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抗性基因报告系统辅助选育厦门霉素A高产菌株
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国家重大科技专项(2018YFA0901900)


Breeding of a high xiamenmycin A-producing strain by random mutagenesis and reporter-based selection
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    摘要:

    【背景】厦门霉素A是厦门链霉菌(Streptomyces xiamenensis) 318菌株的主要次级代谢产物,具有显著的抗纤维化活性及药用潜力。但野生型菌株中厦门霉素A的产量仅有14 mg/L,其生产水平亟待提升。【目的】通过随机诱变-抗性标记筛选获得高产菌株并进行培养基优化,以提高厦门霉素A的产量。【方法】在厦门霉素A的生物合成基因簇后融合一个抗性基因,用于报告整个基因簇的表达水平。对构建的基因工程菌株进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变,从抗性水平高的突变菌株中筛选高产菌株,并通过培养基优化,使厦门霉素A产量显著提升。【结果】构建携带卡那霉素抗性标记的产厦门霉素A的工程菌MT-XN作为出发菌株,对该菌株进行一轮ARTP诱变,使用90 mg/L卡那霉素筛选,得到了厦门霉素A产量为101.7 mg/L的突变菌株MA-8。进一步通过响应面法优化培养基配方,在最佳培养基中MA-8菌株产生的厦门霉素A达到134.2 mg/L,较野生型菌株提高了845.1%。【结论】采用随机诱变-报告基因筛选系统,可快速筛选出厦门霉素A产量大幅提升的高产菌株,为后续的药物开发奠定良好的基础。

    Abstract:

    [Background] Xiamenmycin A, which shows significant anti-fibrotic activity and can serve as a potential medicinal candidate, is a major secondary metabolite of Streptomyces xiamenensis 318. However, the yield of xiamenmycin A in the wild-type strain is only 14 mg/L. Thus, it is urgent to improve the production of this compound in the strain. [Objective] Random mutagenesis and resistance reporter-based selection was used to develop a high xiamenmycin A-producing strain and further improve the yield of the compound through medium optimization. [Methods] An antibiotic resistance gene was fused at the 3' end of the last gene (ximE) of xiamenmycin A biosynthesis gene cluster, and thus the expression of the entire gene cluster could be indicated by antibiotic resistance level. After atmospheric and room temperature plasma (ARTP) treatment, the high-yielding strain was selected as the mutant with high antibiotic resistance level. The yield of xiamenmycin A in the mutant was further enhanced by medium optimization. [Results] A neo -labeled strain MT-XN was constructed as a starting strain. After one round of ARTP mutagenesis, a mutant MA-8 with resistance to 90 mg/L kanamycin and xiamenmycin A yield of 101.7 mg/L was obtained. Moreover, the yield of xiamenmycin A in MA-8 reached 134.2 mg/L with the medium optimized by response surface methodology, 845.1% higher than that in the wild-type strain. [Conclusion] Random mutagenesis combined with reporter-based selection can be used to identify high xiamenmycin A-yielding strain, which lays a foundation for drug development of this compound.

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李嘉欣,步绪亮,张贵振,江向海,徐俊,徐岷涓. 抗性基因报告系统辅助选育厦门霉素A高产菌株[J]. 微生物学通报, 2022, 49(11): 4821-4831

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历史
  • 收稿日期:2022-03-09
  • 最后修改日期:2022-04-04
  • 录用日期:2022-04-04
  • 在线发布日期: 2022-11-07
  • 出版日期: 2022-11-20