School of Life Science, Ningxia University, Yinchuan 750021, China.
Ningxia Key Laboratory for Food Microbial-Applications Technology and Safety Control, School of Food Science and Engineering, Ningxia University, Yinchuan 750021, China.
J Appl Microbiol. 2024 Jun 3;135(6). doi: 10.1093/jambio/lxae133.
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.
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.
ARTP mutagenesis combined with metabolic engineering is an effective method to construct cytidine-producing strains.
胞苷作为抗病毒和抗肿瘤药物化学合成的重要商业前体，市场需求量很大。因此，本研究旨在构建具有高产胞苷能力的微生物细胞工厂。
通过常压室温等离子体（ARTP）诱变结合高通量筛选，获得了一种对尿苷单磷酸结构类似物具有高耐受性和良好遗传稳定性的突变大肠杆菌 NXBG-11-F34。然后，通过 CRISPR/Cas9 基因编辑技术敲除了参与胞苷分解代谢的 udk 和 rihA 基因，构建了重组大肠杆菌 NXBG-13。在 5 L 生物反应器中发酵胞苷的产量、得率和生产率分别为 15.7 g/L、0.164 g/g 和 0.327 g/L·h-1。原始菌株和重组大肠杆菌 NXBG-13 的转录组分析表明，两株菌的基因表达谱发生了显著变化，重组菌的胞苷从头途径基因显著上调。
ARTP 诱变与代谢工程相结合是构建产胞苷菌株的有效方法。
