纳他霉素产生菌产量差异菌株的分析
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摘要
纳他霉素(Natamycin)是一种多烯大环内酯类抗生素,能有效抑制霉菌和酵母菌的生物活性,是一种重要的生物防腐剂和抗真菌药物。
本论文应用基因组重排技术选育得到纳他霉素高产突变株F2-2,其产量与出发菌株SG-2相比提高了101.5%。分别采用DNA随机扩增多态性(RAPD)技术及蛋白质双向电泳联用质谱技术对上述菌株进行分析。
首先,通过单因素和正交试验相结合的方法,建立了适于该放线菌的RAPD扩增条件,即在20μL体系中,模板DNA浓度60～150ng,Taq聚合酶为1.0-1.5U,引物浓度0.3-0.4mM,dNTPs浓度200-250μM,Mg2+浓度2.5-3.0mM。此条件下可扩增出条带数目多且清晰稳定的电泳图谱。RAPD检测结果发现,重排后菌株F2-2出现了一条1500bp左右的特异性条带,将其在大肠杆菌DH5a中克隆并测序,在GenBank上进行序列比对发现该片段上的部分序列(约230bp)与Streptomyces griseus(灰色链霉菌),Streptomyces avermitilis(阿维链霉菌),Streptomyces coelicolor A3(2)(天蓝色链霉菌),Streptomyces lavendulae(淡紫灰链霉菌)中的MarR-family转录调控因子同源性较高,分别为100%,91%,88%,89%。
其次,通过蛋白质双向电泳联用质谱技术发现基因组重排前后产量差异菌株存在着蛋白表达差异,结合已知的纳他霉素生物合成途径及链霉菌2-DE数据库中的信息,选取5个可能与纳他霉素合成相关的差异蛋白进行质谱分析。结果表明,在95%的置信区间内,蛋白点2301的分子量和等电点的偏离范围均符合要求,鉴定为葡萄糖激酶调节蛋白。这些工作为进一步了解纳他霉素的代谢途径进而提高其产量奠定了一定的理论基础。
Natamycin is one kind of macrolide antibiotics with 26-membered ring polyene structure, which is an important bio-antiseptic and antifungal agent for its biological activity to restrain the growth of mold and yeast.
In the paper, genome shuffling was used to breed natamycin high production strain. The strain F2-2 was obtained, whose natamycin production was increased by 105% than the starting strain SG-2. Then random amplified polymorphic DNA and protein two dimensional electrophoresis (2-DE)were used to analyze different production strains of natamycin.
Firstly, the RAPD conditions were optimized by single factor experiment and orthogonal experiment. The results were as follows:DNA 60-150ng, Taq DNA polymerase 1.0～1.5U, primer concentration 0.3-0.4mM, dNTPs concentration 200-250μM, Mg2+ concentration 2.5-3.0mM in 20μL PCR system. Under above optimal conditions, the abundant, stable and clear strips could be obtained. It was found that one specific fragment that is about 1500bp was obtained in shuffling-induced strain. It was cloned in E.Coli DH5 and sequenced. Blaset in GeneBank found that partial sequence of this gene band had a high homology with the MarR-family transcription regulator factor existing of Streptomyces griseus, Streptomyces avermitili, Streptomyces coelicolor A3(2), Streptomyces lavendulae. The homologies were 100%,91%,88%,89%, respectively.
Secondly, through two dimensional electrophoresis and mass spectrometry find that protein expression differences were existed between the starting stain and shuffling-induced strain. Based on natamycin biosynsetic pathway and Streptomyces 2-D database,5 different proteins which may be concerned with natamycin production were analyzed by mass spectrometry. According to biostatistics method, in the 95% confidence interval, the protein 2301 was determined as glucokinase regulator-related protein. This work provides a theoretical basis for understanding metabolic pathway of natamycin and increasing its production.
本论文应用基因组重排技术选育得到纳他霉素高产突变株F2-2,其产量与出发菌株SG-2相比提高了101.5%。分别采用DNA随机扩增多态性(RAPD)技术及蛋白质双向电泳联用质谱技术对上述菌株进行分析。
首先,通过单因素和正交试验相结合的方法,建立了适于该放线菌的RAPD扩增条件,即在20μL体系中,模板DNA浓度60～150ng,Taq聚合酶为1.0-1.5U,引物浓度0.3-0.4mM,dNTPs浓度200-250μM,Mg2+浓度2.5-3.0mM。此条件下可扩增出条带数目多且清晰稳定的电泳图谱。RAPD检测结果发现,重排后菌株F2-2出现了一条1500bp左右的特异性条带,将其在大肠杆菌DH5a中克隆并测序,在GenBank上进行序列比对发现该片段上的部分序列(约230bp)与Streptomyces griseus(灰色链霉菌),Streptomyces avermitilis(阿维链霉菌),Streptomyces coelicolor A3(2)(天蓝色链霉菌),Streptomyces lavendulae(淡紫灰链霉菌)中的MarR-family转录调控因子同源性较高,分别为100%,91%,88%,89%。
其次,通过蛋白质双向电泳联用质谱技术发现基因组重排前后产量差异菌株存在着蛋白表达差异,结合已知的纳他霉素生物合成途径及链霉菌2-DE数据库中的信息,选取5个可能与纳他霉素合成相关的差异蛋白进行质谱分析。结果表明,在95%的置信区间内,蛋白点2301的分子量和等电点的偏离范围均符合要求,鉴定为葡萄糖激酶调节蛋白。这些工作为进一步了解纳他霉素的代谢途径进而提高其产量奠定了一定的理论基础。
Natamycin is one kind of macrolide antibiotics with 26-membered ring polyene structure, which is an important bio-antiseptic and antifungal agent for its biological activity to restrain the growth of mold and yeast.
In the paper, genome shuffling was used to breed natamycin high production strain. The strain F2-2 was obtained, whose natamycin production was increased by 105% than the starting strain SG-2. Then random amplified polymorphic DNA and protein two dimensional electrophoresis (2-DE)were used to analyze different production strains of natamycin.
Firstly, the RAPD conditions were optimized by single factor experiment and orthogonal experiment. The results were as follows:DNA 60-150ng, Taq DNA polymerase 1.0～1.5U, primer concentration 0.3-0.4mM, dNTPs concentration 200-250μM, Mg2+ concentration 2.5-3.0mM in 20μL PCR system. Under above optimal conditions, the abundant, stable and clear strips could be obtained. It was found that one specific fragment that is about 1500bp was obtained in shuffling-induced strain. It was cloned in E.Coli DH5 and sequenced. Blaset in GeneBank found that partial sequence of this gene band had a high homology with the MarR-family transcription regulator factor existing of Streptomyces griseus, Streptomyces avermitili, Streptomyces coelicolor A3(2), Streptomyces lavendulae. The homologies were 100%,91%,88%,89%, respectively.
Secondly, through two dimensional electrophoresis and mass spectrometry find that protein expression differences were existed between the starting stain and shuffling-induced strain. Based on natamycin biosynsetic pathway and Streptomyces 2-D database,5 different proteins which may be concerned with natamycin production were analyzed by mass spectrometry. According to biostatistics method, in the 95% confidence interval, the protein 2301 was determined as glucokinase regulator-related protein. This work provides a theoretical basis for understanding metabolic pathway of natamycin and increasing its production.
引文
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