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

doi:10.13523/j.cb.2302034

China Biotechnology

2023, Vol. 43

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

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

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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

Received: 20 February 2023 Published: 05 September 2023

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	Cong MA
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Cite this article:

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.

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

Table 1 Bacterial strains and plasmids used in this study

Table 2 Primers used in this experiment

Fig.1 Verification results of CRISPR-mediated purR knockout M:2 000 bp DNA marker;1,2:Product after knockout of purR (120 bp);3,4:Pre-knockdown PCR product control (1 005 bp)

Fig.2 Growth curve of starting strain and mutant strain

Fig.3 Sugar consumption of starting strain and mutant strain

Fig.4 Cytidine production of starting strain and mutant strain

Table 3 Sequencing data quality control comparison results

Fig.5 Sample correlation analysis

Fig.6 Differentially expressed gene volcano map

Fig.7 GO enrichment analysis of differentially expressed genes

Table 4 KEGG pathway enrichment analysis of DEGs

Table 5 KEGG pathway functional annotation of DEGs

Fig.8 Schematic representation of gene expression related to cytidine synthesis-related pathway Red representing upward adjustmen, blue representing downward adjustment

Fig.9 DEGs qRT-PCR validation results


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