Pacidamycin Biosynthesis: Identification and Heterologous Expression of the First Uridyl Peptide Antibiotic Gene Cluster

The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3′‐deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact.     

Exploring Chemical Diversity of α‐Pyrone Antibiotics: Molecular Basis of Myxopyronin Biosynthesis

Myxopyronins and corallopyronins are structurally related α‐pyrone antibiotics from myxobacteria. They are thought to represent a highly promising compound class for the development of broad‐spectrum antibacterial therapeutic agents, because of their ability to inhibit RNA polymerase through interaction with the “switch region”, a recently identified novel drug target. Here we describe the identification and characterization of the myxopyronin biosynthetic pathway from Myxococcus fulvus Mx f50. A detailed comparison with the recently identified corallopyronin biosynthetic pathway revealed the genetic and biochemical basis, thus explaining the observed structural differences between the two natural product families. Directed mutagenesis procedures for M. fulvus Mx f50 were developed to enable functional studies and pathway modifications. Our work provided new insights into myxopyronin biosynthesis and led to the production of a novel and unexpected myxopyronin derivative.     

Functional Analysis of MycE and MycF,Two O‐Methyltransferases Involved in the Biosynthesis of Mycinamicin Macrolide Antibiotics

Mg motors : We characterized the in vitro function of MycE and MycF, two O‐methyltransferases involved in the biosynthesis of mycinamicin antibiotics. Each enzyme was confirmed to be an S‐adenosyl‐L ‐methionine (SAM)‐dependent deoxysugar methyltransferase. Their optimal activities require the presence of Mg²⁺. With the reconstituted in vitro assays, the order of mycinamicin VI→III→IV in the post‐PKS (polyketide synthase) tailoring pathway of mycinamicin was unambiguously determined.

     

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.     

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.     

Cloning and Heterologous Expression of the Aurachin RE Biosynthesis Gene Cluster Afford a New Cytochrome P450 for Quinoline N‐Hydroxylation

Aurachin RE is a prenylated quinoline antibiotic that was first isolated from the genus Rhodococcus. It shows potent antibacterial activity against a variety of Gram‐positive bacteria. Here we have identified a minimal biosynthesis gene cluster for aurachin RE in Rhodococcus erythropolis JCM 6824 by using random transposon mutagenesis and heterologous production. The Rhodococcus aurachin (rau) gene cluster consists of genes encoding cytochrome P450 (rauA), prenyltransferase, polyketide synthase, and farnesyl pyrophosphate synthase, as well as others including genes involved in regulation and transport. Markerless gene disruption of rauA resulted in the complete loss of aurachin RE production and in the accumulation of a new aurachin derivative lacking the N‐hydroxy group. When the recombinant RauA was expressed in Escherichia coli, it catalyzed N‐hydroxylation of the derivative to form aurachin RE. This study establishes the biosynthetic pathway of aurachin RE and provides experimental evidence for the role of P450 RauA in catalyzing N‐hydroxylation of the quinoline ring, which is indispensable for the antibacterial activity of aurachin RE.     

Five‐Membered Cyclitol Phosphate Formation by a myo‐Inositol Phosphate Synthase Orthologue in the Biosynthesis of the Carbocyclic Nucleoside Antibiotic Aristeromycin

Aristeromycin is a unique carbocyclic nucleoside antibiotic produced by Streptomyces citricolor. In order to elucidate its intriguing carbocyclic formation, we used a genome‐mining approach to identify the responsible enzyme. In silico screening with known cyclitol synthases involved in primary metabolism, such as myo‐inositol‐1‐phosphate synthase (MIPS) and dehydroqunate synthase (DHQS), identified a unique MIPS orthologue (Ari2) encoded in the genome of S. citricolor. Heterologous expression of the gene cluster containing ari2 with a cosmid vector in Streptomyces albus resulted in the production of aristeromycin, thus indicating that the cloned DNA region (37.5 kb) with 33 open reading frames contains its biosynthetic gene cluster. We verified that Ari2 catalyzes the formation of a novel five‐membered cyclitol phosphate from d ‐fructose 6‐phosphate (F6P) with NAD⁺ as a cofactor. This provides insight into cyclitol phosphate synthase as a member of the MIPS family of enzymes. A biosynthetic pathway to aristeromycin is proposed based on bioinformatics analysis of the gene cluster.     

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.     

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.     

Understanding the Glycopeptide Antibiotic Crosslinking Cascade: In Vitro Approaches Reveal the Details of a Complex Biosynthesis Pathway

The glycopeptide antibiotics (GPAs) are a fascinating example of complex natural product biosynthesis, with the nonribosomal synthesis of the peptide core coupled to a cytochrome P450-mediated cyclisation cascade that crosslinks aromatic side chains within this peptide. Given that the challenges associated with the synthesis of GPAs stems from their highly crosslinked structure, there is great interest in understanding how biosynthesis accomplishes this challenging set of transformations. In this regard, the use of in vitro experiments has delivered important insights into this process, including the identification of the unique role of the X-domain as a platform for P450 recruitment. In this minireview, we present an analysis of the results of in vitro studies into the GPA cyclisation cascade that have demonstrated both the tolerances and limitations of this process for modified substrates, and in turn developed rules for the future reengineering of this important antibiotic class.     

Properties of poly(AAc‐co‐HPMA‐cl‐EGDMA) hydrogel‐bound lipase of Pseudomonas aeruginosa MTCC‐4713 and its use in synthesis of methyl acrylate

Microbial lipases (E.C. 3.1.1.3) are preferred biocatalysts for the synthesis of esters in organic solvents. Various extracellular thermoalkaliphilic lipases have been reported from Pseudomonas sp. In the present study, a purified alkaline thermoalkalophilic extracellular lipase of Pseudomonas aeruginosa MTCC‐4713 was efficiently immobilized onto a synthetic poly(AAc‐co‐HPMA‐cl‐EGDMA) hydrogel by adsorption and the bound lipase was evaluated for its hydrolytic potential towards various p‐nitrophenyl acyl esters varying in their C‐chain lengths. The bound lipase showed optimal hydrolytic activity towards p‐nitrophenyl palmitate (p‐NPP) at pH 8.5 and temperature 45°C. The hydrolytic activity of the hydrogel‐bound lipase was markedly enhanced by the presence of Hg²⁺, Fe³⁺, and NH salt ions in that order. The hydrogel‐immobilized lipase (25 mg) was used to perform esterification in various n‐alkane(s) that resulted in ～ 84.9 mM of methyl acrylate at 45°C in n‐heptane under shaking (120 rpm) after 6 h, when methanol and acrylic acid were used in a ratio of 100 mM:100 mM, respectively. Addition of a molecular sieve (3Å × 1.5 mm) to the reaction system at a concentration of 100 mg/reaction vol (1 mL) resulted in a moderate enhancement in conversion of reactants into methyl acrylate (85.6 mM). During the repetitive esterification under optimum conditions, the hydrogel‐bound lipase produced 71.3 mM of ester after 10th cycle of reuse. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 183–191, 2007     

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.     

Characteristics of poly(AAc5‐co‐HPMA3‐cl‐EGDMA15) hydrogel‐immobilized lipase of Pseudomonas aeruginosa MTCC‐4713

Four series of noble networks were synthesized with acrylic acid (AAc) copolymerized with varying amount of 2‐hydroxy propyl methacrylate or dodecyl methacrylate (AAc/HPMA or AAc/DMA; 5:1 to 5:5, w/w) in the presence of ethylene glycol dimethacrylate (EGDMA; 1, 5, 10, 15, and 20%, w/w) as a crosslinker and ammonium per sulfate (APS) as an initiator. Each of the networks was used to immobilize a purified lipase from Pseudomonas aeruginosa MTCC‐4713. The lipase was purified by successive salting out with (NH₄)₂SO₄, dialysis, and DEAE anion exchange chromatography. Two of the matrices, E_15a, i.e. [poly (AAc₅‐co‐DMA₁‐cl‐EGDMA₁₅)] and I_15c, i.e. [poly (AAc₅‐co‐HPMA₃‐cl‐EGDMA₁₅)], that showed relatively higher binding efficiency for lipase were selected for further studies. I_15c‐hydrogel retained 58.3% of its initial activity after 10th cycle of repetitive hydrolysis of p‐NPP, and I_15c was thus catalytically more stable and efficient than the other matrix. The I_15c‐hydrogel‐immobilized enzyme showed maximum activity at 65°C and pH 9.5. The hydrolytic activity of free and I_15c‐hydrogel‐immobilized enzyme increased profoundly in the presence of 5 mM chloride salts of Hg²⁺, NH₄⁺, Al³⁺, K⁺, and Fe³⁺. The immobilized lipase was preferentially active on medium chain length p‐nitrophenyl acyl ester (C:8, p‐nitrophenyl caprylate). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4636–4644, 2006     

Structure–Activity Relationship Analysis of the Peptide Deformylase Inhibitor 5‐Bromo‐1H‐indole‐3‐acetohydroxamic Acid

SAR by NMR : A series of indole compounds derived from 5‐bromo‐1H‐indole‐3‐acetohydroxamic acid were synthesized. Their inhibitory activities were evaluated against purified peptide deformylases (PDFs), and their antibacterial activities against B. subtilis, E. coli (wild type and tolC), and a variety of pathogens were also determined. The potency of the best inhibitors was related to the NMR footprints of the respective acids with ¹⁵N‐labeled E. coli Ni‐PDF.

     

Reprogramming the Biosynthesis of Cyclodepsipeptide Synthetases to Obtain New Enniatins and Beauvericins

Non‐ribosomal peptide synthetases are complex multimodular biosynthetic machines that assemble various important and medically relevant peptide antibiotics. An interesting subgroup comprises the cyclodepsipeptide synthetases from fungi synthesizing cyclohexa‐ and cyclo‐octadepsipeptides with antibacterial, anthelmintic, insecticidal, and anticancer properties; some are marketed drugs. We exploit the modularity of these highly homologous synthetases by fusing the hydroxy‐acid‐activating module of PF1022 synthetase with the amino‐acid‐activating modules of enniatin and beauvericin synthetase, thus yielding novel hybrid synthetases. The artificial synthetases expressed in Escherichia coli and the fungus Aspergillus niger yielded new cyclodepsipeptides, thus paving the way for the exploration of these derivatives for their bioactivity.     

Diaryl‐Substituted Azolylthioacetamides: Inhibitor Discovery of New Delhi Metallo‐β‐Lactamase‐1 (NDM‐1)

The emergence and spread of antibiotic‐resistant pathogens is a global public health problem. Metallo‐β‐lactamases (MβLs) such as New Delhi MβL‐1 (NDM‐1) are principle contributors to the emergence of resistance because of their ability to hydrolyze almost all known β‐lactam antibiotics including penicillins, cephalosporins, and carbapenems. A clinical inhibitor of MBLs has not yet been found. In this study we developed eighteen new diaryl‐substituted azolylthioacetamides and found all of them to be inhibitors of the MβL L1 from Stenotrophomonas maltophilia (K_i<2 μM ), thirteen to be mixed inhibitors of NDM‐1 (K_i<7 μM ), and four to be broad‐spectrum inhibitors of all four tested MβLs CcrA from Bacteroides fragilis, NDM‐1 and ImiS from Aeromonas veronii, and L1 (K_i<52 μM ), which are representative of the B1a, B1b, B2, and B3 subclasses, respectively. Docking studies revealed that the azolylthioacetamides, which have the broadest inhibitory activity, coordinate to the Zn^II ion(s) preferentially via the triazole moiety, while other moieties interact mostly with the conserved active site residues Lys224 (CcrA, NDM‐1, and ImiS) or Ser221 (L1).