Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis
Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis
复制标题
血浆诱变与基因工程联合应用，从丙城链霉菌中筛选出以5-氧米尔贝霉素A3/A4为主要成分的药物
DOI：
10.1007/s00253-014-5970-6
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发表时间：
2014-12-01
影响因子：
5
通讯作者：
Xiang, Wen-Sheng
中科院分区：
文献类型：
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作者：
Wang, Hai-Yan;Zhang, Ji;Xiang, Wen-Sheng
米尔贝肟已作为有效的驱虫剂在动物健康、农业和人类感染领域中商业化。目前，米尔贝肟是通过两步化学反应合成的，该反应包括使用CrO 3作为催化剂将米尔贝霉素A3/A4酮化以产生中间体5-氧代米尔贝霉素A3/A4。由于米尔贝霉素A3/A4酮化的低效率和环境不友好性，开发替代策略以生产5-氧代米尔贝霉素A3/A4势在必行。本研究首次采用常压和室温等离子体（ARTP）诱变系统对米尔贝霉素产生菌宾城链霉菌（Streptomycesbingchengensis）进行诱变，获得了一株以米尔贝霉素A3、A4、B2和B3为主要成分的突变株BC-120-4，为构建5-氧代米尔贝霉素基因工程菌奠定了基础。重要的是，BC-120-4的米尔贝霉素A3/A4产量达到3，890 ± 52 g/l，比初始菌株BC-109-6（1，326 ± 37 g/l）高约两倍。编码负责米尔贝霉素酮化的C5-酮还原酶的基因milF的随后中断导致产生5-氧代米尔贝霉素A3/A4和消除米尔贝霉素A3、A4、B2和B3的菌株BCJ 60（milF）。BCJ 60的高5-氧代米尔贝霉素A3/A4产量（3，470 ± 147 g/l）和遗传稳定性暗示了在工业上制备用于米尔贝霉素肟的半合成的5-氧代米尔贝霉素A3/A4的潜在用途。
Milbemycin oxime has been commercialized as effective anthelmintics in the fields of animal health, agriculture, and human infections. Currently, milbemycin oxime is synthesized by a two-step chemical reaction, which involves the ketonization of milbemycins A3/A4 to yield the intermediates 5-oxomilbemycins A3/A4 using CrO3 as catalyst. Due to the low efficiency and environmental unfriendliness of the ketonization of milbemycins A3/A4, it is imperative to develop alternative strategies to produce 5-oxomilbemycins A3/A4. In this study, the atmospheric and room temperature plasma (ARTP) mutation system was first employed to treat milbemycin-producing strain Streptomyces bingchenggensis, and a mutant strain BC-120-4 producing milbemycins A3, A4, B2, and B3 as main components was obtained, which favors the construction of genetically engineered strains producing 5-oxomilbemycins. Importantly, the milbemycins A3/A4 yield of BC-120-4 reached 3,890 ± 52 g/l, which was approximately two times higher than that of the initial strain BC-109-6 (1,326 ± 37 g/l). The subsequent interruption of the gene milF encoding a C5-ketoreductase responsible for the ketonization of milbemycins led to strain BCJ60 (∆milF) with the production of 5-oxomilbemycins A3/A4 and the elimination of milbemycins A3, A4, B2, and B3. The high 5-oxomilbemycins A3/A4 yield (3,470 ± 147 g/l) and genetic stability of BCJ60 implied the potential use in industry to prepare 5-oxomilbemycins A3/A4 for the semisynthesis of milbemycins oxime.