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Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l . This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications.

In search for new natural products, which may lead to the development of new drugs for all kind of applications, novel methods are needed. Here we describe the identification of electrophilic natural products in crude extracts via their reactivity against azide as a nucleophile followed by their subsequent enrichment using a cleavable azide-reactive resin (CARR). Using this approach, natural products carrying epoxides and α,β-unsaturated enones as well as several unknown compounds were identified in crude extracts from entomopathogenic bacteria.

The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure-activity relationship of the simple and antistaphylococcal amide nematophin from and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluorescent derivatives, incorporating azaindole moieties was achieved for the first time.

Peptides derived from non-ribosomal peptide synthetases (NRPSs) represent an important class of pharmaceutically relevant drugs. Methods to generate novel non-ribosomal peptides or to modify peptide natural products in an easy and predictable way are therefore of great interest. However, although the overall modular structure of NRPSs suggests the possibility of adjusting domain specificity and selectivity, only a few examples have been reported and these usually show a severe drop in production titre. Here we report a new strategy for the modification of NRPSs that uses defined exchange units (XUs) and not modules as functional units. XUs are fused at specific positions that connect the condensation and adenylation domains and respect the original specificity of the downstream module to enable the production of the desired peptides. We also present the use of internal condensation domains as an alternative to other peptide-chain-releasing domains for the production of cyclic peptides.

The production of natural product compound libraries has been observed in nature for different organisms such as bacteria, fungi and plants; however, little is known about the mechanisms generating such chemically diverse libraries. Here we report mechanisms leading to the biosynthesis of the chemically diverse rhabdopeptide/xenortide peptides (RXPs). They are exclusively present in entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus that live in symbiosis with nematodes delivering them to insect prey, which is killed and utilized for nutrition by both nematodes and bacteria. Chemical diversity of the biologically active RXPs results from a combination of iterative and flexible use of monomodular nonribosomal peptide synthetases including substrate promiscuity, enzyme cross-talk and enzyme stoichiometry as shown by in vivo and in vitro experiments. Together, this highlights several of nature's methods for diversification, or evolution, of natural products and sheds light on the biosynthesis of the bioactive RXPs.

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3′ end of ppzA. We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

Many important natural products are produced by non-ribosomal peptide synthetases (NRPSs). These giant enzyme machines activate amino acids in an assembly line fashion in which a set of catalytically active domains is responsible for the section, activation, covalent binding and connection of a specific amino acid to the growing peptide chain. Since NRPS are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would give access to a diverse range of peptides available biotechnologically. Here we describe a new fusion point inside condensation (C) domains of NRPSs that enables the efficient production of peptides, even containing non-natural amino acids, in yields higher than 280 mg/L. The technology called eXchange Unit 2.0 (XU2.0) also allows the generation of targeted peptide libraries and therefore might be suitable for the future identification of bioactive peptide derivatives for pharmaceutical and other applications.

Abstract The Siberian mammoth steppe ecosystem changed dramatically with the disappearance of large grazers in the Holocene. The concept of Pleistocene rewilding is based on the idea that large herbivore grazing significantly alters plant communities and can be employed to recreate lost ecosystems. On the other hand, modern rangeland ecology emphasizes the often overriding importance of harsh climates. We visited two rewilding projects and three rangeland regions, sampling a total of 210 vegetation relevés in steppe and surrounding vegetation (grasslands, shrublands and forests) along an extensive climatic gradient across Yakutia, Russia. We analyzed species composition, plant traits, diversity indices and vegetation productivity, using partial canonical correspondence and redundancy analysis. Macroclimate was most important for vegetation composition, and microclimate for the occurrence of extrazonal steppes. Macroclimate and soil conditions mainly determined productivity of vegetation. Bison grazing was responsible for small-scale changes in vegetation through trampling, wallowing and debarking, thus creating more open and disturbed plant communities, soil compaction and xerophytization. However, the magnitude of effects depended on density and type of grazers as well as on interactions with climate and site conditions. Effects of bison grazing were strongest in the continental climate of Central Yakutia, and steppes were generally less affected than meadows. We conclude that contemporary grazing overall has rather limited effects on vegetation in northeastern Siberia. Current rewilding practices are still far from recreating a mammoth steppe, although large herbivores like bison can create more open and drier vegetation and increase nutrient availability in particular in the more continental Central Yakutian Plain.

Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.

Abstract Vegetation-plot resurvey data are a main source of information on terrestrial biodiversity change, with records reaching back more than one century. Although more and more data from re-sampled plots have been published, there is not yet a comprehensive open-access dataset available for analysis. Here, we compiled and harmonised vegetation-plot resurvey data from Germany covering almost 100 years. We show the distribution of the plot data in space, time and across habitat types of the European Nature Information System (EUNIS). In addition, we include metadata on geographic location, plot size and vegetation structure. The data allow temporal biodiversity change to be assessed at the community scale, reaching back further into the past than most comparable data yet available. They also enable tracking changes in the incidence and distribution of individual species across Germany. In summary, the data come at a level of detail that holds promise for broadening our understanding of the mechanisms and drivers behind plant diversity change over the last century.