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.
关键词
大肠杆菌/基因组重测序/CRISPR/Cas9基因编辑技术/尿苷生产/工程菌株
Key words
Escherichia coli/resequencing of genome/CR1SPR/Cas9 gene editing technology/uridine production/engineered strains
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