透明颤菌血红蛋白基因在淀粉酶产色链霉菌中的表达及对合成丰加霉素的影响

Expression of Vitreoscilla hemoglobin gene for toyocamycin biosynthesis in Streptomyces diastatochromogenes

摘要:

【目的】丰加霉素(Toyocamycin)是核苷类抗生素家族的重要成员, 其在农业植物病害防治领域具有巨大的应用价值。为改善丰加霉素生产菌淀粉酶产色链霉菌(Streptomyces diastatochromogenes 1628)发酵过程溶氧限制, 旨在实现vgb在S. diastatochromogenes 1628中的表达以促进丰加霉素的生物合成。【方法】首先以gfp为报告基因检测红霉素抗性基因启动子Perm*在S. diastatochromogenes 1628中的转录活性, 再利用PermE*实现vgb的异源表达。【结果】在荧光显微镜下, 重组菌1628-GFP菌丝可发出稳定明亮的绿色荧光, 表明启动子PermE*在菌株1628中可有效启动外源基因的表达; 通过一氧化碳结合差光谱分析显示VHb具有生物学活性; 摇瓶实验表明: 与原始菌株相比, 重组菌可促进丰加霉素产量的提高, 在中度和高度限氧条件下促进效果尤为明显, 提高幅度分别为48.9%和104.5%。PCR和发酵效价检测显示重组菌具有良好的遗传稳定性。【结论】成功实现了vgb在S. diastatochromogenes 1628中的表达, 有效提高了其丰加霉素的合成水平, 为丰加霉素的工业化生产提供了基础条件。

Abstract:

[Objective] Toyocamycin is an important nucleoside antibiotics with diverse bioactivities and potentials for biological control of plant diseases. We expressed the vgb gene encoding Vitreoscilla hemoglobin to alleviate the dissolved oxygen (DO) limitation and to improve the toyocamycin production in Streptomyces diastatochromogenes 1628. [Methods] we used gfp gene as the report gene to test the transcription activity of erythromycin resistance gene promoter PermE* of integrative plasmid pIB139. Then, the promoter PermE* was used to drive the gene vgb expression in S. diastatochromogenes 1628. [Results] Green fluorescent of recombinant strain 1628-GFP was detected by fluorescent microscopy. The promoter PermE* was efficient for heterologous gene expression in S. diastatochromogenes 1628. The vgb gene was also expressed in engineered strain 1628-VHB by the CO-difference spectrum analysis. The engineered strain 1628-VHB improved toyocamycin production under all diverse DO condition. Specially, strain 1628-VHB increased 48.9% toyocamycin production under middle and 104.5% under high limitation DO condition, comparing with the control stain 1628. Meanwhile, strain 1628-VHB was genetically stable by the PCR and toyocamycin yield test from different generation strain. [Conclusion] Expression of vgb gene improved toyocamycin production of strain 1628.