【作者】 张慧敏；

【导师】 姚善泾； 清水和幸；

【作者基本信息】 浙江大学 ， 生物化工， 2005， 博士

【摘要】 随着酵母完整的基因测序已经基本完成,人类第一次获得了真核细胞生物基因组的完整核苷酸序列,酵母基本的代谢过程也已经了解。同时酵母又是一种重要的工业微生物,也是代谢工程的基础研究中一种较好的模式生物。但对于其在不同条件、不同的环境及生长的不同阶段等情况下胞内产生的系统而精细的代谢过程的调控还不是十分清楚,这些方面的研究一直以来就是基础理论与生产实践中的热点问题。本文通过发酵过程生长参数的分析、酶活性检测、蛋白质组分析和代谢通量分析研究了S.cerevisiae利用不同的碳源和不同生长阶段时,胞内代谢体系的响应及细胞自身的这种代谢调控的机理。 考察了酵母中间代谢途径中一些关键酶活性,包括糖酵解途径（丙酮酸激酶,pyk）,磷酸戊糖途径（6-磷酸葡萄糖脱氢酶,G6PDH）,三羧酸循环（异柠檬酸脱氢酶,ICDH;α-酮戊二酸脱氢酶,2-KGDH）,和糖异生途径（FBPase）以及磷酸烯醇式丙酮酸羧化激酶（pck）。采用固相pH梯度-SDS聚丙烯酰胺双向凝胶电泳对酿酒酵母S.cerevisiae利用不同碳源和培养的不同阶段时细胞的总蛋白进行分离,使用2D蛋白质图像分析系统Image master 2D Elite对双向电泳图谱进行定量分析。以MIPS酵母代谢数据库为基础,建立了包括糖酵解途径、磷酸戊糖途径、三羧酸循环、一些回补反应、发酵途径的中间代谢途径和氨基酸生物合成途径等的代谢模型,并根据反应机理,以原子映射矩阵对其反应中的碳原子去向进行描述。对于同种物质相互之间进行的双分子反应,对其原子映射矩阵进行拆分处理,对称分子生成非对称分子的反应,则需按两条反应路线计算。通过同模式标记分子向量和原子映射矩阵来构造同位素映射矩阵。在中间代谢产物平衡的基础上,以分布向量、同位素映射矩阵和反应映射矩阵等建立了同位素分布平衡的非线性限制条件系统。相对于传统的代谢通量分析方法,不考虑不确定性大的辅酶因子NADH、NADPH和ADP、ATP等的平衡关系,以同位素平衡的限制条件来代替以往的最大化比生长速率、最大化生物量、最小化能量需求、能量平衡等限制条件。对该同位素平衡非线性系统的Jacobi矩阵偏微分解析形式做一些理论探讨。采用K.Schmidt等学者提出的均值迭代法（iterative averaging）来计算同位素的稳态分布。以胞外代谢产物的测量值、氨基酸¹³C标记模式分布信息与计算值之间的偏差作为初始适应度函数,对其进行线性定标,根据个体的适应度函数值所度量的优、劣程度决定它所携带的自由通量信息在下一代是被淘汰还是被遗传。结果表明,适应度较大（优良）个体有较大的存在机会,而适应度较小（低劣）的个体继续存在的机会也较小。以自然选择和群体遗传理论为基础,模拟生物进更多还原

【Abstract】 With the completion of genome sequencing of Saccharomyces cerevisiae and a deeper understanding of the basic metabolism of which, S.cerevisiae not only plays an important role in industrial application, but also becomes a very nice model organism for research in many areas. Nevertheless, the in vivo response to various culture conditions and the systematical regulation mechanism of metabolism hereby is still not clearly known. The research on which has always been a focus issue in theoretical or applied study. The present study combine the approach of fermentation characteristics, enzyme assay, proteome analysis and metabolic flux analysis to investigate the regulation of central carbon metabolism of S.cerevisiae under different culture conditions.Some of the enzyme activities were measured for glycolytic pathway (Pyruvate kinase: pyk), oxidative pentose phosphate pathway (Glucose-6-phosphate dehydrogenase: G6PDH), TCA cycle (Isocitrate dehydrogenase: ICDH and 2-Ketoglutaratedehydrogenase: 2KGDH) and gluconeogenesis (FBPase). The activity of PEP carboxykinase (pck) which participates in the anaplerotic reaction was also measured. The proteins of cell utilizing glucose or lactate as carbon sources were seperated by two-dimensional electrophoresis with immobilized pH gradients as the first dimension and SDS-PAGE as the second. About 500 protein spos were detected by imploying the 2D proteome image analysis system Image master 2D Elite and SWISS-2DPAGE preteome database.Most of the protein expressed and involved in the glycolysis, pentose phosphate (PP) pathway, anaplerotic pathway, as well as TCA cycle were analyzed. The metabolism regulation of protein level for Saccharomyces cerevisiae under various carbon sources, as well as during different phase of growth, was studied. It was found that the carbon sources other than glucose were mainly utilized through gluconeogenesis and glyoxylate shunt to enter the central corbon metabolism, the compensatory mechanism of NADPH was also investigated.The fate of each carbon atom of the substrate was traced using Atom Mapping Matrices (AMMs), where its rows and columns correspond to carbon position in the product and reactant molecules, respectively. The labeling patterns in the metabolite pool were coded by numbering the carbon atom positions in a molecule, and the carbon atoms that are labeled or unlabeled are expressed by a sequence of ones andzeros. Thus, the labeling patterns were indexed as elements of the isotopomer distribution vectors(IDVs), which contain mole fractions of metabolite molecules that are labeled in a specific pattern. The IDVs of the product metabolite pools were obtained from reactant pools by Isotopomer Mapping Matrices (IMMs), constructed by summing up all pairs of reactant isotopomers that produce the respective product isotopomer in all positions of the product IDV, together with fluxes through the reactions as the corresponding weight. The set of isotopomer balance equations were solved iteratively by the method of iterative averaging introduced by Schmidt. In the present study, all the IMMs were generated from the databse of AMMs automatically by .the module developed with double nested loops. The IDVs of intermediate metabolites were transferred to NMR multiplet patterns of amino acids by matrices manipulation according to the relationship between precursors and the corresponding amino acids. The carbon flux distribution in the bioreaction network was determined as the best fit to all the measured extracellular fluxes and the relative intensities of the 13C-13C scalar coupling multiplet patterns of proteinogenic amino acids. The algorithm finds the best fit solution which minimizes the quadratic error function. The hybridized genetic algorithm was incorporated in the computer algorithm to minimize the objective function by mutation, crossover, fitness scaling and natural selection. The local search method multidimensional Sequential Simplex Programming (SSP) was combined with the global search algorithm of GA to optimize the procedure.It was found that for cell yields (Yx/s), glycerol and glucose are almost the same, while it is relatively lower for the case where acetate was used as a carbon source. This may be due to the higher CO2 evolution rate and oxygen consumption rate, indicating the increase in the respiratory metabolism. The CO2 evolution rate for the case where glycerol was used as a carbon source was a little higher than the case of glucose used as a carbon source.Glucose-6-phosphate was the biggest carbon flux drain as the precursor of polysaccharides synthesis for the construction of cell wall. When glucose or glycerol is taken as carbon source, instead of phosphoenolpyruvate carboxykinase , the reaction catalyzed by pyruvate carboxylase replenished the TGA cycle with 16.9% of the total carbon flux , playing the main role of anaplerosis. When utilizing glycerol, the reactions between the glucose-6-phosphate and 3-phosphoglycerate in the glycolysis were found to be in the opposite direction to the case of glucose, while ribose-5-phosphate was still provided by the oxidative reaction in the pentose phosphate pathway as the precursor for biosynthesis of histidine, tryptophane andnucleotides. In acetate culture, the flux drained from acetyl CoA accounted for 9.47% of the total carbon flux, which is relatively larger than those for the cases of glucose and glycerol. This may be due to the larger requirement of lipid synthesis from acetyl CoA and the shortest chain from acetate to acetyl CoA for the case of acetate used as the sole carbon source. The TCA cycle is quite active in this case, which is consistent with the fermentation result and enzyme activity result. The flux from oxaloacetate (OAA) to phosphoenolpyruvate (PEP) catalyzed by Pck was utilized to a large extent for gluconeogenesis. Reaction catalyzed by malic enzyme carrying 5.04% of the total carbon flux replenish the shortage of pyruvate caused by the low specific activity of pyruvate kinase.The lack of transhydrogenase catalyzing the exchange between NADH and NADPH implies that most of the NADPH required for biosynthesis was provided mainly by pentose phosphate pathway or by oxidation of isocitrate to a -ketoglutarate through a NADP-dependent isocitrate dehydrogenase. Another potential source of mitochondrial NADPH is the malic enzyme, but the flux of this pathway was significantly low. Since the oxidation of glucose and glycerol in the TCA cycle and respiration pathway do not produce enough NADPH, ribose-5-phosphate and erythrose-4-phosphate were produced through the oxidative branch of the pentose phosphate pathway and through the nonoxidative PP pathway for the case of acetate.The up regulation of the gluconeogenetic reaction via pck for the case of glycerol and acetate used as carbon source as compared with the case of glucose may be due to the increase of ATP-dissipating requirement caused by the increase in oxygen uptake rate and carbon dioxide production rate. Especially in the case of acetate, oxaloacetate was decarboxylated to yield phosphoenolpyruvate via the PEP carboxykinase. which appears to be essential to sustain the cell growth in acetate.Gluconeogeneisis follows the route of steps catalyzed by FBPase and phosphoglucose isomerase to sustain the growth on glycerol and acetate. These regulations further provided the intermediate metabolite of fructose-6-phosphate and 3-phosphoglycerate to be utilized by the non-oxidative branch of pentose phosphate pathway. In acetate culture, carbon flux through zw/reduces and the general direction of reaction catalyzed by transketolase II reverses. This compensatory mechanism was to respond to the depletion of R5P and E4P in the pentose phosphate pathway resulting from the decrease in the activity of glucose-6-phosphate dehydrogenase.In the case of acetate, only a small amount of carbon flux was directed intoacetyl CoA from pyruvate. Reaction catalyzed by malic enzyme was relatively active in this case to provide pyruvate for the synthesis of alanine, leucine, valine and isoleucine. The flux through glyoxylate shunt was absolutely essential to direct the carbon flow through this bypass to generate four carbon precursors for the biosynthesis in acetate metabolism.更多还原