癸酸基因工程菌蔗糖跨膜运输及代谢过程中的调控机制

Decanoic acid can effectively reduce body fat accumulation, improve lipid metabolism and glucose metabolism, but its content is very low in nature. The preparation of these two middle chain fatty acids from vegetable oil is limited by many restrictive factors and leads to high cost of production. It is very interesting to use microorganisms to synthesize them. During the cultivation of the engineered microorganisms, glucose is always serving as carbon source. In order to reduce the production cost, sucrose, available and low cost carbon is considered to replace glucose. However, acetate accumulation is abundant but the production of the target products is lowered by 60% compared with glucose medium by recombinant Escherichia coli scr+. At present, sucrose transport and metabolism mechanism is not clear, which greatly restricts the application of sucrose in industrial fermentation..In this project, on the base of biological system design and metabolic engineering, we intend to figure out the transport and metabolic pathways of sucrose and fructose in recombinant Escherichia coli scr+, and clarify the regulatory mechanisms among sucrose transport and metabolism, glucose metabolism, and fructose transport and metabolism. Meanwhile, the interactions between transport and metabolism of sucrose and biosynthesis of decanoic acid will be illuminated. And then the balanced regulation model of sucrose - sucrose’s hydrolysis – acetic acid – decanoic acid will be built..The yield of octanoic/decanoic acid will be increased directly, and the mechanism of sucrose transport and metabolism can give some important information to other kinds of microorganisms so as to use sucrose effectively, and pave the way for sucrose application in industries.

癸酸可有效减少体脂肪积累、改善脂代谢和糖代谢，但其在自然界中含量低，植物油提取制备的限制因素较多、成本较高，利用微生物合成该物质是当前的研究热点。在工程菌的发酵中，采用来源广泛、价格低廉的蔗糖取代葡萄糖是降低发酵成本的一个有效途径。然而在scr+重组大肠杆菌利用蔗糖合成癸酸时乙酸大量积累，目标产物产量下降60%。目前，蔗糖跨膜运输和代谢机制还不清楚，极大地制约了蔗糖在微生物发酵上的应用。.本研究拟通过生物系统设计和代谢工程手段确定scr+重组大肠杆菌内蔗糖和果糖的运输及代谢途径，阐明蔗糖运输及代谢与葡萄糖代谢、果糖运输及代谢间的调控机制，以及蔗糖运输和代谢与癸酸生物合成间的相互作用机理，建立蔗糖-蔗糖水解物-乙酸-癸酸的平衡代谢调控模型。.该项目研究成果可提高癸酸的产量和产率，并为其他微生物利用蔗糖提供代谢调控信息，推进蔗糖在重组菌工业化发酵上的应用。

本项目旨在解析大肠杆菌中链脂肪酸降解途径的基础上构建出可表达外源椰肉硫酯酶的fadD基因（脂肪酸降解起始基因）敲除菌株，同时优化发酵生产工艺，使癸酸的最终生物累积量得到提升。.课题组通过Red同源重组技术，利用pKD46、pKD3以及pCP20三种质粒，敲除了大肠杆菌MG1655中脂肪酸降解途径的起始基因（fadD）。随后使用TRIzol法提取了椰肉的总DNA，使用PCR技术，以椰肉的总DNA为模板，扩增出椰肉硫酯酶基因（tesB），通过双酶切与连接技术构建出椰肉硫酯酶表达质粒ptrc99a-tesB；随后将椰肉硫酯酶表达质粒ptrc99a-tesB转化入fadD基因缺失的MG1655菌株中，获得了MG1655-△fadD-tesB菌株；将椰肉硫酯酶表达质粒ptrc99a-tesB转化入MG1655菌株中，获得MG1655-tesB菌株。.在使用MG1655-tesB发酵生产过程中，于4 h时加入终浓度为1 mM的异丙基-β-D-硫代半乳糖苷（IPTG），在24 h时获得了59 mg/L的癸酸累积量。在使用MG1655-△fadD-tesB菌株发酵生产过程中，于4 h时加入终浓度为1 mM的IPTG，在24 h时获得了73 mg/L的癸酸累积量。然后在发酵生产过程中的第4 h、6 h及8 h添加终浓度为0.2 mM、0.5 mM、0.8 mM及1.0 mM的IPTG，对比其在第24 h时的癸酸累积量，结果表明在6 h时添加终浓度为0.8 mM的IPTG，其在24 h时的癸酸的累积量最大，为124 mg/L。随后通过在发酵过程中第12 h时调节发酵液的pH值至7.0，使得癸酸的累积量提升了1.25倍，达到了155 mg/L。然后使用定向进化技术，通过向培养基中梯度添加外源癸酸，使得工程菌对癸酸的耐受性得到提升，从而降低发酵过程中癸酸的累积对工程菌的毒性，进化后的工程菌株在24 h时的癸酸最终累积量达到了247 mg/L，累积量提升了1.99倍。随后根据前置优化实验，在6 h时添加终浓度为0.8 mM的IPTG，在发酵过程中第12 h时调节pH至7.0，最终在24 h时得到了284 mg/L的癸酸累积量，相较于未优化时的癸酸累积量提升了3.89倍。