Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting

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.

Effects of heavy‐ion beam irradiation on avermectin B1a and its analogues production by Streptomyces avermitilis

“…Cao et al. established a reliable high‐throughput screening (HTS) strategy integrating fluorescence‐activated cell sorting and random mutagenesis for screening high avermectin titer S. avermitilis mutants. They reported that a high‐titer avermectin producer (G9) was obtained from 5760 mutants after mutagenesis and HTS.…”

“…The ZJAV-Y-HS mutants was found to provide the best production, with maximal production of 4632.17 μg/mL of avermectin and other analogue after 240 h. Among the ZJAV-Y-147 and ZJAV-Y-HS mutants and the wild-type strain, the ZJAV-Y-147 mutant was found to provide the best production, yielding maximal production of 4822.23 μg/mL of the B1a component of avermectin as estimated by HPLC-UV. Cao et al [32] established a reliable high-throughput screening (HTS) strategy integrating fluorescence-activated cell sorting and random mutagenesis for screening high avermectin titer S. avermitilis mutants. They reported that a high-titer avermectin producer (G9) was obtained from 5760 mutants after mutagenesis and HTS.…”

Functional development and performance enhancement of probiotics

“…周扬等人 [97] 通过活跃链霉菌产生的那 西肽对藤黄微球菌具有抑制效果, 根据抑菌圈的大 小经高通量筛选获得了那西肽高产菌株. Cao等人 [98] 通过对孢子进行碘化丙锭(PI)和二乙酸荧光素(FDA) 染色, 利用流式细胞仪分选活性孢子, 高通量筛选了 阿维菌素高产菌株. Zeng等人 [99] Figure 3 (Color online) The process of microbial high throughput screening 型在菌株之间的传递成为可能 [104] .…”

Combinatorial Effect of ARTP Mutagenesis and Ribosome Engineering on an Industrial Strain of Streptomyces albus S12 for Enhanced Biosynthesis of Salinomycin

“…Although ARTP has several outstanding advantages over other conventional methods, including rapid mutation rate and high operational flexibility (Zhang et al, 2014). In order to obtain a desired target, a single ARTP treatment or an iterative ARTP treatment is usually performed (Wang et al, 2010; Zhang et al, 2015; Ren et al, 2017; Cao et al, 2018). Interestingly, in previous studies, ARTP treatment was also combined with other treatments such as single resistance screening (Liang et al, 2015; Ren et al, 2017), or radiation mutagenesis with UV (Zhang et al, 2015) to improve the overall efficiency.…”