Raising the avermectins production in Streptomyces avermitilis by utilizing nanosecond pulsed electric fields (nsPEFs)

Avermectins and its analogs, produced from a gram-positive bacterium named Streptomyces avermitilis, are major commercial antiparasitic agents applied in agriculture, animal health and human anti-infection1. There are eight major avermectin components (A1a, A2a, A1b, A2b, B1a, B2a, B1b and B2b) resulting from structural differences at C5, C22-C23, and C26. Among those compounds, the B1a component has the most effective antiparasitic activity2. Avermectins are a novel class of 16-membered ring macrolide antibiotics, not only exhibiting excellent anthelmintic activity against nematode and arthropod parasites, but having low toxic side effects on the host organism as well2,3. On the other hand, raising the production of avermectins is a big challenge with commercial importance in the biological pesticide market, because its output is remarkably insufficient for human consumption nowadays. Basically, there are three conventional approaches to improve the fermentation efficiency of avermectins in S. avermitilis: utilization of mutagens (such as ultraviolet, atmospheric pressure cold plasma, lithium chloride and nitrosoguanidine)4,5, genetic engineering technology6,7, and optimization of fermentation conditions8,9. In fact, each technique has its disadvantages. For the using of mutagens, positive mutants are produced accompanied by the formation of negative mutants5,10. Referring to genetic engineering technology, the majority of bacterial recombinant strains bearing genes cloned in plasmids are known to be often unstable both in batch and prolonged continuous cultivation in a chemostat11. With respect to optimizing the fermentation conditions, it has limited capacity to improve the fermentation efficiency10,12. Therefore, an alternative effective and easy-to-apply technology to yield amount of avermectins in S. avermitilis has been taken on a high priority. Recently, nanosecond pulsed electric fields (nsPEFs) is emerging as a novel non-thermal technology concerned in the biomedical field, including electro-gene therapy13, cell electrofusion14, tumor therapy15,16,17, bacteria inactivation18, platelet gel formation19, calcium mobilization20 and alteration of cell membrane permeability21. Unlike conventional electroporation, nsPEFs exhibits extremely short pulse durations (nanoseconds), high electric fields (kV/cm), but low energy (mJ/cc) and non-thermal effects22. More recently, an interest is growing in concerning the proliferation effect induced by nsPEFs under relatively low electric field strength. Several researches have been reported that nsPEFs could improve the growth of Arabidopsis thaliana23,24 and Haloxylon ammodendron seeds25 as well as enhance the proliferation and dedifferentiation of chondrocytes26. Inspired by the promising capability of nsPEFs for cell proliferation enhancement, we attempt to introduce this pulsed power technology to stimulate cell growth and to increase avermectins production of S. avermitilis. In this study, nsPEFs was applied to treat the spore suspensions of S. avermitilis. The cell viability of S. avermitilis after treatment was examined via colony forming units (CFU) count, and the morphology change of S. avermitilis was detected by scanning electron microscope (SEM). The cell growth curve and the total avermectins production of S. avermitilis were measured by UV spectrometer at 450 nm and 245 nm, respectively. Furthermore, the changes in the expression of three key genes (aveR, malE and σ25) related to the avermectins production during the fermentation were analyzed by real-time RT-PCR. To better understand the mechanism of nsPEFs induced S. avermitilis proliferation enhancement, the physicochemical properties of nsPEFs-treated phosphate buffer saline (PBS) without spore were evaluated and compared by recording the oxidation reduction potential (ORP), electrical conductivity, pH and temperature.