乙基伊维菌素高产菌株选育及其发酵工艺优化

基因工程菌Streptomyces avermitilis AVE-H39产生的甲基和乙基伊维菌素 (methyl and ethyl ivermectins)，为新型高效低毒的阿维菌素 (avermectin) 衍生物，具有很好的开发应用前景。活性测试结果显示，乙基伊维菌素对朱砂叶螨Tetranychus cinnabarinus和松材线虫Bursaphelenchus xylophilus等农林作物害虫的活性优于甲基伊维菌素。鉴于菌株S. avermitilis AVE-H39的乙基伊维菌素产量低，制约了产业化开发，本研究经过多轮诱变选育，筛选获得了一株乙基伊维菌素发酵单位高的突变菌株，并通过Plackett-Burman 试验设计及Box-Behnken 设计-响应面法 (response surface methodology, RSM)对该突变菌株的发酵培养基进行改良。经优化后的发酵条件为:玉米淀粉149.8 g/L，黄豆粉38.1 g/L，(NH₄)₂SO₄ 3.04 g/L，甘露醇30.0 g/L，酵母抽提物20.0 g/L，CaCO₃ 3.0 g/L，CoCl₂ 0.01 mg/L，FeSO₄ 0.002 mg/L，转速220 r/min，pH 7.2，装液量30 mL/250 mL，接种量5%，发酵温度28 ℃。在此条件下，乙基伊维菌素的发酵效价达到最大值，为4 965 mg/L。研究结果将为开发以乙基伊维菌素为主成分的新型十六元大环内酯农药奠定基础。

Breeding of high-yield strains for producing ethyl ivermectin and the optimization of the fermentation process

Methyl ivermectin and ethyl ivermectin, as a new kind of avermectin derivatives with high efficacy and low toxicity, were obtained from a genetically engineered strain Streptomyces avermitilis AVE-H39 and have a good prospect of development and application. Bioassay results showed that the insecticidal activity of ethyl ivermectin against the agricultural and forestry pests including Tetranychus cinnabarinus and Bursaphelenchus xylophilus was higher than that of methyl ivermectin. However, the industrial development of ethyl ivermectin is restricted by its low fermentation titer. In this research, a mutant strain with a higher fermentation yield of ethyl ivermectin was obtained through multiple rounds of mutagenesis and breeding. The fermentation medium was improved by Plackett-Burman experimental design and Box-Behnken design-response surface methodology (RSM). The optimized fermentation condition was corn starch 149.8 g/L, soybean flour 38.1 g/L, (NH₄)₂SO₄ 3.04 g/L, mannitol 30.0 g/L, yeast extract 20.0 g/L, CaCO₃ 3.0 g/L, CoCl₂ 0.01 mg/L, FeSO₄ 0.002 mg/L, rotation speed 220 r/min, initial pH 7.2, 30 mL medium per 250 mL Erlenmeyer flasks, inoculum volume 5%, fermentation temperature 28 ℃. Under the optimized condition, the fermentation titer of ethyl ivermectin reached the maximum value of 4965 mg/L. This research lay the foundation for the development of the new sixteen-membered macrolide insecticide ethyl ivermectin.