摘要
嘧啶核苷包括尿苷和胞苷,是生产抗病毒和抗肿瘤药物的重要中间体。为获得胞苷高产菌,该研究以高产尿苷的大肠杆菌UR6为出发菌株,利用CRISPR/Cas9介导的基因组编辑技术对其进行了一系列的代谢工程改造。结果显示,cdd、cmk和ygdH 3个基因的敲除阻断了胞苷及其前体物的主要降解途径,使胞苷产量达到了2.8 g/L,而尿苷产量基本无变化;突变体PyrH ecj(D93A)和PyrG cgl(D160E、E162、E168K)的引入结合nudG基因的双拷贝有效增强了胞苷合成途径,使胞苷产量提高至5.6 g/L,尿苷产量降低至9.3 g/L;引入酵母菌的5′-核苷酸酶PHM8使尿苷的产量提高至11.5 g/L,而对胞苷的积累影响不大;乳清酸核苷-5-磷酸脱羧酶和尿苷酸激酶的融合表达,增强了尿苷酸到胞苷的合成通量,使胞苷产量进一步提升至7.2 g/L,尿苷产量降低至8.2 g/L。最终所得菌株可用于胞苷和尿苷联产,具有较好的工业化应用前景。
Pyrimidine nucleosides,including uridine and cytidine,are important intermediates in the production of antiviral and antitumor drugs.To obtain a high-yield cytidine-producing strain,this research took Escherichia coli UR6,a high-yield uridine-producing strain,as the starting strain and carried out a serious of metabolic engineering transformations using CRISPR/Cas9 mediated genome editing technology.The results showed that the knockout of cdd,cmk and ygdH genes blocked the main degradation pathways of cytidine and its precursors and the production of cytosine reached 2.8 g/L,while the uridine production remained basically unchanged.The introduction of mutants PyrH ecj(D93A)and PyrG cgl(D160E,E162,E168K)combined with the double copy of nudG gene effectively enhanced the cytidine synthetic pathway,resulting in the increase of cytidine production to 5.6 g/L,and the uridine production decrease to 9.3 g/L.The introduction of 5′-nucleotidase PHM8 from Saccharomyces cerevisiae increased the uridine production to 11.5 g/L and had little effect on the accumulation of cytidine.The fusion expression of orotidine-5′-phosphate decarboxylase and uridine kinase promoted UMP flow to cytidine synthesis,which further increased the cytidine production to 7.2 g/L and reduced the uridine production to 8.2 g/L.The final strain can be used for co-production of cytidine and uridine and has a good prospect for industrial application.
作者
刘益宁
秦臻
李旋
蒋帅
吴鹤云
谢希贤
LIU Yining;QIN Zhen;LI Xuan;JIANG Shuai;WU Heyun;XIE Xixian(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457,China;National and Local United Engineering Lab of Metabolic Control Fermentation Technology,Tianjin 300457,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2021年第12期10-16,共7页
Food and Fermentation Industries
基金
国家重点研发计划(2018YFA0900300)。
关键词
胞苷
尿苷
代谢工程
大肠杆菌
发酵
cytidine
uridine
metabolic engineering
Escherichia coli
fermentation
