Structural Diversification of Lyngbyatoxin A by Host‐Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster

Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin—lyngbyatoxin A—and its biosynthetic intermediates by heterologous expression of the Streptomyces‐derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk.     

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 Six Ramoplanin Family Gene Clusters and the Lipoglycodepsipeptide Chersinamycin**

Ramoplanins and enduracidins are peptidoglycan lipid intermediate II-binding lipodepsipeptides with broad-spectrum activity against methicillin- and vancomycin-resistant Gram-positive pathogens. Targeted genome mining using probes from conserved sequences within the ramoplanin/enduracidin biosynthetic gene clusters (BGCs) was used to identify six microorganisms with BGCs predicted to produce unique lipodepsipeptide congeners of ramoplanin and enduracidin. Fermentation of Micromonospora chersina yielded a novel lipoglycodepsipeptide, called chersinamycin, which exhibited good antibiotic activity against Gram-positive bacteria (1–2 μg/mL) similar to the ramoplanins and enduracidins. The covalent structure of chersinamycin was determined by NMR spectroscopy and tandem mass spectrometry in conjunction with chemical degradation studies. These six new BGCs and isolation of a new antimicrobial peptide provide much-needed tools to investigate the fundamental aspects of lipodepsipeptide biosynthesis and to facilitate efforts to produce novel antibiotics capable of combating antibiotic-resistant infections.     

Identification of the Biosynthetic Gene Cluster for Himeic Acid A: A Ubiquitin‐Activating Enzyme (E1) Inhibitor in Aspergillus japonicus MF275

Himeic acid A, which is produced by the marine fungus Aspergillus japonicus MF275, is a specific inhibitor of the ubiquitin‐activating enzyme E1 in the ubiquitin–proteasome system. To elucidate the mechanism of himeic acid biosynthesis, feeding experiments with labeled precursors have been performed. The long fatty acyl side chain attached to the pyrone ring is of polyketide origin, whereas the amide substituent is derived from leucine. These results suggest that a polyketide synthase–nonribosomal peptide synthase (PKS‐NRPS) is involved in himeic acid biosynthesis. A candidate gene cluster was selected from the results of genome sequencing analysis. Disruption of the PKS‐NRPS gene by Agrobacterium‐mediated transformation confirms that HimA PKS‐NRPS is involved in himeic acid biosynthesis. Thus, the him biosynthetic gene cluster for himeic acid in A. japonicus MF275 has been identified.     

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.     

The Cremeomycin Biosynthetic Gene Cluster Encodes a Pathway for Diazo Formation

Diazo groups are found in a range of natural products that possess potent biological activities. Despite longstanding interest in these metabolites, diazo group biosynthesis is not well understood, in part because of difficulties in identifying specific genes linked to diazo formation. Here we describe the discovery of the gene cluster that produces the o‐diazoquinone natural product cremeomycin and its heterologous expression in Streptomyces lividans. We used stable isotope feeding experiments and in vitro characterization of biosynthetic enzymes to decipher the order of events in this pathway and establish that diazo construction involves late‐stage N?N bond formation. This work represents the first successful production of a diazo‐containing metabolite in a heterologous host, experimentally linking a set of genes with diazo formation.     

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.     

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.     

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.     

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.     

Characterization of the Nocardiopsin Biosynthetic Gene Cluster Reveals Similarities to and Differences from the Rapamycin and FK‐506 Pathways

Macrolide‐pipecolate natural products, such as rapamycin ( 1 ) and FK‐506 ( 2 ), are renowned modulators of FK506‐binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB‐M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A–D ( 4 – 7 ) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L ‐lysine into the L ‐pipecolic acid incorporated into 4 and 5 . Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP‐binding metabolites and for probing new enzymology in nature's polyketide tailoring arsenal.     

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.     

Characterization of an Environmental DNA‐Derived Gene Cluster that Encodes the Bisindolylmaleimide Methylarcyriarubin

Bisindolylmaleimides represent a naturally occurring class of metabolites that are of interest because of their protein kinase inhibition activity. From a metagenomic library constructed with soil DNA, we identified the four gene mar cluster, a bisindolylmaleimide gene cluster that encodes for methylarcyriarubin ( 1 ) production. Heterologous expression of the mar gene cluster in E. coli revealed that the Rieske dioxygenase MarC facilitates the oxidative decarboxylation of a chromopyrrolic acid (CPA) intermediate to yield the bisindolylmaleimide core. The characterization of the mar cluster defines a new role for CPA in the biosynthesis of structurally diverse bacterial tryptophan dimers.     

Biosynthetic Origin of the Antibiotic Pseudopyronines A and B in Pseudomonas putida BW11M1

Within the framework of our effort to discover new antibiotics from pseudomonads, pseudopyronines A and B were isolated from the plant‐derived Pseudomonas putida BW11M1. Pseudopyronines are 3,6‐dialkyl‐4‐hydroxy‐2‐pyrones and displayed high in vitro activities against several human pathogens, and in our hands also towards the plant pathogen Pseudomonas savastanoi. Here, the biosynthesis of pseudopyronine B was studied by a combination of feeding experiments with isotopically labeled precursors, genomic sequence analysis, and gene deletion experiments. The studies resulted in the deduction of all acetate units and revealed that the biosynthesis of these α‐pyrones occurs with a single PpyS‐homologous ketosynthase. It fuses, with some substrate flexibility, a 3‐oxo‐fatty acid and a further unbranched saturated fatty acid, both of medium chain‐length and provided by primary metabolism.     

Direct Cloning, Genetic Engineering, and Heterologous Expression of the Syringolin Biosynthetic Gene Cluster in E. coli through Red/ET Recombineering

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.