抗性基因报告系统辅助选育厦门霉素A高产菌株

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

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.