产核黄素枯草芽孢杆菌中心碳代谢的代谢工程
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摘要
本文以代谢工程的理论为指导,围绕产核黄素枯草芽孢杆菌的中心碳代谢进行遗传改造,以减少酸性副产物乙酸的生成,提高碳的转化率,并构建了一系列的产核黄素枯草芽孢杆菌的基因工程菌,比较系统地研究中心碳代谢的的遗传改造对枯草芽孢杆菌的核黄素生物合成的影响,得到的主要结果如下:
1.建立产核黄素枯草芽孢杆菌的代谢网络模型,进行了通量平衡分析和代谢通量分析的对比。发现枯草芽孢杆菌的核黄素得率最大时,不产生酸性代谢物乙酸、乳酸和丙酮酸等,并预测进一步遗传改造的靶点。
2.构建了pta基因缺失的产核黄素工程菌,pta基因缺失工程菌与其亲株相比,pta基因缺失使得菌体生长缓慢。代谢分析表明, pta基因缺失减少乙酸的生物合成,伴随着丙酮酸积累。随后在pta基因缺失工程菌中扩增alsS基因,发现alsS基因扩增使得枯草芽孢杆菌有能力将丙酮酸导向3-羟基丁酮生物合成途径,降低丙酮酸的积累,同时改善了菌体的生长,可在0.5%葡萄糖基本培养基积累核黄素54.4 mg/L,比出发菌株RH33::[pRB63]_n提高了85.7%。遗传改造后代谢通量分析表明,中心碳代谢的遗传扰动对代谢网络关键节点的通量分配影响很小,但这些微小变化促进核黄素生物合成途径通量的改善。
3.研究了gdh基因的扩增对枯草芽孢杆菌核黄素生物合成能力的影响。结果表明:扩增gdh基因使得菌株在0.5%葡萄糖基本培养基积累核黄素37.2 mg/L,比出发菌株RH33::[pRB63]_n提高了29.7%,同时降低酸性副产物生成速率,这表明提高葡萄糖酸支路的通量有利于核黄素的积累。
4.对磷酸葡萄糖异构酶靶点进行遗传改造,构建pgi基因缺失工程菌。通过对其发酵和生长的研究得出结论:pgi基因缺失使菌体生长缓慢,利用葡萄糖的能力降低,但当培养基中添加适量酵母抽提物时,可以改善菌体生长和糖的利用,可在0.5%葡萄糖基本培养基积累核黄素35.9 mg/L,比出发菌株提高了25.1%。
5.6-磷酸葡萄糖节点的代谢控制分析表明,降低糖酵解的通量能有效地提高磷酸戊糖途径的通量。
In order to reduce acetate formation and increase carbon yield, A series of genetic modified central carbon metabolism of riboflavin-production Bacillus subtilis were constructed based on the principles of metabolic engineering. The riboflavin producing capabilities of these strains were studied systematically and the major findings are:
In this thesis, a simple metabolic network of riboflavin-producing B.subtilis was constructed. The theoretical flux distribution of maximum riboflavin production was computed through flux balance analysis. Acid metabolites such as acetate, lactate and pyruvate are not formated when maximum riboflavin production in B.subtilis is obtained. After comparisons with the metabolic flux analysis (MFA) results computed from the wet experiments data of riboflavin-producing B.subtilis, the potential improvement targets for further genetic modification were identified and selected.
Riboflavin-producing B.subtilis pta mutant was constrcuted. The lower rates of cell growth and glucose consumption were found in the pta-disrupted mutant while the rate of acetate formation was reduced. Moreover, pyruvate accumulation was found in pta mutant by metabolic analysis. In contrast, the strain has the ablity to redirect pyruvate toward the synthesis of a nonacidic end production, acetoin, when the expression of the gene encoding acetolactate synthase was increased in the pta-disrupted mutant, and cell growth of pta mutant was restored. The riboflavin yield of B. subtilis achieved 54.4mg/l in basic medium containing 0.5% glucose , which was 85.7% enhancement compared to B. subtilis RH33::[pRB63]_n. These resultes from metabolic flux analysis showed that genetic modified central carbon metabolism of riboflavin-production B.subtilis could not significantly affect the flux distribution of key points in metabolic network, but carbon flux through the riboflavin biosynthesis pathway was improved by these changes.
The effects of gdh gene amplification on riboflavin biosynthesis of strain B.subtilis RH33::[pRB63]_n was studied in this thesis. The results showed that riboflavin yield achieved 37.2 mg/L when the expression of gdh gene was obtained, which was 29.7% enhancement compared to the parental strain. Moreover, the formation rate of acid metabolites was reduced. These indicated that the increased carbon flow through the gluconate bypass could improve riboflavin yield.
Riboflavin-producing B.subtilis pgi mutant was constructed by genetic modified phosphoglucose isomerase. Comparesions with the pgi mutant and the parental strain is different in physiological and fermentation products yields. The mutant showed more slowly specific growth rate, lower glucose consumption. The lower rates of cell growth and glucose consumption could be restored when yeast extract was added into the basic fermentation medium containing 0.5% glucose, and riboflavin yield achieved 35.9mg/L, which was 25.1% enhancement compared to the parental strain.
Metabolic control analysis of 6-phosphate glucose branch point showed that carbon flux through HMP pathway could be increased by reducing carbon flux in EMP pathway.
1.建立产核黄素枯草芽孢杆菌的代谢网络模型,进行了通量平衡分析和代谢通量分析的对比。发现枯草芽孢杆菌的核黄素得率最大时,不产生酸性代谢物乙酸、乳酸和丙酮酸等,并预测进一步遗传改造的靶点。
2.构建了pta基因缺失的产核黄素工程菌,pta基因缺失工程菌与其亲株相比,pta基因缺失使得菌体生长缓慢。代谢分析表明, pta基因缺失减少乙酸的生物合成,伴随着丙酮酸积累。随后在pta基因缺失工程菌中扩增alsS基因,发现alsS基因扩增使得枯草芽孢杆菌有能力将丙酮酸导向3-羟基丁酮生物合成途径,降低丙酮酸的积累,同时改善了菌体的生长,可在0.5%葡萄糖基本培养基积累核黄素54.4 mg/L,比出发菌株RH33::[pRB63]_n提高了85.7%。遗传改造后代谢通量分析表明,中心碳代谢的遗传扰动对代谢网络关键节点的通量分配影响很小,但这些微小变化促进核黄素生物合成途径通量的改善。
3.研究了gdh基因的扩增对枯草芽孢杆菌核黄素生物合成能力的影响。结果表明:扩增gdh基因使得菌株在0.5%葡萄糖基本培养基积累核黄素37.2 mg/L,比出发菌株RH33::[pRB63]_n提高了29.7%,同时降低酸性副产物生成速率,这表明提高葡萄糖酸支路的通量有利于核黄素的积累。
4.对磷酸葡萄糖异构酶靶点进行遗传改造,构建pgi基因缺失工程菌。通过对其发酵和生长的研究得出结论:pgi基因缺失使菌体生长缓慢,利用葡萄糖的能力降低,但当培养基中添加适量酵母抽提物时,可以改善菌体生长和糖的利用,可在0.5%葡萄糖基本培养基积累核黄素35.9 mg/L,比出发菌株提高了25.1%。
5.6-磷酸葡萄糖节点的代谢控制分析表明,降低糖酵解的通量能有效地提高磷酸戊糖途径的通量。
In order to reduce acetate formation and increase carbon yield, A series of genetic modified central carbon metabolism of riboflavin-production Bacillus subtilis were constructed based on the principles of metabolic engineering. The riboflavin producing capabilities of these strains were studied systematically and the major findings are:
In this thesis, a simple metabolic network of riboflavin-producing B.subtilis was constructed. The theoretical flux distribution of maximum riboflavin production was computed through flux balance analysis. Acid metabolites such as acetate, lactate and pyruvate are not formated when maximum riboflavin production in B.subtilis is obtained. After comparisons with the metabolic flux analysis (MFA) results computed from the wet experiments data of riboflavin-producing B.subtilis, the potential improvement targets for further genetic modification were identified and selected.
Riboflavin-producing B.subtilis pta mutant was constrcuted. The lower rates of cell growth and glucose consumption were found in the pta-disrupted mutant while the rate of acetate formation was reduced. Moreover, pyruvate accumulation was found in pta mutant by metabolic analysis. In contrast, the strain has the ablity to redirect pyruvate toward the synthesis of a nonacidic end production, acetoin, when the expression of the gene encoding acetolactate synthase was increased in the pta-disrupted mutant, and cell growth of pta mutant was restored. The riboflavin yield of B. subtilis achieved 54.4mg/l in basic medium containing 0.5% glucose , which was 85.7% enhancement compared to B. subtilis RH33::[pRB63]_n. These resultes from metabolic flux analysis showed that genetic modified central carbon metabolism of riboflavin-production B.subtilis could not significantly affect the flux distribution of key points in metabolic network, but carbon flux through the riboflavin biosynthesis pathway was improved by these changes.
The effects of gdh gene amplification on riboflavin biosynthesis of strain B.subtilis RH33::[pRB63]_n was studied in this thesis. The results showed that riboflavin yield achieved 37.2 mg/L when the expression of gdh gene was obtained, which was 29.7% enhancement compared to the parental strain. Moreover, the formation rate of acid metabolites was reduced. These indicated that the increased carbon flow through the gluconate bypass could improve riboflavin yield.
Riboflavin-producing B.subtilis pgi mutant was constructed by genetic modified phosphoglucose isomerase. Comparesions with the pgi mutant and the parental strain is different in physiological and fermentation products yields. The mutant showed more slowly specific growth rate, lower glucose consumption. The lower rates of cell growth and glucose consumption could be restored when yeast extract was added into the basic fermentation medium containing 0.5% glucose, and riboflavin yield achieved 35.9mg/L, which was 25.1% enhancement compared to the parental strain.
Metabolic control analysis of 6-phosphate glucose branch point showed that carbon flux through HMP pathway could be increased by reducing carbon flux in EMP pathway.
引文
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