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

【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。

[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(xim E)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.