乳酸乳球菌合成2’-岩藻糖乳糖的调控机制研究

The function of human milk oligosaccharides (HMOs) to promote the formation of beneficial microflora in the intestinal tract was deeply researched. And the amount of market demand for HMOs as the additive of formula milk powder and functional foods is increasing. The safe and efficient synthesis of HMOs became the focus of attention. To solve the current 2’-fucosyllactose synthesis problems existed in E.coli of glycosyl donor GDP-L-fucose synthetic insufficiency, the unbalanced distribution of coenzyme, fucosyltransferase low catalytic activity, as well as the security problem of product, this project will be carried out. As the food grade strain, Lactococcus lactis will be used as the host strain. Firstly, the synthesis pathway of 2’-fucosyllactose will be construction with markless knockout method and highly efficent exprssion system in L.lactis cells. The distribution proportion of glucose metabolism will be altered by changing coenzyme the proportion of NAD (P) H and NAD (P). The expression level of key enzymes will be optimized by the promoter and gene copies control. To optimize the synthesis rate, fucosyltransferase will be studied by structure prediction, rational design and directed transformation to improve the substrate affinity and activity. Combined with the analysis of the oligosaccharides molecules structure, the synthesis pathway and the molecular structure of fucosyltransferase will be further optimized. The methods of coenzyme engineering, transcriptomics and metabolomics will be applied to elaborate the relationship between 2’-fucosyllactose synthesis and glucose metabolism, and energy metabolism, and cell growth. The results will provide a theoretical basis for the safe and controllable synthesis of 2’-fucosyllactose and other fucosylated oligosaccharides.

随着人乳寡糖（HMOs）促进肠道有益微生物菌群形成的功能研究的深入，以及婴儿配方奶粉和功能性食品对HMOs需求量的不断增加，HMOs安全、高效合成成为了人们关注的焦点。为解决目前大肠杆菌合成HMOs结构2’-岩藻糖乳糖过程中存在的糖基供体合成量不足、辅酶分配不平衡、岩藻糖基转移酶的催化活性低、以及产品的安全风险等问题，本项目拟在食品级安全菌株乳酸乳球菌中构建2’-岩藻糖乳糖的合成途径，优化启动子和基因拷贝数以控制关键酶的表达水平，通过改变葡萄糖代谢分配比例以改变细胞内的辅酶和能量水平；通过岩藻糖基转移酶的结构预测、理性设计和定向改造，阐述酶的结构和催化活性之间的关系，应用辅酶工程、转录组学、以及代谢组学等方法全面阐述辅酶和关键酶表达水平、岩藻糖基转移酶的结构与活性、糖的利用与菌体生长等对2’-岩藻糖乳糖合成的调控机制，为安全、高效合成2’-岩藻糖乳糖及其它结构岩藻糖基化寡糖提供理论依据。

2′-岩藻糖乳糖（2′-FL）是存在于人乳中含量最丰富的功能性寡糖，利用微生物全细胞合成2′-FL是目前最有潜力实现大规模生产2′-FL的方法。乳酸乳球菌作为公认安全的食品级微生物，可直接用于乳制品中，而且在表达酶类和活性蛋白等方面具有优势。本课题通过在L.lactis中构建2′-FL的从头合成途径和补救途径，实现了乳酸乳球菌对2′-FL的有效合成，显著提高其合成2′-FL的水平。. 首先，分别在L.lactis NZ3900和NZ9000中构建2′-FL补救合成途径，确定NZ9000为最优底盘，又在L.lactis NZ9000中构建了2′-FL的从头合成途径。将基因manA、manB、gmd和wcaG整合至L.lactis NZ9000染色体的upp位点，将关键酶基因manC、futC和lacF克隆到质粒pNZ8148上进行表达，同时利用不同强度启动子对酶的表达水平进行组合调控，对发酵培养基中的氯化高铁血红素、胰蛋白胨和酵母粉的浓度进行了优化，以葡萄糖和乳糖为底物，在摇瓶中合成了1.58 g/L的2′-FL，为相关报道的较高水平。. 同时，在大肠杆菌MG1655基因组中增加manA、manB、gmd和wcaG拷贝数，敲除分解乳糖的β-半乳糖苷酶基因lacZ，过表达乳糖透过酶lacY基因，过表达manC和futC。利用质粒pSB4K5分别过表达ManA、ManB、ManC、Gmd、WcaG、FutC，确定限速步骤并组合表达限速酶。在此基础上，利用不同强度的启动子调控合成途径通量及辅酶NADPH的水平，获得一株在5 L发酵罐中合成46.06 g/L 2′-FL的高产菌株，随后对该菌株进行适应性进化，获得了一株细胞耐受性提高的突变菌株（rpoC*），将2'-FL产量提升至61.06 g/L，是目前报道的最高水平。. 另外，还建立2'-FL合成的酶体外级联催化体系（MECCS），对关键酶FutC的性质进行研究。在优化后的MECCS中添加TeFucT催化40 h后获得27.07 g/L的2′-FL，生产速率0.67 g/L/h，添加HpFucT进行至35 h，2′-FL产量为25.88 g/L，生产速率为0.73 g/L/h，为后续补救合成途径的调控及其他类型人乳寡糖的合成提供参考。