Abstract
Avermectins, produced by Streptomyces avermitilis, are important antiparasitic agents. The use of traditional microbial breeding methods for this organism has been limited by the low-throughput shake flask-based screening process. The unique growth cycle of actinomycetes makes the establishment of a reliable high-throughput screening (HTS) process difficult. To enhance the efficiency of screening strains with high yields of avermectin, a HTS process aided by fluorescence-activated cell sorting (FACS) was established. Four different spore solutions were investigated for maintaining a relatively high viability of spores. Propidium iodide (PI) and fluorescein diacetate (FDA) were used to discriminate between dead and live spores using the FACS system. Spores stained with 7-μg/mL PI and 15-μg/mL FDA at 4 °C in the dark for 30 min resulted in optimum sorting. Spores were treated by atmospheric and room temperature plasma (ARTP). Single live spores were sorted and sprayed into 96-well microtiter plates containing 50 μL of solid agar culture medium. Solid-liquid combinatorial microculture was used for high-throughput avermectin culture. A high-titer avermectin producer (G9) was obtained from 5760 mutants after mutagenesis and HTS. Compared with the original strain, the titer was improved by 18.9% on flask culture and 20.6% on fermenter, respectively. The HTS process established in this study could easily be transferred to other similar target products produced by actinomycetes.
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Funding
This work was supported by the Major State Basic Research Development Program of China (973 Program, 2013CB733602), the National Natural Science Foundation of China (21390204, 21406087), the Key Research and Development Program of Jiangsu Province (BE2016689), the Fundamental Research Funds for the Central Universities (JUSRP51701A), the Six Talent Peaks Project in Jiangsu Province (2015-JY-005), the Distinguished Professor Project of Jiangsu Province, and the 111 Project (111-2-06).
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Cao, X., Luo, Z., Zeng, W. et al. Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting. Appl Microbiol Biotechnol 102, 703–712 (2018). https://doi.org/10.1007/s00253-017-8658-x
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DOI: https://doi.org/10.1007/s00253-017-8658-x