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.     

Core Steps to the Azaphilone Family of Fungal Natural Products

Azaphilones are a family of polyketide-based fungal natural products that exhibit interesting and useful bioactivities. This minireview explores the literature on various characterised azaphilone biosynthetic pathways, which allows for a proposed consensus scheme for the production of the core azaphilone structure, as well as identifying early diversification steps during azaphilone biosynthesis. A consensus understanding of the core enzymatic steps towards a particular family of fungal natural products can aid in genome-mining experiments. Genome mining for novel fungal natural products is a powerful technique for both exploring chemical space and providing new insights into fungal natural product pathways.     

Strategies for the Discovery of New Natural Products by Genome Mining

New drugs from silent gene clusters : Analysis of genome sequence data has identified numerous “cryptic” gene clusters encoding novel natural product biosynthetic assembly lines; this suggests that many new bioactive metabolites remain to be discovered, even in extensively investigated organisms. Several related and complementary strategies for identifying the products of these clusters have emerged recently and revitalized the search for novel bioactive natural products.

     

Biosynthesis of Sinapigladioside,an Antifungal Isothiocyanate from Burkholderia Symbionts

Sinapigladioside is a rare isothiocyanate-bearing natural product from beetle-associated bacteria (Burkholderia gladioli) that might protect beetle offspring against entomopathogenic fungi. The biosynthetic origin of sinapigladioside has been elusive, and little is known about bacterial isothiocyanate biosynthesis in general. On the basis of stable-isotope labeling, bioinformatics, and mutagenesis, we identified the sinapigladioside biosynthesis gene cluster in the symbiont and found that an isonitrile synthase plays a key role in the biosynthetic pathway. Genome mining and network analyses indicate that related gene clusters are distributed across various bacterial phyla including producers of both nitriles and isothiocyanates. Our findings support a model for bacterial isothiocyanate biosynthesis by sulfur transfer into isonitrile precursors.     

Identification of Genes Essential for Fluorination and Sulfamylation within the Nucleocidin Gene Clusters of Streptomyces calvus and Streptomyces virens

The gene cluster in Streptomyces calvus associated with the biosynthesis of the fluoro- and sulfamyl-metabolite nucleocidin was interrogated by systematic gene knockouts. Out of the 26 gene deletions, most did not affect fluorometabolite production, nine abolished sulfamylation but not fluorination, and three precluded fluorination, but had no effect on sulfamylation. In addition to nucI, nucG, nucJ, nucK, nucL, nucN, nucO, nucQ and nucP, we identified two genes (nucW_, nucA), belonging to a phosphoadenosine phosphosulfate (PAPS) gene cluster, as required for sulfamyl assembly. Three genes (orf(−3), orf2 and orf3) were found to be essential for fluorination, although the activities of their protein products are unknown. These genes as well as nucK, nucN, nucO and nucPNP, whose knockouts produced results differing from those described in a recent report, were also deleted in Streptomyces virens – with confirmatory outcomes. This genetic profile should inform biochemistry aimed at uncovering the enzymology behind nucleocidin biosynthesis.     

雷莫拉宁的分离纯化工艺研究

以聚酰胺树脂为分离介质,研究了从发酵液中分离纯化雷莫拉宁的工艺过程,对吸附及解吸条件进行了优化。结果表明,在雷莫拉宁浸提液浓度为1600～2000μg.mL-1、吸附流速为1.0BV.h-1、乙醇作洗脱剂(先用30%或50%乙醇洗脱,再用70%乙醇洗脱)的条件下,可得到总收率超过80%、纯度大于95%的雷莫拉宁,该方法简单易行、安全性高,适于工业化生产。     

Reporter-Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis

Most natural product biosynthetic gene clusters that can be observed bioinformatically are silent. This insight has prompted the development of several methodologies for inducing their expression. One of the more recent methods, termed reporter-guided mutant selection (RGMS), entails creation of a library of mutants that is then screened for the desired phenotype via reporter gene expression. Herein, we apply a similar approach to Burkholderia thailandensis and, using transposon mutagenesis, mutagenize three strains, each carrying a fluorescent reporter in the malleilactone (mal), capistruin (cap), or an unidentified ribosomal peptide (tomm) gene cluster. We show that even a small library of <500 mutants can be used to induce expression of each cluster. We also explore the mechanism of activation and find that inhibition of pyrimidine biosynthesis is linked to the induction of the mal cluster. Both a transposon insertion into pyrF as well as small-molecule-mediated inhibition of PyrF trigger malleilactone biosynthesis. Our results pave the way toward the broad application of RGMS and related approaches to Burkholderia spp.     

Targeted Rediscovery and Biosynthesis of the Farnesyl-Transferase Inhibitor Pepticinnamin E

The natural product pepticinnamin E potently inhibits protein farnesyl transferases and has potential applications in treating cancer and malaria. Pepticinnamin E contains a rare N-terminal cinnamoyl moiety as well as several nonproteinogenic amino acids, including the unusual 2-chloro-3-hydroxy-4-methoxy-N-methyl-L-phenylalanine. The biosynthesis of pepticinnamin E has remained uncharacterized because its original producing strain is no longer available. Here we identified a gene cluster (pcm) for this natural product in a new producer, Actinobacteria bacterium OK006, by means of a targeted rediscovery strategy. We demonstrated that the pcm cluster is responsible for the biosynthesis of pepticinnamin E, a nonribosomal peptide/polyketide hybrid. We also characterized a key O-methyltransferase that modifies 3,4-dihydroxy-l -phenylalanine. Our work has identified the gene cluster for pepticinnamins for the first time and sets the stage for elucidating the unique chemistry required for biosynthesis.     

Gene Discovery in Gelsemium Highlights Conserved Gene Clusters in Monoterpene Indole Alkaloid Biosynthesis

Genome mining is a routine technique in microbes for discovering biosynthetic pathways. In plants, however, genomic information is not commonly used to identify novel biosynthesis genes. Here, we present the genome of the medicinal plant and oxindole monoterpene indole alkaloid (MIA) producer Gelsemium sempervirens (Gelsemiaceae). A gene cluster from Catharanthus roseus, which is utilized at least six enzymatic steps downstream from the last common intermediate shared between the two plant alkaloid types, is found in G. sempervirens, although the corresponding enzymes act on entirely different substrates. This study provides insights into the common genomic context of MIA pathways and is an important milestone in the further elucidation of the Gelsemium oxindole alkaloid pathway.     

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.     

Genome Mining of Streptomyces sp. Tü 6176: Characterization of the Nataxazole Biosynthesis Pathway

Streptomyces sp. Tü 6176 produces the cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of 38 putative secondary‐metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, the antibiotic hygromycin B, and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster was identified and characterized: it lacks the O‐methyltransferase gene required to convert AJI9561 into nataxazole. This O‐methyltransferase activity might act as a resistance mechanism, as AJI9561 shows antibiotic activity whereas nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the production of AJI9561. Nataxazole biosynthesis requires the shikimate pathway to generate 3‐hydroxyanthranilate and an iterative type I PKS to generate 6‐methylsalicylate. Production of nataxazole was improved up to fourfold by disrupting one regulatory gene in the cluster. An additional benzoxazole, 5‐hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5‐Hydroxynataxazole derives from nataxazole by the activity of an as yet unidentified oxygenase; this implies cross‐talk between the nataxazole biosynthesis pathway and an unknown pathway.     

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.     

Total Synthesis and Structural Revision of Kasumigamide,and Identification of a New Analogue

Kasumigamide is an antialgal hybrid peptide–polyketide isolated from the freshwater cyanobacterium Microcystis aeruginosa (NIES-87). The biosynthetic gene cluster was identified from not only the cyanobacterium but also Candidatus “Entotheonella”, associated with the Japanese marine sponge Discodermia calyx. Therefore, kasumigamide is considered to play a key role in microbial ecology, regardless of the terrestrial and marine habitats. We now report synthetic studies on this intriguing natural product that have led to a structural revision and the first total synthesis. During this study, a new analogue, deoxykasumigamide, was also isolated and structurally validated. This study confirmed the presence of the unusual pathway in the biosynthesis of a hybrid peptide–polyketide natural product.     

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.     

Discovery and Elucidation of the Biosynthesis of Aspernidgulenes: Novel Polyenes from Aspergillus Nidulans by Using Serial Promoter Replacement

Through serial promoter exchanges, we isolated several novel polyenes, the aspernidgulenes, from Aspergillus nidulans and uncovered their succinct biosynthetic pathway involving only four enzymes. An enoyl reductase (ER)-less highly reducing polyketide synthase (HR-PKS) putatively produces a 5,6-dihydro-α-pyrone polyene, which undergoes bisepoxidation, epoxide ring opening, cyclization, and hydrolytic cleavage by three tailoring enzymes to generate aspernidgulene A1 and A2. Our findings demonstrate the prowess of fungal-tailoring enzymes to transform a polyketide scaffold concisely and efficiently into complex structures. Moreover, comparison with citreoviridin and aurovertin biosynthesis suggests that methylation of the α-pyrone hydroxy group by methyltransferase (CtvB or AurB) is the branching point at which the biosynthesis of these two classes of compounds diverge. Therefore, scanning for the presence or absence of the gatekeeping α-pyrone methyltransferase gene in homologous clusters might be a potential way to classify the product bioinformatically as belonging to methylated α-pyrone polyenes or polyenes containing rings derived from the cyclization of the unmethylated 5,6-dihydro-α-pyrone, such as 2,3-dimethyl-γ-lactone and oxabicyclo[2.2.1]heptane.     

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.     

Discovery of the Tiancilactone Antibiotics by Genome Mining of Atypical Bacterial Type II Diterpene Synthases

Although genome mining has advanced the identification, discovery, and study of microbial natural products, the discovery of bacterial diterpenoids continues to lag behind. Herein, we report the identification of 66 putative producers of novel bacterial diterpenoids, and the discovery of the tiancilactone (TNL) family of antibiotics, by genome mining of type II diterpene synthases that do not possess the canonical DXDD motif. The TNLs, which are broad‐spectrum antibiotics with moderate activities, are produced by both Streptomyces sp. CB03234 and Streptomyces sp. CB03238 and feature a highly functionalized diterpenoid skeleton that is further decorated with chloroanthranilate and γ‐butyrolactone moieties. Genetic manipulation of the tnl gene cluster resulted in TNL congeners, which provided insights into their biosynthesis and structure–activity relationships. This work highlights the biosynthetic potential that bacteria possess to produce diterpenoids and should inspire continued efforts to discover terpenoid natural products from bacteria.     

Genome Mining of the Lipopeptide Biosynthesis of Paenibacillus polymyxa E681 in Combination with Mass Spectrometry: Discovery of the Lipoheptapeptide Paenilipoheptin

Paenibacillus polymyxa strains are qualified for agro‐biotechnological uses such as plant growth promotion and for biocontrol strategies against deleterious phytopathogenic competitors in the soil depending on their attractive arsenal of bioactive compounds. Moreover, they are potent producers of antibiotics for medical applications. To identify new products of such organisms, genome mining strategies in combination with mass spectrometry are the methods of choice. Herein, we performed such studies with the Paenibacillus strain E681. Bioinformatic evaluation of its genome sequence revealed four gene clusters A–D encoding nonribosomal peptide synthetases (NRPSs). Accordingly, four lipopeptide families were detected by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Clusters A and D codify the well known fusaricidins and polymyxins. A yet‐unknown lipoheptapeptide was discovered and structurally characterized by de novo sequencing by using MALDI‐LIFT‐TOF/TOF MS. It was designated as paenilipoheptin. From structure predictions we infer that the production of this agent is encoded by gene cluster C. Gene cluster B encodes the synthesis of tridecaptins, a family of open‐chain lipotridecapeptides. Strain E681 produces new subspecies of such compounds (tridecaptins E) showing variations both in their fatty‐acid part as well as in their peptide part.