Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures.

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N-N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H-15N heteronuclear multiple bond correlation (HMBC) along with 1H-15N heteronuclear single quantum coherence (HSQC) and 1H-15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade-structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.

Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams.

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or ß-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and ß-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a ß-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 µM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.

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.

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.

Nyuzenamide C, an Antiangiogenic Epoxy Cinnamic Acid-Containing Bicyclic Peptide from a Riverine Streptomyces sp.

A new nonribosomal peptide, nyuzenamide C (1), was discovered from riverine sediment-derived Streptomyces sp. DM14. Comprehensive analysis of the spectroscopic data of nyuzenamide C (1) revealed that 1 has a bicyclic backbone composed of six common amino acid residues (Asn, Leu, Pro, Gly, Val, and Thr) and four nonproteinogenic amino acid units, including hydroxyglycine, ß-hydroxyphenylalanine, p-hydroxyphenylglycine, and 3,ß-dihydroxytyrosine, along with 1,2-epoxypropyl cinnamic acid. The absolute configuration of 1 was proposed by J-based configuration analysis, the advanced Marfey's method, quantum mechanics-based DP4 calculations, and bioinformatic analysis of its nonribosomal peptide synthetase biosynthetic gene cluster. Nyuzenamide C (1) displayed antiangiogenic activity in human umbilical vein endothelial cells and induced quinone reductase in murine Hepa-1c1c7 cells.

Taeanamides A and B, Nonribosomal Lipo-Decapeptides Isolated from an Intertidal-Mudflat-Derived Streptomyces sp.

Two new lipo-decapeptides, namely taeanamides A and B (1 and 2), were discovered from the Gram-positive bacterium Streptomyces sp. AMD43, which was isolated from a mudflat sample from Anmyeondo, Korea. The exact molecular masses of 1 and 2 were revealed by high-resolution mass spectrometry, and the planar structures of 1 and 2 were elucidated using NMR spectroscopy. The absolute configurations of 1 and 2 were determined using a combined analysis of 1H-1H coupling constants and ROESY correlations, the advanced Marfey's method, and bioinformatics. The putative nonribosomal peptide synthetase pathway for the taeanamides was identified by analyzing the full genome sequence data of Streptomyces sp. AMD43. We also found that taeanamide A exhibited mild anti-tuberculosis bioactivity, whereas taeanamide B showed significant bioactivity against several cancer cell lines.

Systems and synthetic biology to elucidate secondary metabolite biosynthetic gene clusters encoded in Streptomyces genomes.

Covering: 2010 to 2020 Over the last few decades, Streptomyces have been extensively investigated for their ability to produce diverse bioactive secondary metabolites. Recent advances in Streptomyces research have been largely supported by improvements in high-throughput technology 'omics'. From genomics, numerous secondary metabolite biosynthetic gene clusters were predicted, increasing their genomic potential for novel bioactive compound discovery. Additional omics, including transcriptomics, translatomics, interactomics, proteomics and metabolomics, have been applied to obtain a system-level understanding spanning entire bioprocesses of Streptomyces, revealing highly interconnected and multi-layered regulatory networks for secondary metabolism. The comprehensive understanding derived from this systematic information accelerates the rational engineering of Streptomyces to enhance secondary metabolite production, integrated with the exploitation of the highly efficient 'Design-Build-Test-Learn' cycle in synthetic biology. In this review, we describe the current status of omics applications in Streptomyces research to better understand the organism and exploit its genetic potential for higher production of valuable secondary metabolites and novel secondary metabolite discovery.

The yeast platform engineered for synthetic gRNA-landing pads enables multiple gene integrations by a single gRNA/Cas9 system.

Saccharomyces cerevisiae is a versatile microbial platform to build synthetic metabolic pathways for production of diverse chemicals. To expedite the construction of complex metabolic pathways by multiplex CRISPR-Cas9 genome-edit, eight desirable intergenic loci, located adjacent to highly expressed genes selected from top 100 expressers, were identified and fully characterized for three criteria after integrating green fluorescent protein (GFP) gene - CRISPR-mediated GFP integration efficiency, expression competency assessed by levels of GFP fluorescence, and assessing growth rates of GFP integrated strains. Five best performing intergenic loci were selected to build a multiplex CRISPR platform, and a synthetic 23-bp DNA comprised of 20-bp synthetic DNA with a protospacer adjacent motif (PAM) was integrated into the five loci using CRISPR-Cas9 in a sequential manner. This process resulted in five different yeast strains harbouring 1-5 synthetic gRNA-binding sites in their genomes. Using these pre-engineered yeast strains, simultaneous integrations of 2-, 3-, 4-, or 5-genes to the targeted loci were demonstrated with efficiencies from 85% to 98% using beet pigment betalain (3-gene pathway), hygromycin and geneticin resistance markers. Integrations of the multiple, foreign genes in the targeted loci with 100% precision were validated by genotyping. Finally, we further developed the strain to have 6th synthetic gRNA-binding site, and the resulting yeast strain was used to generate a yeast strain producing a sesquiterpene lactone, kauniolide by simultaneous 6-gene integrations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in yeast.

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.

Biosynthesis of Nonimmunosuppressive ProlylFK506 Analogues with Neurite Outgrowth and Synaptogenic Activity.

Separating the immunosuppressive activity of FK506 (1) from its neurotrophic activity is required to develop FK506 analogues as drugs for the treatment of neuronal diseases. Two new FK506 analogues, 9-deoxo-36,37-dihydro-prolylFK506 (2) and 9-deoxo-31-O-demethyl-36,37-dihydro-prolylFK506 (3) containing a proline moiety instead of the pipecolate ring at C-1 and modifications at the C-9/C-31 and C-36-C-37 positions, respectively, were biosynthesized, and their biological activities were evaluated. The proline substitution in 9-deoxo-36,37-dihydroFK506 and 9-deoxo-31-O-demethyl-36,37-dihydroFK506 reduced immunosuppressive activity by more than 120-fold, as previously observed. Compared with FK506 (1), 2 and 3 exhibited ∼1.2 × 105- and 2.2 × 105-fold reductions in immunosuppressive activity, respectively, whereas they retained almost identical neurite outgrowth activity. Furthermore, these compounds significantly increased the strength of synaptic transmission, confirming that replacement of the pipecolate ring with a proline is critical to reduce the strong immunosuppressive activity of FK506 (1) while enhancing its neurotrophic activity.

Enhanced production of clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain.

Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important ß-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3% (5.21 ± 0.26 g/l) in a flask culture and 17.4% (6.11 ± 0.36 g/l) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

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.

Structure Revision and the Biosynthetic Pathway of Tripartilactam.

Tripartilactam (1) is a natural macrocyclic lactam originally reported to have a unique [18,8,4]-tricyclic framework. However, the validity of this structure has been contested since niizalactam C (2), bearing a [18,6,6]-tricyclic skeleton, was proposed as an alternative structure in 2015. In the present study, a comprehensive reinvestigation of NMR spectroscopic data and a 13C-13C COSY NMR experiment identified direct 13C-13C coupling, thus leading to the unequivocal revision of the structure of tripartilactam as niizalactam C (2). In addition, whole-genome sequencing analysis of the tripartilactam-producing bacterial strain and subsequent bioinformatics and mutagenesis analyses identified its biosynthetic pathway, which probably utilizes one of the type I polyketide synthase (PKS) modules iteratively during its biosynthesis and exhibits spontaneous [4+2] cycloaddition from the precursor compound, sceliphrolactam, in the post-PKS process.

Formicolides A and B, Antioxidative and Antiangiogenic 20-Membered Macrolides from a Wood Ant Gut Bacterium.

Two new macrolides, formicolides A (1) and B (2), were isolated from Streptomyces sp. BA01, a gut bacterial strain of the wood ant (Formica yessensis). Their 20-membered macrocyclic lactone structures were established using NMR and mass spectrometric data. The relative configurations of the formicolides were determined by J-based configuration analysis utilizing ROESY, HETLOC, and HECADE NMR spectroscopic data. Genomic and bioinformatics analysis of the bacterial strain enabled us to identify the type-I polyketide synthase pathway employing a trans-acyltransferase system. The absolute configurations of 1 and 2 are proposed based on detailed analysis of the sequences of the ketoreductases in the modular gene cluster and statistical comparative analysis of the experimental NMR chemical shifts and quantum mechanical calculations. Formicolides A and B (1 and 2) induced quinone reductase activity in murine Hepa-1c1c7 cells and antiangiogenic activity by suppression of tube formation in human umbilical vein endothelial cells.

Absolute Configuration and Antibiotic Activity of Piceamycin.

The cultivation of a Streptomyces sp. SD53 strain isolated from the gut of the silkworm Bombyx mori produced two macrolactam natural products, piceamycin (1) and bombyxamycin C (2). The planar structures of 1 and 2 were identified by a combination of NMR, MS, and UV spectroscopic analyses. The absolute configurations were assigned based on chemical and chromatographic methods as well as ECD calculations. A new chromatography-based experimental method for determining the configurations of stereogenic centers ß to nitrogen atoms in macrolactams was established and successfully applied in this report. These compounds exhibited significant bioactivities against the silkworm entomopathogen Bacillus thuringiensis and various human pathogens as well as human cancer cell lines. In particular, piceamycin potently inhibited Salmonella enterica and Proteus hauseri with MIC values of 0.083 µg/mL and 0.025 µg/mL, respectively. The biosynthetic pathway involved in the formation of the cyclopentenone moiety in piceamycin is discussed.