Glycodiversification of gentamicins through glycosyltransferase swapping enabled the creation of novel hybrid aminoglycoside antibiotics with potent activity and low ototoxicity.

Aminoglycosides (AGs) are a class of antibiotics with a broad spectrum of activity. However, their use is limited by safety concerns associated with nephrotoxicity and ototoxicity, as well as drug resistance. To address these issues, semi-synthetic approaches for modifying natural AGs have generated new generations of AGs, however, with limited types of modification due to significant challenges in synthesis.

Efomycine U, a new -asymmetric elaiophylin derivative from DSM 4137.

Chemical examination of an actinomycete strain DSM 4137 derived from a soil sample derived isolate . DSM 3816, yielded a new -asymmetric elaiophylin derivative efomycine U () and a known analogue halichoblelide D (). These structures were unambiguously elucidated on the basis of extensive NMR spectroscopic and mass spectrometric analyses.

Insights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition.

Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER inside module 1/2 and the ketosynthase domain KS inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis.

Enzyme-Catalyzed Spiroacetal Formation in Polyketide Antibiotic Biosynthesis.

A key step in the biosynthesis of numerous polyketides is the stereospecific formation of a spiroacetal (spiroketal). We report here that spiroacetal formation in the biosynthesis of the macrocyclic polyketides ossamycin and oligomycin involves catalysis by a novel spiroacetal cyclase. OssO from the ossamycin biosynthetic gene cluster (BGC) is homologous to OlmO, the product of an unannotated gene from the oligomycin BGC.

Efophylins A and B, Two -Asymmetric Macrodiolide Immunosuppressants from .

Genomics-inspired isolation led to the identification of two new natural congeneric -asymmetric macrodiolide immunosuppressants, named efophylins A () and B (), from DSM 4137. Their structures were elucidated by spectroscopic and computational methods and were in agreement with biosynthetic predictions from the efophylin gene cluster. Compound exhibited potent immunosuppressive activity and demonstrated to inhibit the activation of the NFAT and block NFAT dephosphorylation .

The crystal structure of AjiA1 reveals a novel structural motion mechanism in the adenylate-forming enzyme family.

Adenylate-forming enzymes (AFEs) are a mechanistic superfamily of proteins that are involved in many cellular roles. In the biosynthesis of benzoxazole antibiotics, an AFE has been reported to play a key role in the condensation of cyclic molecules. In the biosynthetic gene cluster for the benzoxazole AJI9561, AjiA1 catalyzes the condensation of two 3-hydroxyanthranilic acid (3-HAA) molecules using ATP as a co-substrate.

The biosynthetic pathway to tetromadurin (SF2487/A80577), a polyether tetronate antibiotic.

The type I polyketide SF2487/A80577 (herein referred to as tetromadurin) is a polyether tetronate ionophore antibiotic produced by the terrestrial Gram-positive bacterium Actinomadura verrucosospora. Tetromadurin is closely related to the polyether tetronates tetronasin (M139603) and tetronomycin, all of which are characterised by containing a tetronate, cyclohexane, tetrahydropyran, and at least one tetrahydrofuran ring. We have sequenced the genome of Actinomadura verrucosospora to identify the biosynthetic gene cluster responsible for tetromadurin biosynthesis (the mad gene cluster).

Cross-Module Enoylreduction in the Azalomycin F Polyketide Synthase.

The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module.

Biosynthesis of depsipeptides with a 3-hydroxybenzoate moiety and selective anticancer activities involves a chorismatase.

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of , here we discovered four new neoantimycin analogs, unantimycins B-E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety.

Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis.

Enzymes catalysing remarkable Diels-Alder-like [4+2] cyclisations have been previously implicated in the biosynthesis of spirotetronate and spirotetramate antibiotics. Biosynthesis of the polyether antibiotic tetronasin is not anticipated to require such steps, yet the tetronasin gene cluster encodes enzymes Tsn11 and Tsn15, homologous to authentic [4+2] cyclases. Here we show that deletion of Tsn11 led to accumulation of a late-stage intermediate, in which the two central rings of tetronasin, and four of its 12 asymmetric centres, remain unformed.

Sarpeptins A and B, Lipopeptides Produced by sp. KO-7888 Overexpressing a Specific SARP Regulator.

Whole genome analysis of sp. KO-7888 has revealed various pathway-specific transcriptional regulatory genes associated with silent biosynthetic gene clusters. A antibiotic regulatory protein gene, , located adjacent to a novel nonribosomal peptide synthetase (NRPS) gene cluster, was overexpressed in the wild-type strain.

The biosynthetic pathway to ossamycin, a macrocyclic polyketide bearing a spiroacetal moiety.

Ossamycin from Streptomyces hygroscopicus var. ossamyceticus is an antifungal and cytotoxic polyketide and a potent inhibitor of the mitochondrial ATPase. Analysis of a near-complete genome sequence of the ossamycin producer has allowed the identification of the 127-kbp ossamycin biosynthetic gene cluster.

C-Nucleoside Formation in the Biosynthesis of the Antifungal Malayamycin A.

Malayamycin A is an unusual bicyclic C-nucleoside, with interesting antiviral, antifungal, and anticancer bioactivity. We report here the discovery and characterization of the biosynthetic pathway to malayamycin by using genome mining of near-identical clusters both from the known producer Streptomyces malaysiensis and from Streptomyces chromofuscus. The key precursor 5'-pseudouridine monophosphate (5'-Ψ-MP) is supplied chiefly through the action of MalD, a TruD-like pseudouridine synthase.

Hidden Specificities in Enzyme Catalysis: Structural Basis of Substrate Structure-Selectivity Relationship of a Ketoreductase.

Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity; yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate-enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated β-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae.

Directed Accumulation of Anticancer Depsipeptides by Characterization of Neoantimycins Biosynthetic Pathway and an NADPH-Dependent Reductase.

Neoantimycins (NATs) are members of antimycin-types of depsipeptides with outstanding anticancer activities. We isolated NAT-A (1) and -F (2) from the fermentation extract of Streptomyces conglobatus. The NAT biosynthetic gene cluster ( nat BGC) was identified by genome sequencing and bioinformatics analysis.

Methyltransferases of gentamicin biosynthesis.

Gentamicin C complex from remains a globally important antibiotic, and there is revived interest in the semisynthesis of analogs that might show improved therapeutic properties. The complex consists of five components differing in their methylation pattern at one or more sites in the molecule. We show here, using specific gene deletion and chemical complementation, that the gentamicin pathway up to the branch point is defined by the selectivity of the methyltransferases GenN, GenD1, and GenK.

Sulfation and amidinohydrolysis in the biosynthesis of giant linear polyenes.

Clethramycin from DSM4137, and mediomycins (produced together with clethramycin from ), are near-identical giant linear polyenes apparently constructed from, respectively, a 4-guanidinobutanoate or 4-aminobutanoate starter unit and 27 polyketide extender units, and bearing a specific -sulfonate modification at the C-29 hydroxy group. We show here that mediomycins are actually biosynthesised not by use of a different starter unit but by direct late-stage deamidination of (desulfo)clethramycin. A gene () encoding a candidate sulfotransferase has been located in both gene clusters.

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines.

Structural Basis of the Selectivity of GenN, an Aminoglycoside N-Methyltransferase Involved in Gentamicin Biosynthesis.

Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterized as an S-adenosyl-l-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3″-N-methylation of 3″-dehydro-3″-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex.

An Iterative Module in the Azalomycin F Polyketide Synthase Contains a Switchable Enoylreductase Domain.

Detailed analysis of the modular Type I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycin F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension-module 1 showed that this module catalyzes two successive elongations with different outcomes.

Isoafricanol synthase from Streptomyces malaysiensis.

Genome sequencing of Streptomyces malaysiensis DSM 4137 revealed the presence of four terpene cyclase genes, one of which was characterised as (+)-isoafricanol synthase. Its cyclisation mechanism was extensively studied using isotopically labelled precursors. Several enzymes with high homology that likely also function as (+)-isoafricanol synthases are encoded in a number of other genome sequenced streptomycetes.

Chemical probing of thiotetronate bio-assembly.

Chemical 'chain termination' probes were utilised for the investigation of thiotetronate antibiotic biosynthesis in the filamentous bacteria Lentzea sp. and Streptomyces thiolactonus NRRL 15439. The use of these tools led to the capture of biosynthetic intermediates involved in the thiotetronate polyketide backbone assembly, providing first insights into substrate specificity and in vivo intermediate processing by unusual iterative synthases.

Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase.

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from DSM4137 and Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension.