Unveiling the Biosynthetic Pathway of the Ribosomally Synthesized and Post‐translationally Modified Peptide Ustiloxin B in Filamentous Fungi

The biosynthetic machinery of the first fungal ribosomally synthesized and post‐translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side‐chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.     

Radical S‐Adenosyl Methionine Epimerases: Regioselective Introduction of Diverse D‐Amino Acid Patterns into Peptide Natural Products

PoyD is a radical S‐adenosyl methionine epimerase that introduces multiple D ‐configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel post‐translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D ‐amino acid patterns into all‐L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering.     

Leader Peptide‐Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease‐Targeted Libraries

Microviridins are a family of ribosomally synthesized and post‐translationally modified peptides with a highly unusual architecture featuring non‐canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP‐grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT‐type N‐acetyltransferase. The method facilitates the efficient in vitro one‐pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo‐enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure–activity relationships for respective target proteases.     

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine‐rich snakin‐1 and ‐2 antimicrobial peptides by using a combination of solid‐phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40–50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin‐2 compared to natural snakin‐2, we demonstrated that synthetic snakin‐2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin‐2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.     

In Vitro Reconstitution Reveals a Central Role for the N-Oxygenase PvfB in (Dihydro)pyrazine-N-oxide and Valdiazen Biosynthesis

The Pseudomonas virulence factor (pvf) operon is essential for the biosynthesis of two very different natural product scaffolds: the (dihydro)pyrazine-N-oxides and the diazeniumdiolate, valdiazen. PvfB is a member of the non-heme diiron N-oxygenase enzyme family that commonly convert anilines to their nitroaromatic counterparts. In contrast, we show that PvfB catalyzes N-oxygenation of the α-amine of valine, first to the hydroxylamine and then the nitroso, while linked to the carrier protein of PvfC. PvfB modification of PvfC-tethered valine was observed directly by protein NMR spectroscopy, establishing the intermediacy of the hydroxylamine. This work reveals a central role for PvfB in the biosynthesis of (dihydro)pyrazine-N-oxides and valdiazen.     

Xanthomonins I–III: A New Class of Lasso Peptides with a Seven‐Residue Macrolactam Ring

Lasso peptides belong to the class of ribosomally synthesized and post‐translationally modified peptides. Their common distinguishing feature is an N‐terminal macrolactam ring that is threaded by the C‐terminal tail. This lasso fold is maintained through steric interactions. The isolation and characterization of xanthomonins I–III, the first lasso peptides featuring macrolactam rings consisting of only seven amino acids, is now presented. The crystal structure of xanthomonin I and the NMR structure of xanthomonin II were also determined. A total of 25 variants of xanthomonin II were generated to probe different aspects of the biosynthesis, stability, and fold maintenance. These mutational studies reveal the limits such a small ring imposes on the threading and show that every plug amino acid larger than serine is able to maintain a heat‐stable lasso fold in the xanthomonin II scaffold.     

Structure and Biosynthesis of Crocagins: Polycyclic Posttranslationally Modified Ribosomal Peptides from Chondromyces crocatus

Secondary metabolome mining efforts in the myxobacterial multiproducer of natural products, Chondromyces crocatus Cm c5, resulted in the isolation and structure elucidation of crocagins, which are novel polycyclic peptides containing a tetrahydropyrrolo[2,3-b]indole core. The gene cluster was identified through an approach combining genome analysis, targeted gene inactivation in the producer, and in vitro experiments. Based on our findings, we developed a biosynthetic scheme for crocagin biosynthesis. These natural products are formed from the three C-terminal amino acids of a precursor peptide and thus belong to a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs). We demonstrate that crocagin A binds to the carbon storage regulator protein CsrA, thereby inhibiting the ability of CsrA to bind to its cognate RNA target.     

Biosynthesis of the Enterotoxic Pyrrolobenzodiazepine Natural Product Tilivalline

The nonribosomal enterotoxin tilivalline was the first naturally occurring pyrrolobenzodiazepine to be linked to disease in the human intestine. Since the producing organism Klebsiella oxytoca is part of the intestinal microbiota and the pyrrolobenzodiazepine causes the pathogenesis of colitis it is important to understand the biosynthesis and regulation of tilivalline activity. Here we report the biosynthesis of tilivalline and show that this nonribosomal peptide assembly pathway initially generates tilimycin, a simple pyrrolobenzodiazepine with cytotoxic properties. Tilivalline results from the non‐enzymatic spontaneous reaction of tilimycin with biogenetically generated indole. Through a chemical total synthesis of tilimycin we could corroborate the predictions made about the biosynthesis. Production of two cytotoxic pyrrolobenzodiazepines with distinct functionalities by human gut resident Klebsiella oxytoca has important implications for intestinal disease.     

Biosynthesis and Chemical Synthesis of Presilphiperfolanol Natural Products

Presilphiperfolanols constitute a family of biosynthetically important sesquiterpenes which can rearrange to diverse sesquiterpenoid skeletons. While the origin of these natural products can be traced to simple linear terpene precursors, the details of the enzymatic cyclization mechanism that forms the stereochemically dense tricyclic skeleton has required extensive biochemical, computational, and synthetic investigation. Parallel efforts to prepare the unique and intriguing structures of these compounds by total synthesis have also inspired novel strategies, thus resulting in four synthetic approaches and two completed syntheses. While the biosynthesis and chemical synthesis studies performed to date have provided much insight into the role and properties of these molecules, emerging questions regarding the biosynthesis of newer members of the family and subtle details of rearrangement mechanisms have yet to be explored.     

Mechanistic Insights into the Radical S‐adenosyl‐l‐methionine Enzyme NosL From a Substrate Analogue and the Shunt Products

The radical S‐adenosyl‐l ‐methionine (SAM) enzyme NosL catalyzes the transformation of l ‐tryptophan into 3‐methyl‐2‐indolic acid (MIA), which is a key intermediate in the biosynthesis of a clinically interesting antibiotic nosiheptide. NosL catalysis was investigated by using the substrate analogue 2‐methyl‐3‐(indol‐3‐yl)propanoic acid (MIPA), which can be converted into MIA by NosL. Biochemical assays with different MIPA isotopomers in D₂O and H₂O unambiguously indicated that the 5′‐deoxyadenosyl (dAdo)‐radical‐mediated hydrogen abstraction is from the amino group of l ‐tryptophan and not a protein residue. Surprisingly, the dAdo‐radical‐mediated hydrogen abstraction occurs at two different sites of MIPA, thereby partitioning the substrate into different reaction pathways. Together with identification of an α,β‐unsaturated ketone shunt product, our study provides valuable mechanistic insight into NosL catalysis and highlights the remarkable catalytic flexibility of radical SAM enzymes.     

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.     

Production of the Bengamide Class of Marine Natural Products in Myxobacteria: Biosynthesis and Structure–Activity Relationships

The bengamides, sponge‐derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M. virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M. xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half‐life in mice and antitumor efficacy in a melanoma mouse model.     

Structure Elucidation and Activity of Kolossin A,the D‐/L‐Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D ‐/ L ‐peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness.     

Structure and Biosynthesis of Xenoamicins from Entomopathogenic Xenorhabdus

During the search for novel natural products from entomopathogenic Xenorhabdus doucetiae DSM17909 and X. mauleonii DSM17908 novel peptides named xenoamicins were identified in addition to the already known antibiotics xenocoumacin and xenorhabdin. Xenoamicins are acylated tridecadepsipeptides consisting of mainly hydrophobic amino acids. The main derivative xenoamicin A ( 1 ) was isolated from X. mauleonii DSM17908, and its structure elucidated by detailed 1 D and 2 D NMR experiments. Detailed MS experiments, also in combination with labeling experiments, confirmed the determined structure and allowed structure elucidation of additional derivatives. Moreover, the xenoamicin biosynthesis gene cluster was identified and analyzed in X. doucetiae DSM17909, and its participation in xenoamicin biosynthesis was confirmed by mutagenesis. Advanced Marfey’s analysis of 1 showed that the absolute configuration of the amino acids is in agreement with the predicted stereochemistry deduced from the nonribosomal peptide synthetase XabABCD. Biological testing revealed activity of 1 against Plasmodium falciparum and other neglected tropical diseases but no antibacterial activity.     

Epigenetic Genome Mining of an Endophytic Fungus Leads to the Pleiotropic Biosynthesis of Natural Products

The small‐molecule biosynthetic potential of most filamentous fungi has remained largely unexplored and represents an attractive source for the discovery of new compounds. Genome sequencing of Calcarisporium arbuscula, a mushroom‐endophytic fungus, revealed 68 core genes that are involved in natural product biosynthesis. This is in sharp contrast to the predominant production of the ATPase inhibitors aurovertin B and D in the wild‐type fungus. Inactivation of a histone H3 deacetylase led to pleiotropic activation and overexpression of more than 75 % of the biosynthetic genes. Sampling of the overproduced compounds led to the isolation of ten compounds of which four contained new structures, including the cyclic peptides arbumycin and arbumelin, the diterpenoid arbuscullic acid A, and the meroterpenoid arbuscullic acid B. Such epigenetic modifications therefore provide a rapid and global approach to mine the chemical diversity of endophytic fungi.     

Type S Non-Ribosomal Peptide Synthetases for the Rapid Generation of Tailormade Peptide Libraries**

Bacterial natural products in general, and non-ribosomally synthesized peptides in particular, are structurally diverse and provide us with a broad range of pharmaceutically relevant bioactivities. Yet, traditional natural product research suffers from rediscovering the same scaffolds and has been stigmatized as inefficient, time-, labour- and cost-intensive. Combinatorial chemistry, on the other hand, can produce new molecules in greater numbers, cheaper and in less time than traditional natural product discovery, but also fails to meet current medical needs due to the limited biologically relevant chemical space that can be addressed. Consequently, methods for the high throughput generation of new natural products would offer a new approach to identifying novel bioactive chemical entities for the hit to lead phase of drug discovery programs. As a follow-up to our previously published proof-of-principle study on generating bipartite type S non-ribosomal peptide synthetases (NRPSs), we now envisaged the de novo generation of non-ribosomal peptides (NRPs) on an unreached scale. Using synthetic zippers, we split NRPSs in up to three subunits and rapidly generated different bi- and tripartite NRPS libraries to produce 49 peptides, peptide derivatives, and de novo peptides at good titres up to 145 mg L⁻¹. A further advantage of type S NRPSs not only is the possibility to easily expand the created libraries by re-using previously created type S NRPS, but that functions of individual domains as well as domain-domain interactions can be studied and assigned rapidly.