In vitro Synthesis of New Cyclodepsipeptides of the PF1022‐Type: Probing the α‐D‐Hydroxy Acid Tolerance of PF1022 Synthetase

The nonribosomal peptide synthetase PF1022‐synthetase (PFSYN) synthesises the cyclooctadepsipeptide PF1022 from the building blocks D ‐lactate, D ‐phenyllactate and N‐methylleucine. The substrate tolerance of PFSYN for hydroxy acids was probed by in vitro screening of a set of aliphatic and aromatic α‐D ‐hydroxy acids with various structural modifications in the side chain. Thus, new PF1022 derivatives for example, propargyl‐D ‐lactyl‐PF1022 and β‐thienyl‐D ‐lactyl‐PF1022 were generated. The promiscuous behaviour of PFSYN towards aliphatic and aromatic α‐D ‐hydroxy acids is considerably larger than that of related enniatin synthetase (ESYN) and thus gives rise to the enzymatic generation of various new PF1022 derivatives.     

Mechanistic Probes for the Epimerization Domain of Nonribosomal Peptide Synthetases

Nonribosomal peptide synthetases (NRPSs) are responsible for the synthesis of a variety of bioactive natural products with clinical and economic significance. Interestingly, these large multimodular enzyme machineries incorporate nonproteinogenic d -amino acids through the use of auxiliary epimerization domains, converting l -amino acids into d -amino acids that impart into the resulting natural products unique bioactivity and resistance to proteases. Due to the large and complex nature of NRPSs, several questions remain unanswered about the mechanism of the catalytic domain reactions. We have investigated the use of mechanism-based crosslinkers to probe the mechanism of an epimerization domain in gramicidin S biosynthesis. In addition, MD simulations were performed, showcasing the possible roles of catalytic residues within the epimerization domain.     

Functional characterization of the recombinant N-methyltransferase domain from the multienzyme enniatin synthetase

A 51 kDa fusion protein incorporating the N-methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S-adenosyl methionine (AdoMet) as demonstrated by cross-linking experiments with (14)C-methyl-AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H(2) and H(8) of the adenine moiety, H(1') of D-ribose, and S-CH(3) group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) of L-Val, L-Ile, and L-Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4'-phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl-SNAC and (14)C-methyl-AdoMet for various lengths of time, for up to 30 min. N-[(14)C-Methyl]-aminoacyl-SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N-[(14)C-methyl] amino acids. Further proof for the formation of N-(14)C-methyl-aminoacyl-SNACs came from MALDI-TOF mass spectrometry which yielded 23 212 Da for N-methyl-valyl-SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L-Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D-Val was not accepted as a substrate; this indicates selectivity for the L isomer.     

Antifungal Peptides and Proteins to Control Toxigenic Fungi and Mycotoxin Biosynthesis

The global challenge to prevent fungal spoilage and mycotoxin contamination on food and feed requires the development of new antifungal strategies. Antimicrobial peptides and proteins (AMPs) with antifungal activity are gaining much interest as natural antifungal compounds due to their properties such as structure diversity and function, antifungal spectrum, mechanism of action, high stability and the availability of biotechnological production methods. Given their multistep mode of action, the development of fungal resistance to AMPs is presumed to be slow or delayed compared to conventional fungicides. Interestingly, AMPs also accomplish important biological functions other than antifungal activity, including anti-mycotoxin biosynthesis activity, which opens novel aspects for their future use in agriculture and food industry to fight mycotoxin contamination. AMPs can reach intracellular targets and exert their activity by mechanisms other than membrane permeabilization. The mechanisms through which AMPs affect mycotoxin production are varied and complex, ranging from oxidative stress to specific inhibition of enzymatic components of mycotoxin biosynthetic pathways. This review presents natural and synthetic antifungal AMPs from different origins which are effective against mycotoxin-producing fungi, and aims at summarizing current knowledge concerning their additional effects on mycotoxin biosynthesis. Antifungal AMPs properties and mechanisms of action are also discussed.     

Identifying the Minimal Enzymes Required for Biosynthesis of Epoxyketone Proteasome Inhibitors

Epoxyketone proteasome inhibitors have attracted much interest due to their potential as anticancer drugs. Although the biosynthetic gene clusters for several peptidyl epoxyketone natural products have recently been identified, the enzymatic logic involved in the formation of the terminal epoxyketone pharmacophore has been relatively unexplored. Here, we report the identification of the minimal set of enzymes from the eponemycin gene cluster necessary for the biosynthesis of novel metabolites containing a terminal epoxyketone pharmacophore in Escherichia coli, a versatile and fast‐growing heterologous host. This set of enzymes includes a non‐ribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), and an acyl‐CoA dehydrogenase (ACAD) homologue. In addition to the in vivo functional reconstitution of these enzymes in E. coli, in vitro studies of the eponemycin NRPS and ¹³C‐labeled precursor feeding experiments were performed to advance the mechanistic understanding of terminal epoxyketone formation.     

Synthetic Biology: A New Tool for the Trade

Protein–protein interactions are fundamental to many biological processes. Yet, the weak and transient noncovalent bonds that characterize most protein–protein interactions found in nature impose limits on many bioengineering experiments. Here, a new class of genetically encodable peptide–protein pairs—isopeptag‐N/pilin‐N, isopeptag/pilin‐C, and SpyTag/SpyCatcher—that interact through autocatalytic intermolecular isopeptide bond formation is described. Reactions between peptide–protein pairs are specific, robust, orthogonal, and able to proceed under most biologically relevant conditions both in vitro and in vivo. As fusion constructs, they provide a handle on molecules of interest, both organic and inorganic, that can be grasped with an iron grip. Such stable interactions provide robust post‐translational control over biological processes and open new opportunities in synthetic biology for engineering programmable and self‐assembling protein nanoarchitectures.     

Directed Biosynthesis of Phytotoxic Alkaloids in the Cyanobacterium Nostoc 78–12A

Out of the green! Precursor‐directed biosynthesis allowed for the production of new nostocarboline derivatives that display phytotoxic and algicidal properties—in a phototrophic organism. The mechanism of action includes downregulation of photosynthesis, as demonstrated by chlorophyll‐a fluorescence imaging.

     

Deciphering Pactamycin Biosynthesis and Engineered Production of New Pactamycin Analogues

Pactamycin is an aminocyclopentitol‐derived natural product that has potent antibacterial and antitumor activities. Sequence analysis of an 86 kb continuous region of the chromosome from Streptomyces pactum ATCC 27456 revealed a gene cluster involved in the biosynthesis of pactamycin. Gene inactivation of the Fe‐S radical SAM oxidoreductase (ptmC) and the glycosyltransferase (ptmJ), individually abrogated pactamycin biosynthesis; this confirmed the involvement of the ptm gene cluster in pactamycin biosynthesis. The polyketide synthase gene (ptmQ) was found to support 6‐methylsalicylic acid (6‐MSA) synthesis in a heterologous host, S. lividans T7. In vivo inactivation of ptmQ in S. pactum impaired pactamycin and pactamycate production but led to production of two new pactamycin analogues, de‐6‐MSA‐pactamycin and de‐6‐MSA‐pactamycate. The new compounds showed equivalent cytotoxic and antibacterial activities with the corresponding parent molecules and shed more light on the structure–activity relationship of pactamycin.     

Discovery and Biosynthesis of Ureidopeptide Natural Products Macrocyclized via Indole N-acylation in Marine Microbulbifer spp. Bacteria

Commensal bacteria associated with marine invertebrates are underappreciated sources of chemically novel natural products. Using mass spectrometry, we had previously detected the presence of peptidic natural products in obligate marine bacteria of the genus Microbulbifer cultured from marine sponges. In this report, the isolation and structural characterization of a panel of ureidohexapeptide natural products, termed the bulbiferamides, from Microbulbifer strains is reported wherein the tryptophan side chain indole participates in a macrocyclizing peptide bond formation. Genome sequencing identifies biosynthetic gene clusters encoding production of the bulbiferamides and implicates the involvement of a thioesterase in the indolic macrocycle formation. The structural diversity and widespread presence of bulbiferamides in commensal microbiomes of marine invertebrates point toward a possible ecological role for these natural products.     

Interrogating the Tailoring Steps of Pactamycin Biosynthesis and Accessing New Pactamycin Analogues

Pactamycin is a bacteria‐derived aminocyclitol antibiotic with a wide‐range of biological activity. Its chemical structure and potent biological activities have made it an interesting lead compound for drug discovery and development. Despite its unusual chemical structure, many aspects of its formation in nature remain elusive. Using a combination of genetic inactivation and metabolic analysis, we investigated the tailoring processes of pactamycin biosynthesis in Streptomyces pactum. The results provide insights into the sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. We also identified two new pactamycin analogues that have better selectivity indexes than pactamycin against malarial parasites.     

A Chemoproteomics Approach to Investigate Phosphopantetheine Transferase Activity at the Cellular Level

Phosphopantetheinylation is an essential post‐translational protein modification to primary and secondary metabolic pathways that ensures bacterial cell viability and virulence, and it is used in the production of many pharmaceuticals. Traditional methods have not provided a comprehensive understanding of these modifications. By using chemical proteomic probes for adenylation and thiolation domains in nonribosomal peptide synthetases (NRPSs), chemoproteomics has been applied to survey and validate the cellular activity of 4‐[3‐chloro‐5‐(trifluoromethyl)pyridin‐2‐yl]‐N‐(4‐methoxypyridin‐2‐yl)piperazine‐1‐carbothioamide (ML267), which is a potent and selective small‐molecule 4′‐phosphopantetheinyl transferase (PPTase) inhibitor that attenuates secondary metabolism and viability of bacterial cells. ML267 inhibited Sfp‐type PPTase and antagonized phosphopantetheinylation in cells, which resulted in a decrease in phosphopantetheinylated NRPSs and the attenuation of Sfp‐PPTase‐dependent metabolite production. These results indicate that this chemoproteomics platform should enable a precise interpretation of the cellular activities of Sfp‐type PPTase inhibitors.     

Biosynthesis of the Peptide Antibiotic Feglymycin by a Linear Nonribosomal Peptide Synthetase Mechanism

Feglymycin, a peptide antibiotic produced by Streptomyces sp. DSM 11171, consists mostly of nonproteinogenic phenylglycine‐type amino acids. It possesses antibacterial activity against methicillin‐resistant Staphylococcus aureus strains and antiviral activity against HIV. Inhibition of the early steps of bacterial peptidoglycan synthesis indicated a mode of action different from those of other peptide antibiotics. Here we describe the identification and assignment of the feglymycin (feg) biosynthesis gene cluster, which codes for a 13‐module nonribosomal peptide synthetase (NRPS) system. Inactivation of an NRPS gene and supplementation of a hydroxymandelate oxidase mutant with the amino acid l ‐Hpg proved the identity of the feg cluster. Feeding of Hpg‐related unnatural amino acids was not successful. This characterization of the feg cluster is an important step to understanding the biosynthesis of this potent antibacterial peptide.