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.     

How Nature Morphs Peptide Scaffolds into Antibiotics

The conventional notion that peptides are poor candidates for orally available drugs because of protease‐sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide‐based frameworks that are synthesized and utilized by Nature. Several of these antibiotics, including penicillin and vancomycin, are employed to treat bacterial infections in humans and have been best‐selling therapeutics for decades. Others might provide new platforms for the design of novel therapeutics to combat emerging antibiotic‐resistant bacterial pathogens.     

Biosynthesis and Genome Mining Potentials of Nucleoside Natural Products

Nucleoside natural products show diverse biological activities and serve as leads for various application purposes, including human and veterinary medicine and agriculture. Studies in the past decade revealed that these nucleosides are biosynthesized through divergent mechanisms, in which early steps of the pathways can be classified into two types (C5' oxidation and C5' radical extension), while the structural diversity is created by downstream tailoring enzymes. Based on this biosynthetic logic, we investigated the genome mining discovery potentials of these nucleosides using the two enzymes representing the two types of C5' modifications: LipL-type α-ketoglutarate (α-KG) and Fe-dependent oxygenases and NikJ-type radical S-adenosyl-L-methionine (SAM) enzymes. The results suggest that this approach allows discovery of putative nucleoside biosynthetic gene clusters (BGCs) and the prediction of the core nucleoside structures. The results also revealed the distribution of these pathways in nature and implied the possibility of future genome mining discovery of novel nucleoside natural products.     

Parallel Post‐Polyketide Synthase Modification Mechanism Involved in FD‐891 Biosynthesis in Streptomyces graminofaciens A‐8890

To isolate a key polyketide biosynthetic intermediate for the 16‐membered macrolide FD‐891 ( 1 ), we inactivated two biosynthetic genes coding for post‐polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD‐892 ( 2 ), which lacks the epoxide moiety at C8–C9, the hydroxy group at C10, and the O‐methyl group at O‐25 of FD‐891, was isolated from the gfsF/gfsG double‐knockout mutant. In addition, 25‐O‐methyl‐FD‐892 ( 3 ) and 25‐O‐demethyl‐FD‐891 ( 4 ) were isolated from the gfsF and gfsG mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of 2 and 4 in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8‐C9 of 2 and 3 and subsequent hydroxylation at C10, to afford 4 and 1 , respectively. These results suggest that a parallel post‐PKS modification mechanism is involved in FD‐891 biosynthesis.     

Functional Characterization of PyrG,an Unusual Nonribosomal Peptide Synthetase Module from the Pyridomycin Biosynthetic Pathway

Pyridomycin is an antimycobacterial cyclodepsipeptide assembled by a nonribosomal peptide synthetase/polyketide synthase hybrid system. Analysis of its cluster revealed a nonribosomal peptide synthetase (NRPS) module, PyrG, that contains two tandem adenylation domains and a PKS‐type ketoreductase domain. In this study, we biochemically validated that the second A domain recognizes and activates α‐keto‐β‐methylvaleric acid (2‐KVC) as the native substrate; the first A domain was not functional but might play a structural role. The KR domain catalyzed the reduction of the 2‐KVC tethered to the peptidyl carrier protein of PyrG in the presence of the MbtH family protein, PyrH. PyrG was demonstrated to recognize many amino acids. This substrate promiscuity provides the potential to generate pyridomycin analogues with various enolic acids moiety; this is important for binding InhA, a critical enzyme for cell‐wall biosynthesis in Mycobacterium tuberculosis.     

Molecular Cross‐Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions

Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N‐terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post‐translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.     

Post‐translational Modification in Microviridin Biosynthesis

Cyanobacteria are prolific producers of bioactive natural products that mostly belong to the nonribosomal peptide and polyketide classes. We show here how a linear precursor peptide of microviridin K, a new member of the microviridin class of peptidase inhibitors, is processed to become the mature tricyclic peptidase inhibitor. The microviridin (mvd) biosynthetic gene cluster of P. agardhii comprises six genes encoding microviridin K, an apparently unexpressed second microviridin, two RimK homologues, an acetyltransferase, and an ABC transporter. We have over‐expressed three enzymes of this pathway and have demonstrated their biochemical function in vitro through chemical degradation and mass spectrometry. We show that a prepeptide undergoes post‐translational modification through cross‐linking by ester and amide bond formation by the RimK homologues MvdD and MvdC, respectively. In silico analysis of the mvd gene cluster suggests the potential for widespread occurrence of microviridin‐like compounds in a broad range of bacteria.     

Role of Two Exceptional trans Adenylation Domains and MbtH-like Proteins in the Biosynthesis of the Nonribosomal Peptide WS9324A from Streptomyces calvus ATCC 13382

Nonribosomal peptide synthetases (NRPS) are organized in a modular arrangement. Usually, the modular order corresponds to the assembly of the amino acids in the respective peptide, following the collinearity rule. The WS9326A biosynthetic gene cluster from Streptomyces calvus shows deviations from this rule. Most interesting is the presence of two trans adenylation domains that are located downstream of the modular NRPS arrangement. Adenylation domains are responsible for the activation of their respective amino acids. In this study, we confirmed the involvement of the trans adenylation domains in WS9326A biosynthesis by performing gene knockout experiments and by observing the selective adenylation of their predicted amino acid substrates in vitro. We conclude that the trans adenylation domains are essential for WS9326A biosynthesis. Moreover, both adenylation domains are observed to have MbtH-like protein dependency. Overall, we conclude that the trans adenylation domains are essential for WS9326A biosynthesis.     

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.     

Biosynthetic Gene Cluster of a d‐Tryptophan‐Containing Lasso Peptide,MS‐271

MS‐271, produced by Streptomyces sp. M‐271, is a lasso peptide natural product comprising 21 amino acid residues with a d ‐tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS‐271, especially the mechanism of d ‐Trp introduction, is of great interest. The MS‐271 biosynthetic gene cluster was identified by draft genome sequencing of the MS‐271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C‐terminal tryptophan. This suggested that the d ‐Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS‐271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS‐271 production including a gene for a new peptide epimerase. Furthermore, a gene‐deletion experiment indicated that MslB1, ‐B2, ‐C and ‐H were indispensable for MS‐271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS‐271.     

Tailoring Enzyme Stringency Masks the Multispecificity of a Lyngbyatoxin (Indolactam Alkaloid) Nonribosomal Peptide Synthetase

Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.     

Insect‐Specific Production of New GameXPeptides in Photorhabdus luminescens TTO1, Widespread Natural Products in Entomopathogenic Bacteria

Discovery of new natural products by heterologous expression reaches its limits, especially when specific building blocks are missing in the heterologous host or the production medium. Here, we describe the insect‐specific production of the new GameXPeptides E–H ( 5 – 8 ) from Photorhabdus luminescens TTO1, which can be produced heterologously from expression of the GameXPeptide synthetase GxpS only upon supplementation of the production media with the missing building blocks, and thus must be regarded as the true natural products under natural conditions.     

Rational Design,Synthesis, and Biological Evaluation of Lactam‐Bridged Gramicidin A Analogues: Discovery of a Low‐Hemolytic Antibacterial Peptide

A linear peptide, gramicidin A (GA), folds into a β^6.3‐helix, functions as an ion channel in the cell membrane, and exerts antibacterial activity. Herein we describe the rational design, synthesis, and biological evaluation of lactam‐bridged GA analogues. The GA analogue with a 27‐membered macrolactam was found to adopt a stable β^6.3‐helical conformation and exhibits higher ion‐exchange activity than GA. Furthermore, this GA analogue retains the potent antibiotic activity of GA, but its hemolytic activity and toxicity toward mammalian cells are significantly lower than those of GA. This study thus dissociates the antibacterial and hemolytic/cytotoxic activities of GA, and charts a rational path forward for the development of new ion‐channel‐based antibiotics.     

Expanding Substrate Promiscuity by Engineering a Novel Adenylating‐Methylating NRPS Bifunctional Enzyme

Nonribosomal peptides synthetases (NRPSs), which are multifunctional mega‐enzymes producing many biologically active metabolites, are ideal targets for enzyme engineering. NRPS adenylation domains play a critical role in selecting/activating the amino acids to be transferred to downstream NRPS domains in the biosynthesis of natural products. Both monofunctional and bifunctional A domains interrupted with an auxiliary domain are found in nature. Here, we show that a bifunctional interrupted A domain can be uninterrupted by deleting its methyltransferase auxiliary domain portion to make an active monofunctional enzyme. We also demonstrate that a portion of an auxiliary domain with almost no sequence identity to the original auxiliary domain can be insert into naturally interrupted A domain to develop a new active bifunctional A domain with increased substrate profile. This work shows promise for the creation of new interrupted A domains in engineered NRPS enzymes.