College of Life Science, Northeast Agricultural University, Harbin, 150030, People's Republic of China.
J Ind Microbiol Biotechnol. 2013 Aug;40(8):877-89. doi: 10.1007/s10295-013-1280-8. Epub 2013 May 9.
Genome shuffling is an efficient approach for the rapid engineering of microbial strains with desirable industrial phenotypes. In this study, a strategy of incorporating streptomycin resistance screening into genome shuffling (GS-SR) was applied for rapid improvement of doramectin production by Streptomyces avermitilis NEAU1069. The starting mutant population was generated through treatment of the spores with N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet (UV) irradiation, respectively, and five mutants with higher productivity of doramectin were selected as starting strains for GS-SR. Finally, a genetically stable strain F4-137 was obtained and characterized to be able to yield 992 ± 4.4 mg/l doramectin in a shake flask, which was 7.3-fold and 11.2-fold higher than that of the starting strain UV-45 and initial strain NEAU1069, respectively. The doramectin yield by F4-137 in a 50-l fermentor reached 930.3 ± 3.8 mg/l. Furthermore, the factors associated with the improved doramectin yield were investigated and the results suggested that mutations in ribosomal protein S12 and the enhanced production of cyclohexanecarboxylic coenzyme A may contribute to the improved performance of the shuffled strains. The random amplified polymorphic DNA analysis showed a genetic diversity among the shuffled strains, which confirmed the occurrence of genome shuffling. In conclusion, our results demonstrated that GS-SR is a powerful method for enhancing the production of secondary metabolites in Streptomyces.
基因组改组是一种快速工程化具有理想工业表型的微生物菌株的有效方法。在这项研究中，我们将链霉素抗性筛选纳入基因组改组（GS-SR）策略，用于快速提高阿维菌素产生菌阿维链霉菌 NEU1069 的生产能力。起始突变体群体是通过分别用 N-甲基-N'-硝基-N-亚硝基胍和紫外线（UV）照射孢子产生的，然后选择五个具有更高阿维菌素生产能力的突变体作为 GS-SR 的起始菌株。最终，获得了一个遗传稳定的菌株 F4-137，其能够在摇瓶中产生 992±4.4mg/L 的阿维菌素，分别比起始菌株 UV-45 和初始菌株 NEU1069 提高了 7.3 倍和 11.2 倍。F4-137 在 50 升发酵罐中的阿维菌素产量达到 930.3±3.8mg/L。此外，还研究了与提高阿维菌素产量相关的因素，结果表明核糖体蛋白 S12 的突变和环己烷羧酸辅酶 A 的增强生产可能有助于提高改组菌株的性能。随机扩增多态性 DNA 分析显示改组菌株之间存在遗传多样性，证实了基因组改组的发生。总之，我们的结果表明，GS-SR 是一种增强链霉菌中次生代谢产物生产的有效方法。
