诺西肽产生菌的菌种选育及溶氧条件的改善
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摘要
本论文采用基因组重排的方法对诺西肽产生菌活跃链霉菌Streptomyces actuosus进行了菌种选育，以及通过转入透明颤菌血红蛋白基因vgb来改善菌体的溶氧状况，从而获得适合工业化生产的高产菌株。
在基因组重排育种中，首先选用利福霉素和乙硫氨酸作为抗性标记，分别筛选得到了产抗明显提高的突变菌株。考察了原生质体的制备及再生、原生质体灭活和原生质体融合等条件后，将利福霉素抗性菌株与乙硫氨酸抗性菌株进行了第一轮原生质体融合，筛选得到了六株诺西肽产量提高的菌株。然后又将该六株菌进行了第二轮原生质体融合，最终筛选得到了比出发菌株提高200％的诺西肽产生菌。由此可见，利用基因组重排育种是一种非常有效的菌种选育的方法。
质粒pSPU240携带有透明颤菌血红蛋白基因，由于带有attP整合位点，可使其整合到宿主菌的染色体上，利用PmerR启动子组成型表达血红蛋白基因。本论文利用接合转移的方法将质粒pSPU240转入活跃链霉菌中，经PCR验证表明vgb已整合到活跃链霉菌染色体中；CO结合差示光谱分析表明vgb在此菌中表达出了有生物活性的透明颤菌血红蛋白；摇瓶发酵结果表明，在溶氧较低的情况下vgb的表达可以明显提高菌体发酵时的溶氧水平，进而提高诺西肽的产量；稳定性考察表明，重组菌稳定，适合工业化生产的需要。
In order to obtain the industrial nosiheptide producting strains, gonome shuffling was utilized to screen out the high-producting Streptomyces actuosus, and introduction of Vitrescilla hemoglobin gene into Streptomyces actuosus by genetic engineering technologies promoted the dissolved oxygen uptake in fermentation industry in this paper.
In the experiment of genome shuffling we chose the rifamycin and DL-ethionine to screen out the high-producting resistance strains at first. Then the conditions of the protoplasts preparation, regeneration, inactivation and fusion of Streptomyces actuosus were optimized. After protoplasts fusions of optimal resistance strains two times we obtained the optimal strain which titer was increased by 200% than the original strain. Therefore, genome shuffling is a good method to accelerate the process of strain breeding.
The plasmid containing Vitrescilla hemoglobin gene was introduced into Streptomyces actuosus through conjugal transfer, which was integrated into the chromosome of Streptomyces actuosus by the attP attachment site and expressed constructively Vitrescilla hemoglobin gene by the constructive strong promoter PmerR. PCR analysis indicated vgb had been integrated into the chromosome of Streptomyces actuosus, and CO binding difference spectrum revealed that vgb had been expressed successfully in Streptomyces actuosus, and the fermentation results showed that expression of vgb could dramatically promote the dissolved oxygen uptake in the lower level of dissolved oxygen and promote biosynthesis of nosiheptide. At the same time the recombinant strains were stable and suitable for industrial application.
在基因组重排育种中，首先选用利福霉素和乙硫氨酸作为抗性标记，分别筛选得到了产抗明显提高的突变菌株。考察了原生质体的制备及再生、原生质体灭活和原生质体融合等条件后，将利福霉素抗性菌株与乙硫氨酸抗性菌株进行了第一轮原生质体融合，筛选得到了六株诺西肽产量提高的菌株。然后又将该六株菌进行了第二轮原生质体融合，最终筛选得到了比出发菌株提高200％的诺西肽产生菌。由此可见，利用基因组重排育种是一种非常有效的菌种选育的方法。
质粒pSPU240携带有透明颤菌血红蛋白基因，由于带有attP整合位点，可使其整合到宿主菌的染色体上，利用PmerR启动子组成型表达血红蛋白基因。本论文利用接合转移的方法将质粒pSPU240转入活跃链霉菌中，经PCR验证表明vgb已整合到活跃链霉菌染色体中；CO结合差示光谱分析表明vgb在此菌中表达出了有生物活性的透明颤菌血红蛋白；摇瓶发酵结果表明，在溶氧较低的情况下vgb的表达可以明显提高菌体发酵时的溶氧水平，进而提高诺西肽的产量；稳定性考察表明，重组菌稳定，适合工业化生产的需要。
In order to obtain the industrial nosiheptide producting strains, gonome shuffling was utilized to screen out the high-producting Streptomyces actuosus, and introduction of Vitrescilla hemoglobin gene into Streptomyces actuosus by genetic engineering technologies promoted the dissolved oxygen uptake in fermentation industry in this paper.
In the experiment of genome shuffling we chose the rifamycin and DL-ethionine to screen out the high-producting resistance strains at first. Then the conditions of the protoplasts preparation, regeneration, inactivation and fusion of Streptomyces actuosus were optimized. After protoplasts fusions of optimal resistance strains two times we obtained the optimal strain which titer was increased by 200% than the original strain. Therefore, genome shuffling is a good method to accelerate the process of strain breeding.
The plasmid containing Vitrescilla hemoglobin gene was introduced into Streptomyces actuosus through conjugal transfer, which was integrated into the chromosome of Streptomyces actuosus by the attP attachment site and expressed constructively Vitrescilla hemoglobin gene by the constructive strong promoter PmerR. PCR analysis indicated vgb had been integrated into the chromosome of Streptomyces actuosus, and CO binding difference spectrum revealed that vgb had been expressed successfully in Streptomyces actuosus, and the fermentation results showed that expression of vgb could dramatically promote the dissolved oxygen uptake in the lower level of dissolved oxygen and promote biosynthesis of nosiheptide. At the same time the recombinant strains were stable and suitable for industrial application.
引文
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