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 wildtype 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 σ 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. The relevant residues were identified to center in the conserved regions. Engineering of the hrdB gene can not only elicit the overexpression of aveR but also allows for simultaneous transcription of many other genes. The results indicate that manipulating the key genes revealed by reverse engineering can effectively improve the yield of the target metabolites, providing a route to optimize production in these complex regulatory systems.precision engineering | RNA polymerase | overproduction

BackgroundDiamagnetic levitation is a technique that uses a strong, spatially varying magnetic field to simulate an altered gravity environment, as in space. In this study, using Streptomyces avermitilis as the test organism, we investigate whether changes in magnetic field and altered gravity induce changes in morphology and secondary metabolism. We find that a strong magnetic field (12T) inhibit the morphological development of S. avermitilis in solid culture, and increase the production of secondary metabolites.Methodology/Principal Findings S. avermitilis on solid medium was levitated at 0 g*, 1 g* and 2 g* in an altered gravity environment simulated by diamagnetic levitation and under a strong magnetic field, denoted by the asterix. The morphology was obtained by electromicroscopy. The production of the secondary metabolite, avermectin, was determined by OD245 nm. The results showed that diamagnetic levitation could induce a physiological response in S. avermitilis. The difference between 1 g* and the control group grown without the strong magnetic field (1 g), showed that the magnetic field was a more dominant factor influencing changes in morphology and secondary metabolite production, than altered gravity.Conclusion/SignificanceWe have discovered that magnetic field, rather than altered gravity, is the dominant factor in altered gravity simulated by diamagnetic levitation, therefore care should to be taken in the interpretation of results when using diamagnetic levitation as a technique to simulate altered gravity. Hence, these results are significant, and timely to researchers considering the use of diamagnetic levitation to explore effects of weightlessness on living organisms and on physical phenomena.

Mutant libraries of avermectin-producing Streptomyces avermitilis strains were constructed by different mutagenesis strategies. A metric was applied to assess the mutation spectrum by calculating the distribution of average phenotypic distance of each population. The results showed for the first time that a microgravity environment could introduce larger phenotype distribution and diversity than UV and N-methyl-N-nitro-Nnitrosoguanidine (NTG) could.Induced mutagenesis is a classical and successful method for improving strains to increase the productivity of commercially significant microbial metabolites. To evaluate different induced-mutagenesis approaches, Klein-Marcuschamer and Stephanopoulos presented a metric based on the quantification of phenotypic diversity to evaluate strain improvement approaches (14).New approaches of inducing mutagenesis emerged with the development of biotechnology, and of these new approaches, spaceflight-induced mutagenesis has led to great progress in strain improvement (6,15,26). In outer space, cosmic rays, high vacuum, intense magnetic field, and microgravity induced chromosomal aberrations, which lead to genetic mutations in microorganisms (13). However, it is difficult to carry out spaceflight-induced mutagenesis extensively owing to the limitations of high cost and few chances to board spaceships. Therefore, ground-based simulated experiments have greater practical significance, and high-magnetogravity experiments are a good choice to simulate the space environment (16).Avermectins and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents for animal health, agriculture, and human infections (7). A variety of mutagenesis methods have been developed to increase the productivity of S. avermitilis (18,19,(21)(22)(23)25). Though most of them can produce higher mutation rates, the potential of their success in strain improvement is different.In this study, mutant libraries of S. avermitilis strains were constructed by three mutagenesis-inducing strategies: UV, Nmethyl-N-nitro-N-nitrosoguanidine (NTG), and high-magnetogravitational environment (HMGE). For each population, the distribution of average phenotypic distance was calculated on the basis of the modified version (15) of the metric of Klein-Marcuschamer and Stephanopoulos (14). The mutation rate was also calculated. A good correlation between the distribution of average phenotypic distance and the percent improvement was found and analyzed. In this way, the potential to produce mutations among different induced-mutagenesis approaches was evaluated to find the most effective one for S. avermitilis.The industrial avermectin-producing S. avermitilis 3-115 strain and the mutants derived from strain 3-115 were grown on YMG agar medium (10). For diversity quantification and preliminary screening, fermentation was carried out in highthroughput format at 28°C. For confirmation of results and secondary screening, mutants that exhibited a higher yield than the wild-type strain were inoculated i...