Retinestatin, a Polyol Polyketide from a Termite Nest-Derived Streptomyces sp.

A new polyol polyketide, named retinestatin (1), was obtained and characterized from the culture of a Streptomyces strain, which was isolated from a subterranean nest of the termite Reticulitermes speratus kyushuensis Morimoto. The planar structure of 1 was elucidated on the basis of the cumulative analysis of ultraviolet, infrared, mass spectrometry, and nuclear magnetic resonance spectroscopic data. The absolute configuration of 1 at 12 chiral centers was successfully assigned by employing a J-based configuration analysis in combination with ROESY correlations, a quantum mechanics-based computational approach to calculate NMR chemical shifts, and a 3 min flash esterification by Mosher's reagents followed by NMR analysis. Biological evaluation of retinestatin (1) using an in vitro model of Parkinson's disease revealed that 1 protected SH-SY5Y dopaminergic cells from MPP+-induced cytotoxicity, indicating its neuroprotective effects.

Modular polyketide synthase-derived insecticidal agents: from biosynthesis and metabolic engineering to combinatorial biosynthesis for their production.

Covering: up to 2022Polyketides derived from actinomycetes are a valuable source of eco-friendly biochemical insecticides. The development of new insecticides is urgently required, as the number of insects resistant to more than one drug is rapidly increasing. Moreover, significant enhancement of the production of such biochemical insecticides is required for economical production. There has been considerable improvement in polyketide insecticidal agent production and development of new insecticides. However, most commercially important biochemical insecticides are synthesized by modular type I polyketide synthases (PKSs), and their structural complexities make chemical modification challenging. A detailed understanding of the biosynthetic mechanisms of potent polyketide insecticides and the structure-activity relationships of their analogs will provide insight into the comprehensive design of new insecticides with improved efficacies. Further metabolic engineering and combinatorial biosynthesis efforts, reinvigorated by synthetic biology, can eventually produce designed analogs in large quantities. This highlight reviews the biosynthesis of representative insecticides produced by modular type I PKSs, such as avermectin, spinosyn, and spectinabilin, and their insecticidal properties. Metabolic engineering and combinatorial biosynthetic strategies for the development of high-yield strains and analogs with insecticidal activities are emphasized, proposing a way to develop a next-generation insecticide.

Discovery and structure elucidation of glycosyl and 5-hydroxy migrastatins from dung beetle gut Kitasatospora sp.

Two new macrocyclic secondary metabolites, glycosyl-migrastatin (1) and 5-hydroxy-migrastatin (2), were isolated from a gut bacterium Kitasatospora sp. JL24 in dung beetle Onthophagus lenzii. Based on a comprehensive analysis of the nuclear magnetic resonance (NMR), MS, and UV spectroscopic data, the planar structures of 1 and 2 were successfully identified as new derivatives of migrastatin. Compound 1 was the first glycosylated member of the migrastatin family. The absolute configuration of the sugar moiety was determined to be d-glucose through the analysis of coupling constants and ROESY correlations, followed by chemical derivatization and chromatographic comparison with authentic d- and l-glucose. Compound 2, identified as 5-hydroxy-migrastatin possessing an additional hydroxy group with a previously unreported chiral center, was assigned using Mosher's method through 19F NMR chemical shifts and confirmed with the modified Mosher's method. Genomic analysis of Kitasatospora sp. strain JL24 revealed a putative biosynthetic pathway involving an acyltransferase-less type I polyketide synthase biosynthetic gene cluster. ONE-SENTENCE SUMMARY: Two secondary metabolites, glycosyl-migrastatin (1) and 5-hydroxy-migrastatin (2), were discovered from the gut bacterium Kitasatospora sp. JL24 in the dung beetle Onthophagus lenzii.

Whole-genome sequencing and analysis of Streptomyces strains producing multiple antinematode drugs.

BACKGROUND: Nematodes are parasitic animals that cause over 100 billion US dollars loss in agricultural business. The whole-genomes of two Streptomyces strains, Streptomyces spectabilis KCTC9218T and Streptomyces sp. AN091965, were sequenced. Both strains produce spectinabilin, an antinematode drug. Its secondary metabolism was examined to aid the development of an efficient nematicidal drug-producing host strain. RESULTS: The whole-genome sequences of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 were analyzed using PacBio and Illumina sequencing platforms, and assembled using hybrid methodology. The total contig lengths for KCTC9218T and AN091965 were 9.97 Mb and 9.84 Mb, respectively. A total of 8,374 and 8,054 protein-coding genes, as well as 39 and 45 secondary metabolite biosynthetic gene clusters were identified in KCTC9218T and AN091965, respectively. 18.4 ± 6.45 mg/L and 213.89 ± 21.30 mg/L of spectinabilin were produced by S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Pine wilt disease caused by nematode was successfully prevented by lower concentration of spectinabilin injection than that of abamectin recommended by its manufacturer. Production of multiple antinematode drugs, including spectinabilin, streptorubin B, and undecylprodigiosin was observed in both strains using high-resolution liquid chromatography mass spectrometry (LC-MS) analysis. CONCLUSIONS: Whole-genome sequencing of spectinabilin-producing strains, coupled with bioinformatics and mass spectrometry analyses, revealed the production of multiple nematicidal drugs in the KCTC9218T and AN091965 strains. Especially, Streptomyces sp. AN091965 showed high production level of spectinabilin, and this study provides crucial information for the development of potential nematicidal drug producers.

Publisher Correction: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

In the version of this article originally published, reference to another structure of GenB1 was omitted (Dow, G. T., Thoden, J. B., & Holden, H. M. The three-dimensional structure of NeoB: an aminotransferase involved in the biosynthesis of neomycin. Protein Sci. 27, 945-956 (2018)). This paper is now cited as reference 32, and "Another structure of GenB1 was also reported independently during the revision of this article32" was added to the text in the Discussion section. This error has been corrected in the PDF and HTML versions of the article.

Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

Gentamicin B (GB), a valuable starting material for the preparation of the semisynthetic aminoglycoside antibiotic isepamicin, is produced in trace amounts by the wild-type Micromonospora echinospora. Though the biosynthetic pathway to GB has remained obscure for decades, we have now identified three hidden pathways to GB production via seven hitherto unknown intermediates in M. echinospora. The narrow substrate specificity of a key glycosyltransferase and the C6'-amination enzymes, in combination with the weak and unsynchronized gene expression of the 2'-deamination enzymes, limits GB production in M. echinospora. The crystal structure of the aminotransferase involved in C6'-amination explains its substrate specificity. Some of the new intermediates displayed similar premature termination codon readthrough activity but with reduced toxicity compared to the natural aminoglycoside G418. This work not only led to the discovery of unknown biosynthetic routes to GB, but also demonstrated the potential to mine new aminoglycosides from nature for drug discovery.

Azetidine-Bearing Non-Ribosomal Peptides, Bonnevillamides D and E, Isolated from a Carrion Beetle-Associated Actinomycete.

Two new nonribosomal peptides, bonnevillamides D and E (1 and 2), have been discovered in Streptomyces sp. UTZ13 isolated from the carrion beetle, Nicrophorus concolor. Combinational analysis of the UV, MS, and NMR spectroscopic data revealed that their planar structures were comprised of dichlorinated linear peptides containing nonproteinogenic amino acid residues, such as 4-methylazetidinecarboxylic acid and 4-O-acetyl-5-methylproline. The configurations of bonnevillamides D and E (1 and 2) were determined based on ROESY correlations, the advanced Marfey's method, phenylglycine methyl ester derivatization, molecular modeling, and circular dichroism spectroscopy. The nonribosomal peptide synthetase biosynthetic pathway of bonnevillamides D and E has been proposed using bioinformatic analysis of the whole-genome sequence data of Streptomyces sp. UTZ13. Their biological activity toward the aggregation of amyloid-ß, which is one of the key pathogenic proteins in Alzheimer's disease, was evaluated using a thioflavin T assay and gel electrophoresis. Bonnevillamides D and E reversed the fibril formation by inducing the monomerization of amyloid-ß aggregates.

Structures and Biosynthetic Pathway of Coprisamides C and D, 2-Alkenylcinnamic Acid-Containing Peptides from the Gut Bacterium of the Carrion Beetle Silpha perforata.

Coprisamides C and D (1 and 2) were isolated from a gut bacterium, Micromonospora sp. UTJ3, of the carrion beetle Silpha perforata. Based on the combined analysis of UV, MS, and NMR spectral data, the planar structures of 1 and 2 were elucidated to be unreported derivatives of coprisamides A and B, cyclic depsipeptides bearing a 2-alkenylcinnamic acid unit and the unusual amino acids ß-methylaspartic acid and 2,3-diaminopropanoic acid. The absolute configuration of 1 was determined using the advanced Marfey's method, phenylglycine methyl ester derivatization, and J-based configuration analysis. The biosynthetic gene clusters for the coprisamides were investigated based on genomic data from coprisamide-producing strains Micromonospora sp. UTJ3 and Streptomyces sp. SNU533. Coprisamide C (1) was active against the Mycobacterium tuberculosis mc2 6230 strain.

Unprecedented Noncanonical Features of the Nonlinear Nonribosomal Peptide Synthetase Assembly Line for WS9326A Biosynthesis.

Systematic inactivation of nonribosomal peptide synthetase (NRPS) domains and translocation of the thioesterase (TE) domain revealed several unprecedented nonlinear NRPS assembly processes during the biosynthesis of the cyclodepsipeptide WS9326A in Streptomyces sp. SNM55. First, two sets of type ΙΙ TE (TEΙΙ)-like enzymes mediate the shuttling of activated amino acids between two sets of stand-alone adenylation (A)-thiolation (T) didomain modules and an "A-less" condensation (C)-T module with distinctive specificities and flexibilities. This was confirmed by the elucidation of the affinities of the A-T didomains for the TEΙΙs and its structure. Second, the C-T didomain module operates iteratively and independently from other modules in the same protein to catalyze two chain elongation cycles. Third, this biosynthetic pathway includes the first example of module skipping, where the interpolated C and T domains are required for chain transfer.

Minor components of aminoglycosides: recent advances in their biosynthesis and therapeutic potential.

Covering: up to 2019 There is significant demand for new aminoglycoside antibiotics due to the widespread emergence of multidrug-resistant Gram-negative bacteria and their high toxicity, but these are not easily accessible in nature because their biosynthetic gene clusters are less commonly found in actinomycetes than are other natural products. Mining minor aminoglycoside components whose pharmacological activity has not yet been assessed could be an alternative approach for the development of next-generation antibiotics for use in the post-antibiotic era. Here, we review the biosynthetic steps responsible for the structural diversity of aminoglycosides and highlight current developments regarding the use of natural minor and semi-synthetic aminoglycosides as promising therapeutic leads or candidates.

High-yield production of multiple O-methylated phenylpropanoids by the engineered Escherichia coli-Streptomyces cocultivation system.

BACKGROUND: O-Methylated phenylpropanoids, which are generally present in small amounts in plants, have improved or distinct biological activities and pharmacological properties as opposed to their unmethylated counterparts. Although microbial production could be a useful tool for the efficient and environment-friendly production of methylated phenylpropanoids, a high-yield microbial production of neither tri-methylated stilbenes nor di-/tri-methylated flavonoids has been achieved to date. RESULTS: A methyltransferase from Streptomyces avermitilis (SaOMT2), which has been known to possess 7-O-methylation activity toward several flavonoids, exhibited more diverse regiospecificity and catalyzed mono-, di-, and tri-methylation of stilbene, flavanone, and flavone when it was expressed in Streptomyces venezuelae. For the efficient production of multi-methylated phenylpropanoids, a cocultivation system was developed by employing engineered Escherichia coli strains producing pterostilbene, naringenin, and apigenin, respectively, along with SaOMT2-expressing S. venezuelae mutant. Consequently, high-yield microbial production of tri-methylated stilbenes and di-/tri-methylated flavonoids (including 3,5,4'-trimethoxystilbene, 5-hydroxy-7,4'-dimethoxyflavanone, 4'-hydroxy-5,7-dimethoxyflavanone, 5,7,4'-trimethoxyflavanone, 5-hydroxy-7,4'-dimethoxyflavone, and 5,7,4'-trimethoxyflavone) has been demonstrated for the first time. CONCLUSIONS: This cocultivation system based on the phenylpropanoid-producing E. coli and SaOMT2-expressing S. venezuelae provides an efficient tool for producing scarce and potentially valuable multi-methylated phenylpropanoids and will enable further development of these compounds as pharmaceuticals and nutraceuticals.

Camporidines A and B: Antimetastatic and Anti-inflammatory Polyketide Alkaloids from a Gut Bacterium of Camponotus kiusiuensis.

Chemical studies of gut bacteria of the carpenter ant Camponotus kiusiuensis led to the discovery of two new alkaloids, camporidines A and B (1 and 2), from Streptomyces sp. STA1. The structures of 1 and 2 were established as new polyketide alkaloids bearing a piperidine-cyclopentene-epoxide 6/5/3 tricyclic system based on NMR spectroscopic and mass spectrometric analysis. The relative configurations of the camporidines were determined by their 1H-1H NOESY/ROESY and 1D NOE NMR correlations. The experimental ECD spectra of 1 and 2 were compared with their calculated ECD spectra to assign their absolute configurations. Camporidine A (1) displayed antimetastatic activity by suppression of cell invasion against the metastatic breast cancer cell line MDA-MB-231 and showed an anti-inflammatory effect by suppressing nitric oxide production induced by lipopolysaccharide. In addition, the putative biosynthetic gene cluster of the camporidines was identified, and the biosynthetic pathway of the camporidines was proposed based on bioinformatic analysis of the full genome of Streptomyces sp. STA1. Camporidines A and B (1 and 2) could be biosynthesized by a modular type I PKS containing an acyl transferase domain that accepts an unusual extender unit, which becomes the (C1'-C6') hexyl side chain. The post-PKS modification enzymes were predicted to perform an amination and an oxidation along with spontaneous Schiff base formation and generate the unique piperidine-cyclopentene-epoxide 6/5/3 tricyclic framework.

The biosynthetic pathway of FK506 and its engineering: from past achievements to future prospects.

FK506, a 23-membered macrolide produced by several Streptomyces species, is an immunosuppressant widely used to prevent the rejection of transplanted organs. In addition, FK506 and its analogs possess numerous promising therapeutic potentials including antifungal, neuroprotective, and neuroregenerative activities. Herein, we introduce the biological activities and mechanisms of action of FK506 and discuss recent progress made in understanding its biosynthetic pathway, improving production, and in the mutasynthesis of diverse analogs. Perspectives highlighting further strain improvement and structural diversification aimed at generating more analogs with improved pharmaceutical properties will be emphasized.

Characterization and engineering of the ethylmalonyl-CoA pathway towards the improved heterologous production of polyketides in Streptomyces venezuelae.

Streptomyces venezuelae has an inherent advantage as a heterologous host for polyketide production due to its fast rate of growth that cannot be endowed easily through metabolic engineering. However, the utility of S. venezuelae as a host has been limited thus far due to its inadequate intracellular reserves of the (2S)-ethylmalonyl-CoA building block needed to support the biosynthesis of polyketides preventing the efficient production of the desired metabolite, such as tylactone. Here, via precursor supply engineering, we demonstrated that S. venezuelae can be developed into a more efficient general heterologous host for the quick production of polyketides. We first identified and functionally characterized the ethylmalonyl-CoA pathway which plays a major role in supplying the (2S)-ethylmalonyl-CoA extender unit in S. venezuelae. Next, S. venezuelae was successfully engineered to increase the intracellular ethylmalonyl-CoA concentration by the deletion of the meaA gene encoding coenzyme B12-dependent ethylmalonyl-CoA mutase in combination with ethylmalonate supplementation and was engineered to upregulate the expression of the heterologous tylosin PKS by overexpression of the pathway specific regulatory gene pikD. Thus, a dramatic increase (∼10-fold) in tylactone production was achieved. In addition, the detailed insights into the role of the ethylmalonyl-CoA pathway, which is present in most streptomycetes, provides a general strategy to increase the ethylmalonyl-CoA supply for polyketide biosynthesis in the most prolific family of polyketide-producing bacteria.

2-Deoxystreptamine-containing aminoglycoside antibiotics: recent advances in the characterization and manipulation of their biosynthetic pathways.

The 2-deoxystreptamine-containing aminoglycosides, such as neomycin, kanamycin and gentamicin, are an important class of antibiotics. A detailed understanding of the complete biosynthetic pathway of aminoglycosides and their biosynthetic enzymes will allow us to not only generate more robust antibiotic agents or drugs with other altered biological activities, but also to produce clinically important semi-synthetic antibiotics by direct fermentation. This Highlight focuses on recent advances in the characterization of their biosynthetic enzymes and pathway as well as some chemo-enzymatic and metabolic engineering approaches for the biological production of natural, semi-synthetic, and novel aminoglycosides.

Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation.

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.

Characterization of FK506 biosynthetic intermediates involved in post-PKS elaboration.

The post-PKS modification steps of FK506 biosynthesis include C9-oxidation and 31-O-methylation, but the sequence of these reactions and the exact route have remained unclear. This study details the post-PKS modification pathways in FK506 biosynthesis through the identification of all intermediates and in vitro enzymatic reactions of the cytochrome P450 hydroxylase FkbD and the methyltransferase FkbM. These results complete our understanding of post-PKS modification steps to FK506 showing the substrate flexibility of two enzymes involved and the existence of two parallel biosynthetic routes to FK506.

Achievements and impacts of glycosylation reactions involved in natural product biosynthesis in prokaryotes.

Bioactive natural products, such as polyketides, flavonoids, glycopeptides, and aminoglycosides, have been used as therapeutic agents. Many of them contain structurally diverse sugar moieties attached to the aglycone core structures. Glycosyltransferases (GTs) catalyze the attachment of nucleotide-activated sugar substrates to acceptor aglycones. Because these sugar moieties are usually essential for biological activity, in vivo pathway engineering in prokaryotic hosts and in vitro enzymatic approaches coupled with GT engineering are currently being used to synthesize novel glycosylated derivatives, and some of them exhibited improved biological activities compared to the parent molecules. Therefore, harnessing the potential of diverse glycosylation reactions in prokaryotes will increase the structural diversity of natural products and the possibility to generate new bioactive products.

Chemo-enzymatic Synthesis of Pseudo-trisaccharide Aminoglycoside Antibiotics with Enhanced Nonsense Read-through Inducer Activity.

Aminoglycosides (AGs) are broad-spectrum antibiotics used to treat bacterial infections. Over the last two decades, studies have reported the potential of AGs in the treatment of genetic disorders caused by nonsense mutations, owing to their ability to induce the ribosomes to read through these mutations and produce a full-length protein. However, the principal limitation in the clinical application of AGs arises from their high toxicity, including nephrotoxicity and ototoxicity. In this study, five novel pseudo-trisaccharide analogs were synthesized by chemo-enzymatic synthesis by acid hydrolysis of commercially available AGs, followed by an enzymatic reaction using recombinant substrate-flexible KanM2 glycosyltransferase. The relationships between their structures and biological activities, including the antibacterial, nephrotoxic, and nonsense readthrough inducer (NRI) activities, were investigated. The absence of 1-N-acylation, 3',4'-dideoxygenation, and post-glycosyl transfer modifications on the third sugar moiety of AGs diminishes their antibacterial activities. The 3',4'-dihydroxy and 6'-hydroxy moieties regulate the in vitro nephrotoxicity of AGs in mammalian cell lines. The 3',4'-dihydroxy and 6'-methyl scaffolds are indispensable for the ex vivo NRI activity of AGs. Based on the alleviated in vitro antibacterial properties and nephrotoxicity, and the highest ex vivo NRI activity among the five compounds, a kanamycin analog (6'-methyl-3''-deamino-3''-hydroxykanamycin C) was selected as a novel AG hit for further studies on human genetic disorders caused by premature transcriptional termination.