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题名	酿酒酵母代谢工程产C4二元羧酸的研究
作者	闫道江
学位类别	博士
答辩日期	2014-06
授予单位	中国科学院研究生院
导师	邢建民
关键词	C4二元羧酸   富马酸酶   低pH   碳酸酐酶   甘油   丙酮酸羧化酶   磷酸烯醇式丙酮酸羧激酶   酿酒酵母
其他题名	Metabolic engineering of Saccharomyces cerevisiae for the production of C4 dicarboxylic acids
学位专业	生物化工
中文摘要	C4二元羧酸包括苹果酸、富马酸和丁二酸，是具有重要应用价值的生物基平台化合物。微生物发酵法生产C4二元羧酸具有使用可再生物质为原料，可固定CO2等优点，具有替代传统石化生产法的潜力。酿酒酵母发酵过程副产物种类少，发酵在酸性条件下进行，具有较强的工业化潜能。本论文通过C4二元羧酸合成途径的设计、构建和优化，利用代谢工程的手段构建了一系列生产丁二酸、苹果酸和富马酸的酿酒酵母工程菌株，分别以葡萄糖和甘油为碳源进行了C4二元羧酸的发酵研究，并实现了低pH条件下C4二元羧酸的发酵。 在丁二酸的合成途径中（TCA氧化途径、乙醛酸途径等），还原途径具有可固定CO2，理论产率高的优点。然而在酿酒酵母中，富马酸酶（FUM）不可逆催化富马酸到苹果酸，成为通过还原途径生产丁二酸的主要障碍。本文首先通过敲除FUM和引入与FUM催化偏好相反的外源FUM 两种方式，探讨了酿酒酵母利用还原途径生产丁二酸的可行性。 首先，在酿酒酵母内敲除FUM1，并引入合成丁二酸的还原途径，实现了丁二酸的生产。通过敲除甘油脱氢酶基因GPD1使工程菌的丁二酸产量提高到 8.09±0.28 g/L，进一步调节了碳酸钙、尿素和生物素水平，使工程菌的丁二酸产量达到了9.98±0.31 g/L，得率为0.32 mol/mol葡萄糖。对丁二酸合成途径分析表明，还原途径为该菌株的主要合成途径，另一部分的产量（15%）则由TCA氧化为富马酸后再经还原为丁二酸所得。该合成途径的优势是所需NADH较少（1.5 mol/mol 丁二酸），缺点是部分的还原碳代谢流的丢失。 其次，研究了引入的大肠杆菌富马酸酶FumB与酿酒酵母富马酸酶的竞争关系，在不敲除FUM1的条件下，实现了由还原途径直接生产丁二酸。对还原途径的苹果酸脱氢酶和富马酸还原酶进行了不同活性的组合。所得的菌株SA10具有丁二酸产量高，中间副产物（草酰乙酸、苹果酸等）积累少等优点。随后，在超表达内源丙酮酸羧化酶PYC2后，还原碳代谢流得以提高，工程菌的丁二酸产量达到了48 g/L，摩尔得率为0.38 mol/mol葡萄糖。 研究了工程菌在低pH条件下发酵生产丁二酸的过程。比较了不同pH条件下的发酵过程，确定pH 3.8为丁二酸发酵的pH值。在此条件下，通过调节CO2的供给，实现了丁二酸的低pH发酵，丁二酸产量为12.97±0.42 g/L，产率为0.21 mol/mol葡萄糖。还原途径生产C4二元羧酸的限制性步骤为由丙酮酸羧酸酶（PYC）催化的丙酮酸羧化反应，PYC具有固定CO2的特点，然而PYC的直接底物为HCO3-而不是CO2。在低pH条件下，酿酒酵母胞内pH下降会导致胞质HCO3-的减少。初步研究了外源碳酸酐酶的表达对酿酒酵母HCO3-供给和C4二元羧酸生成的影响。结果表明，通过蓝藻碳酸酐酶的引入强化了胞内碳酸氢根离子的供应，促进了碳代谢流流向还原途径产C4二元羧酸的方向。在优化外源碳酸钙的供给条件下，苹果酸得率可以达到0.62 mol/mol葡萄糖，较出发菌株提高了19%。 分别构建了以甘油为碳源，利用还原途径可发酵生产苹果酸、富马酸和丁二酸的酿酒酵母工程菌株。苹果酸的产量为0.82±0.17 g/L，富马酸的产量为0.27±0.08 g/L，丁二酸的产量为0.89±0.26 g/L。在这些工程菌中超表达了甘油透性酶基因GUP1，使苹果酸、富马酸和丁二酸的产量分别达到了1.14±0.49 g/L，0.49±0.13 g/L 和1.24±0.31 g/L，比对照菌株产量上升了39%、81%和51%。研究了PYC2分别与大肠杆菌PEPC、产丁二酸放线杆菌PEPCK协同表达对于丁二酸生产的影响，PYC2与AsPEPCK共表达可以使丁二酸的产量提高至2.17±0.52 g/L，优于PYC2与EcPEPC的协同表达。
英文摘要	C4 dicarboxylic acids，including malate, fumarate and succinate, are most valuable bio-based chemicals. The fermentative production of C4 dicarboxylic acids by microbial organisms as a green technology has the advantages of CO2-fixation and using biomass as the feedstock, which is considered an effective method to replace the traditional petroleum-based chemical process. It’s sole alcohol fermentation and less kinds of byproduct conducted by S. cerevisiae. More importantly, it has the industrial potential to using yeast to produce succinate since the fermentation is done in acidic condition. In this study, a series of engineering strains capable of producing malate, fumarate and succinate were constructed with the strategy of metabolic engineering. The synthetic pathways were designed, constructed and optimized to produce C4 dicarboxylic acids from glucose and glycerol under low pH in S. cerevisiae. Reductive pathway for succinate production has the characteristics of fixation of CO2 and the highest theoretical yield. However, fumarase is an irreversible enzyme that catalytic the direction of fumaric acid to malic acid, which becomes the main obstacle for succinate production through the reduction pathway in S. cerevisiae. In this study, we investigated the possibility of succinate production using the reductive pathway through two strategies which are deletion of FUM1 and introduction of exogenous FUM that has the opposite catalytic preference. Firstly, the reductive pathway for succinate production was introduced in a fum1-deficient S. cerevisiae strain. The fermentation results suggested that succinate can be effectively produced through the synthetic pathway. The succinate titer was increased to 8.09±0.28 g/L by the deletion of GPD1 and even higher to 9.98±0.31 g/L with a yield of 0.32 mol/mol glucose by the regulation of the levels of CaCO3, urea and biotin. The analysis for succinate production route showed that reductive pathway was the main synthetic route. Part of the succinate level (about 15%) was donated by TCA oxidative and followed by fumarate reduction. The advantage of using this strategy to produce succinate is less NADH needed (1.5 mol/mol succinate as calculated). However, part of the reductive carbon flux was lost which reduced the succinate yield. Secondly, the relationship between the activity of fumarase and succinate was investigated by introduction of FumB from E. coli to the FUM1-undeleted S. cerevisiae. The engineered strain could directly produce through reductive pathway. In addition, activities of cytosolic malate dehydrogenase and fumarate reductase were optimized by overexpression of their corresponding genes under different promoters and copy number plasmids. The resulted strain produced the highest level of succinate with minimum accumulation of intermediates involving in reductive pathway (oxaloacetate, malate et al.). In addition, endogenous pyruvate carboxylase gene PYC2 was overexpressed to increase the reductive carbon flux. The engineered strain SA19 produced 48 g/L of succinate with a yield of 0.38 mol/mol glucose. Succinate production was investigated in a bioreactor under different pH values. Under optimal supplemental CO2 conditions in a bioreactor, the engineered strain produced 12.97±0.42 g/L succinate with a yield of 0.21 mol/ mol glucose under pH 3.8. The limiting step for C4 dicarboxylic acids production through the reductive pathway is pyruvate carboxylation that catalyzed by PYC. The direct substrate form of PYC is HCO3- instead of CO2. Cytosolic concentration of HCO3- was decreased due to drop of endocellular pH under low pH condition. Thus, the effect of expression of exogenous CA on HCO3- supplement and production of C4 dicarboxylic acid was investigated in S. cerevisiae. A heterogeneous carbonic anhydrase encoded by cyanobacterium Anabaena sp. 7120 ecaA gene was introduced to S. cerevisiae. The supply of cytosolic HCO3- was enhanced for malate synthesis in this strain.
语种	中文
公开日期	2015-07-08
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/15520]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	闫道江. 酿酒酵母代谢工程产C4二元羧酸的研究[D]. 中国科学院研究生院. 2014.

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