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食品与发酵工业  2021, Vol. 47 Issue (12): 10-16    DOI: 10.13995/j.cnki.11-1802/ts.026576
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
胞苷合成途径改造对大肠杆菌嘧啶核苷发酵的影响
刘益宁1, 秦臻1, 李旋1, 蒋帅1, 吴鹤云2, 谢希贤1,3*
1(天津科技大学 生物工程学院,天津,300457)
2(天津科技大学 食品科学与工程学院,天津,300457)
3(代谢控制发酵技术国家地方联合工程实验室,天津,300457)
Effect of cytidine synthesis pathway modification on the fermentation of pyrimidine nucleoside by Escherichia coli
LIU Yining1, QIN Zhen1, LI Xuan1, JIANG Shuai1, WU Heyun2, XIE Xixian1,3*
1(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China)
2(College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457,China)
3(National and Local United Engineering Lab of Metabolic Control Fermentation Technology,Tianjin 300457,China)
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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,cddcmkygdH 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PyrHecj(D93A)和PyrGcgl(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。
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刘益宁
秦臻
李旋
蒋帅
吴鹤云
谢希贤
关键词:  胞苷  尿苷  代谢工程  大肠杆菌  发酵    
Abstract: 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 PyrHecj(D93A) and PyrGcgl(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.
Key words:  cytidine    uridine    metabolic engineering    Escherichia coli    fermentation
收稿日期:  2020-12-30      修回日期:  2021-02-15           出版日期:  2021-06-25      发布日期:  2021-07-22      期的出版日期:  2021-06-25
基金资助: 国家重点研发计划(2018YFA0900300)
作者简介:  硕士研究生(谢希贤教授为通讯作者,E-mail:xixianxie@tust.edu.cn)
引用本文:    
刘益宁,秦臻,李旋,等. 胞苷合成途径改造对大肠杆菌嘧啶核苷发酵的影响[J]. 食品与发酵工业, 2021, 47(12): 10-16.
LIU Yining,QIN Zhen,LI Xuan,et al. Effect of cytidine synthesis pathway modification on the fermentation of pyrimidine nucleoside by Escherichia coli[J]. Food and Fermentation Industries, 2021, 47(12): 10-16.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.026576  或          http://sf1970.cnif.cn/CN/Y2021/V47/I12/10
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