基于转录组学分析大肠杆菌中<i>purR</i>基因缺失对胞苷合成代谢的影响<sup>*</sup>

doi:10.13523/j.cb.2302034

中国生物工程杂志

2023, Vol. 43

Issue (8): 52-62 DOI: 10.13523/j.cb.2302034

研究报告

基于转录组学分析大肠杆菌中purR基因缺失对胞苷合成代谢的影响^*

马聪^1,²,张想军^1,²,叶彤^1,²,柳凤敏^1,²,张皓杰^1,²,刘慧燕^1,^2,^**(

),方海田^1,^2,^**(

)

1 宁夏大学食品与葡萄酒学院银川 750021
2 宁夏食品微生物应用技术与安全控制重点实验室银川 750021

Transcriptomics-based Analysis of the Effect of purR Gene Deletion on Cytidine Anabolism in Escherichia coli

MA Cong^1,²,ZHANG Xiang-jun^1,²,YE Tong^1,²,LIU Feng-min^1,²,ZHANG Hao-jie^1,²,LIU Hui-yan^1,^2,^**(

),FANG Hai-tian^1,^2,^**(

)

1 School of Food and Wine, Ningxia University, Yinchuan 750021, China
2 Ningxia Key Laboratory of Food Microbiology Application Technology and Safety Control, Yinchuan 750021, China

全文: PDF(1594 KB) HTML

摘要：

胞苷可作为功能营养化学品与药物合成原料,具有重要的应用价值。大肠杆菌中由purR基因编码的DNA结合转录抑制因子对胞苷合成代谢有重要调控作用。采用CRISPR/Cas9技术敲除大肠杆菌purR基因,并通过转录组学分析突变菌株基因表达的差异。结果表明,从出发菌株E. coli NXBG-12基因组上成功敲除了purR基因,获得了突变菌株E. coli NXBG-17P。对突变菌株E. coli NXBG-17P与E. coli NXBG-12的转录组学结果进行对比分析,发现有534个差异基因,其中上调基因302个、下调基因232个;GO分析显示,差异表达基因(DEGs)主要富集于细胞膜、ATP结合、DNA结合和水解酶活性的代谢过程;KEGG分析表明,上调基因主要富集在果糖和甘露糖代谢、嘧啶代谢和磷酸转移酶系统,下调基因主要富集在氧化磷酸化、半乳糖代谢和肽聚糖的生物合成。同时,突变菌株E. coli NXBG-17P在37℃摇瓶发酵40 h,测定胞苷浓度为(3.21±0.01) g/L,是出发菌株E. coli NXBG-12的1.58倍。这表明突变菌株E. coli NXBG-17P胞苷产量升高,可能是purR基因缺失导致葡萄糖磷酸转移酶系统(PTS)转运和磷酸戊糖途径中关键基因表达上调,合成了大量胞苷所需能量NADPH和前体物质PRPP。

关键词： 大肠杆菌; 胞苷; purR基因; 基因敲除; 转录组分析

Abstract:

Cytidine is used as a raw material for drug synthesis from functional nutritional chemicals and has important application value. DNA-binding transcriptional repressors encoded by the purR gene in Escherichia coli are important regulators of cytidine anabolism. In this study, the E. coli purR gene was knocked down using CRISPR/Cas9 technology and the differences in gene expression of mutant strains were analyzed by transcriptomics. The results showed that the purR gene was successfully knocked out from the genome of the starting strain E. coli NXBG-12, and the mutant strain E. coli NXBG-17P was obtained. Comparative analysis of transcriptomic results from mutant strains E. coli NXBG-17P and E. coli NXBG-12 revealed 534 differential genes, including 302 up-regulated genes and 232 down-regulated genes. GO analysis showed that differentially expressed genes (DEGs) were mainly enriched in the metabolic processes of cytoplasmic membrane, ATP binding, DNA binding and hydrolase activity; KEGG analysis showed that up-regulated genes were mainly enriched in fructose and mannose metabolism, pyrimidine metabolism and phosphotransferase system, and down-regulated genes were mainly enriched in oxidative phosphorylation, galactose metabolism and peptidoglycan biosynthesis. Meanwhile, mutant strain E. coli NXBG-17P was fermented in a shake flask at 37℃ for 40 h. The cytidine concentration was determined to be (3.21±0.01) g/L, which was 1.58 times the level of the starting strain E. coli NXBG-12. It is well demonstrated that purR gene deletion enhances PTS (glucose phosphotransferase system) transport and pentose phosphate pathway, which can provide more NADPH and pyrimidine nucleoside precursors PRPP for cytidine synthesis pathway.

Key words: Escherichia coli Cytidine purR gene Gene knockout Transcriptome analysis

收稿日期: 2023-02-20 出版日期: 2023-09-05

ZTFLH:

Q815

基金资助: 国家自然科学基金(31860020);宁夏食品微生物应用技术与安全控制重点实验室平台建设项目(2021JCTJ0030)

通讯作者: **电子信箱:liuhy@nxu.edu.cn; fanght@nxu.edu.cn

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引用本文:

马聪, 张想军, 叶彤, 柳凤敏, 张皓杰, 刘慧燕, 方海田. 基于转录组学分析大肠杆菌中purR基因缺失对胞苷合成代谢的影响^*[J]. 中国生物工程杂志, 2023, 43(8): 52-62.

MA Cong, ZHANG Xiang-jun, YE Tong, LIU Feng-min, ZHANG Hao-jie, LIU Hui-yan, FANG Hai-tian. Transcriptomics-based Analysis of the Effect of purR Gene Deletion on Cytidine Anabolism in Escherichia coli. China Biotechnology, 2023, 43(8): 52-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2302034 或 https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I8/52

表1 本研究中使用的菌株和质粒

表2 本实验所用引物

图1 CRISPR介导的purR敲除验证结果

图2 出发菌株和突变菌株生长曲线图

图3 出发菌株和突变菌株耗糖量

图4 出发菌株和突变菌株胞苷产量

表3 测序数据质控对比结果

图5 样品相关性分析

图6 差异表达基因火山图

图7 差异表达基因的GO富集分析

表4 DEGs的KEGG通路富集分析

表5 DEGs的KEGG通路功能挖掘

图8 胞苷合成相关通路相关基因表达示意图

图9 DEGs qRT-PCR验证结果

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