新型高产富马酸米根霉代谢途径重构及其机理研究

Fumarate is one of the most important additives and bulky chemicals in food, medical and chemical industry. Currently, the raw material for fumarate is still petroleum. Due to the shortage of petroleum resources, a novel route for biological production of fumarate from renewable resources is described in this project. A novel metabolic pathway in a strain Rhizopus oryzae is established by genetic reconstruction and regulation based on genomics, genetic engineering, enzymology and metabolomics, to increase carbon source to fumarate for improved yield. The advantage of this strain is low requirement on nutrition and low contamination risk. The fumarate related genes will be recombined under control by a strong promoter for enhanced expression in cytosolic; the by-products pathways will be reduced, to obtain a new strain with improved fumarate yield. Meanwhile, the metabolic pathways of different modified strains will be investigated by metabolic profile analysis based on metabolomics, to find out the key nodes for regulation and improving, to establish high yield platform of fumarate. The success of this project provides an economic genetically modified Rhizopus oryzae strain, with highly theoretic meaning and applied value; and offers references for further construction more genetically modified Rhizopus oryzae with different applications.

富马酸是一种在食品、医药和化工领域有重要用途的添加剂和工业原料。目前工业上生产富马酸主要是利用石油基化学品作为原料。由于石油资源的紧缺，本项目提出一种通过生物法利用可再生资源生产富马酸的新思路。运用基因组学、基因工程、酶学与代谢组学技术对一株具有较高富马酸产量的出发菌米根霉进行基因改造和代谢途径调控，建立一条全新的代谢途径，用以增加碳源流向富马酸的代谢通量，提高其产量。该菌株具有营养要求简单且不易染菌等工业化优点。通过基因重组技术，将其胞质富马酸相关基因连接到强启动子的下游以增强其表达，并弱化丙酮酸代谢的副产物途径，获得富马酸高产的新菌株。同时，运用代谢组学技术进行代谢轮廓分析不同改造后菌株代谢途径，找到关键节点，并对其进行调控、优化，以建立富马酸高产平台。本项目的完成，提供了一株实用的米根霉基因工程菌，具有很高的理论意义及实用价值，并为构建更多其它用途的米根霉基因工程菌提供借鉴意义.

富马酸别名叫做延胡索酸，化学上的名称是反丁烯二酸。作为一种四碳二元酸，富马酸在医药、食品、化工、树脂等领域有广泛应用。目前工业上生产富马酸主要是利用石油基化学品作为原料。由于石油资源的紧缺，本项目提出一种通过生物法利用可再生资源生产富马酸的新思路。本项目通过将丙酮酸羧化酶（PC）、胞质苹果酸脱氢酶（MDH）及富马酸酶（Tfum）基因整合到毕赤酵母染色体上。并通过检测不同基因拷贝数对于不同酶活性以及富马酸产量的影响。这部分结果显示，丙酮酸羧化酶与胞质苹果酸脱氢酶活性的增加有利于富马酸的积累，但是富马酸酶活性的增加反而导致了富马酸产量的下降。另一方面，本项目以实验室保藏的米根霉作为发酵的原始菌株，通过溴甲酚绿和丙烯醇平板的复壮筛选得到优良菌株米根霉RH-7-13。通过对种子培养条件的正交试验，找到了米根霉种子培养阶段形成小球的最佳条件：尿素浓度1.5g/L，接种的孢子密度为6.2×107个/mL，pH 2.5。同时，本项目进一步探索研究了不同材料固定化米根霉发酵对富马酸产量的影响。以葡萄糖为碳源，采用棉球和丝瓜瓤吸附米根霉产富马酸，富马酸的产量分别可达29.33g/L和38.94g/L。