集胞藻PCC 6803高效合成三羟基丙酸（3-HP）及其对异源途径代谢响应机制的研究
发布时间：2018-08-25 10:01
【摘要】：作为一种重要的平台化合物,三羟基丙酸(3-HP)的应用非常广泛。目前三羟基丙酸主要是依靠化学合成的方法进行生产,这将对环境保护和可持续发展产生不利影响。蓝细菌拥有可以直接固定CO2的能力,使其成为适合燃料以及化学品生产的自养型微生物“细胞工厂”。在本研究中,我们在蓝细菌集胞藻Synechocystis sp.PCC 6803中构建了三羟基丙酸的生物合成途径,并通过以下方法对该生物合成系统进行了优化:1)使用不同启动子提高丙二酰辅酶A还原酶(MCR)基因表达量以及培养条件的优化;2)过表达乙酰辅酶A羧化酶和生物素酰化酶基因提高前体物丙二酰辅酶A产量;3)过表达NAD(P)转氢酶基因改善NADPH供应;4)通过敲除PHA以及乙酸合成竞争通路使得碳源更多的流入3-HP合成通路。通过优化,经过6天的培养Synechocystis中3-HP产量可以达到837.18 mg L-1(348.8 mg/g细胞干重)。另外,过表达来自鱼腥藻Anabaena sp.PCC7120和聚球藻Synechococcus sp.PCC 7942中核酮糖-1,5-二磷酸羧化酶/加氧酶基因并没有增加3-HP产量,这表明CO2固定也许并不是影响3-HP的生物合成的主要因素。目前对于蓝细菌细胞内部3-HP生物合成对代谢的响应机制的研究尚无报道。为此,在Synechocystis中成功构建了3-HP代谢通路后,我们利用同位素标记相对和绝对定量技术与液相二级质谱联用技术(iTRAQ-LC-MS/MS)对3-HP生产株与野生型中蛋白表达谱进行了比较。经过比对,蛋白组分析共检出2,264个独立蛋白,占Synechocystis基因组预测蛋白总数的63%。在所有检测到的蛋白中上调蛋白为204个,下调蛋白为123个。我们还利用液相色谱与质谱联用技术(LC-MS)对细胞内24种重要中心代谢物进行了检测,综合蛋白组学与代谢组学的数据可以看出Synechocystis中有关光合作用,氧化磷酸化作用,中心碳代谢过程的蛋白质以及转运结合蛋白,蛋白翻译过程,细胞调控过程的蛋白以及中心碳代谢过程中24种重要代谢物中有14种代谢物上调。综合蛋白组和代谢组的数据我们发现向集胞藻中引入外源3-HP合成途径后,由于大量生产3-HP,细胞需要通过代谢调控机制对系统内所消耗的前体物以及能量、还原力进行补充,并通过自我调节降低3-HP产生毒性的影响,以保证了细胞在正常生长。我们还通过RT-qPCR分析和相关基因的过表达对蛋白组数据以及代谢调控机制进行了验证,结果进一步证明了蛋白组数据的可靠性以及机理分析的准确性。通过对3-HP生产株与野生型的代谢和网络分析比较使我们对Synechocystis sp.PCC 6803中3-HP的代谢响应机制有了更深刻的认识。
[Abstract]:As an important platform compound, 3-hydroxypropionic acid (3-HP) is widely used. At present, the production of trihydroxypropionic acid mainly depends on chemical synthesis, which will have adverse effects on environmental protection and sustainable development. Cyanobacteria have the ability to fix CO2 directly, making it suitable for fuel and chemical production. In this study, we constructed the biosynthetic pathway of trihydroxypropionic acid in Synechocystis sp. PCC 6803, and optimized the biosynthetic system by the following methods: 1) using different promoters to increase the expression of malonyl coenzyme A reductase (MCR) gene and the expression of MCR gene. Optimization of culture conditions; 2) Overexpression of acetyl-CoA carboxylase and biotin acylase genes increased the production of the precursor malonyl-CoA; 3) Overexpression of NAD (P) transhydrogenase gene improved the supply of NADPH; 4) Knocking out the competition pathway of PHA and acetic acid synthesis increased the flow of carbon into the 3-HP synthesis pathway. In addition, overexpression of ribose-1,5-diphosphate carboxylase/oxygenase gene from Anabaena sp. PCC7120 and Synechococcus sp. PCC 7942 did not increase 3-HP production, suggesting that CO2 fixation may not be the main factor affecting the biosynthesis of 3-HP. Essential factors. Up to now, no studies have been reported on the mechanism of 3-HP biosynthesis in cyanobacterial cells in response to metabolism. A total of 2,264 independent proteins were detected by proteomic analysis, accounting for 63% of the total predicted proteins in the Synechocystis genome. Of all the proteins detected, 204 were up-regulated and 123 were down-regulated. Liquid chromatography-mass spectrometry (LC-MS) was also used to identify 24 important intracellular proteins. Cardiac metabolites were detected. Proteomics and metabonomics data showed that 24 important metabolites in Synechocystis, including photosynthesis, oxidative phosphorylation, central carbon metabolism proteins and transport-binding proteins, protein translation, cell-regulated proteins, and central carbon metabolism, were involved. 14 metabolites are up-regulated. Data from proteomics and metabolomics show that after introducing exogenous 3-HP biosynthesis pathway into Syncystis spp., cells need to replenish the precursors and energy consumed by the system through metabolic regulation mechanism, and reduce the toxicity of 3-HP by self-regulation. We also validated the proteomic data and metabolic regulation mechanism by RT-q PCR analysis and overexpression of related genes. The results further proved the reliability of proteomic data and the accuracy of mechanism analysis. We have a deeper understanding of the metabolic response mechanism of 3-HP in Synechocystis sp.PCC 6803.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946;TQ225.4
本文编号：2202542
[Abstract]:As an important platform compound, 3-hydroxypropionic acid (3-HP) is widely used. At present, the production of trihydroxypropionic acid mainly depends on chemical synthesis, which will have adverse effects on environmental protection and sustainable development. Cyanobacteria have the ability to fix CO2 directly, making it suitable for fuel and chemical production. In this study, we constructed the biosynthetic pathway of trihydroxypropionic acid in Synechocystis sp. PCC 6803, and optimized the biosynthetic system by the following methods: 1) using different promoters to increase the expression of malonyl coenzyme A reductase (MCR) gene and the expression of MCR gene. Optimization of culture conditions; 2) Overexpression of acetyl-CoA carboxylase and biotin acylase genes increased the production of the precursor malonyl-CoA; 3) Overexpression of NAD (P) transhydrogenase gene improved the supply of NADPH; 4) Knocking out the competition pathway of PHA and acetic acid synthesis increased the flow of carbon into the 3-HP synthesis pathway. In addition, overexpression of ribose-1,5-diphosphate carboxylase/oxygenase gene from Anabaena sp. PCC7120 and Synechococcus sp. PCC 7942 did not increase 3-HP production, suggesting that CO2 fixation may not be the main factor affecting the biosynthesis of 3-HP. Essential factors. Up to now, no studies have been reported on the mechanism of 3-HP biosynthesis in cyanobacterial cells in response to metabolism. A total of 2,264 independent proteins were detected by proteomic analysis, accounting for 63% of the total predicted proteins in the Synechocystis genome. Of all the proteins detected, 204 were up-regulated and 123 were down-regulated. Liquid chromatography-mass spectrometry (LC-MS) was also used to identify 24 important intracellular proteins. Cardiac metabolites were detected. Proteomics and metabonomics data showed that 24 important metabolites in Synechocystis, including photosynthesis, oxidative phosphorylation, central carbon metabolism proteins and transport-binding proteins, protein translation, cell-regulated proteins, and central carbon metabolism, were involved. 14 metabolites are up-regulated. Data from proteomics and metabolomics show that after introducing exogenous 3-HP biosynthesis pathway into Syncystis spp., cells need to replenish the precursors and energy consumed by the system through metabolic regulation mechanism, and reduce the toxicity of 3-HP by self-regulation. We also validated the proteomic data and metabolic regulation mechanism by RT-q PCR analysis and overexpression of related genes. The results further proved the reliability of proteomic data and the accuracy of mechanism analysis. We have a deeper understanding of the metabolic response mechanism of 3-HP in Synechocystis sp.PCC 6803.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946;TQ225.4
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