Microansamycins J and K from <i>Micromonospora</i> sp. HK160111mas13OE

Microansamycins were novel pentaketide ansamycins isolated from Micromonospora sp. HK160111mas13OE with AHBA-C2-C2-C3-C3 skeleton and diverse post-PKS modifications. In this paper, two new congeners, namely microansamycins J (1) and K (2), were identified based on their NMR, HRESIMS data and compared with those of microansamycins F and E. Neither showed antibacterial activity against Staphy­lococcus aureus ATCC25923 and Escherichia coli at 40 µg/mL.</p

Activation of a Cryptic Gene Cluster in <i>Lysobacter enzymogenes</i> Reveals a Module/Domain Portable Mechanism of Nonribosomal Peptide Synthetases in the Biosynthesis of Pyrrolopyrazines

<i>Lysobacter</i> are considered “peptide specialists”. However, many of the nonribosomal peptide synthetase genes are silent. Three new compounds were identified from <i>L. enzymogenes</i> upon activating the six-module-containing <i>led</i> cluster by the strong promoter <i>P</i>_HSAF. Although <i>ledD</i> was the first gene under <i>P</i>_HSAF control, the second gene <i>ledE</i> was expressed the highest. Targeted gene inactivation showed that the two-module LedE and the one-module LedF were selectively used in pyrrolopyrazine biosynthesis, revealing a module/domain portable mechanism

Identification and Characterization of the 28‑<i>N</i>‑Methyltransferase Involved in HSAF Analogue Biosynthesis

Polycyclic tetramate macrolactams (PoTeMs) are a family of structurally intriguing bioactive natural products. Although the presence of the N-28 methyl group is known to affect bioactivities of some PoTeMs, the mechanism for this methylation remains unclear. We report here the identification and characterization of the 28-N-methyltransferase for HSAF analogues, which is encoded by a gene located outside the HSAF (heat-stable antifungal factor) cluster in Lysobacter enzymogenes C3. Our data suggested that 28-N-methyltransferase utilizes S-adenosylmethionine (SAM) to methylate HSAF analogues, and acts after the dicyclic and tricyclic ring formation and prior to C-3 hydroxylation. Kinetic analysis showed that the optimal substrate for the enzyme is 3-dehydroxy HSAF (3-deOH HSAF). Moreover, it could also accept PoTeMs bearing a 5–6 or 5–6–5 polycyclic system as substrates. This is the first N-methyltransferase identified in the family of PoTeMs, and the identification of this enzyme provides a new tool to generate new PoTeMs as antibiotic lead compounds

Hygrocins C–G, Cytotoxic Naphthoquinone Ansamycins from <i>gdmAI</i>-Disrupted <i>Streptomyces</i> sp. LZ35

Six hygrocins, polyketides of ansamycin class, were isolated from the <i>gdmAI</i>-disrupted Streptomyces sp. LZ35. The planar structure of hygrocins C–E (<b>1</b>–<b>3</b>) was determined by one-dimensional and two-dimensional NMR spectroscopy and high-resolution mass spectrometry. They are derivatives of hygrocin A but differ in the configuration at C-2 and the orientation of the C-3,4 double bond. Hygrocin F­(<b>4</b>) and G­(<b>5</b>) were shown to be isomers of hygrocin C (<b>1</b>) and B (<b>6</b>), respectively, due to the different alkyl oxygen participating in the macrolide ester linkage. Hygrocins C, D, and F were found to be toxic to human breast cancer MDA-MB-431 cells (IC₅₀ = 0.5, 3.0, and 3.3 μM, respectively) and prostate cancer PC3 cells (IC₅₀ = 1.9, 5.0, and 4.5 μM, respectively), while hygrocins B, E, and G were inactive

Unusual Activities of the Thioesterase Domain for the Biosynthesis of the Polycyclic Tetramate Macrolactam HSAF in <i>Lysobacter enzymogenes</i> C3

HSAF is an antifungal natural product with a new mode of action. A rare bacterial iterative PKS-NRPS assembles the HSAF skeleton. The biochemical characterization of the NRPS revealed that the thioesterase (TE) domain possesses the activities of both a protease and a peptide ligase. Active site mutagenesis, circular dichroism spectra, and homology modeling of the TE structure suggested that the TE may possess uncommon features that may lead to the unusual activities. The iterative PKS-NRPS is found in all polycyclic tetramate macrolactam gene clusters, and the unusual activities of the TE may be common to this type of hybrid PKS-NRPS

Juanlimycins A and B, Ansamycin Macrodilactams from <i>Streptomyces</i> sp.

Ansamycins are a family of macrolactams characterized by an aromatic chromophore with an aliphatic chain (<i>ansa</i> chain) connected back to a nonadjacent position through an amide bond. This family has shown a high degree of druggability exemplified by rifamycins, maytansinoids, and geldanamycins. In this study, the isolation of two novel ansamycin macrodilactams with unprecedented features, juanlimycins A (<b>1</b>) and B (<b>2</b>), from <i>Streptomyces</i> sp. LC6 were reported. The structures of <b>1</b> and <b>2</b> were assigned on the basis of analysis of NMR spectroscopic data and X-ray single crystal diffraction

Activating a Cryptic Ansamycin Biosynthetic Gene Cluster To Produce Three New Naphthalenic Octaketide Ansamycins with <i>n</i>‑Pentyl and <i>n</i>‑Butyl Side Chains

Genome mining is a rational approach to discovering new natural products. The genome sequence analysis of <i>Streptomyces</i> sp. LZ35 revealed the presence of a putative ansamycin gene cluster (<i>nam</i>). Constitutive overexpression of the pathway-specific transcriptional regulatory gene <i>nam1</i> successfully activated the <i>nam</i> gene cluster, and three novel naphthalenic octaketide ansamycins were discovered with unprecedented <i>n</i>-pentylmalonyl-CoA or <i>n</i>-butylmalonyl-CoA extender units. This study represents the first example of discovering novel ansamycin scaffolds via activation of a cryptic gene cluster

Heterocyclic Aromatic <i>N</i>‑Oxidation in the Biosynthesis of Phenazine Antibiotics from Lysobacter antibioticus

Heterocyclic aromatic <i>N</i>-oxides often have potent biological activities, but the mechanism for aromatic <i>N</i>-oxidation is unclear. Six phenazine antibiotics were isolated from Lysobacter antibioticus OH13. A 10 gene cluster was identified for phenazine biosynthesis. Mutation of <i>LaPhzNO1</i> abolished all <i>N</i>-oxides, while non-oxides markedly increased. LaPhzNO1 is homologous to Baeyer–Villiger flavoproteins but was shown to catazlye phenazine <i>N</i>-oxidation. LaPhzNO1 and LaPhzS together converted phenazine 1,6-dicarboxylic acid to 1,6-dihydroxyphenazine <i>N</i>5,<i>N</i>10-dioxide. LaPhzNO1 also catalyzed <i>N</i>-oxidation of 8-hydroxyquinoline

Transgenic overexpression of PABA synthase improves thermotolerance of strain <i>8213</i>.

<p>(A) Relative mRNA level of <i>Pabs</i> gene of strains <i>02</i>, <i>8213</i> and two <i>Pabs</i>-overexpressing transgenic strains <i>TB-2</i> and <i>TB-3</i> (derived from <i>8213</i>) under normal temperature (23°C) and heat stress (33°C). The mRNA of corresponding samples was extracted and analyzed after 24 hours of treatment. (B) The PABA content of strains <i>02</i>, <i>8213</i> and <i>TB-2</i> and <i>TB-3</i> under normal temperature (23°C) and heat stress (33°C) for 3 days. The PABA content of corresponding samples was extracted and measured after 3 days of treatment. (C) The mycelia growth of Strains <i>02</i>, <i>8213</i> and <i>TB-2</i> and <i>TB-3</i> under normal temperature (23°C) and heat stress (33°C). Mycelia cultures were photographed after 2 weeks of treatment. (D) The mycelia elongation of strains <i>02</i>, <i>8213</i> and <i>TB-2</i> and <i>TB-3</i> under normal temperature (23°C) and heat stress (33°C). The mycelia length is measured after 14 and 21 days of treatment. Three independent biological replicates were performed for each analysis. Data are expressed as average ± SEM. Unpaired t-tests were performed between strain <i>8213</i> and all other strains as indicated within each treatment condition, ns: P>0.05, *: P<0.05, **: P<0.01.</p