Biosynthetic Gene Cluster of Linaridin Peptides Contains Epimerase Gene

Salinipeptins belong to the type-A linaridin class of ribosomally synthesized and post-translationally modified peptides (RiPPs) comprising 22 amino acid residues with multiple D -amino acids. Although chirality of other type-A linaridins, such as grisemycin and cypemycin, has not been reported, the biosynthetic gene clusters of type-A linaridins have identical gene organization. Here, we report heterologous expression of grisemycin biosynthetic gene cluster (grm) and show that grisemycin contains multiple D -amino acids, similar to salinipeptins. The heterologous expression experiments also confirm the involvement of a novel peptide epimerase in grisemycin biosynthesis. Gene-deletion experiments indicate that grmL, a single gene with unknown function, is indispensable for grisemycin production. We also show that the presence of D -amino acids is likely a common feature of linaridin natural products by analyzing two other type-A linaridin clusters.     

Discovery of Ubonodin,an Antimicrobial Lasso Peptide Active against Members of the Burkholderia cepacia Complex

We report the heterologous expression, structure, and antimicrobial activity of a lasso peptide, ubonodin, encoded in the genome of Burkholderia ubonensis. The topology of ubonodin is unprecedented amongst lasso peptides, with 18 of its 28 amino acids found in the mechanically bonded loop segment. Ubonodin inhibits RNA polymerase in vitro and has potent antimicrobial activity against several pathogenic members of the Burkholderia genus, most notably B. cepacia and B. multivorans, causative agents of lung infections in cystic fibrosis patients.     

Two Opposing d‐Amino Acids Give Zigzag Hairpin Epitopes an Additional Kink to Create Antibody‐Selective Peptide Antigens

We have developed peptides that are able to distinguish between subgroups of polyclonal antibodies. These β‐hairpin peptides act as conformational epitopes with specific shape and flexibility; they have been analyzed by NMR and CD spectroscopy, and have been shown to identify known disease markers. As a standalone mini β‐sheet, a hairpin is stabilized by alternating pairs of hydrogen‐bonded and non‐bonded amino acids on its two opposing peptide strands. A single d mutation disrupts this secondary structure, the correlated double‐d mutation of two opposing amino acids compensates for this destabilizing effect. The designed kink was introduced into both hydrogen‐bonded and ‐non‐bonded positions of an all‐l hairpin that is a known conformational epitope in molecular recognition. Our peptides enabled the discrimination of different human rheumatoid arthritis autoantibodies in an ELISA assay.     

Site‐Directed and Global Incorporation of Orthogonal and Isostructural Noncanonical Amino Acids into the Ribosomal Lasso Peptide Capistruin

Expansion of the structural diversity of peptide antibiotics was performed through two different methods. Supplementation‐based incorporation (SPI) and stop‐codon suppression (SCS) approaches were used for co‐translational incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the lasso peptide capistruin. Two ncAAs were employed for the SPI method and five for the SCS method; each of them probing the incorporation of ncAAs in strategic positions of the molecule. Evaluation of the assembly by HR‐ESI‐MS proved more successful for the SCS method. Bio‐orthogonal chemistry was used for post‐biosynthetic modification of capistruin congener Cap_Alk10 containing the ncAA Alk (Nε‐Alloc‐L ‐lysine) instead of Ala. A second‐generation Hoveyda–Grubbs catalyst was used for an in vitro metathesis reaction with Cap_Alk10 and an allyl alcohol, which offers options for post‐biosynthetic modifications. The use of synthetic biology allows for the in vivo production of new peptide‐based antibiotics from an expanded amino acid repertoire.     

Biosynthetic Gene Cluster for Surugamide A Encompasses an Unrelated Decapeptide,Surugamide F

Genome mining is a powerful method for finding novel secondary metabolites. In our study on the biosynthetic gene cluster for the cyclic octapeptides surugamides A–E (inhibitors of cathepsin B), we found a putative gene cluster consisting of four successive non‐ribosomal peptide synthetase (NRPS) genes, surA, surB, surC, and surD. Prediction of amino acid sequence based on the NRPSs and gene inactivation revealed that surugamides A–E are produced by two NRPS genes, surA and surD, which were separated by two NRPS genes, surB and surC. The latter genes are responsible for the biosynthesis of an unrelated peptide, surugamide F. The pattern of intercalation observed in the sur genes is unprecedented. The structure of surugamide F, a linear decapeptide containing one 3‐amino‐2‐methylpropionic acid (AMPA) residue, was determined by spectroscopic methods and was confirmed by solid‐phase peptide synthesis.     

Genome Mining of the Hitachimycin Biosynthetic Gene Cluster: Involvement of a Phenylalanine‐2,3‐aminomutase in Biosynthesis

Hitachimycin is a macrolactam antibiotic with (S)‐β‐phenylalanine (β‐Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β‐Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine‐2,3‐aminomutase (PAM), five polyketide synthases, four β‐amino‐acid‐carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)‐β‐Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.     

Discovery of the Class I Antimicrobial Lasso Peptide Arcumycin

Lasso peptides are a structurally diverse superfamily of conformationally constrained peptide natural products, of which a subset exhibits broad antimicrobial activity. Although advances in bioinformatics have increased our knowledge of strains harboring the biosynthetic machinery for lasso peptide production, relating peptide sequence to bioactivity remains a continuous challenge. To this end, genome mining investigation of Actinobacteria-produced antimicrobial lasso peptides was performed to correlate predicted structure with antibiotic activity. Bioinformatic evaluation revealed eight putative novel class I lasso peptide sequences. Fermentation of one of these hits, Streptomyces NRRL F-5639, resulted in the production of a novel class I lasso peptide, arcumycin. Arcumycin exhibited antibiotic activity against Gram-positive bacteria including Bacillus subtilis (4 μg/mL), Staphylococcus aureus (8 μg/mL), and Micrococcus luteus (8 μg/mL). Arcumycin treatment of B. subtilis liaI-β-gal promoter fusion reporter strain resulted in upregulation of the liaRS system by the promoter liaI, indicating arcumycin interferes with lipid II biosynthesis. Cumulatively, the results illustrate the relationship between phylogenetically related lasso peptides and their bioactivity as validated through the isolation, structural determination, and evaluation of bioactivity of the novel class I antimicrobial lasso peptide arcumycin.     

Unusual Post-Translational Modifications in the Biosynthesis of Lasso Peptides

Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) and feature the threaded, lariat knot-like topology. The basic post-translational modifications (PTMs) of lasso peptide contain two steps, including the leader peptide removal of the ribosome-derived linear precursor peptide by an ATP-dependent cysteine protease, and the macrolactam cyclization by an ATP-dependent macrolactam synthetase. Recently, advanced bioinformatic tools combined with genome mining have paved the way to uncover a rapidly growing number of lasso peptides as well as a series of PTMs other than the general class-defining processes. Despite abundant reviews focusing on lasso peptide discoveries, structures, properties, and physiological functionalities, few summaries concerned their unique PTMs. In this review, we summarized all the unique PTMs of lasso peptides uncovered to date, shedding light on the related investigations in the future.     

The Role of a Nonribosomal Peptide Synthetase in l‐Lysine Lactamization During Capuramycin Biosynthesis

Capuramycins are one of several known classes of natural products that contain an l ‐Lys‐derived l ‐α‐amino‐?‐caprolactam (l ‐ACL) unit. The α‐amino group of l ‐ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP‐independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP‐dependent amide‐bond‐forming catalyst responsible for the biosynthesis of the remaining amide bond present in l ‐ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l ‐Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS‐mediated mechanism is employed in the biosynthesis of other l ‐ACL‐containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS‐derived peptides that contain an unmodified l ‐Lys residue.     

Harnessing the Synthetic Capabilities of Glycopeptide Antibiotic Tailoring Enzymes: Characterization of the UK‐68,597 Biosynthetic Cluster

In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81‐kb biosynthetic cluster for the unusual sulfated glycopeptide UK‐68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram‐positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK‐68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.     

Initial Molecular Recognition Steps of McjA Precursor during Microcin J25 Lasso Peptide Maturation

Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post‐translational modification of the precursor McjA by the ATP‐dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three‐dimensional folding of MccJ25. We further showed that the N‐terminal region of the leader peptide is involved in interaction with both maturation enzymes and identified a predominant interaction of V43–S55 in the core McjA sequence with McjC. Moreover, we demonstrated that residues K23–Q34 in the N‐terminal McjA leader peptide tend to adopt a helical conformation in the presence of membrane mimics, implying a role in directing McjA to the membrane in the vicinity of the lasso synthetase/export machinery. These data provide valuable insights into the initial molecular recognition steps in the MccJ25 maturation process.     

Characterization of the Actinonin Biosynthetic Gene Cluster

The hydroxamate moiety of the natural product actinonin mediates inhibition of metalloproteinases because of its chelating properties towards divalent cations in the active site of those enzymes. Owing to its antimicrobial activity, actinonin has served as a lead compound for the development of new antibiotic drug candidates. Recently, we identified a putative gene cluster for the biosynthesis of actinonin. Here, we confirm and characterize this cluster by heterologous pathway expression and gene‐deletion experiments. We assigned the biosynthetic gene cluster to actinonin production and determine the cluster boundaries. Furthermore, we establish that ActI, an AurF‐like oxygenase, is responsible for the N‐hydroxylation reaction that forms the hydroxamate warhead. Our findings provide the basis for more detailed investigations of actinonin biosynthesis.     

The Phormidolide Biosynthetic Gene Cluster: A trans‐AT PKS Pathway Encoding a Toxic Macrocyclic Polyketide

Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16‐membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans‐type acyltransferase (trans‐AT) ORFs along with KS‐AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans‐AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain.     

Bioactivity‐Guided Genome Mining Reveals the Lomaiviticin Biosynthetic Gene Cluster in Salinispora tropica

The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico‐chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo‐ and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora tropica CNB‐440 by a DNA interference bioassay to isolate DNA‐targeting enediyne polyketides. An organic extract of S. tropica showed DNA‐interference activity that surprisingly was not abolished in genetic mutants of the targeted enediyne pathways, ST_pks1 and spo. Instead we showed that the product of the orphan type II polyketide synthase pathway, ST_pks2, is solely responsible for the DNA‐interfering activity of the parent strain. Subsequent comparative metabolic profiling revealed the lomaiviticins, glycosylated diazofluorene polyketides, as the ST_pks2 products. This study marks the first report of the 59 open reading frame lomaiviticin gene cluster (lom) and supports the biochemical logic of their dimeric construction through a pathway related to the kinamycin monomer.     

Generation of New Complestatin Analogues by Heterologous Expression of the Complestatin Biosynthetic Gene Cluster from Streptomyces chartreusis AN1542

The heterologous expression of the biosynthetic gene cluster (BGC) of natural products enables the production of complex metabolites in a well‐characterized host, and facilitates the generation of novel analogues by the manipulation of the genes. However, the BGCs of glycopeptides such as vancomycin, teicoplanin, and complestatin are usually too large to be directly cloned into a single cosmid. Here, we describe the heterologous expression of the complestatin BGC. The 54.5 kb cluster was fully reconstituted from two overlapping cosmids into one cosmid by λ‐RED recombination‐mediated assembly. Heterologous expression of the assembled gene cluster in Streptomyces lividans TK24 resulted in the production of complestatin. Deletion of cytochrome P450 monooxygenase genes (open reading frames 10 and 11) and heterologous expression of the modified clusters led to the production of two new monocyclic and linear derivatives, complestatins M55 and S56.     

Identification of the Tirandamycin Biosynthetic Gene Cluster from Streptomyces sp. 307‐9

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyl‐tetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307–9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid polyketide synthase–nonribosomal peptide synthetase gene cluster with a colinear domain organization, which is entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI, which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any post assembly‐line oxidative modifications.