Avermectin B1a

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are widely used in the field of animal health, agriculture, and human health. Here we have adopted a practical approach to successfully improve avermectin production in an industrial overproducer. Transcriptional levels of the wild-type strain and industrial overproducer in production cultures were monitored using microarray analysis. The avermectin biosynthetic genes, especially the pathway-specific regulatory gene, aveR, were up-regulated in the high-producing strain. The upstream promoter region of aveR was predicted and proved to be directly recognized by sigma(hrdB) in vitro. A mutant library of hrdB gene was constructed by error-prone PCR and selected by high-throughput screening. As a result of evolved hrdB expressed in the modified avermectin high-producing strain, 6.38 g/L of avermectin B1a was produced with over 50% yield improvement, in which the transcription level of aveR was significantly increased.

Microbial samples, including our library of known microbial compounds (ca. 300) and microbial culture broths (ca. 9000), were screened for small molecules affecting the phenotype of Caenorhabditis elegans. As a result, seven known compounds were found to induce phenotypic abnormality of C. elegans. Staurosporine exhibited morphological defects in the vulva and tail of C. elegans, avermectin B1a exhibited hatching inhibition of starting eggs on day 1 at 25-100 µM and growth inhibition at 0.01-12.5 µM, siccanin and antimycin A inhibited the growth of C. elegans, and fluorouracil inhibited hatching of eggs newly spawned by adult C. elegans. Toromycin induced morphological defects in the intestine. 5-(4-Methoxyphenyl)-oxazole, isolated as a fungal metabolite for the first time, inhibited the hatching of eggs newly spawned by adult C. elegans.

Many γ-butyrolactone-autoregulator receptors control the production of secondary metabolites in Streptomyces spp. Hence, AvaR1, an autoregulator receptor protein in Streptomyces avermitilis, was characterized as a negative regulator of avermectin (Ave) production. Deletion of AvaR1 in a high-producing strain increased production of Ave B1a approx. 1.75 times (~700 μg/ml) compared with the parent strain. Semi-quantitative RT-PCR and electrophoretic mobility shift assays revealed that AvaR1 regulates the biosynthesis of Ave but not through the aveR pathway-specific regulatory gene. A special signaling molecule, avenolide, increased production of Ave. This study has refined our understanding of how avenolide regulates the production of Aves which is promising for developing new methods to improve the production of antibiotics in industrial strains.

Nuclear magnetic resonance is defined as a quantitative spectroscopic tool that enables a precise determination of the number of substances in liquids as well as in solids. There is few report demonstrating the application of NMR in the quantification of avermectin B1a (AVB1a ); here, a proton nuclear magnetic resonance spectroscopy ((1) H NMR) using benzene [1-methoxy-4-(2-nitroethyl) (PMN)] as an internal standard and deuterochloroform as an NMR solvent was tested for the quantitative determination of AVB1a . The integrated signal of AVB1a at 5.56 ppm and the signal of PMN at 8.14 ppm in the (1) H NMR spectrum were used for quantification purposes. Parameters of specificity, linearity, accuracy, precision, intermediate precision, range, limit of detection (LOD), limit of quantification (LOQ), stability and robustness were validated. The established method was accurate and precise with good recovery (98.86%) and relative standard deviation (RSD) of assay (0.