【作者】 李晓静；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2006， 博士

【摘要】 本文围绕核黄素生物合成反应对产核黄素枯草芽孢杆菌进行了代谢工程改造,构建了一系列产核黄素枯草芽孢杆菌基因工程菌。研究了核黄素操纵子启动子替换及ribA基因的扩增对产核黄素工程菌核黄素生物合成的影响。通过bd氧化酶缺失对枯草芽孢杆菌呼吸链进行了改造。建立了枯草芽孢杆菌的生化反应网络,计算了工程菌的理论得率,并对呼吸链改造前后的工程菌在间歇培养过程中的代谢通量进行了比较分析。最后,通过原生质融合技术改进了产核黄素基因工程菌的性能。得到的主要结果如下:利用枯草芽孢杆菌中组成型启动子SPO2和P43成功替换了核黄素操纵子的一级启动子ribP1。利用不同的整合型载体通过不同整合方式构建了系列核黄素操纵子一级启动子替换的产核黄素基因工程菌。在构建的所有工程菌中,以pUC18为克隆载体所构建的含核黄素操纵子整合型载体转化受体菌得到的工程菌B. subtilis PK具有最高的核黄素生物合成能力,与出发菌相比提高了10倍。建立了产核黄素枯草芽孢杆菌生化反应网络模型,计算了工程菌B. subtilis PK的理论得率,得出该工程菌核黄素的最大合成速率为0.48 mmol葡萄糖/g (CDW)/h,生物量的最大得率为0.47 g cell C/g glucose C。研究了ribA基因的扩增对工程菌B. subtilis RH13、B. subtilis PK和B. subtilis PK/pXJ96核黄素生物合成能力的影响。结果表明:在核黄素操纵子表达量低的工程菌中,ribA基因的表达是核黄素过量合成的限制因素。构建了系列cyd基因缺失的产核黄素工程菌。对比cyd基因缺失工程菌与其受体菌发现,cyd基因的缺失可使工程菌生长速率加快,最大生物量增加,菌体维持能降低,核黄素生物合成能力增强。通过对发酵副产物分析表明:cyd基因缺失工程菌中,乙酸含量明显降低,三羟基丁酮含量明显升高。对工程菌B. subtilis PK/pXJ96和B. subtilis PK cyd /pXJ96通量分布的计算结果表明:cyd基因缺失可使工程菌中PP途径通量增强,用于生物量合成的葡萄糖百分比增加,并在一定程度上削弱了溢流代谢。基于基因组重排原理改进了产核黄素工程菌的生产性状,通过基因组重排得到的B. subtilis RP/pXJ96在摇瓶发酵条件下可产核黄素5.6 g/l,比B. subtilis RH33/pMX45提高了33%。B. subtilis RP/pXJ96的核黄素产率达到0.104 g核黄素/g葡萄糖,B. subtilis PK/pXJ96相比提高了42%。在5 L发酵罐流加发酵条件下发酵48小时,B. subtilis RP/pXJ96核黄素产量可达16 g/l,产率为0.06 g核黄素/g葡萄糖。更多还原

【Abstract】 Based on riboflavin biosynthetic reaction, a series of genetic engineering Bacillus subtilis for riboflavin over production were constructed in this study. The effects of promoter replacement and the amplification of ribA on riboflavin biosynthesis were studied. The respiratory chain was reconstructed by knocking out the bd oxidase. A biochemical reaction network was assembled with metabolites, and then the comparison analysis of metabolic fluxes in batch cultures was carried out between the bd mutant and the control. The traits of riboflavin overproducing Bacillus subtilis were improved by protoplast fusion. The main results presented in this work are as follows:The primary promoter of riboflavin operon was successfully substituted with the constitutive strong promoter P43 and SPO2 respectively. A series of ribP1 substituted Bacillus subtilis were constructed by different integration mechanism using different integration vectors. B. subtilis PK constructed by integration of the pUC18 carried P43-rib fragment into B. subtilis RH13 chromosome via compell-type mechanism made a showy display of its riboflavin biosynthetic ability which was about ten times higher than the host strain.A biochemical reaction network of engineered riboflavin overproducing Bacillus subtilis was constructed. The theoretical yields with riboflavin biosynthetic rate of 0.48 mmol (glucose)/g (CDW)/h and growth yield of 0.47 g cell C/g glucose C were obtained in B. subtilis PK by linear programming using Lingo software.The effects of ribA amplification on riboflavin biosynthesis of strain B. subtilis RH13、B. subtilis PK and B. subtilis PK/pXJ96 showed that the expression of ribA gene was the limit factor for riboflavin over production in the strain with lower riboflavin operon expression level.A series of cyd mutants were constructed. The mutants showed more rapidly specific growth rate, higher biomass yield, lower maintenance requirement, and higher riboflavin biosynthetic ability than the control. Compared to by-products formation, a discernible shift with less acetate and more acetoin in cyd mutant was observed.Comparative flux analysis between B. subtilis PK/pXJ96 and B. subtilis PK cyd/pXJ96 showed that the fluxes in PP pathway and also the fluxes used for biomass biosynthesis were enhanced in cyd mutant. The overflow metabolism was also更多还原