基于基因组重测序的高产尿苷大肠杆菌工程菌株的构建

Construction of engineered E.coli strains with high uridine yield based on genome-resequencing

以大肠杆菌高产胞苷E.coli XD10为出发菌株,首先对其进行基因组重测序,分析其相较于野生型菌株的尿苷合成及降解相关基因变化情况。应用CRISPR/Cas9基因编辑技术,通过依次构建T7 RNA聚合酶基因T7 gene 1、使用异源T7启动子的催化胞苷向尿苷转化的胞苷脱氨酶基因cdd以及催化尿苷降解的核苷酸-5′磷酸核苷酶基因ppnN的敲除质粒及其同源修复供体DNA片段,将敲除质粒及相应同源修复供体共转化大肠杆菌感受态细胞,PCR和测序验证正确的转化子进行打靶质粒和pCas质粒的消除,获得基因编辑大肠杆菌工程菌株E.coli URI-1、URI-2和URI-3。半定量PCR实验结果表明,染色体敲入的T7 gene 1和cdd基因在工程菌株中均进行了有效表达。使用T7转录系统过表达cdd基因的URI-2菌株摇瓶发酵产物的HPLC检测结果显示,其发酵液中尿苷产量为8.32 g/L,而出发菌株XD10发酵液尿苷产量为0.80 g/L。进一步敲除ppnN基因的工程菌株URI-3发酵液尿苷HPLC检测产量为10.76 g/L,显著高于URI-2发酵液尿苷产量(P<0.05)。本研究获得的高产尿苷大肠杆菌工程菌株,为尿苷的工业化发酵生产提供了菌种保障。

In this study,Escherichia coli XD10 with high cytidine yield was used as the starting strain,and its genome was resequenced and the changes in genes related to uridine synthesis and degradation were analyzed by comparing with the genome of wild-type E.coli strain.Using CRISPR/Cas9 gene editing technology,the knockout plasmids were constructed for the T7 RNA polymerase-encoding gene T7 gene 1,cytidine deaminase-encoding gene cdd that catalyzes the conversion of cytidine to uridine,and the nucleotide-5'phosphonucleosidase gene ppnN that catalyzes uridine degradation,among which cdd and ppnN gene knockout constructs were driven by the heterologous T7 promoter.The homologous repair donor DNA fragments and the knockout plasmid were co-transformed into E.coli competent cells.The knockout plasmid and pCas plasmid in the correct transformant were eliminated by PCR and sequencing verification,identifying the engineered strains E.coli URI-1,URI-2,and URI-3.Semi-quantitative PCR identification results showed that both T7 gene 1 and cdd performed efficient expression in engineered E.coli strains.The HPLC detection results of the shaking flask fermentation culture of the URI-2 strain overexpressing the cdd gene showed that the yield of uridine was 8.32 g/L,while the uridine yield of the starting strain XD10 was 0.80 g/L.The uridine yield in the fermentation culture of the engineered strain E.coli URI-3 in which the ppnN gene was further knocked out was 10.76 g/L,which was significantly higher than that of the strain E.coli URI-2.The engineered E.coli strain with high yield uridine constructed in this study would provide a strain guarantee for the industrial fermentation production of uridine.