【作者】 侯进；

【导师】 鲍晓明； Lisbeth Olsson；

【作者基本信息】 山东大学 ， 微生物学， 2009， 博士

【摘要】 氧化还原辅酶在合成代谢,分解代谢以及能量生产过程中都发挥着重要的作用。维持细胞内辅酶的平衡对于细胞生长,代谢以及产物的产生都非常关键。尽管代谢工程的研究工作已经广泛开展,但代谢工程改造依然常常不能得到预期的效果,这与人们对细胞内的代谢过程,调控过程还不够了解有着很大的关系。辅酶在细胞中涉及的反应广泛,细胞内辅酶浓度的改变,也会引起代谢的广泛改变,同时影响产物的形成,这对于深入研究细胞的氧化还原,糖代谢和能量代谢都有很重要的作用,同时也为我们对细胞工厂的理性代谢工程改造提供理论基础。在酿酒酵母细胞内,NADH/NAD⁺和NADPH/NADP⁺有着不同的功能并参与不同的代谢过程,此外,由于它们不能穿越线粒体内膜,在线粒体和细胞质中的辅酶代谢基本上也是各自独立的。NADH主要参与分解代谢,生物质合成过程会产生大量NADH,好氧条件下细胞质或者线粒体的NADH通过呼吸链的内部或外部NADH脱氧酶氧化并产生ATP。在高浓度的葡萄糖存在时,由于葡萄糖的有氧氧化能力弱于其酵解能力,造成了乙醇的产生,导致Crabtree效应,但乙醇的产生过程为氧化还原中性,不能减少NADH积累,而代谢中产生的过量NADH,需要通过磷酸二羟丙酮产生甘油的过程还原。而NADPH主要参与合成代谢,氨基酸,脂类以及核苷酸的合成都需要NADPH提供还原力。酿酒酵母中NADPH的产生的途径不多,在细胞质主要通过磷酸戊糖途径葡萄糖-6-磷酸转化成核糖-5-磷酸的过程产生,在线粒体中则由TCA循环的NADP⁺依赖型异柠檬酸脱氢酶产生。另外,NADP⁺依赖型乙醛脱氢酶,NADP⁺依赖型苹果酸酶以及NADH激酶也是产生NADPH的来源。虽然NADH和NADPH分别在分解代谢和合成代谢中发挥作用,但在木糖代谢过程需要两种辅酶的共同参与。在木糖代谢过程中木糖还原酶（XR）利用NADPH转化木糖为木糖醇,然后木糖醇脱氢酶（XDH）以NAD⁺为辅酶转化木糖醇为木酮糖,由于这两步反应所需的辅酶不同,造成了辅酶的氧化还原不平衡,导致中间产物木糖醇积累,乙醇得率不高,为研究和解决该问题,本论文分别尝试了两种不同的策略。一个策略为变改变Pichia stipitis来源的木糖醇脱氢酶的辅酶结合特异性,研究对木糖代谢的影响。通过对木糖醇脱氢酶进行定点突变,改变辅酶结合结构域关键位点D207A/I208R/F209S,另外引入增加热稳定性的锌指结构S96C/S99C/Y102C/D207A/I208R/F209S,改变木糖醇脱氢酶的辅酶结合特异性,由原来严格的NAD⁺依赖型,变成了倾向于以NADP⁺为辅酶,在酿酒酵母中表达后,使木糖还原酶和木糖醇脱氢酶所利用的辅酶能够偶联起来,从发酵结果分析,突变菌株（D207A/I208R/F209S）,可以有效减少木糖醇和甘油的积累,但木糖利用率和乙醇产生也减少。而突变菌株（S96C/S99C/Y102C/D207A/I208R/F209S）,由于XDH/XR的比率的过低,反而导致木糖醇积累增加,木糖利用率和乙醇产率的降低。这表明,改变XDH的辅酶偏好性是缓解木糖醇积累的有效途径,但是高活性的XDH以及XR/XDH合适的比率,也是影响木糖代谢以及乙醇产生的重要原因。除了辅酶结合特异性改变对木糖代谢影响的研究,我们还利用辅酶工程策略研究木糖代谢,通过NADH激酶在木糖代谢重组酿酒酵母的引入,研究其对代谢的影响。将来源于酿酒酵母线粒体的ATP介导的NADH激酶（由核基因POS5编码）分别在线粒体或细胞质中进行超表达,以增加可以利用的NADPH,并减少NADH的积累,并研究其对葡萄糖和木糖代谢的影响。实验结果表明,线粒体NADH激酶的超表达略微增加了菌株的比生长速率,对代谢并没有太大的影响。相反细胞质NADH激酶的超表达明显影响了代谢流向。在好氧或厌氧葡萄糖培养时,它可以减少碳源的损失,提高乙醇的产生,使代谢流从CO₂转向乙醇。而在厌氧葡萄糖木糖混合培养条件下,细胞质NADH激酶反而对发酵产生了负面的影响,增加了木糖醇的积累,使碳流向由乙醇产生转向了积累木糖醇。尽管NADH激酶的超表达不能提高木糖的代谢,但该研究让我们对木糖代谢与辅酶的关系有了更深的了解。辅酶对木糖代谢的制约,是影响代谢过程的一个典型例子。除此之外,辅酶的扰动对产物生成,能量代谢以及胞内代谢流都会造成广泛的影响。因此本论文围绕酿酒酵母辅酶的扰动对代谢流的影响也展开了深入的研究。由于NADH,NADPH等不能穿越线粒体内膜,因此线粒体和细胞质中的辅酶代谢是独立的,需要分区进行研究。本文通过在酿酒酵母中引入可以直接实现辅酶间的转变,而不涉及其他代谢途径的酶类,研究了改变线粒体或者细胞质NADH或NADPH的浓度,对葡萄糖的代谢流以及能量代谢的影响。利用NADH氧化酶,alternative氧化酶,ATP介导的NADH激酶和可溶性转氢酶在酿酒酵母细胞内不同分区的表达,改变辅酶的水平,利用好氧分批培养,好氧碳源限制的accelerostat培养,研究对代谢的影响。发现,减少细胞质NADH的浓度会减少甘油的产生,而线粒体NADH的浓度的降低则降低了乙醇的产生,缓解了Crabtree效应。但是,当NADH的减少偶联NADPH产生的时候,产物变化的幅度就会缓和很多。通过胞内代谢物的测定,发现辅酶的扰动对胞内代谢流也造成了广泛的影响。根据葡萄糖代谢模型的计算结果,我们还发现线粒体NADH浓度的降低,增加了氧化磷酸化的效率,暗示线粒体中过高的NADH会抑制氧化磷酸化。在分析了减少NADH的浓度对代谢的影响之后,本文又研究了增加细胞内NADH的浓度对代谢流的改变。由于没有能够直接实现NAD⁺还原而不产生其他代谢物的酶类,我们利用甲酸脱氢酶可以催化甲酸完成NADH的还原,但其产物却不参与其他代谢途径的特点,在一个甲酸脱氢酶缺陷的酿酒酵母菌株中,表达内源NAD⁺依赖型甲酸脱氢酶（由FDH1基因编码）,或者融合CYB2的线粒体导肽序列,使其分别在细胞质或者线粒体中定位表达。在好氧碳源限制的恒化培养中添加甲酸后,重组菌株消耗甲酸并导致代谢流向改变,即使在葡萄糖解抑制的条件下也开始进行发酵代谢,产生代谢产物。甲酸脱氢酶住线粒体中的表达,导致乙醇作为主要的发酵产物的产生。而当甲酸脱氢酶在细胞质中的表达时,会有甘油和少量的乙醇形成。这与本文前一部分的研究结果是相辅相成的。另外,通过胞内代谢物的测定分析,发现磷酸果糖激酶,甘油醛-3-磷酸脱氢酶以及α-酮戊二酸脱氢酶催化的反应是对NADH的扰动最为敏感的反应。作为利用辅酶工程对细胞工厂改造的例子,本文利用甘油产生途径的甘油-3-磷酸脱氢酶基因的启动子（GPD1 promoter）调控NADH氧化酶,有效控制NADH水平,使菌株只氧化代谢中过量的NADH。在NADH氧化酶调控表达后,好氧葡萄糖培养的甘油积累降低了57%,同时使菌株的比生长速率和生物质产率都显著增加,从而实现了NADH/NAD⁺比率的优化。本论文通过辅酶扰动对木糖或葡萄糖代谢流影响的研究,深入分析了辅酶的浓度与产物形成,能量代谢以及细胞内代谢的关系,为细胞工厂进行代谢工程改造提供了理论基础。尽管上述研究集中于酿酒酵母,但对其他工业微生物的研究也有借鉴作用。更多还原

【Abstract】 Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation in the overall process of metabolism.There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output.Engineering the level of metabolic cofactors to induce alternate regulatory pathways is emerging to be an attractive strategy for bioprocess applications.In the present work,we targeted cofactors,not only because of its high degree of connectivity in the metabolic network,but also because product formation is the consequence of redox homeostasis.We demonstrate that altering cofactors metabolism is a powerful strategy in affecting the carbon fluxes in Saccharomyces cerevisiae.NADH is predominantly produced in the catabolism of glucose and redox homeostasis is maintained by the action of NADH dehydrogenases.Since NADH cannot traverse the mitochondrial membrane,S.cerevisiae has two external and one internal NADH dehydrogenases.Glucose cannot be oxidized as fast as it can be consumed and a portion of it is fermented to ethanol.This phenomenon which is called Crabtree effect is often attributed to the inhibition of respiration.Ethanol production is a redox-neutral process and hence,does not contribute to NADH accumulation.The additional NADH generated from concomitant biomass synthesis is oxidized via glycerol production.NADPH has a greater role in anabolism as many of the reactions involved in the biosynthesis of amino acids,lipids and nucleotides use NADPH as the reducing agent.In S.cerevisiae,a majority of the NADPH is generated in the oxidative pathway of the pentose phosphate pathway,in the conversion of glucose-6-phosphate to ribose-5-phosphate in the cytosol. Mitochondrial NADPH is synthesized mainly from the NADP⁺-dependent isocitrate dehydrogenase in the TCA cycle.Besides these,there are other reactions that might produce NADPH.such as the NADP⁺-dependent acetaldehyde dehydrogenase.NADP⁺-dependant malate dehydrogenase.S.cerevisiae also has NADH kinase mediate the ATP-driven conversion of NADH into NADPH.During growth of Saccharomyces cerevisiae on glucose,the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis,respectively.However, the metabolism of xylose by recombinant S.cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH,and creates cofactor imbalance during growth on xylose.In this study,we demonstrated two possible solutions to overcoming this imbalance.One strategy is changing the cofactor specificity of XDH.The genes XYL2 （D207A/1208R/F209S） and XYL2（S96C/S99C/Y102C/D207A/I208R/F209S） were introduced to construct xylose metabolism S.cerevisiae,respectively.The specific activities of mutated XDH in both strains showed a distinct increase in NADP⁺-dependent activity. During xylose fermentation,the strain with XDH（D207A/1208R/F209S） had a large decrease in xylitol and glycerol yield,while the xylose consumption and ethanol yield were decreased. In the strain with XDH（S96C/S99C/Y102C/D207A/1208R/F209S）,the xylose consumption and ethanol yield were also decreased,and the xylitol yield was increased,due to low XDH activity.The results showed that changing xylitol dehydrogenase cofactor specificity was a sufficient method for reducing the production of xylitol,but high activity of XDH and the high XDH/XR ratio were also required for improved ethanol formation.As another possible solution to overcoming cofactor imbalance,the effect of overexpressing the native NADH kinase（encoded by the POS5 gene） in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated.The physiology of the NADH kinase containing strains was also evaluated during growth on glucose.Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose.However,cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption,by channeling carbon flow from ethanol to xylitol.In contrast,overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent.Overall,although NADH kinase did not increase the rate of xylose consumption. we believe that it can be an important strategy to engineer the redox metabolism in yeast.Xylose metabolism is a typical example which is limited by cofactors.Beside it.we also presented a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering.First,we enhanced NADH oxidation by introducing NADH oxidase or alternative oxidase,its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble,non proton-translocating bacterial transhydrogenase.Decreased cytosolic NADH level decreased glycerol production,while decreased mitochondrial NADH decreased ethanol production.However,when these reactions were coupled with NADPH production,the metabolic changes were more moderated.The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations of corresponding cofactors.Besides the cofactors,the concentration of other intracellular metabolites also varied to counteract the perturbations. These changes in product formation profile and concentration of intracellular metabolites were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation,as estimated from a simple stoichiometric model for glucose metabolism to different products（biomass,CO₂,ethanol and glycerol）.The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications.Then,we demonstrated that increasing NADH level is a powerful strategy in affecting the carbon fluxes in Saccharomyces cerevisiae.In a S.cerevisiae strain that was disabled to consume formate,we overexpressed the native NAD⁺-dependent formate dehydrogenase either in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence from the CYB2 gene.Upon exposure to formate,the mutant strains readily consumed formate and induced fermentative metabolism even under glucose de-repressed conditions.Ethanol was the main by-product when formate dehydrogenase was directed into the mitochondria while we observed glycerol and some ethanol when it was overexpressed in the cytosol.Clearly.these results point towards strong compartmental regulation of redox homeostasis.By following the intracellular metabolite profiles during the pulse,we identified phosphofructokinase,glyceraldehyde-3-phosphate dehydrogenase andα-ketoglutarate dchydrogenase as the most sensitive enzymes to NADH perturbation.As an example,we demonstrated an effective cofactor engineering strategy and expressed NADH oxidase in Saccharomyces cerevisiae under the control of the glycerol pathway by GPD2 promoter to modulate the decrease in cytosolic NADH to the right level where the heterologous enzyme does not compete with oxidative phosphorylation while at the same time,decreasing glycerol production.The metabolic design eliminated glycerol production by 57%and did not decreased ethanol production.In the meanwhile,it increased the specific growth rate of S.cerevisiae by 14%and biomass yield by 9%.It indicated that the strategy optimize the NADH/NAD⁺ ration in S.cerevisiae.Although we demonstrated the amenability of cofactors in dictating product profile and metabolism profile only in S.cerevisiae,we believe that this concept could be used in other industrially relevant microorganisms as well.更多还原