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Updated: May 5, 2026
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Improving the level of the cytidine biosynthesis in E. coli through atmospheric room temperature plasma mutagenesis
Xiangjun Zhang1, Lu Liu2, Cong Ma2
1School of Life Science, Ningxia University, Yinchuan 750021, China.
Aims:
Cytidine, as an important commercial precursor in the chemical synthesis of antiviral and antitumor drugs, is in great demand in the market. Therefore, the purpose of this study is to build a microbial cell factory with high cytidine production.
Methods And Results:
A mutant E. coli NXBG-11-F34 with high tolerance to uridine monophosphate structural analogs and good genetic stability was obtained by atmospheric room temperature plasma (ARTP) mutagenesis combined with high-throughput screening. Then, the udk and rihA genes involved in cytidine catabolism were knocked out by CRISPR/Cas9 gene editing technology, and the recombinant strain E. coli NXBG-13 was constructed. The titer, yield, and productivity of cytidine fermented in a 5 l bioreactor were 15.7 g l-1, 0.164 g g-1, and 0.327 g l-1 h-1, respectively. Transcriptome analysis of the original strain and the recombinant strain E. coli NXBG-13 showed that the gene expression profiles of the two strains changed significantly, and the cytidine de novo pathway gene of the recombinant strain was up-regulated significantly.
Conclusions:
ARTP mutagenesis combined with metabolic engineering is an effective method to construct cytidine-producing strains.
