γ-Butyrolactone autoregulators and receptor proteins in non-<Emphasis Type="Italic">Streptomyces</Emphasis> actinomycetes producing commercially important secondary metabolites

The presence of -butyrolactone autoregulators and their receptor proteins were investigated in five representative strains of non-Streptomyces actinomycetes producing commercially important secondary metabolites. Ethyl acetate extracts of culture were assayed using wild-type S. virginiae for virginiae butanolide, S. lavendulae FRI-5 for IM-2, and S. griseus HH1 for A-factor. Actinoplanes teichomyceticus and Amycolatopsis mediterranei were shown to produce autoregulators. Corresponding autoregulator-binding activities were found in the crude cell-free lysates of these strains, using the binding assay with tritium-labeled autoregulator analogues as ligands, which suggests that non-Streptomyces actinomycetes might have autoregulator-dependent signaling cascades.     

Design and synthesis of γ-butyrolactone derivatives as potential spermicidal agents

A series of γ-butyrolactone derivatives has been designed and synthesized from commercially available 2-acetyl butyrolactone (3-acetyldihydrofuran-2(3H)-one, 1) by aminoalkylating its active methylene followed by condensation with different aldehydes. Compounds having amino group were further converted to their respective tartrate salts and were evaluated for spermicidal activity against human sperm in vitro. Compounds showing appreciable spermicidal activity at ⩽0.5% [3c, 4d (0.5%); 2c, 3d (0.1%); 2d, 4c (0.05%)] were tested for safety studies against human cervical (HeLa) cell line. These compounds were found safer than, Nonoxynol-9. One of the two most active compounds was also found to be the safest (IC₅₀ = 961 μg/ml; 4c), while the second compound exhibited lower safety against HeLa (IC₅₀ = 269 μg/ml; 2d). The compound 4c significantly reduced the number of free thiols on human sperm. All the compounds were inactive against Trichomonas vaginalis.     

Aspernolides A and B, butenolides from a marine-derived fungus Aspergillus terreus

Two aromatic butenolides, aspernolides A and B along with the known metabolites, butyrolactone I, terrein and physcion were isolated from the fermentation broth of a soft coral derived fungus Aspergillus terreus. The structures of these metabolites were assigned on the basis of detailed spectroscopic analysis. The absolute stereochemistry of aspernolides A (1) and B (2) was established by their preparation from the known butyrolactone I. Biogenetically aspernolides A and B must be derived from butyrolactone I, a well known specific inhibitor of cyclin dependent kinase (cdk) from A. terreus. When tested, aspernolide A exhibited mild cytotoxicity against cancer cell lines.     

Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called ‘butyrolactone autoregulators’ which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R,6R)- and (2S,3S,6S)-enantiomers] were also produced from (±)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S,6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.     

Inducing secondary metabolite production by the soil-dwelling fungus Aspergillus terreus through bacterial co-culture

The soil-dwelling fungus Aspergillus terreus was isolated from sediment collected from the lake of Wadi EI Natrun in Egypt. Co-cultivation of A. terreus with the bacteria Bacillus subtilis and Bacillus cereus on solid rice medium resulted in an up to 34-fold increase in the accumulation of constitutively present fungal natural products (4–15) compared to axenic cultures of A. terreus. The fungal products included two new butyrolactone derivatives, isobutyrolactone II (1) and 4-O-demethylisobutyrolactone II (2), together with the known N-(carboxymethyl)anthranilic acid (3) that were not present in axenic fungal controls and were only detected during co-cultivation with B. subtilis or with B. cereus. The structures of all compounds were unambiguously elucidated by 1D and 2D NMR spectroscopy, and by HRESIMS measurements, as well as by comparison with the literature. In a second set of experiments, A. terreus was co-cultured with Streptomyces lividans and with Streptomyces coelicolor. These co-cultivation experiments failed to induce fungal natural product accumulation in contrast to co-cultures with Bacillus sp. Compounds 5 and 14 showed weak inhibition of B. cereus with minimal inhibitory concentrations (MICs) of 64 μg/mL, whereas only 8 showed moderate cytotoxicity against the murine lymphoma (L5178Y) cell line with inhibition of 80% at a dose of 10 μg/mL.