An Enzymatic Assay for High-Throughput Screening of Cytidine-Producing Microbial Strains

Dong, Huina; Liu, Yongfei; Zu, Xin; Li, Ning; Li, Feiran; Zhang, Dawei

doi:10.1371/journal.pone.0121612

Cited by 11 publications

(3 citation statements)

References 32 publications

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“…The evolution of NH4 + by deamination of the nucleic acid substrates was observed using a phenol-hypochlorite reaction to produce indophenol in a clear 96-well microtiter plate and modified from Dong et al 2015 [102]. The work of Ngo et al [103] was considered when designing the lysis buffer so as not to interfere with the phenolhypochlorite reaction.…”

Section: Ammonium Detectionmentioning

confidence: 99%

A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

et al. 2021

Preprint

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The El Tor biotype of Vibrio cholerae is responsible for perpetuating the longest cholera pandemic in recorded history (1961-current). The genomic islands VSP-1 and -2 are two understudied genetic features that distinguish El Tor from previous pandemics. To understand their utility, we calculated the co-occurrence of VSP genes across bacterial genomes. This analysis predicted the previously uncharacterized vc0175, herein renamed deoxycytidylate deaminase Vibrio (dcdV), is in a gene network with dncV, a cyclic GMP-AMP synthase involved in phage defense. DcdV consists of two domains, a P-loop kinase and a deoxycytidylate deaminase, that are required for the deamination of dCTP and dCMP, inhibiting phage predation by corrupting cellular nucleotide concentrations. Additionally, DcdV is post-translationally inhibited by a unique noncoding RNA encoded 5-prime of the dcdV locus. DcdV homologs are conserved in bacteria and eukaryotes and our results identify V. cholerae DcdV as the founding member of a previously undescribed bacterial phage defense system.

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Section: Ammonium Detectionmentioning

confidence: 99%

A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

et al. 2021

Preprint

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“…As a pyrimidine nucleoside, cytidine is a structural component of RNA in organisms that are involved in several cellular processes [1]. Cytidine has recently been a research hotspot, primarily as an intermediate in various antiviral, anticancer, and anti-AIDS medicines [2][3][4].…”

Section: Introductionmentioning

confidence: 99%

The Effect of E. coli Uridine-Cytidine Kinase Gene Deletion on Cytidine Synthesis and Transcriptome Analysis

Liu

Zhang

et al. 2022

Fermentation

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Cytidine is an antiviral and anticancer drug intermediate, its primary method of manufacture being fermentation. Uridine-cytidine kinase (UCK) catalyzes the reverse process of phosphorylation of cytidine to produce cytidylic acid, which influences cytidine accumulation in the Escherichia coli cytidine biosynthesis pathway. The cytidine-producing strain E. coli NXBG-11 was used as the starting strain in this work; the udk gene coding UCK was knocked out of the chromosomal genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. The mutant strain E. coli NXBG-12 was obtained; its transcriptomics were studied to see how udk gene deletion affected cytidine synthesis and cell-wide transcription. The mutant strain E. coli NXBG-12 generated 1.28 times more cytidine than the original strain E. coli NXBG-11 after 40 h of shake-flask fermentation at 37 °C. The udk gene was knocked out, and transcriptome analysis showed that there were 1168 differentially expressed genes between the mutant and original strains, 559 upregulated genes and 609 downregulated genes. It was primarily shown that udk gene knockout has a positive impact on the cytidine synthesis network because genes involved in cytidine synthesis were significantly upregulated (p < 0.05) and genes related to the cytidine precursor PRPP and cofactor NADPH were upregulated in the PPP and TCA pathways. These results principally demonstrate that udk gene deletion has a favorable impact on the cytidine synthesis network. The continual improvement of cytidine synthesis and metasynthesis is made possible by this information, which is also useful for further converting microorganisms that produce cytidine.

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“…The challenge is to develop efficient screening methods to acquire mutants with desired phenotypes. Although combining cytidine deaminase (CDA) and indophenol method have been performed in 96-well microplate for the high-throughput screening of cytidine producing strains, the two-step reactions and purified CDA required in the process are expensive and time-consuming [ 20 ]. Recently, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis has been successfully applied to the microbial production of lactic acid, arachidonic acid and ε-poly-L-lysine [ 21 – 23 ].…”

Section: Introductionmentioning

confidence: 99%

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening

Fan

et al. 2017

PLoS ONE

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In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5′-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain.

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An Enzymatic Assay for High-Throughput Screening of Cytidine-Producing Microbial Strains

Cited by 11 publications

References 32 publications

A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

The Effect of E. coli Uridine-Cytidine Kinase Gene Deletion on Cytidine Synthesis and Transcriptome Analysis

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening

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