大肠杆菌肌苷酸节点代谢优化高效合成肌苷  

Inosinic acid node metabolism optimization for efficient synthesis of inosine in Escherichia coli

作　　者：刘铁重 高志强 侯德欣 谢希贤[1,2] 吴鹤云 LIU Tiezhong;GAO Zhiqiang;HOU Dexin;XIE Xixian;WU Heyun(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin University of Science and Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]工业发酵微生物教育部重点实验室(天津科技大学),天津300457

出　　处：《食品与发酵工业》2024年第4期44-50,共7页Food and Fermentation Industries

基　　金：国家重点研发计划(2018YFA0900300)。

摘　　要：该研究以产肌苷的大肠杆菌INO4-5为出发菌,针对其发酵过程中副产物含量高、肌苷产量低以及需要额外添加腺嘌呤等问题,对肌苷酸节点代谢流进行了优化。首先使用不同启动子和核糖体结合位点对枯草芽孢杆菌来源的purA_(bsu)基因进行了表达,以强化腺苷合成支路,使菌株5 L发酵罐上的肌苷产量达到了29.5 g/L,且无需额外添加腺嘌呤。接着通过替换guaB基因的原有启动子以弱化鸟苷合成支路,并过表达guaC基因促使鸟苷酸回流至肌苷酸,使菌株副产物含量显著降低且肌苷产量提升至35.1 g/L;最后对不同5′-核苷酸酶进行了筛选和强化,发现引入来自酿酒酵母的特异性5′-核苷酸酶基因(introduction of specific 5′-nucleotidase gene,ISN1)可有效促进肌苷生产。最终所得菌株INO7-6在5 L发酵罐上发酵,肌苷产量为41.2 g/L,转化率为0.17 g/g葡萄糖,生产强度为0.86 g/(L·h),为目前报道的基因工程菌株的最高产量。In this study,an inosine-producing strain Escherichia coli INO4-5 was used as the starting strain,and the metabolic flow of inosinic acid(IMP)node was optimized in view of the high content of byproducts,low inosine production and the requirement of additional adenine during fermentation.Firstly,the purA_(bsu) gene from Bacillus subtilis was expressed using different promoters and ribosome binding sites to strengthen the adenosine synthesis branch,which enabled the strain to produce 29.5 g/L inosine in 5 L fermentor without extra addition of adenine.Then,the original promoter of guaB gene was replaced to weaken the guanosine synthesis branch and guaC gene was overexpressed to promote guanylic acid reflux to IMP,which significantly reduced the byproduct content of the strain and increased the inosine production to 35.1 g/L.Finally,different 5′-nucleotidases were screened and enhanced,and the introduction of specific 5′-nucleotidase gene(ISN 1)from Saccharomyces cerevisiae was found to effectively promote inosine production.The resulting strain INO7-6 produced 41.2 g/L inosine with yield of 0.17 g/g glucose and productivity of 0.86 g/(L·h)in 5 L fermentor.The inosine production of this strain is the highest within the genetically engineered strains reported so far,laying a good foundation for the industrial production of inosine.

关 键 词：肌苷 肌苷酸 大肠杆菌 代谢工程 发酵 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]