Heterologous biosynthesis of myxobacterial lanthipeptides melittapeptins.

The myxobacteria are an attractive bioresource for bioactive compounds since the large size genome contains many biosynthetic gene clusters of secondary metabolites. The genome of the myxobacterium Melittangium boletus contains three biosynthetic gene clusters for lanthipeptide production. One of the gene clusters includes genes coding lanthipeptide precursor (melA), class II lanthipeptide synthetase (melM), and transporter (melT). The amino acid sequence of melA indicated similarity with that of known lanthipeptides mersacidin and lichenicidin A1 by the alignment. To perform heterologous production of new lanthipeptides, the expression vector containing the essential genes (melA and melM) was constructed by utilizing codon-optimized synthetic genes. The co-expression of two genes in the host bacterial cells of Escherichia coli BL21 (DE3) afforded new lanthipeptides named melittapeptins A-C. The structures of melittapeptins A-C including lanthionine/methyllanthionine bridge pattern were proposed based on protease digestion and MS/MS experiments. The native strain of M. boletus did not produce melittapeptins A-C, so heterologous production using the biosynthetic gene cluster was effective in obtaining the lanthipeptides. Melittapeptins A-C showed specific and potent antibacterial activity to the Gram-positive bacterium Micrococcus luteus. To the best of our knowledge, this is the first report of antibacterial lanthipeptides derived from myxobacterial origin. KEY POINTS: • New lanthipeptides melittapeptins were heterologously produced in Escherichia coli. • Melittapeptins showed specific antibacterial activity against Micrococcus luteus. • Melittapeptins were the first antibacterial lanthipeptides of myxobacterial origin.

Enhypyrazinones A and B, Pyrazinone Natural Products from a Marine-Derived Myxobacterium Enhygromyxa sp.

To date, studies describing myxobacterial secondary metabolites have been relatively scarce in comparison to those addressing actinobacterial secondary metabolites. This realization suggests the immense potential of myxobacteria as an intriguing source of secondary metabolites with unusual structural features and a wide array of biological activities. Marine-derived myxobacteria are especially attractive due to their unique biosynthetic gene clusters, although they are more difficult to handle than terrestrial myxobacteria. Here, we report the discovery of two new pyrazinone-type molecules, enhypyrazinones A and B, from a marine-derived myxobacterium Enhygromyxa sp. Their structures were elucidated by HRESIMS and comprehensive NMR data analyses. Compounds 1 and 2, which contain a rare trisubstituted-pyrazinone core, represent a unique class of molecules from Enhygromyxa sp.

An Unusual Diterpene-Enhygromic Acid and Deoxyenhygrolides from a Marine Myxobacterium, Enhygromyxa sp.

Three new compounds, enhygromic acid (1) and deoxyenhygrolides A (2) and B (3), were isolated from a marine myxobacterium, Enhygromyxa sp. Compound 1 was found to be an acrylic acid derivative with a rare polycyclic carbon skeleton, decahydroacenaphthylene, by spectroscopic analyses. Compounds 2 and 3 were deoxy analogs of the known γ-alkylidenebutenolides, enhygrolides. Compound 1 exhibited cytotoxicity against B16 melanoma cells and anti-bacterial activity against Bacillus subtilis, and enhanced the NGF-induced neurite outgrowth of PC12 cells.

Isolation and Biosynthetic Analysis of Haliamide, a New PKS-NRPS Hybrid Metabolite from the Marine Myxobacterium Haliangium ochraceum.

Myxobacteria of marine origin are rare and hard-to-culture microorganisms, but they genetically harbor high potential to produce novel antibiotics. An extensive investigation on the secondary metabolome of the unique marine myxobacterium Haliangium ochraceum SMP-2 led to the isolation of a new polyketide-nonribosomal peptide hybrid product, haliamide (1). Its structure was elucidated by spectroscopic analyses including NMR and HR-MS. Haliamide (1) showed cytotoxicity against HeLa-S3 cells with IC50 of 12 µM. Feeding experiments were performed to identify the biosynthetic building blocks of 1, revealing one benzoate, one alanine, two propionates, one acetate and one acetate-derived terminal methylene. The biosynthetic gene cluster of haliamide (hla, 21.7 kbp) was characterized through the genome mining of the producer, allowing us to establish a model for the haliamide biosynthesis. The sulfotransferase (ST)-thioesterase (TE) domains encoded in hlaB appears to be responsible for the terminal alkene formation via decarboxylation.

Heterologous production of a new lanthipeptide boletupeptin using a cryptic biosynthetic gene cluster of the myxobacterium Melittangium boletus.

Myxobacteria have comparatively large genomes that contain many biosynthetic genes with the potential to produce secondary metabolites. Based on genome mining, we discovered a new biosynthetic gene cluster of class III lanthipeptide in the genome of the myxobacterium Melittangium boletus. The biosynthetic gene cluster contained a precursor peptide-coding gene bolA, and a class III lanthipeptide synthetase-coding gene bolKC. The expression vector containing bolA and bolKC was constructed using synthetic DNA with codon-optimized sequences based on the commercially available vector pET29b. Co-expression of the two genes in the host Escherichia coli BL21(DE3) yielded a new class III lanthipeptide named boletupeptin. The structure of boletupeptin was proposed to have one unit of labionin, as determined by mass spectrometry experiments after reductive cleavage. This is the first report of a class III lanthipeptide from a myxobacterial origin.

Identification of a Novel Antibiotic from Myxobacterium Stigmatella Eracta WXNXJ-B and Evaluation of its Antitumor Effects I n - vitro.

This work was to isolate and identify the bioactive secondary metabolite which was produced by myxobacterium Stigmatella eracta WXNXJ-B, and to evaluate its antitumor and apoptosis-inducing effects. The results showed that one novel compound (molecular formula C29H25NO3) was isolated, purified by Sephadex LH-20 column chromatography and preparative RP-HPLC, and identified as 5-(6-benzyl-quinolin-3-ylmethyl)-6- phenyl-3,7-dioxa- bicycle [4.1.0] heptan-3-one (named as quinoxalone) according to its UV, IR, HRMS and NMR spectra. The compound showed strong antitumor activity on B16, HepG2, MCF-7, SGC-7901, MDA-MB231 and CT-26 six tumor cell lines in-vitro. Nevertheless, it showed less cytotoxic to the mouse normal spleen cells (IC50 was 836.27 ± 13.02 µg mL(-1)). The cytotoxic study on HepG2 cells in-vitro showed that quinoxalone could induce the change of cell nuclear and arrested the cell division in the S and G2/M phase. Our results suggest that quinoxalone could be a potential anti-cancer agent.