产尿嘧啶核苷酸的酿酒酵母重组菌的构建及其代谢调控的研究

Uridine monophosphate (UMP)，as one of important nucleotides, is widely used in food industry, agriculture and pharmacy. Using recombinant strain to synthesize UMP, the problems in traditional production as high cost, difficult extraction and heavy pollution will be resolved. However, the UMP yield is normally lower, while the metabolism of recombinant strain is still unclear. This study is concentrated to solve problems addressed above. First, to realize a further strain improvement, the knockout of URA6 gene will be performed to reduce the UMP consumption,which is based on the recombinant Saccharomyces cerevisiae strain constructed before to enhance the UMP yield. Secondly, a complete metabolic network of recombinant strain will be constructed to reveal the metabolism mechanism of UMP biosynthesis. The influence of different substance level, carbon resource concentration and ventilation level to metabolic flux distribution will be considered, especially the variation of phophoribosy pyrophosphate rate. The key nodes and limiting step will be determined. Thirdly, the metabolic flux distribution towards the ideal UMP accumulation is compared with that in the real situation by using linear programming method. As a result, the crucial control mechanism for UMP accumulation will be exposed. Finally, the metabolic regulation for UMP biosynthesis is established. The research provides solid technical foundation for producing nucleotides by recombinant strain and theoretical basis for enhancing the nucleotides yield by metabolic regulation.

以尿嘧啶核苷酸（Uridine monophosphate，UMP）为代表的核苷酸类产品在食品、农业、制药等领域应用前景广阔。利用重组菌生产核苷酸有利于解决传统方法成本高、提取难、污染重等问题，但菌种代谢机制不明朗，产品得率较低。针对该问题，本研究拟以前期构建的产UMP的酿酒酵母重组菌为出发菌，敲除URA6基因来降低UMP的消耗，完善菌种的改良；建立重组菌胞内代谢网络的计量学模型，分析底物水平、碳源浓度及通气量对重组菌代谢流分布的影响，解析关键代谢中间体5-磷酸核糖焦磷酸代谢速率的变化，确定关键节点和限速步骤，阐明UMP合成的代谢机理；利用线性规划方法分析实际与最佳代谢流分布的差异，解析促进UMP积累的关键分子调控机制，在此基础上建立UMP合成的代谢调控方法。研究结果为利用工程菌发酵生产核苷酸奠定坚实的技术基础，并为通过代谢调控提高核苷酸类产品产率提供理论依据。

尿嘧啶核苷酸及其代表的核苷酸类产品广泛应用于食品、农业、制药等领域。现有核苷酸生产技术存在成本高、提取难、污染重等问题，利用重组菌有助于上述问题的解决。但是，由于重组菌代谢机制不明确，无法实施卓有成效的发酵调控手段，致使产品得率较低。针对上述情况，本项目首先以前期构建的高表达OPRTase和ODCase的酿酒酵母重组菌为出发菌株，成功敲除URA6基因以阻断下游代谢途径中UDP的生成。并通过响应面优化、发酵动力学等方法优化了发酵条件，初步提高了重组菌产尿苷酸的能力。然后，在优化合成培养基的基础上，成功的建立了多反应参与的UMP生物合成的重组菌胞内代谢网络计量学模型，通过分析碳源浓度及通气量对重组菌代谢通量分布的影响，解析关键节点代谢通量的变化，阐明了UMP合成代谢机理。基于线性规划法求解了理想状态下最优代谢流分布与实际代谢流的差异，解析促进产物积累的代谢流流向与调控的关键分子机制。结果表明，通过适当增大HMP流量，减小EMP流量可增大合成尿苷酸的流量。故提出代谢调控的策略：一是发酵代谢水平的调控，即在发酵后期添加糖酵解抑制剂碘乙酸和氟化钠，抑制进入EMP流量，二是分子水平的调控，即通过分子生物学手段过表达碳流进入HMP的关键酶6-磷酸葡萄糖脱氢酶（EC 1.1.1.49，G6PDH）以增大HMP的流量。调控完成后，最终结果表明，发酵48h添加甲苯0.5%（v/v），糖酵解抑制剂氟化钠0.18mg/L，反应30h后UMP合成量最大约55mg/L，提高57%；过表达关键酶G6PDH基因zwf1，转化后酶活力提高1.2倍，且转化株UMP产量为80.59mg/L，较转化前提高42%。上述调控方法有望实施于工业化生产中，提升工业化菌株发酵水平和尿苷酸得率。