Abstract

Milbemycin, a group of 16-membered macrolide antibiotics produced by Streptomyces milbemycinicus, has been widely used as an insecticide and an anthelmintic. To enhance the production of milbemycin, ribosomal engineering with heterologous expression of a regulatory gene was used to screen for a high milbemycin-producing mutant. The mutant S. milbemycinicus R2-6-5 isolated after the treatment of S. milbemycinicus A2079 with 6 μg/mL streptomycin produced 172.5 and 163.1% of milbemycins A3 and A4, respectively, compared with original strain. Analysis of the gene rsmG revealed a frameshift mutation, one cytidine unit being inserted into the 21 position (21C → 21CC). The heterologous regulatory gene aveR, which belongs to the LAL-family was integrated into the genome of S. milbemycinicus R2-6-5 denoted S. milbemycinicus J37 to enhance production of milbemycin. The production of milbemycins A3 and A4 in S. milbemycinicus J37 reached 758.9 and 279.0 μg/g respectively, representing 142 and 61% higher yields over S. milbemycinicus R2-6-5. The combination of ribosomal engineering and heterologous regulatory gene expression in S. milbemycinicus J37 resulted in an increase by 12.4-fold for milbemycin A3 and 11.7-fold for milbemycin A4, respectively, when compared to the original strain. Overall, these results demonstrate that combining available technologies for strain modification such as ribosome engineering technology and heterologous regulatory gene expression is an effective approach for development of high milbemycin-producing strains.

中文翻译：

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将核糖体工程与调控基因的异源表达相结合以提高米尔倍霉素链霉菌 A2079 中米尔倍霉素的产量

摘要

Milbemycin 是由Streptomyces milbemycinicus产生的一组 16 元大环内酯类抗生素，已被广泛用作杀虫剂和驱虫剂。为了提高米尔倍霉素的产量，使用具有调节基因异源表达的核糖体工程来筛选高米尔倍霉素产生突变体。所述突变体S. milbemycinicus R2-6-5的治疗后，分离S. milbemycinicus A2079与6微克/ mL链霉素产生172.5和米尔倍霉素A3和A4分别163.1％，与原始菌株相比。对基因rsmG 的分析揭示了移码突变，一个胞苷单元被插入到 21 位（21C → 21CC）。异源调控基因aveR，属于 LAL 家族，被整合到S. milbemycinicus R2-6-5的基因组中，表示为S. milbemycinicus J37，以提高 milbemycin 的产量。米尔倍霉素J37中米尔倍霉素 A3 和 A4 的产量分别达到 758.9 和 279.0 μg/g，比S. milbemycinicus R2-6-5 的产量高 142% 和 61% 。密氏链球菌核糖体工程与异源调控基因表达的结合与原始菌株相比，J37 分别使米尔倍霉素 A3 增加了 12.4 倍，米尔倍霉素 A4 增加了 11.7 倍。总的来说，这些结果表明，结合现有的菌株修饰技术，如核糖体工程技术和异源调控基因表达，是开发高产米尔倍霉素菌株的有效方法。