade abrogate exercise capacity. These findings indicate that the rewarding properties of exercise are influenced by gut-derived interoceptive circuits and provide a microbiome-dependent explanation for interindividual variability in exercise performance. Our study also suggests that interoceptomimetic molecules that stimulate the transmission of gut-derived signals to the brain may enhance the motivation for exercise.

Navigating and expanding the roadmap of natural product genome mining tools.

Natural products are structurally highly diverse and exhibit a wide array of biological activities. As a result, they serve as an important source of new drug leads. Traditionally, natural products have been discovered by bioactivity-guided fractionation. The advent of genome sequencing technology has resulted in the introduction of an alternative approach towards novel natural product scaffolds: Genome mining. Genome mining is an in-silico natural product discovery strategy in which sequenced genomes are analyzed for the potential of the associated organism to produce natural products. Seemingly universal biosynthetic principles have been deciphered for most natural product classes that are used to detect natural product biosynthetic gene clusters using pathway-encoded conserved key enzymes, domains, or motifs as bait. Several generations of highly sophisticated tools have been developed for the biosynthetic rule-based identification of natural product gene clusters. Apart from these hard-coded algorithms, multiple tools that use machine learning-based approaches have been designed to complement the existing genome mining tool set and focus on natural product gene clusters that lack genes with conserved signature sequences. In this perspective, we take a closer look at state-of-the-art genome mining tools that are based on either hard-coded rules or machine learning algorithms, with an emphasis on the confidence of their predictions and potential to identify non-canonical natural product biosynthetic gene clusters. We highlight the genome mining pipelines' current strengths and limitations by contrasting their advantages and disadvantages. Moreover, we introduce two indirect biosynthetic gene cluster identification strategies that complement current workflows. The combination of all genome mining approaches will pave the way towards a more comprehensive understanding of the full biosynthetic repertoire encoded in microbial genome sequences.

Antiprotozoal activity of different Xenorhabdus and Photorhabdus bacterial secondary metabolites and identification of bioactive compounds using the easyPACId approach.

Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.

Complex peptide natural products: Biosynthetic principles, challenges and opportunities for pathway engineering.

Complex peptide natural products exhibit diverse biological functions and a wide range of physico-chemical properties. As a result, many peptides have entered the clinics for various applications. Two main routes for the biosynthesis of complex peptides have evolved in nature: ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic pathways and non-ribosomal peptide synthetases (NRPSs). Insights into both bioorthogonal peptide biosynthetic strategies led to the establishment of universal principles for each of the two routes. These universal rules can be leveraged for the targeted identification of novel peptide biosynthetic blueprints in genome sequences and used for the rational engineering of biosynthetic pathways to produce non-natural peptides. In this review, we contrast the key principles of both biosynthetic routes and compare the different biochemical strategies to install the most frequently encountered peptide modifications. In addition, the influence of the fundamentally different biosynthetic principles on past, current and future engineering approaches is illustrated. Despite the different biosynthetic principles of both peptide biosynthetic routes, the arsenal of characterized peptide modifications encountered in RiPP and NRPS systems is largely overlapping. The continuous expansion of the biocatalytic toolbox of peptide modifying enzymes for both routes paves the way towards the production of complex tailor-made peptides and opens up the possibility to produce NRPS-derived peptides using the ribosomal route and vice versa.

Antifungal activity of different Xenorhabdus and Photorhabdus species against various fungal phytopathogens and identification of the antifungal compounds from X. szentirmaii.

Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of Steinernema and Heterorhabditis nematodes, respectively. These bacteria produce an extensive set of natural products (NPs) with antibacterial, antifungal, antiprotozoal, insecticidal, or other bioactivities when vectored into insect hemocoel by nematodes. We assessed the in vitro activity of different Xenorhabdus and Photorhabdus cell-free supernatants against important fungal phytopathogens, viz., Cryphonectria parasitica, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum and identified the bioactive antifungal compound/s present in the most effective bacterial supernatant using the easyPACId (easy promoter-activated compound identification) approach against chestnut blight C. parasitica. Our data showed that supernatants from Xenorhabdus species were comparatively more effective than extracts from Photorhabdus in suppressing the fungal pathogens; among the bacteria assessed, Xenorhabdus szentirmaii was the most effective species against all tested phytopathogens especially against C. parasitica. Subsequent analysis revealed fabclavines as antifungal bioactive compounds in X. szentirmaii, generated by a polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) hybrid system. Fabclavines are broad-spectrum, heat-stable NPs that have great potential as biological control compounds against fungal plant pathogens. More studies are needed to assess the potential phytotoxicity of these compounds and their effects on non-target organisms before commercialization. KEY POINTS: • Chemical fungicides have toxic effects on humans and other non-target organisms. • Alternatives with novel modes of action to supplant current fungicide are needed. • A novel bioactive antifungal compound from Xenorhabdus szentirmaii was identified.

Structure and biosynthesis of deoxy-polyamine in Xenorhabdus bovienii.

Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans.

Fabclavine diversity in Xenorhabdus bacteria.

The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on so-called secondary or specialized metabolites (SMs), identified in large screening approaches. To continue this successful strategy, new sources for bioactive compounds are required, such as the bacterial genera Xenorhabdus or Photorhabdus. In these strains, fabclavines are widely distributed SMs with a broad-spectrum bioactivity. Fabclavines are hybrid SMs derived from nonribosomal peptide synthetases (NRPS), polyunsaturated fatty acid (PUFA), and polyketide synthases (PKS). Selected Xenorhabdus and Photorhabdus mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine (fcl) biosynthesis gene cluster (BGC), followed by the analysis of the occurring fabclavines. Subsequently, known and unknown derivatives were identified and confirmed by MALDI-MS and MALDI-MS2 experiments in combination with an optimized sample preparation. This led to a total number of 22 novel fabclavine derivatives in eight strains, increasing the overall number of fabclavines to 32. Together with the identification of fabclavines as major antibiotics in several entomopathogenic strains, our work lays the foundation for the rapid fabclavine identification and dereplication as the basis for future work of this widespread and bioactive SM class.

Fabclavine biosynthesis in X. szentirmaii: shortened derivatives and characterization of the thioester reductase FclG and the condensation domain-like protein FclL.

Fabclavines, unusual peptide-polyketide-polyamine hybrids, show broad-spectrum bioactivity against a variety of different organism like Gram-positive and -negative bacteria, fungi and protozoa. We elucidated the biosynthesis of these NRPS-PKS hybrids in Xenorhabdus szentirmaii by deletion of most genes encoded in the fabclavine BGC and subsequent analysis of produced fabclavine or polyamine intermediates. Thereby, we identified shortened fabclavines similar to the bioactive zeamines. Furthermore, we analyzed the thioester reductase FclG and the free-standing condensation domain-like protein FclL in detail and observed low substrate specificity for both enzymes.