Nonribosomal Peptide Assembly Research Articles

Genome Mining Reveals Endopyrroles from a Nonribosomal Peptide Assembly Line Triggered in Fungal-Bacterial Symbiosis.

The bacterial endosymbiont (Burkholderia rhizoxinica) of the rice seedling blight fungus (Rhizopus microsporus) harbors a large number of cryptic biosynthesis gene clusters. Genome mining and sequence similarity networks based on an encoded nonribosomal peptide assembly line and the associated pyrrole-forming enzymes in the symbiont indicated that the encoded metabolites are unique among a large number of tentative pyrrole natural products in diverse and unrelated bacterial phyla. By performing comparative metabolic profiling using a mutant generated with an improved pheS Burkholderia counterselection marker, we found that the symbionts' biosynthetic pathway is mainly activated under salt stress and exclusively in symbiosis with the fungal host. The cryptic metabolites were fully characterized as novel pyrrole-substituted depsipeptides (endopyrroles). A broader survey showed that endopyrrole production is a hallmark of geographically distant endofungal bacteria, which produce the peptides solely under symbiotic conditions.

Fusaoctaxin A, an Example of a Two-Step Mechanism for Non-Ribosomal Peptide Assembly and Maturation in Fungi.

Fungal non-ribosomal peptide synthetase (NRPS) clusters are spread across the chromosomes, where several modifying enzyme-encoding genes typically flank one NRPS. However, a recent study showed that the octapeptide fusaoctaxin A is tandemly synthesized by two NRPSs in Fusarium graminearum. Here, we illuminate parts of the biosynthetic route of fusaoctaxin A, which is cleaved into the tripeptide fusatrixin A and the pentapeptide fusapentaxin A during transport by a cluster-specific ABC transporter with peptidase activity. Further, we deleted the histone H3K27 methyltransferase kmt6, which induced the production of fusaoctaxin A.

Fimsbactin and Acinetobactin Compete for the Periplasmic Siderophore Binding Protein BauB in Pathogenic Acinetobacter baumannii.

Environmental and pathogenic microbes produce siderophores as small iron-binding molecules to scavenge iron from natural environments. It is common for microbes to produce multiple siderophores to gain a competitive edge in mixed microbial environments. Strains of human pathogenic Acinetobacter baumannii produce up to three siderophores: acinetobactin, baumannoferrin, and fimsbactin. Production of acinetobactin and baumannoferrin is highly conserved among clinical isolates while fimsbactin production appears to be less common. Fimsbactin is structurally related to acinetobactin through the presence of catecholate and phenolate oxazoline metal-binding motifs, and both are derived from nonribosomal peptide assembly lines with similar catalytic domain orientations and identities. Here we report on the chemical, biochemical, and microbiological investigation of fimsbactin and acinetobactin alone and in combination. We show that fimsbactin forms a 1:1 complex with iron(III) that is thermodynamically more stable than the 2:1 acinetobactin ferric complex. Alone, both acinetobactin and fimsbactin stimulate A. baumannii growth, but in combination the two siderophores appear to compete and collectively inhibit bacterial growth. We show that fimsbactin directly competes with acinetobactin for binding the periplasmic siderophore-binding protein BauB suggesting a possible biochemical mechanism for the phenomenon where the buildup of apo-siderophores in the periplasm leads to iron starvation. We propose an updated model for siderophore utilization and competition in A. baumannii that frames the molecular, biochemical, and cellular interplay of multiple iron acquisition systems in a multidrug resistant Gram-negative human pathogen.

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.

Novel chemical probes for the investigation of nonribosomal peptide assembly.

Chemical probes were devised and evaluated for the capture of biosynthetic intermediates involved in the bio-assembly of the nonribosomal peptide echinomycin. Putative intermediate peptide species were isolated and characterised, providing fresh insights into pathway substrate flexibility and paving the way for novel chemoenzymatic approaches towards unnatural peptides.

Correction: Novel chemical probes for the investigation of nonribosomal peptide assembly.

Correction for 'Novel chemical probes for the investigation of nonribosomal peptide assembly' by Y. T. Candace Ho et al., Chem. Commun., 2017, 53, 7088-7091.

Recent advances in engineering nonribosomal peptide assembly lines.

Nonribosomal peptides are amongst the most widespread and structurally diverse secondary metabolites in nature with many possessing bioactivity that can be exploited for therapeutic applications. Due to the major challenges associated with total- and semi-synthesis, bioengineering approaches have been developed to increase yields and generate modified peptides with improved physicochemical properties or altered bioactivity. Here we review the major advances that have been made over the last decade in engineering the biosynthesis of nonribosomal peptides. Structural diversity has been introduced by the modification of enzymes required for the supply of precursors or by heterologous expression of tailoring enzymes. The modularity of nonribosomal peptide synthetase (NRPS) assembly lines further supports module or domain swapping methodologies to achieve changes in the amino acid sequence of nonribosomal peptides. We also review the new synthetic biology technologies promising to speed up the process, enabling the creation and optimisation of many more assembly lines for heterologous expression, offering new opportunities for engineering the biosynthesis of novel nonribosomal peptides.

Glycopeptide antibiotic biosynthesis

Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.

Heterologous Expression and Structural Characterisation of a Pyrazinone Natural Product Assembly Line

Through a number of strategies nonribosomal peptide assembly lines give rise to a metabolic diversity not possible by ribosomal synthesis. One distinction within nonribosomal assembly is that products are elaborated on an enzyme-tethered substrate, and their release is enzyme catalysed. Reductive release by NAD(P)H-dependent catalysts is one observed nonribosomal termination and release strategy. Here we probed the selectivity of a terminal reductase domain by using a full-length heterologously expressed nonribosomal peptide synthetase for the dipeptide aureusimine and were able to generate 17 new analogues. Further, we generated an X-ray structure of aureusimine terminal reductase to gain insight into the structural details associated with this enzymatic domain.

Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line

Nonribosomal peptide synthetases (NRPSs) usually catalyze the biosynthesis of peptide natural products by sequential selection, activation, and condensation of amino acid precursors. It was reported that some fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines as an alternative to amino acids. Ecteinascidin 743 (ET-743), naphthyridinomycin (NDM), and quinocarcin (QNC) are three important antitumor natural products belonging to the tetrahydroisoquinoline family. Although ET-743 has been approved as an anticancer drug, the origin of an identical two-carbon (C(2)) fragment among these three antibiotics has not been elucidated despite much effort in the biosynthetic research in the past 30 y. Here we report that two unexpected two-component transketolases (TKases), NapB/NapD in the NDM biosynthetic pathway and QncN/QncL in QNC biosynthesis, catalyze the transfer of a glycolaldehyde unit from ketose to the lipoyl group to yield the glycolicacyl lipoic acid intermediate and then transfer the C(2) unit to an acyl carrier protein (ACP) to form glycolicacyl-S-ACP as an extender unit for NRPS. Our results demonstrate a unique NRPS extender unit directly derived from ketose phosphates through (α,β-dihydroxyethyl)-thiamin diphosphate and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis, all of which also highlights the biosynthesis of ET-743. This hybrid system and precursor are distinct from the previously described universal modes involving the NRPS machinery. They exemplify an alternate strategy in hybrid NRPS biochemistry and enrich the diversity of precursors for NRPS combinatorial biosynthesis.

Biosynthesis and function of secondary metabolites

Natural products have long been used by humans owing to their beneficial effects. Indulgences such as coffee and tea, with their moderate stimulatory properties, have a long cultural tradition and are today some of the most important agricultural products worldwide. Other drugs have much stronger impacts on the central nervous system. For example, coca was originally used by the Inca civilisation because of its strong stimulatory effects, which result in feelings of happiness and euphoria. Opium was used by many ancient cultures as an efficient analgesic, but this also illustrates one of the first examples of drug abuse as it was used by warriors as an anxiolytic when girding their loins. Besides their pleasant traits these highly active drugs have adverse side effects, including most importantly an extremely high potential to cause addiction in combination with physical and mental prostration upon long-term abuse. Therefore, the production, importation, trading, and possession of these drugs are today under strict control by public authorities. Another historically interesting example of drug misuse is given by the death penalty imposed on Socrates in 399 B.C., carried out by the forced ingestion of the proverbial cup of hemlock. These traditional and historical examples of the usage of bioactive natural products all originated in the ancient cultures, without any knowledge about the underlying chemistry. This situation started to change at the beginning of the 19th century, when Friedrich Wilhelm Adam Serturner (1783–1841) first isolated morphine from opium, naming it after Morpheus, the Greek god of dreams and demonstrating its activity in self-tests, as was typical practice during these early times of chemistry. A likewise important discovery was made by Alexander Fleming (1881–1955), who isolated the antibiotic penicillin from the mould Penicillium notatum in a groundbreaking contribution, which was awarded the Nobel Prize in 1945. The discovery of new bioactive natural products is still a fascinating field in organic chemistry as demonstrated by the recent paradigms of the anticancer drug epothilon, the immunosuppressant rapamycin, or the proteasome inhibitor salinosporamide, to name but a few of hundreds of possible examples. Finding new secondary metabolites is a prerequisite for the development of novel pharmaceuticals, and this is an especially urgent task in the case of antibiotics due to the rapid spreading of bacterial resistances and the emergence of multiresistant pathogenic strains, which poses severe clinical problems in the treatment of infectious diseases. This Thematic Series on the biosynthesis and function of secondary metabolites deals with the discovery of new biologically active compounds from all kinds of sources, including plants, bacteria, and fungi, and also with their biogenesis. Biosynthetic aspects are closely related to functional investigations, because a deep understanding of metabolic pathways to natural products, not only on a chemical, but also on a genetic and enzymatic level, allows for the expression of whole biosynthetic gene clusters in heterologous hosts. This technique can make interesting, new secondary metabolites available from unculturable microorganisms, or may be used to optimise their availability by fermentation, for further research and also for production in the pharmaceutical industry. Especially fascinating is the intrinsic logic of the polyketide and nonribosomal peptide biosynthetic machineries, which is strongly correlated with the logic of fatty acid biosynthesis as part of the primary metabolism. Insights into the mechanisms of modular polyketide and nonribosomal peptide assembly lines open up the possibility for direct modifications, e.g., of oxidation states of the natural product’s carbon backbone by simple domain knockouts within the responsible megasynthases, or the introduction of a variety of alternative biosynthetic starters by mutasynthesis approaches, thus leading to new variants of known metabolites, which may have improved properties for therapeutic use. Another interesting aspect is the usage of enzymes in chemical transformations, which can provide synthetic organic chemists with an efficient access route to typically chiral building blocks that may otherwise be difficult to obtain. It is my great pleasure to thank all of the contributors to this Thematic Series, the staff of the Beilstein-Institut, and my esteemed colleague and teacher Prof. Henning Hopf (Braunschweig) for opening the opportunity to guest-edit the present issue. Enjoy reading it! Jeroen S. Dickschat Braunschweig, October 2011

TRNA-dependent peptide bond formation by the transferase PacB in biosynthesis of the pacidamycin group of pentapeptidyl nucleoside antibiotics

Pacidamycins are a family of uridyl tetra/pentapeptide antibiotics with antipseudomonal activities through inhibition of the translocase MraY in bacterial cell wall assembly. The biosynthetic gene cluster for pacidamycins has recently been identified through genome mining of the producer Streptomyces coeruleorubidus, and the highly dissociated nonribosomal peptide assembly line for the uridyl tetrapeptide scaffold of pacidamycin has been characterized. In this work a hypothetical protein PacB, conserved in known uridyl peptide antibiotics gene clusters, has been characterized by both genetic deletion and enzymatic analysis of the purified protein. PacB catalyzes the transfer of the alanyl residue from alanyl-tRNA to the N terminus of the tetrapeptide intermediate yielding a pentapeptide on the thio-templated nonribosomal peptide synthetase (NRPS) assembly line protein PacH. PacB thus represents a new group of tRNA-dependent peptide bond-forming enzymes in secondary metabolite biosynthesis in addition to the recently identified cyclodipeptide synthases. The characterization of PacB completes the assembly line reconstitution of pacidamycin pentapeptide antibiotic scaffolds, bridging the primary and secondary metabolic pathways by hijacking an aminoacyl-tRNA to the antibiotic biosynthetic pathway.

An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in the biosynthesis of calcium-dependent lipopeptide antibiotics

Nonribosomal peptides contain a wide range of unusual non-proteinogenic amino acid residues. As a result, these complex natural products are amongst the most structurally diverse secondary metabolites in nature, and possess a broad spectrum of biological activities. beta-Hydroxylation of amino acid precursors or peptidyl residues and their subsequent processing by downstream tailoring enzymes are some of the most common themes in the biosynthetic diversification of these therapeutically important peptides. Identification and characterization of the biosynthetic intermediates and enzymes involved in these processes are thus pivotal in understanding nonribosomal peptide assembly and modification. To this end, the putative asparaginyl oxygenase- and 3-hydroxyasparaginyl phosphotransferase-encoding genes hasP and asnO were separately deleted from the calcium-dependent antibiotic (CDA) biosynthetic gene cluster of Streptomyces coelicolor. Whilst the parent strains produce a number of 3-hydroxyasparagine- and 3-phosphohydroxyasparagine-containing CDAs, the DeltahasP mutants produce exclusively non-phosphorylated CDAs. On the other hand, DeltaasnO mutants produce several new Asn-containing CDAs not present in the wild-type, which retain calcium-dependent antimicrobial activity. This confirms that AsnO and HasP are required for the beta-hydroxylation and phosphorylation of the Asn residue within CDA.

Timing of epimerization and condensation reactions in nonribosomal peptide assembly lines: kinetic analysis of phenylalanine activating elongation modules of tyrocidine synthetase B.

The cyclic decapeptide antibiotic tyrocidine has D-Phe residues at positions 1 and 4, produced during peptide chain growth from L-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2(-3) bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (L-Phe-L-Phe-S-TycB3 if L-Phe-D-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over time were determined by hydrolysis and chiral TLC separations, allowing for the clear conclusion that epimerization in the internal TycB3 module occurs preferentially on the peptidyl-S-enzyme rather than the aminoacyl-S-enzyme, by a factor of about 3000/1. In turn, this imposes constraints on the chiral selectivity of the condensation (C) domains immediately upstream and downstream of E domains. The stereoselectivity of the upstream C domain was shown to be L-selective at both donor and acceptor sites ((L)C(L)) by site-directed mutagenesis studies of an E domain active site residue and using the small-molecule surrogate D-Phe-Pro-L-Phe-N-acetylcysteamine thioester (D-Phe-Pro-L-Phe-SNAC) and D-Phe-Pro-D-Phe-SNAC as donor probes.