利用代谢酶学和模型技术改善谷氨酸发酵的稳定性和糖酸转化率

Enhancing Glutamate Fermentation Stability and Yield by Techniques of Metabolic Enzymology and Model

作者: 专业:发酵工程 导师:史仲平 年度:2013 学位:博士 

关键词
代谢酶学 发酵稳定性 谷氨酸发酵 生物素 糖酸转化率

Keywords
metabolic enzymology, fermentative stability, glutamate fermentation, biotin, glutamate conversion yield
        谷氨酸是世界上产量最大的氨基酸。国内普遍采用生物素缺陷型谷氨酸棒杆菌(Corynebacterium glutamicum)通过发酵法生产谷氨酸,谷氨酸产量一般为10%~12%,糖酸转化率为55%~60%。国内谷氨酸生产技术基本成熟,但是,工艺水平、特别是转化率远远低于理论水平(81%)和国外先进水平(68%)。谷氨酸工业发酵配料工艺原始,极易导致培养基成分(生物素)的波动、严重影响发酵生产的稳定性。另外,菌种自身的发酵特性也经常会发生变化,引起菌体抵抗环境变化能力和产酸能力的下降,出现补糖后谷氨酸合成便停止、发酵性能不稳定的现象。本论文以C. glutamicum S9114作为谷氨酸发酵的实验菌株,以代谢酶学和模型技术为手段,提出了提高糖酸转化率、稳定发酵性能(应对培养基成分初期波动和菌种特性变化)的有效方法和策略,并对利用上述策略改善发酵性能的机制机理进行了理论分析和探究。论文主要研究内容如下:(1)研究分析了初始生物素含量不当,以及初始含量不当、采取补救措施后,主要代谢节点丙酮酸(PYR)、异柠檬酸(ICIT)和α-酮戊二酸(α-KG)处的关键酶活性变化规律。生物素不足时,异柠檬酸脱氢酶(ICDH)活性降低,合成谷氨酸的前体物质减少,α-酮戊二酸脱氢酶(ODHC)完全失活,能量代谢主要靠乙醛酸循环维持。当发现生物素不足并补加生物素后,ICDH活性得到强化,TCA重新成为主要供能途径。生物素过量时,与谷氨酸合成相关的酶(IDHC和谷氨酸脱氢酶)活性降低,而与能量运转相关的酶(ODHC)被激活。当发现生物素过量并添加吐温40后,丙酮酸脱氢酶(PDH)和ICDH依旧保持很高活性,ODHC和异柠檬酸裂解酶(ICL)的活性下降。采用上述补救措施可以挽救因初始生物素含量不当所引起的异常发酵,终酸浓度均可恢复到正常水平(75~80g·L-1)。(2)研究了不同生物素浓度及发酵途中添加吐温40条件下,谷氨酸合成关键酶和谷氨酸运输蛋白的基因转录水平。初始生物素不足时,在谷氨酸主合成期内,所有谷氨酸合成关键酶和运输蛋白的表达量均有降低、特别是ICDH,导致终酸浓度很低(53g·L-1),但谷氨酸分泌不受影响。生物素过量时,ICDH依旧很低,但其它关键酶的转录水平均与对照相当,运输蛋白表达量约为对照的10倍,但是,由于细胞没有正常转型,细胞膜(壁)不具备通透能力,谷氨酸无法正常分泌到胞外,谷氨酸在胞内和胞外均无法积累。初始生物素过量、发酵途中添加吐温40刺激了所有关键酶及运输蛋白的表达,同时诱导了细胞转型,胞内谷氨酸含量升高、谷氨酸可正常分泌到胞外,终酸浓度达到正常水平(75~80g·L-1)。(3)提出了使用混合碳源改善谷氨酸发酵稳定性的策略。共混流加质量比为5:1的葡萄糖/山梨醇混合液或葡萄糖/甘油混合液,或者于初始培养基中加入适量的山梨醇或甘油(10~15g·L-1),可以缓解因菌种发酵特性变化所引起的发酵性能不稳定现象。谷氨酸合成可以在补料之后正常进行,终酸浓度基本恢复到正常水平,发酵稳定性得到改善。此时,胞内NAD+/NADH比、ORP、PDH、ICDH、和细胞色素c氧化酶活性均维持在较高水平。研究结果表明,山梨醇和甘油不能用作谷氨酸发酵的碳源,它们可以认为是谷氨酸发酵的保护剂,起到提高细胞抵抗环境变化能力和维持关键酶活性的作用。(4)提出了协同调节pH和添加NaHCO3的新型发酵工艺,以提高谷氨酸发酵的糖酸转化率。比较了单独添加NaHCO3、调节pH及两者协同操作条件下的发酵性能,结果表明:在同时升高pH和添加NaHCO3,或先升高pH、再添加NaHCO3这两种条件下,葡萄糖消耗量和CO2释放量大幅下降,糖酸转化率比对照(无NaHCO3添加和pH调节)提高了34%~36%,且其浓度也可达到对照水平。关键酶活性分析结果表明,单独提高丙酮酸羧化酶(PC,CO2固定反应的催化酶)的活性并不能提高转化率,只有在各关键酶相互协同作用的条件下,转化率才能得到有效提高。(5)在谷氨酸代谢网络的基础上,提出了一种活用生物酶酶活数据的新型代谢网络模型。该模型将酶活数据和有向信号线图理论有机地结合起来,可以用来估算不同操作条件下的糖酸转化率、解释转化率提高的内在原因、提出实现谷氨酸最优操作的理论酶学调控体系。理论计算结果验证了关键酶相互协同作用的重要性:关键酶组合对PC/PDH、ICDH/ICL和GDH/ODHC的相对酶活比只有同时维持在5~6:4~5、7~8:2~3和7~8:2~3的水平上,转化率才能得到有效提高。而同时升高pH和添加NaHCO3,或先升高pH、再添加NaHCO3这两种条件下,关键酶组合对PC/PDH、ICDH/ICL和GDH/ODHC的相对酶活比与上述“最优”条件比较接近,实验现象得到了理论解释或证实。在3维空间上,通过对上述酶组合对的相对酶活比和转化率的实验数据进行聚类分析,新型代谢模型的有效性和通用性得到验证。
    Glutamate is the amino acid with the largest production in the world. In China, biotinauxotroph Corynebacterium glutamicum strain is widely used in glutamate production,glutamate concentration and conversion yield (from sugar) vary at the levels of10%~12%and55%~60%. Glutamate production technology in China has a history of more than50years,but the major technological index, conversion yield in particular, is much lower thantheoretical value (81%) and the levels (68%) in advanced foreign countries. In industrialglutamate fermentation, the relatively rough method for medium preparation easily causes thefluctuations in medium components (biotin), and thus severe deteriorates the fermentationstability. In addition, the fermentation features or characteristics of the strain sometimes variesbatch by batch, leading to the lower resistant ability against the environmental change and thedecreased glutamate synthesis ability, which results in glutamate synthesis stoppage aftercarbon source feeding and thus fermentation instability. In this thesis, the efficient strategiesfor increasing the conversion yield and for stabilizing the fermentations to deal with the initialmedium components variations and change of strain characteristics, were proposed andexperimentally testified, using C. glutamicum S9114with the aids of metabolic enzymologyand model techniques. The mechanisms of the fermentation improvements when adopting theproposed strategies were also analyzed and explored theoretically. The main results of thisdissertation were summarized as follows:(1) Activities changes of the key enzymes at metabolic nodes of pyruvate, isocitrate andα-ketoglutarate, when initial biotin content varied, and when the content was at improperlevel but faults-rescue measures were adopted, were investigated. When initial biotin wasin shortage, isocitrate dehydrogenase (ICDH) activity was weakened and amount ofglutamate precursor reduced. α-oxoglutarate dehydrogenase complex (ODHC) activitywas inactivated and energy metabolism completely relied on glyoxylate shuttle. Wheninitial biotin was in shortage but biotin was adaptively supplemented, ICDH acitivityrebounded to higher level, and TCA cycle turned to be the main energy metabolism routeonce again. When initial biotin was in excess, activities of the key enzymes associatedwith glutamate synthesis (ICDH and glutamate dehydrogenase) decreased but that relatedwith energy metabolism (ODHC) was stimulated. When biotin was in excess and Tween40was adaptively added, pyruvate dehydrogenase (PDH) and ICDH remained at highlevels, but activities of ODHC and isocitrate lyase (ICL) declined to the normal levels.With the aids of the faults-rescue measures, the failure-likelihood fermentations due toimproper initial biotin variations could be recovered back to normal, and final glutamateconcentrations could reach the normal levels of75~80g·L-1.(2) The transcriptional levels of key enzymes for glutamate synthesis and glutamate transportprotein (TP) under varied initial biotin contents and adaptively adding Tween40, wereinvestigated. When initial biotin was in shortage, the transcriptional levels of genesencoding the key enzymes and TP were all down-regulated during the main production phase, especially that of ICDH, resulting in a very low glutamate concentration (53g·L-1),although glutamate efflux was not affected. When biotin was in excess, the transcriptionallevels of key enzymes were all at comparable levels as those of control but with lowICDH. In this case, the impermeable cellular membrane stopped the vitro glutamatesecretion even though TP expression was about10-fold of control, glutamate could not beaccumulated intracellularly and extracellularly. When initial biotin was in excess butadaptively adding Tween40stimulated the expression of all key enzymes and TP, inducedcell morphological transformation, increased intracellular glutamate content, resultingfinal glutamate concentration back to normal level (75~80g·L-1).(3) The strategy of mixed-carbon sources was proposed to stabilize fermentation performance.The results demonstrated that, if co-feeding glucose with sorbitol/glycerol at a weightratio of5:1or adding10~15g·L-1of sorbitol/glycerol in the initial medium, glutamatesynthesis could continue after substrate(s) feeding and final glutamate concentration couldbe recovered back to normal level. Under these environments, the NAD+/NADH ratio,ORP, the activities of PDH, ICDH and cytochrome c oxidase could be maintained athigher levels. Sorbitol and glycerol could not be used as carbon sources for thefermentation, they were considered to be the effective protective agents to increase cellsresistant ability against environmental changes and maintain key enzymes activities.(4) A new fermentation technology of adaptively regulating pH and NaHCO3addition wasproposed, aiming at increasing glutamate conversion yield from sugar. Fermentationswhen singly and coordinately regulating pH or/and NaHCO3addition were conducted andtheir performance was compared. The results indicated that, the amounts of glucoseconsumption and CO2released decreased significantly and the conversion yield increasedby34%~36%as compared with control, by raising pH before or at the same time as thecommencement of NaHCO3addition. At the same time, comparably high glutamateproductivity could be maintained. Enzymatic activities analysis revealed that increasingPC activity alone could not increase the yield and the yield could be enhanced only whenall key enzymes for glutamate synthesis worked coordinately.(5) A novel metabolic model integrating directed signal flow diagram and enzymatic activitiesdata was proposed to interpret the yield enhancement. The simulation and experimentalresults revealed that singly regulating each individual enzyme could not increase theconversion yield, and the yield could be enhanced only when six key enzymes of PC,PDH, ICDH, ICL, GDH and ODHC works in a coordinated way. Namely, relativeactivities ratios of enzymatic pairs of PC/PDH should be controlled at moderate level of6:4, while those of ICDH/ICL and GDH/ODHC at higher level of8:2simultaneously. Themodel could cluster data pairs of conversion yields and enzymatic activities obtainedunder different operation conditions into different categories, indicating its abilities inguiding optimal enzyme regulation ways for fermentations characterized with multipleenzymatic reactions and closed reaction loops.
        

利用代谢酶学和模型技术改善谷氨酸发酵的稳定性和糖酸转化率

摘要3-5
Abstract5-6
第一章 绪论10-22
    1.1 概述10-12
        1.1.1 谷氨酸物化性质及生产发展历史10
        1.1.2 谷氨酸主要用途10-11
        1.1.3 国内外谷氨酸产业现状11-12
    1.2 国内外研究进展12-19
        1.2.1 谷氨酸发酵菌种12-13
        1.2.2 谷氨酸发酵工艺13-14
        1.2.3 谷氨酸发酵条件的优化14-15
        1.2.4 谷氨酸分泌15
        1.2.5 谷氨酸发酵的代谢分析15-17
        1.2.6 谷氨酸发酵在线控制优化17-18
        1.2.7 谷氨酸大量合成的机制研究18-19
        1.2.8 谷氨酸提取19
    1.3 本论文主要研究内容19-22
        1.3.1 谷氨酸发酵过程中存在的科学问题19-20
        1.3.2 本论文主要研究内容20-22
第二章 初始生物素含量波动时谷氨酸发酵特征、故障排除及关键酶变化模式22-37
    2.1 前言22-23
    2.2 材料与方法23-26
        2.2.1 主要仪器与设备23
        2.2.2 实验菌株23
        2.2.3 培养基23-24
        2.2.4 分析方法24
        2.2.5 发酵参数及条件控制24-25
        2.2.6 关键酶活性测定25-26
    2.3 结果与讨论26-35
        2.3.1 不同初始生物素浓度条件下发酵性能比较26-28
        2.3.2 初始生物素不足及补加生物素条件下关键酶的活性变化28-30
        2.3.3 初始生物素过量及添加吐温 40 条件下关键酶的活性变化30-31
        2.3.4 初始生物素浓度不当采取补救措施的最适时间的研究31-33
        2.3.5 谷氨酸发酵最优操作条件的探讨33-35
    2.4 本章小结35-37
第三章 初始生物素含量对谷氨酸合成关键酶转录水平及谷氨酸分泌的影响37-45
    3.1 前言37-38
    3.2 材料与方法38-40
        3.2.1 实验菌株38
        3.2.2 培养基38
        3.2.3 发酵条件控制38
        3.2.4 分析方法38-40
    3.3 结果与讨论40-44
        3.3.1 不同初始生物素浓度及添加吐温 40 条件下关键酶的转录水平40-43
        3.3.2 不同生物素浓度及吐温 40 对谷氨酸分泌的影响43-44
    3.4 本章小结44-45
第四章 混合使用葡萄糖/山梨醇或甘油改善谷氨酸发酵稳定性45-58
    4.1 前言45-46
    4.2 材料与方法46-49
        4.2.1 实验菌株46
        4.2.2 培养基46
        4.2.3 分析方法46
        4.2.4 发酵条件控制46-47
        4.2.5 NAD+/NADH 比计算模型的建立47-49
    4.3 结果与讨论49-56
        4.3.1 不正常与正常发酵批次的性能比较49-52
        4.3.2 添加山梨醇条件下的发酵性能52-53
        4.3.3 添加甘油条件下的发酵性能53-54
        4.3.4 添加山梨醇或甘油稳定发酵性能的机理探讨54-55
        4.3.5 工业应用可行性分析55-56
    4.4 本章小结56-58
第五章 协同调节 pH 和添加 NaHCO3提高谷氨酸发酵糖酸转化率58-68
    5.1 前言58-59
    5.2 材料与方法59-60
        5.2.1 实验菌株59
        5.2.2 培养基59
        5.2.3 分析方法59
        5.2.4 发酵条件控制59-60
    5.3 结果与讨论60-67
        5.3.1 NaHCO3最适添加量的确定及不同碳酸(氢)盐对谷氨酸发酵的影响60-61
        5.3.2 单独添加 NaHCO3或调节 pH 条件下的发酵性能和关键酶活性变化61-64
        5.3.3 协同调节 pH 和添加 NaHCO3条件下的发酵性能和关键酶活性变化64-67
    5.4 本章小结67-68
第六章 利用基于有向信号线图理论和酶学数据的代谢模型预测糖酸转化率68-82
    6.1 前言68-69
    6.2 材料与方法69-74
        6.2.1 实验菌株69
        6.2.2 培养基69
        6.2.3 分析方法69
        6.2.4 发酵条件控制69
        6.2.5 结合有酶学数据和有向信号线图理论的代谢模型的构建69-73
        6.2.6 节点间传递函数的确定73
        6.2.7 利用聚类分析方法评价模型的通用能力73-74
    6.3 结果与讨论74-80
        6.3.1 各关键代谢节点处分支路径的代谢强度对 TG和 TCO2影响74-75
        6.3.2 利用聚类分析探寻对应于高糖酸转化率的关键酶组合对的相对酶活比75-77
        6.3.3 以不同组合方式调节 pH 和添加 NaHCO3时糖酸转化率的估算77-78
        6.3.4 提高谷氨酸发酵整体性能的探讨78-80
        6.3.5 协同调节 pH 和添加 NaHCO3操作的工业应用潜力80
    6.4 本章小结80-82
主要结论与展望82-84
    主要结论82-83
    展望83-84
论文主要创新点84-85
致谢85-86
参考文献86-93
附录: 作者在攻读博士学位期间发表的论文93-94
附录: 缩略语一览表94


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