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CN-122012443-A - Deoxycytidine acid hydroxylase and application thereof

CN122012443ACN 122012443 ACN122012443 ACN 122012443ACN-122012443-A

Abstract

The invention discloses deoxycytidine acid hydroxylase AfhB and application thereof. The dCMP hydroxylase AfhB obtained by screening the genome of the bacteriophage HY126 can be synthesized green or fermented to produce 5hdCMP, so that the environmental pollution caused by the traditional chemical synthesis method is greatly reduced, and a novel enzyme element and a novel path are provided for the development of a gene editing technology and the fermentation production of 5hdCMP and derivatives thereof in industrial fermentation.

Inventors

  • WANG LIANRONG
  • CHEN SHI
  • HE YU

Assignees

  • 深圳市儿童医院

Dates

Publication Date
20260512
Application Date
20260317

Claims (10)

  1. 1. A deoxycytidine monophosphate hydroxylase characterized by having the amino acid sequence shown below: 1 MGKHNTMKYI KAADFQSAFK AVNREILENP QFVTDSRIGR CNEIGSMTVV VDTPSSFKMT 61 DPRINRISYE YAEDFWKFMI SGGTDAKEAF KAYPNVAKFI SKPKSDALPA NFNTFYGPRI 121 AAQLPALLKE LKEKPNSRRV VFQILESSDQ ALLDSDETLE YPCTDSVTYY IRDGKLYTHC 181 HMRSQNCAVV MQLDFYLQGK LLHYIANECG VEVGDYTHTM VSAHVFERDF DYVKGFLD。
  2. 2. The deoxycytidine monophosphate hydroxylase according to claim 1, which is a derivative protein having dCMP hydroxylase activity in which the amino acid sequence shown in claim 1 has been substituted, deleted or added with one or more amino acids.
  3. 3. An isolated nucleic acid molecule encoding the deoxycytidine monophosphate hydroxylase of claim 1 or 2, the nucleic acid molecule having a nucleotide sequence as set forth in seq id no: 1 ATGGGAAAAC ATAACACAAT GAAATATATT AAAGCCGCAG ATTTTCAATC CGCATTTAAA 61 GCTGTCAATC GTGAAATTTT AGAAAATCCA CAGTTCGTTA CTGATTCTCG TATTGGACGT 121 TGTAATGAAA TTGGGTCTAT GACAGTAGTT GTTGATACCC CTTCATCATT CAAAATGACC 181 GACCCACGTA TTAATCGCAT CTCTTATGAA TATGCTGAAG ATTTCTGGAA ATTTATGATT 241 TCTGGTGGTA CTGATGCAAA AGAAGCATTT AAGGCATATC CTAACGTCGC TAAATTTATT 301 TCTAAACCTA AATCAGACGC TTTGCCTGCA AACTTCAACA CTTTCTATGG TCCTCGCATT361 GCGGCCCAAT TACCAGCTCT TCTAAAAGAA CTTAAAGAGA AGCCTAACTC CCGAAGAGTT 421 GTGTTCCAAA TTCTAGAAAG TTCCGATCAG GCTTTGCTTG ATTCAGACGA AACGCTTGAA 481 TATCCATGTA CTGACTCAGT GACTTATTAT ATTCGTGATG GTAAGCTTTA TACCCACTGT 541 CATATGCGTT CTCAGAACTG TGCTGTTGTT ATGCAGTTGG ATTTTTATCT CCAAGGAAAA 601 TTACTTCATT ATATCGCTAA CGAATGTGGT GTAGAAGTTG GTGATTATAC TCATACCATG 661 GTAAGTGCCC ATGTGTTTGA ACGTGATTTT GATTATGTTA AAGGATTCCT TGACTAA。
  4. 4. a recombinant expression vector comprising the nucleic acid molecule of claim 3.
  5. 5. An engineered cell, characterized in that, the engineered cell comprising the recombinant expression vector of claim 4.
  6. 6. The engineered cell of claim 5, wherein the host cell of the engineered cell is a prokaryotic cell or a eukaryotic cell.
  7. 7. A method for producing 5-hydroxydeoxycytidine monophosphate (5 hdCMP), comprising the steps of (1) culturing the engineered cell of claim 5 or 6, inducing expression of deoxycytidine monophosphate hydroxylase, and (2) collecting and isolating 5hdCMP from the culture.
  8. 8. A method for the enzymatic synthesis of 5-hydroxydeoxycytidine monophosphate (5 hdCMP) in vitro, characterized in that dCMP is used as substrate and the reaction is carried out in the presence of deoxycytidine monophosphate hydroxylase according to claim 1 or 2 and cofactor to produce 5hdCMP.
  9. 9. The method of claim 8, wherein the cofactor comprises Flavin Adenine Dinucleotide (FAD) and a reducing power provided by NADH and NAD (P) H-flavin reductase, or by Ti 3+ .
  10. 10. Use of a deoxycytidine monophosphate hydroxylase according to claim 1 or 2, or a recombinant expression vector according to claim 4, or an engineered cell according to claim 5 or 6, for the preparation of a biological agent or kit for the production of 5-hydroxydeoxycytidine monophosphate (5 hdCMP).

Description

Deoxycytidine acid hydroxylase and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a phage-encoded deoxycytidylate hydroxylase AfhB and application thereof in green synthesis, fermentation production, gene editing, apparent modification research and the like of hydroxylated cytosine nucleotides. Background Apparent modification is an important cell physiological regulation means, and cells can finely regulate gene expression through the apparent modification. Common apparent modifications include 6-methyladenine, 5-methylcytosine, 5-hydroxymethylcytosine, and the like (Rosaura Esteve-Puig et al 2020). 6-methyladenine, one of the most widely distributed apparent modifications, plays an important role in regulating DNA replication initiation, mismatch repair, phage defense, and the like (Beifang Lu et al, 2025). Other apparent modifications can also regulate the relevant physiological processes to varying degrees. Under genomic replication and various deleterious circumstances, nucleotides can produce unnatural modifications, most of which are deleterious to the cell itself, which can be cleared by the cell via specific pathways (Andrea j. Lee et al, 2017). 5-hydroxycytosine is a more specific class of these. The structural formula of hydroxycytosine is as follows: The cytosine base on the cytoplasm or DNA can be oxidized or catalyzed by a bacterial/phage enzyme to produce 5-hydroxycytosine. When cytosine is oxidized in the cytoplasm to 5-hydroxycytosine, it can be further phosphorylated to form its triphosphate derivative, which is incorporated into the genome as a substrate for DNA synthesis. In situ oxidation of cytosine on DNA can also produce 5-hydroxycytosine. The base pairing property of 5-hydroxycytosine is different from that of ordinary cytosine and more prone to pairing with dA, thus causing C.fwdarw.T mutation and thus potentially detrimental to genome stability. Cells evolved specific enzymes such as Endonuclease VIII (Nei) and endonucleose III (Nth) to scavenge 5-hydroxycytosine. However, for some phages, 5-hydroxycytosine is an essential component of its DNA. For example, the substitution of all cytosines in the genome of the Rhizobium phage RL38JI with ultra-modified 5-hydroxycytosine suggests that the modification may exist stably in the genome and may assist the phage in breaking through the host defense system. In addition, 5-hydroxycytosine is also a natural modification of the 2501 st site of E.coli 23S rRNA (abbreviated as ho5C 2501). The modified abundance is dynamic and reaches the highest in the bacterial stage. High abundance of 5-hydroxycytosine can affect translation efficiency, reduce protein synthesis, but help E.coli adapt to oxidative stress. The related gene deletion mutant strain can not grow under the stress of hydrogen peroxide with millimolar concentration, and the strain retaining the modification can grow normally. Recent studies have also found that substitution of 5-hydroxycytosine for cytosine in antisense oligonucleotides or siRNAs targeting mRNA can reduce hepatotoxicity while maintaining antisense activity. These studies reveal the importance and complexity of 5-hydroxycytosine in physiological activities. At present, a common method for introducing hydroxyl into cytosine nucleoside or deoxynucleoside is a bromohydrolysis method, namely, substitution is carried out on the 5-position of cytosine by utilizing a bromomonomer, and then alkaline hydrolysis is carried out to obtain a hydroxylation product. The technology is chemical synthesis, toxic and strong-irritation bromine simple substance and a large amount of organic reagents are needed in the process, and the safety and the economic benefit are poor. Disclosure of Invention The invention aims to overcome the defects and shortcomings of the existing technology for synthesizing 5-hydroxycytosine nucleotide (5 hdCMP), provides 5hdCMP hydroxylase AfhB and application thereof, and can produce 5hdCMP through in-vivo fermentation or in-vitro enzyme catalysis of escherichia coli. In order to achieve the above purpose, the present invention adopts the following technical scheme: AfhB proteins and their functions were discovered in the present invention. In the phage HY126 genome of E.coli JM109, afhB expression can produce 5hCMP in JM109 cytoplasm. The invention provides a system for specifically synthesizing 5hCMP, which comprises AfhB, wherein the nucleotide sequence of 5hdCMP hydroxylase is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The invention provides a vector pWHU5053 which is used for heterologously expressing afhB genes in escherichia coli to produce 5hdCMP by fermentation. The invention also provides a vector pWHU5054 for expressing recombinant AfhB protein in escherichia coli BL21 (DE 3). The dCMP hydroxylase can be expressed in escherichia coli and used for fermenting and producing 5hdCMP or purify