The Molecular Basis of Conjugated Polyyne Biosynthesis in Phytopathogenic Bacteria

Polyynes (polyacetylenes), which are produced by a variety of organisms, play important roles in ecology. Whereas alkyne biosynthesis in plants, fungi, and insects has been studied, the biogenetic origin of highly unstable bacterial polyynes has remained a riddle. Transposon mutagenesis and genome sequencing unveiled the caryoynencin (cay) biosynthesis gene cluster in the plant pathogen B. caryophylli, and homologous gene clusters were found in various other bacteria by comparative genomics. Gene inactivation and phylogenetic analyses revealed that novel desaturase/acetylenase genes mediate bacterial polyyne assembly. A cytochrome P450 monooxygenase is involved in the formation of the allylic alcohol moiety, as evidenced by analysis of a fragile intermediate, which was stabilized by an in situ click reaction. This work not only grants first insight into bacterial polyyne biosynthesis but also demonstrates that the click reaction can be employed to trap fragile polyynes from crude mixtures.     

Genome Mining,Heterologous Expression,Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost-Associated Bacillus subtilis fmb60

Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D–F (1–3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D–F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS⁺ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated.     

Functional analysis of type II thioesterase of Streptomyces lydicus AS 4.2501

Constructing a mutant strain of single gene disruption is the basis for the study of gene function and metabolomics. Systematic and complete genome sequencing is the basis of genetic manipulation. In the case of a little knowledge about the Streptomyces lydicus genome and the speculation that polyketide synthases (type I) might be responsible for the polyketide side chain biosynthesis of streptolydigin, a 588-bp fragment was amplified by polymerase chain reaction (PCR) according to the homology existing in the same functional genes among Streptomyces. A mutant strain of this gene was constructed by single crossover homologous recombination. The results of sequence analysis as well as the metabolite analysis of the mutant and the original strain by liquid chromatography/mass spectroscopy indicated that this fragment was part of type II thioesterase (TE) gene, which was required for streptolydigin biosynthesis like other type II TEs function in related antibiotics biosynthesis. Furthermore, targeted gene manipulation based on PCR was a powerful tool for studying gene function and metabolomics, especially when little was known about the genomic sequence of streptomyces.     

Biosynthesis of the Apoptolidins in Nocardiopsis sp. FU 40

The apoptolidins are 20/21-membered macrolides produced by Nocardiopsis sp. FU40. Several members of this family are potent and remarkably selective inducers of apoptosis in cancer cell lines, likely via a distinct mitochondria associated target. To investigate the biosynthesis of this natural product, the complete genome of the apoptolidin producer Nocardiopsis sp. FU40 was sequenced and a 116 kb region was identified containing a putative apoptolidin biosynthetic gene cluster. The apoptolidin gene cluster comprises a type I polyketide synthase, with 13 homologating modules, apparently initiated in an unprecedented fashion via transfer from a methoxymalonyl-acyl carrier protein loading module. Spanning approximately 39 open reading frames, the gene cluster was cloned into a series of overlapping cosmids and functionally validated by targeted gene disruption experiments in the producing organism. Disruption of putative PKS and P₄₅₀ genes delineated the roles of these genes in apoptolidin biosynthesis and chemical complementation studies demonstrated intact biosynthesis peripheral to the disrupted genes. This work provides insight into details of the biosynthesis of this biologically significant natural product and provides a basis for future mutasynthetic methods for the generation of non-natural apoptolidins.     

A Unique Tryptophan C‐Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

Cyanobactins are a rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES‐88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES‐88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C‐3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan‐prenylated cyclic peptides.     

Production of New Cladosporin Analogues by Reconstitution of the Polyketide Synthases Responsible for the Biosynthesis of this Antimalarial Agent

The antimalarial agent cladosporin is a nanomolar inhibitor of the Plasmodium falciparum lysyl‐tRNA synthetase, and exhibits activity against both blood‐ and liver‐stage infection. Cladosporin can be isolated from the fungus Cladosporium cladosporioides, where it is biosynthesized by a highly reducing (HR) and a non‐reducing (NR) iterative type I polyketide synthase (PKS) pair. Genome sequencing of the host organism and subsequent heterologous expression of these enzymes in Saccharomyces cerevisiae produced cladosporin, confirming the identity of the putative gene cluster. Incorporation of a pentaketide intermediate analogue indicated a 5+3 assembly by the HR PKS Cla2 and the NR PKS Cla3 during cladosporin biosynthesis. Advanced‐intermediate analogues were synthesized and incorporated by Cla3 to furnish new cladosporin analogues. A putative lysyl‐tRNA synthetase resistance gene was identified in the cladosporin gene cluster. Analysis of the active site emphasizes key structural features thought to be important in resistance to cladosporin.     

Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate ‘Streptomyces maritimus’: evidence for the derailment of an aromatic polyketide synthase

Background: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-β-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain ‘Streptomyces maritimus’ deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides.Results: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from ‘S. maritimus’ has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis.Conclusions: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.     

Structure and Biosynthesis of Xenoamicins from Entomopathogenic Xenorhabdus

During the search for novel natural products from entomopathogenic Xenorhabdus doucetiae DSM17909 and X. mauleonii DSM17908 novel peptides named xenoamicins were identified in addition to the already known antibiotics xenocoumacin and xenorhabdin. Xenoamicins are acylated tridecadepsipeptides consisting of mainly hydrophobic amino acids. The main derivative xenoamicin A ( 1 ) was isolated from X. mauleonii DSM17908, and its structure elucidated by detailed 1 D and 2 D NMR experiments. Detailed MS experiments, also in combination with labeling experiments, confirmed the determined structure and allowed structure elucidation of additional derivatives. Moreover, the xenoamicin biosynthesis gene cluster was identified and analyzed in X. doucetiae DSM17909, and its participation in xenoamicin biosynthesis was confirmed by mutagenesis. Advanced Marfey’s analysis of 1 showed that the absolute configuration of the amino acids is in agreement with the predicted stereochemistry deduced from the nonribosomal peptide synthetase XabABCD. Biological testing revealed activity of 1 against Plasmodium falciparum and other neglected tropical diseases but no antibacterial activity.     

Paenilamicin: Structure and Biosynthesis of a Hybrid Nonribosomal Peptide/Polyketide Antibiotic from the Bee Pathogen Paenibacillus larvae

The spore‐forming bacterium Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a fatal disease of honey bees that occurs worldwide. Previously, we identified a complex hybrid nonribosomal peptide/polyketide synthesis (NRPS/PKS) gene cluster in the genome of P. larvae. Herein, we present the isolation and structure elucidation of the antibacterial and antifungal products of this gene cluster, termed paenilamicins. The unique structures of the paenilamicins give deep insight into the underlying complex hybrid NRPS/PKS biosynthetic machinery. Bee larval co‐infection assays reveal that the paenilamicins are employed by P. larvae in fighting ecological niche competitors and are not directly involved in killing the bee larvae. Their antibacterial and antifungal activities qualify the paenilamicins as attractive candidates for drug development.     

An Unprecedented Cyclization Mechanism in the Biosynthesis of Carbazole Alkaloids in Streptomyces

Carquinostatin A (CQS), a potent neuroprotective substance, is a unique carbazole alkaloid with both an ortho‐quinone function and an isoprenoid moiety. We identified the entire gene cluster responsible for CQS biosynthesis in Streptomyces exfoliatus through heterologous production of CQS and gene deletion. Biochemical characterization of seven CQS biosynthetic gene products (CqsB1–7) established the total biosynthetic pathway of CQS. Reconstitution of CqsB1 and CqsB2 showed that the synthesis of the carbazole skeleton involves CqsB1‐catalyzed decarboxylative condensation of an α‐hydroxyl‐β‐keto acid intermediate with 3‐hydroxybutyryl‐ACP followed by CqsB2‐catalyzed oxidative cyclization. Based on crystal structures and mutagenesis‐based biochemical assays, a detailed mechanism for the unique deprotonation‐initiated cyclization catalyzed by CqsB2 is proposed. Finally, analysis of the substrate specificity of the biosynthetic enzymes led to the production of novel carbazoles.     

Paenibacillus polymyxa PKB1 produces variants of polymyxin B-type antibiotics

Polymyxins are cationic lipopeptide antibiotics active against many species of Gram-negative bacteria. We sequenced the gene cluster for polymyxin biosynthesis from Paenibacillus polymyxa PKB1. The 40.8 kb gene cluster comprises three nonribosomal peptide synthetase-encoding genes and two ABC transporter-like genes. Disruption of a peptide synthetase gene abolished all antibiotic production, whereas deletion of one or both transporter genes only reduced antibiotic production. Computational analysis of the peptide synthetase modules suggested that the enzyme system produces variant forms of polymyxin B (1 and 2), with D-2,4-diaminobutyrate instead of L-2,4-diaminobutyrate in amino acid position 3. Two antibacterial metabolites were resolved by HPLC and identified by high-resolution mass spectrometry and MS/MS sequencing as the expected variants 3 and 4 of polymyxin B₁ (1) and B₂ (2). Stereochemical analysis confirmed the presence of both D-2,4-diaminobutyrate and L-2,4-diaminobutyrate residues.     

Identification of the C‐Glycoside Synthases during Biosynthesis of the Pyrazole‐C‐Nucleosides Formycin and Pyrazofurin

C‐Nucleosides are characterized by a C?C rather than a C?N linkage between the heterocyclic base and the ribofuranose ring. While the biosynthesis of pseudouridine‐C‐nucleosides has been studied, less is known about the pyrazole‐C‐nucleosides such as the formycins and pyrazofurin. Herein, genome screening of Streptomyces candidus NRRL 3601 led to the discovery of the pyrazofurin biosynthetic gene cluster pyf. In vitro characterization of gene product PyfQ demonstrated that it is able to catalyze formation of the C‐glycoside carboxyhydroxypyrazole ribonucleotide (CHPR) from 4‐hydroxy‐1H‐pyrazole‐3,5‐dicarboxylic acid and phosphoribosyl pyrophosphate (PRPP). Similarly, ForT, the PyfQ homologue in the formycin pathway, can catalyze the coupling of 4‐amino‐1H‐pyrazole‐3,5‐dicarboxylic acid and PRPP to form carboxyaminopyrazole ribonucleotide. Finally, PyfP and PyfT are shown to catalyze amidation of CHPR to pyrazofurin 5′‐phosphate thereby establishing the latter stages of both pyrazofurin and formycin biosynthesis.     

Food-Poisoning Bacteria Employ a Citrate Synthase and a Type II NRPS To Synthesize Bolaamphiphilic Lipopeptide Antibiotics**

Mining the genome of the food-spoiling bacterium Burkholderia gladioli pv. cocovenenans revealed five nonribosomal peptide synthetase (NRPS) gene clusters, including an orphan gene locus (bol). Gene inactivation and metabolic profiling linked the bol gene cluster to novel bolaamphiphilic lipopeptides with antimycobacterial activity. A combination of chemical analysis and bioinformatics elucidated the structures of bolagladin A and B, lipocyclopeptides featuring an unusual dehydro-β-alanine enamide linker fused to an unprecedented tricarboxylic fatty acid tail. Through a series of targeted gene deletions, we proved the involvement of a designated citrate synthase (CS), priming ketosynthases III (KS III), a type II NRPS, including a novel desaturase for enamide formation, and a multimodular NRPS in generating the cyclopeptide. Network analyses revealed the evolutionary origin of the CS and identified cryptic CS/NRPS gene loci in various bacterial genomes.     

Use of a Phosphonate Methyltransferase in the Identification of the Fosfazinomycin Biosynthetic Gene Cluster

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for “stable isotope labeling of phosphonates in extract” (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S‐adenosyl methionine. Mass‐guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.     

Discovery and Biosynthesis of Bolagladins: Unusual Lipodepsipeptides from Burkholderia gladioli Clinical Isolates**

Two Burkholderia gladioli strains isolated from the lungs of cystic fibrosis patients were found to produce unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-β-alanine residue. The gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In-frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of about 45 % of B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S assay indicated that these metabolites bind ferric iron, which suppresses their production when added to the growth medium. Moreover, a gene encoding a TonB-dependent ferric-siderophore receptor is adjacent to the biosynthetic genes, suggesting that these metabolites may function as siderophores in B. gladioli.     

Discovery of New Thioviridamide‐Like Compounds with Antitumor Activities

Thioviridamide is a structurally unique compound with potent antitumor activity. The biosynthesis of thioviridamide follows a typical pathway as ribosomally synthesized and post‐translationally modified peptides, making the genome mining‐based discovery of thioviridamide‐like compounds rational. Taking advantage of the linkage between the precursor peptide and the metabolite skeleton, we identified a new biosynthetic gene cluster in Streptomyces sp. NRRL S‐87 that could encode thioviridamide analogues. Overexpression of the whole gene cluster led to the isolation and structure elucidation of TVA‐YJ‐4 and TVA‐YJ‐5, two novel thioviridamide‐like compounds featuring N‐terminal capping groups. Chemical screening of the fermentation extracts also detected TVA‐YJ‐6, another new thioviridamide‐like compound with representative methionine sulfoxide. Detailed analysis further revealed that these structural modifications were introduced during the compound extraction process instead of through genuine enzymatic reactions. TVA‐YJ‐4 and TVA‐YJ‐5 display slightly reduced cytotoxic activities against a panel of tumor cell lines in comparison with their parental natural product, TVA‐YJ‐2. Our work will expand the membership of this rare class of compounds and promote related biosynthetic studies.     

Identification and characterization of xiamycin A and oxiamycin gene cluster reveals an oxidative cyclization strategy tailoring indolosesquiterpene biosynthesis

Xiamycin A (XMA) and oxiamycin (OXM) are bacterial indolosesquiterpenes featuring rare pentacyclic ring systems and are isolated from a marine-derived Streptomyces sp. SCSIO 02999. The putative biosynthetic gene cluster for XMA/OXM was identified by a partial genome sequencing approach. Eighteen genes were proposed to be involved in XMA/OXM biosynthesis, including five genes for terpene synthesis via a non-mevalonate pathway, eight genes encoding oxidoreductases, and five genes for regulation and resistance. Targeted disruptions of 13 genes within the xia gene cluster were carried out to probe their encoded functions in XMA/OXM biosynthesis. The disruption of xiaK, encoding an aromatic ring hydroxylase, led to a mutant producing indosespene and a minor amount of XMA. Feeding of indosespene to XMA/OXM nonproducing mutants revealed indosespene as a common precursor for XMA/OXM biosynthesis. Most notably, the flavin dependent oxygenase XiaI was biochemically characterized in vitro to convert indosespene to XMA, revealing an unusual oxidative cyclization strategy tailoring indolosesquiterpene biosynthesis.     

Discovery of the Azaserine Biosynthetic Pathway Uncovers a Biological Route for α-Diazoester Production

Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in α-diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for α-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α-diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.     

Reconstituted biosynthesis of fungal meroterpenoid andrastin A

Andrastins (andrastin A-D), produced by several Penicillium species, exhibit inhibitory activity against ras farnesyltransferase, suggesting that these compounds could be promising leads for antitumor agents. Although the genome sequence of Penicillium chrysogenum, an andrastin-producing species, is available, the genetic and molecular bases for the biosynthesis of andrastins have not been elucidated. Here we report the identification and characterization of the gene cluster for andrastin biosynthesis. We reconstituted the biosynthetic pathway in Aspergillus oryzae, a fungal expression host, by the co-expression of five genes, including that of a terpene cyclase, and of four genes encoding the tailoring enzymes, required for the generation of andrastins. Remarkably, we successfully obtained andrastin A, the most complex andrastin molecule, as the metabolite of nine gene products, thus confirming the potential of the fungal expression system to synthesize useful natural products.