Genetically engineered bacterium for producing 2 '-deoxyuridine as well as construction method and application of genetically engineered bacterium

Abstract

The invention relates to the technical production field of metabolic engineering and genetic engineering, and particularly provides a genetically engineered bacterium for producing 2 '-deoxyuridine as well as a construction method and application of the genetically engineered bacterium. According to the strain, deoxyuridine kinase tdk is knocked out on an E.coli W3110 genome, and T7 RNA polymerase is integrated; an aspartate transaminase gene aspC, a glutamine synthetase gene glnA, a 5 '-deoxynucleotidase gene yfbR and a uridine monophosphate kinase gene pyrH are subjected to overexpression; the method comprises the following steps: introducing a ribonucleoside diphosphate reductase gene nrdBCA from Escherichia phage T4 in a heterologous series connection manner; the CTP synthase gene pyrG * of Corynebacterium glutamicum is introduced in a heterologous mode, and the CTP synthase gene pyrG * of Corynebacterium glutamicum is introduced in a heterologous mode. The safe, efficient and low-cost production of the 2 '-deoxyuridine is realized.

Description

Technical Field

[0001] The present application relates to the field of metabolic engineering and genetic engineering technology production, and specifically provides a genetically engineered bacterium for producing 2'-deoxyuridine, a construction method thereof, and an application thereof. Background Art

[0002] 2'-deoxyuridine plays an important role in biochemistry. It is a single nucleotide in RNA and DNA and is involved in the synthesis and storage of nucleic acids. In addition, 2'-deoxyuridine is an important intermediate of the highly selective, efficient and low-toxic anti-tumor drug 2'-deoxy-5-fluorouridine. Therefore, 2'-deoxyuridine is an important antiviral drug. It is a raw material for the production of anti-tumor drugs FUDR, antiviral drugs IDUR and BrDUR, and is used as a biochemical reagent. It is also used to synthesize enzyme inhibitors, antiviral agents and anticancer agents. In short, 2'-deoxyuridine has an irreplaceable role in clinical applications, and seeking a more efficient synthesis method has always been a hot topic for scientific researchers. At present, the main method for producing 2'-deoxyuridine is chemical synthesis. Most of these methods use uridine or other intermediates as raw materials and use catalysts for catalysis. The production cost is high, and the production process of some catalysts is dangerous, which is not suitable for large-scale industrial production. The microbial fermentation method for producing 2'-deoxyuridine mainly uses the biosynthetic pathway of microbial strains to synthesize 2'-deoxyuridine from scratch. Its advantage is that it uses glucose as raw material, has low production cost, high efficiency, and a simple and safe production process. Therefore, constructing a 2'-deoxyuridine production strain with a clear genetic background and a relatively considerable yield is a technical problem that needs to be solved urgently. In view of this, this application is hereby filed. Summary of the invention

[0003] One of the purposes of the present application is to provide a genetically engineered bacterium for producing 2'-deoxyuridine and a construction method and application thereof, so as to realize the industrial production of 2'-deoxyuridine by microbial fermentation. In order to achieve the above purpose, this application adopts the following technical solutions: A genetically engineered bacterium for producing 2'-deoxyuridine, wherein the genetically engineered bacterium is a bacterium of Escherichia coli. Escherichia coli W3110 is the starting strain constructed by genetic modification: exist E.coli Deoxyuridine kinase knockout in the W3110 genome tdk Integrate T7 RNA polymerase and overexpress aspartate aminotransferase gene aspC , glutamine synthetase gene glnA , 5'-deoxynucleotidase gene yfbR and uridylate kinase genepyrH ; Heterologous tandem introduction from Escherichia phage T4 ribonucleoside diphosphate reductase gene nrdBCA ; Heterologous introduction of Corynebacterium glutamicum Corynebacterium glutamicum CTP synthase gene pyrG * . Furthermore, the deoxyuridine kinase gene tdk The nucleotide sequence of the T7 RNA polymerase is shown in SEQ ID NO.1; the nucleotide sequence of the T7 RNA polymerase is shown in SEQ ID NO.2.

[0004] Furthermore, in the genome rph Aspartate aminotransferase gene aspC , and with promoter P trc Regulation; the aspartate aminotransferase gene aspC The nucleotide sequence is shown in SEQ ID NO.3; Preferably, in the genome ylbE Glutamine synthetase gene glnA , and with promoter P trc Regulation; The glutamine synthetase gene glnA The nucleotide sequence is shown in SEQ ID NO.4; Preferably, in the genome ilvG 5'-deoxynucleotidase gene yfbR , and with promoter P trc Regulation; the 5'-deoxynucleotidase gene yfbR The nucleotide sequence is shown in SEQ ID NO.5; Preferably, in the genome ycdN Urydinate kinase gene pyrH , and with promoter P T7 Regulation; The uridylate kinase gene pyrH The nucleotide sequence is shown in SEQ ID NO.6.

[0005] Furthermore, in the genome yeeP Tandem integration at the locus Escherichia phage T4 ribonucleoside diphosphate reductase gene nrdBCA , connected by RBS and driven by the same promoter P trc Regulation; Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strain nrdB The nucleotide sequence is shown in SEQ ID NO.7; Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strainnrdC The nucleotide sequence is shown in SEQ ID NO.8; Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strain nrdA The nucleotide sequence is shown in SEQ ID NO.9; Preferably, in the genome mbhA Corynebacterium glutamicum Corynebacterium glutamicum CTP synthase gene pyrG * , using promoter P trc Regulation: The CTP synthase gene of the Corynebacterium glutamicum strain pyrG * The nucleotide sequence is shown in SEQ ID NO.10.

[0006] Furthermore, the P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.11; T7 The nucleotide sequence of the promoter is shown in SEQ ID NO.12; the nucleotide sequence of the RBS is shown in SEQ ID NO.13. The method for constructing the above-mentioned genetically engineered bacteria comprises the following steps: (1) By using the starting strain Escherichia coli Deoxyuridine kinase knockout in W3110 genome tdk And integrate T7 RNA polymerase to block the degradation pathway of 2'-deoxyuridine; (2) By rph , ylbE , ilvG Overexpression at each site aspC , glnA and yfbR , and use P trc The promoters are regulated respectively to enhance the expression of key enzyme genes in the 2'-deoxyuridine synthesis pathway; (3) Through the genome ycdN Overexpression at the site pyrH , and use P T7 Promoter regulation enhances the metabolic flow of UMP to the target pathway; (4) Through the genome yeeP , mbhA ribonucleoside diphosphate reductase gene nrdBCA and CTP synthase gene pyrG* and regulated separately by the Ptrc promoter to strengthen the cytidine diphosphate deoxyreduction pathway, weaken CTP feedback inhibition and strengthen the cytidine triphosphate ribozyme.

[0007] The application of the above-mentioned genetically engineered bacteria for producing 2'-deoxyuridine in the fermentation production of 2'-deoxyuridine. A method for producing 2'-deoxyuridine comprises: fermenting the above-mentioned genetic engineering bacteria for producing 2'-deoxyuridine to obtain 2'-deoxyuridine.

[0008] Further, 2'-deoxyuridine is produced by fermentation in a fermenter, and the specific steps are as follows: (1) Activation of bacterial strains: Inoculate the bacterial solution from the bacterial preservation tube into the slant culture medium for activation and culture for 10 to 14 hours; transfer the colonies from the slant culture medium to the eggplant flask culture medium and continue to activate and expand the culture for 10 to 12 hours at a temperature of 30 to 34°C.

[0009] (2) Seed culture: 24%~26% ammonia solution is added automatically through the fermenter to maintain the pH value of the culture process at 7.0~7.2; the dissolved oxygen is maintained at 30%~50% by adjusting the stirring speed or ventilation volume of the fermenter; the culture temperature is maintained at 32~36℃, and the inoculation requirements are met when the OD is 18~22; (3) Fermentation culture: Inoculate at an inoculum rate of 18% to 22%. During the fermentation process, the pH was controlled at 7.1±0.1, the temperature was 32 to 36°C, and the dissolved oxygen was 30% to 50%. During the culture process, 80% glucose solution was added to maintain the carbon source required by the fermentation process. The fermentation cycle was 36 to 44 hours.

[0010] Furthermore, the seed culture medium used in step (2) is: yeast extract powder 6 g / L, peptone 4 g / L, MgSO4·7H2O 1 g / L, ammonium sulfate 2 g / L, magnesium sulfate 1 g / L, VH 1 mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 2 mg / L each, 1 g / L monosodium glutamate, 50 mg / L kanamycin sulfate, and the rest water; the fermentation medium used in step (3) is: 6 g / L yeast extract powder, 2 g / L peptone, 2 g / L citric acid, 3 g / L MgSO4·7H2O, 6 g / L KH2PO4, 20 mg / L FeSO4·7H2O, 10 mg / L MnSO4, Vb 1、 Vb 3、 Vb 5、 Vb 12 3 mg / L each, VH 1 mg / L, kanamycin sulfate 50 mg / L, sodium hexametaphosphate 3 g / L, glycine 2 g / L, and the rest is water.

[0011] The above culture media can be prepared using standard methods. Compared with the prior art, the technical effects of this application are: The 2'-deoxyuridine genetic engineering bacteria provided in this application have the following main innovative features: (1) Overexpression of uridylate kinase gene pyrH , and with the strong promoter P T7 Regulate and enhance the metabolic flow of UMP to the target pathway; (2) introduce heterologous T4 phage ribonucleoside diphosphate reductase nrdBCA , strengthen the cytidine diphosphate deoxygenation reduction pathway; (3) introduce heterologous Corynebacterium glutamicum Corynebacterium glutamicum Mutant CTP synthase gene pyrG * , weakening CTP feedback inhibition while strengthening cytidine triphosphate ribozyme. The obtained strain does not contain plasmids, has good genetic stability, and does not need to add antibiotics and defective substances during the production process, effectively reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 2'-deoxyuridine engineered strain E.coli Illustration of the construction method of ur7; Figure 2 It is a strain E.coli Fermentation process curve of ur7 in 5L fermenter. DETAILED DESCRIPTION

[0013] In order to enable those skilled in the art to better understand the technical solution of the present application, the technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0014] The 2'-deoxyuridine production strain provided in this application is a strain produced by genetic modification in Escherichia coli. Escherichia coli W3110 was further modified. Escherichia coli W3110 (Escherichia coli E.coli W3110 or wild-type Escherichia coli E.coli W3110) deposit number ATCC 27325.

[0015] The specific construction contents include: E.coli Deoxyuridine kinase knockout in the W3110 genome tdk Integrate T7 RNA polymerase and overexpress aspartate aminotransferase gene aspC , glutamine synthetase gene glnA , 5'-deoxynucleotidase gene yfbR and uridylate kinase gene pyrH ; Heterologous tandem introduction from Escherichia phage T4 ribonucleoside diphosphate reductase gene nrdBCA ; Heterologous introduction of Corynebacterium glutamicum Corynebacterium glutamicum CTP synthase gene pyrG* .

[0016] It should be noted that the present invention knocks out deoxyuridine kinase tdk And integrate T7 RNA polymerase to block the degradation pathway of 2'-deoxyuridine; by overexpression aspC , glnA and yfbR, Enhance the expression of key enzyme genes in the 2'-deoxyuridine synthesis pathway; by overexpression pyrH , enhancing the metabolic flux of UMP to the target pathway; by introducing heterologous ribonucleoside diphosphate reductase genes nrdBCA and CTP synthase gene pyrG* , strengthen the cytidine diphosphate deoxygenation reduction pathway, weaken CTP feedback inhibition and strengthen the cytidine triphosphate ribozyme. Therefore, as long as the strains constructed can achieve the above functions, they are all 2'-deoxyuridine production strains described in this application. At the same time, those skilled in the art can use conventional experimental designs and technical means in the art to achieve the above functions to obtain 2'-deoxyuridine production strains.

[0017] In some embodiments, the knockout deoxyuridine kinase tdk The nucleotide sequence is shown in SEQ ID NO.1 ( tdk ; NCBI-GeneID: 945834); the integrated T7 RNA polymerase nucleotide sequence is shown in SEQ ID NO.2.

[0018] In some embodiments, to overexpress the aspartate aminotransferase gene aspC , glutamine synthetase gene glnA , 5'-deoxynucleotidase gene yfbR and uridylate kinase gene pyrH , the following solutions can be adopted: In one embodiment, in the genome rph Aspartate aminotransferase gene aspC , and with promoter P trc Regulation; the aspartate aminotransferase gene aspC The nucleotide sequence is shown in SEQ ID NO.3 ( aspC ; NCBI-GeneID: 945553).

[0019] In one embodiment, in the genome ylbE Glutamine synthetase gene glnA , and with promoter P trc Regulation; The glutamine synthetase gene glnA The nucleotide sequence is shown in SEQ ID NO.4 ( glnA; NCBI-Gene ID: 948370). In one embodiment, in the genome ilvG 5'-deoxynucleotidase gene yfbR , and with promoter P trc Regulation; the 5'-deoxynucleotidase gene yfbR The nucleotide sequence is shown in SEQ ID NO.5 ( yfbR ; NCBI-GeneID: 946771).

[0020] In one embodiment, in the genome ycdN Urydinate kinase gene pyrH , and with promoter P T7 Regulation; The uridylate kinase gene pyrH The nucleotide sequence is shown in SEQ ID NO.6 ( pyrH ; NCBI-GeneID: 944989). In some embodiments, in order to introduce heterologous tandem Escherichia phage T4 ribonucleoside diphosphate reductase gene nrdBCA ; Heterologous introduction of Corynebacterium glutamicum Corynebacterium glutamicum CTP synthase gene pyrG * , the following technical solutions can be adopted: In one embodiment, in the genome yeeP Tandem integration at the locus Escherichia phage T4 ribonucleoside diphosphate reductase gene nrdBCA , connected by RBS and driven by the same promoter P trc Regulation; Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strain nrdB The nucleotide sequence is shown in SEQ ID NO.7 ( nrdB ; NCBI-GeneID: 1258601); Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strain nrdC The nucleotide sequence is shown in SEQ ID NO.8 ( nrdC ; NCBI-GeneID: 1258540); Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strain nrdA The nucleotide sequence is shown in SEQ ID NO.9 ( nrdA ; NCBI-GeneID: 1258795).

[0021] In one embodiment, in the genome mbhACorynebacterium glutamicum Corynebacterium glutamicum CTP synthase gene pyrG * , using promoter P trc Regulation: The CTP synthase gene of the Corynebacterium glutamicum strain pyrG * The nucleotide sequence is shown in SEQ ID NO.10 ( pyrG ; NCBI-GeneID: 69621931).

[0022] In one embodiment, the P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.11; T7 The nucleotide sequence of the promoter is shown in SEQ ID NO.12; the nucleotide sequence of the RBS is shown in SEQ ID NO.13. The present application provides a method for constructing a genetically engineered bacterium, comprising the following steps: (1) By using the starting strain Escherichia coli Deoxyuridine kinase knockout in W3110 genome tdk And integrate T7 RNA polymerase to block the degradation pathway of 2'-deoxyuridine; (2) By rph , ylbE , ilvG Overexpression at each site aspC , glnA and yfbR , and use P trc The promoters are regulated respectively to enhance the expression of key enzyme genes in the 2'-deoxyuridine synthesis pathway; (3) Through the genome ycdN Overexpression at the site pyrH , and use P T7 Promoter regulation enhances the metabolic flow of UMP to the target pathway; (4) Through the genome yeeP , mbhA ribonucleoside diphosphate reductase gene nrdBCA and CTP synthase gene pyrG* and regulated separately by the Ptrc promoter to strengthen the cytidine diphosphate deoxyreduction pathway, weaken CTP feedback inhibition and strengthen the cytidine triphosphate ribozyme.

[0023] It should be noted that there is no strict requirement for the order of the above steps, as long as the above purposes and functions can be achieved. The application of the above-mentioned genetically engineered bacteria for producing 2'-deoxyuridine in producing 2'-deoxyuridine. The 2'-deoxyuridine production strain constructed in the present application can realize the production of 2'-deoxyuridine and is suitable for industrial production. The present application also provides a method for producing 2'-deoxyuridine. The 2'-deoxyuridine production strain provided in the present application can produce 2'-deoxyuridine through fermentation. The fermentation process can be carried out with reference to conventional fermentation methods in the art.

[0024] The specific method of fermentation tank culture is: (1) Activation of bacterial strains: Inoculate the bacterial solution from the bacterial preservation tube into the slant culture medium for activation and culture for 12 hours; transfer the colonies from the slant culture medium to the eggplant flask culture medium and continue to activate and expand the culture for 10-12 hours at 32°C. (2) Seed culture: 25% ammonia solution is added automatically through the fermenter to maintain the pH at 7.1 during the culture process; the dissolved oxygen is maintained at 30%-50% by adjusting the fermenter stirring speed or ventilation volume; the culture temperature is maintained at 34°C, and the inoculation requirement is met when the OD reaches 20; (3) Fermentation culture: The inoculation volume was 20%. During the fermentation process, the pH was controlled at 7.1±0.1, the temperature was 34°C, and the dissolved oxygen was 30%-50%. During the culture process, 80% glucose solution was added to maintain the carbon source required by the fermentation process. The fermentation cycle was 40 h.

[0025] The slant and eggplant culture medium used in step (1) is: glucose 2g / L, yeast extract powder 5g / L, peptone 10g / L, NaCl 2.5g / L, KH2PO4 1g / L, MgSO4·7H2O 0.2g / L, agar powder 2.5g / L, and the rest is water.

[0026] The seed culture medium used in step (2) is: yeast extract powder 6g / L, peptone 4g / L, MgSO4·7H2O 1g / L, ammonium sulfate 2g / L, magnesium sulfate 1g / L, VH 1 mg / L, Vb1, Vb 3, Vb 5, Vb 12 each 2mg / L, monosodium glutamate 1g / L, kanamycin sulfate 50mg / L, and the rest is water. The fermentation medium used in step (3) is: yeast extract powder 6g / L, peptone 2g / L, citric acid 2g / L, MgSO4·7H2O 3g / L, KH2PO4 6g / L, FeSO4·7H2O 20mg / L, MnSO4 10mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 3 mg / L each, VH 1 mg / L, kanamycin sulfate 50 mg / L, sodium hexametaphosphate 3 g / L, glycine 2 g / L, and the rest is water.

[0027] The percentage sign "%" involved in the embodiments, unless otherwise specified, refers to mass percentage. The percentage of a solution refers to the number of grams of a solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0028] The starting strain used in the embodiment is E.coli W3110, with the accession number of ATCC 27325; strain T4 bacteriophage (DSM4505) is a commercially available strain; strain Corynebacterium glutamicum ( DSM 20300) is a commercially available strain; the corresponding promoter and genes are shown in the sequence listing.

[0029] Example 1 The gene editing method used is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). The engineering plasmid pGRB involved in this method is based on pUC18 as the backbone, including the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence, and ampicillin resistance (working concentration: 100 mg / L). The professional terms such as gene integration and plasmid construction involved in the following examples can be explained in this article. The primers used in the strain construction process are shown in Table 1.

[0030] Table 1 Primers involved in strain construction Primer name Primer sequence (5'-3') Sequence number pGRB--s AGTCCTAGGTATAATACTAGTGCAGCCAATACTTACTGGCAGTTTTAGAGCTAGA SEQ ID NO.14 pGRB--a TTCTAGCTCTAAAACTGCCAGTAAGTATTGGCTGCACTAGTATTATACCTAGGACT SEQ ID NO.15 -up-s GTGAATAGTTACCAGCAGTCATTTACCC SEQ ID NO.16 -up-a AATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAACGAACTGACTTTCCCGGCAC SEQ ID NO.17 T7 RNA polymerase - s GCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGAACACGATTAACATCGCTAAGAACGAC SEQ ID NO.18 T7 RNA polymerase - a CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACGCGAACGCGAAGTCC SEQ ID NO.19 -s TTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGATCAAGCAGGCAGACCTTATAAC SEQ ID NO.20 -a CGACGATGTATAACGCCTAAACACAG SEQ ID NO.21 pGRB--s AGTCCTAGGTATAATACTAGTGGCTGGATCACCGCAGAGTAGTTTTAGAGCTAGAA SEQ ID NO.22 pGRB--a TTCTAGCTCTAAAACTACTCTGCGGTGATCCAGCCACTAGTATTATACCTAGGACT SEQ ID NO.23 -up-s ATAGCGCAGGGTACATTCCACT SEQ ID NO.24 -up-a AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACA SEQ ID NO.25 -s GTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTTGAGAACATTACCGCCG SEQ ID NO.26 aspC - a ACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCACTGCCACAATCG SEQ ID NO.27 -s AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGCCGCAGAGACCGACAT SEQ ID NO.28 -a ACAGCGGTTGTGGTGGCA SEQ ID NO.29 pGRB--s AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA SEQ ID NO.30 pGRB--a TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT SEQ ID NO.31 -up-s ACCCAACCTTACGCAACCAG SEQ ID NO.32 -up-a TATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT SEQ ID NO.33 -s TAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTCCGCTGAACACGTACTG SEQ ID NO.34 -a AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGACGCTGTAGTACAGCTCAAACTC SEQ ID NO.35 -s CCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA SEQ ID NO.36 -a GGCGTAACTCAGCAGGCAG SEQ ID NO.37 pGRB--s AGTCCTAGGTATAATACTAGTGCGTGGAAATCATCATGGCTGTTTTAGAGCTAGAA SEQ ID NO.38 pGRB--a TCTAGCTCTAAAACAGCCATGATGATTTCCACGCACTAGTATTATACCTAGGACT SEQ ID NO.39 -up-s GATTTTGACGCCACCAACACC SEQ ID NO.40 -up-a TAAACAAAATTATTTCTAGACCCTATAGTGAGTCGTATTACCAATCCACATCACACAATCCAT SEQ ID NO.41 -s ATAGGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGCTACCAATGCAAAACCCG SEQ ID NO.42 -a CCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTCCGTGATTAAAGTCCCTTCTTTTTCACC SEQ ID NO.43 -s TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTGAAGGGATTTTTGGCTATCAGGA SEQ ID NO.44 -a CATATCGTATTCGCCAGGCTG SEQ ID NO.45 pGRB--s AGTCCTAGGTATAATACTAGTTATCGGCACTGACGCATTTCGTTTTAGAGCTAGAA SEQ ID NO.46 pGRB--a TTCTAGCTCTAAAACGAAATGCGTCAGTGCCGATAACTAGTATTATACCTAGGACT SEQ ID NO.47 -up-s ACCGAGGAGCAGACAATGAATAA SEQ ID NO.48 -up-a AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGA SEQ ID NO.49 -s TATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAACAGAGCCATTTTTTTGCCC SEQ ID NO.50 -a AACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCGGTGAATCCTGGC SEQ ID NO.51 -s AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGT SEQ ID NO.52 -a GCGCTGGCTAACATGAGGAA SEQ ID NO.53 pGRB--s AGTCCTAGGTATAATACTAGTACTGCAGGACGAGCTGCGCAGTTTTAGAGCTAGAA SEQ ID NO.54 pGRB--a TTCTAGCTCTAAAACTGCGCAGCTCGTCCTGCAGTACTAGTATTATACCTAGGACT SEQ ID NO.55 pGRB-4#-s AGTCCTAGGTATAATACTAGTTGCGCTGGTTGATTTCTTCTAGGGTTTTAGAGCTAGAA SEQ ID NO.56 pGRB-4#-a TTCTAGCTCTAAAACCCTAGAAGAAATCAACCAGCGCAACTAGTATTATACCTAGGACT SEQ ID NO.57 pGRB-1#-s AGTCCTAGGTATAATACTAGTATGAACATAACTCAATTTGTAGGGTTTTAGAGCTAGAA SEQ ID NO.58 pGRB-1#-a TTCTAGCTCTAAAACCCTACAAATTGAGTTATGTTCATACTAGTATTATACCTAGGACT SEQ ID NO.59 GGTCAGGAGGTAACTTATCAGCG SEQ ID NO.60 CCACACATTATACGAGCCGGATGATTAATTGTCAAATGGCAGGGCTCCGTTTT SEQ ID NO.61 first-s ATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCCATGAGCACCGTGTTTAACACCAAC SEQ ID NO.62 irst-4#-a CCTAGAAGAAATCAACCAGCGCAGTATCGCGGCCCAGTTTGG SEQ ID NO.63 second-s GGCGCAGATTGATAACGATGTGG SEQ ID NO.64 second-1#-a CCTACAAATTGAGTTATGTTCATAAACTGCACATAAATGCGCGC SEQ ID NO.65 third-s TGGAACGCTTTGGCAAAAACG SEQ ID NO.66 third-a CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGAGCTTTTACAGTTTGCACGCCGCGCAA SEQ ID NO.67 dw-4#-s TGCGCTGGTTGATTTCTTCTAGGGAACTGGATTTTCTTCTGAACCTGT SEQ ID NO.68 dw-1#-s ATGAACATAACTCAATTTGTAGGGAACTGGATTTTCTTCTGAACCTGT SEQ ID NO.69 dw-trc-s TTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAACTGGATTTTCTTCTGAACCTGT SEQ ID NO.70 dw-a ACGATGTCAGCAGCCAGCA SEQ ID NO.71 pGRB--s AGTCCTAGGTATAATACTAGTGCGTGATGTGAATGAGAAAAGTTTTAGAGCTAGAA SEQ ID NO.72 pGRB--a TTCTAGCTCTAAAACTTTTCTCATTCACATCACGCACTAGTATTATACCTAGGACT SEQ ID NO.73 up-s GCCAGCACGAACATAATCCC SEQ ID NO.74 up-a AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACACGGTGGCAGGTTTTGG SEQ ID NO.75 up-1 TCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACCTCTAGTCGAAAAGTCCGT SEQ ID NO.76 -dw-2 TTAAGGAATGGCTGGGATGCAATCTCACCGACGGTGCCACCG SEQ ID NO.77 up-3 GAGATTGCATCCCAGCCATTCCTTAAAGCAGCTCGCCAGGTACG SEQ ID NO.78 dw-4 AACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTAAGGGTGGACACGCAGCT SEQ ID NO.79 dw-s AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC SEQ ID NO.80 dw-a CGGCGTAATCACAAACTGGC SEQ ID NO.81 like Figure 1 As shown, 2'-deoxyuridine engineered strains E.coli The construction method of ur7 is as follows: first, in the starting strain E.coli Deoxyuridine kinase knockout in the genome of W3110 tdk and integrate T7 RNA polymerase; in the genome rph Aspartate aminotransferase gene aspC , using promoter P trc Regulation; in the genome ylbE Glutamine synthetase gene glnA , using promoter P trc Regulation; in the genome ilvG 5'-deoxynucleotidase gene yfbR , using promoter P trc Regulation; in the genome ycdN Urydinate kinase genepyrH , using promoter P T7 Regulation; in the genome yeeP Tandem integration at the locus Escherichia phage T4 ribonucleoside diphosphate reductase gene nrdBCA , and driven by the same promoter P trc Regulation; in the genome mbhA Corynebacterium glutamicum Corynebacterium glutamicum CTP synthase gene pyrG * , using promoter P trc Regulation; finally obtain the transformed target strain E.coli ur7. The specific method is as follows: 1.1 Gene tdk Knockout of T7 RNA polymerase gene pGRB-tdk-s and pGRB-tdk-a were used as primers to construct the pGRB-tdk plasmid. Then tdk-up-s, tdk-up-a, T7-s, T7-a, tdk-dw-s, and tdk-dw-a were used as primers to construct the pGRB-tdk plasmid. E.coli The genome of W3110 was used as a template to obtain the upstream homology arm tdk-up, the downstream homology arm tdk-dw, and the target gene T7 RNA polymerase gene; then the fusion fragment T7::tdk was obtained. Finally, the pGRB-tdk plasmid and the overlapping fragment T7::tdk were electroporated into E.coli W3110 electroporated competent cells; then tdk-up-s and tdk-dw-a were used as identification primers to screen positive transformants and obtain strain ur-1.

[0031] 1.2 Genes aspC Integration pGRB-rph-s and pGRB-rph-a were used as primers to construct the pGRB-rph plasmid. aspC -s, aspC -a, rph-dw-s, rph-dw-a are primers, E.coli The genome of W3110 was used as a template to obtain the upstream homology arm rph-up, the downstream homology arm rph-dw, and the target gene aspC ; and then obtain the fusion fragment rph:: aspC Finally, the pGRB-rph plasmid was combined with the overlapping fragment rph:: aspC The strain was electroporated into the electroporation competent cells of ur-1; rph-up-s and rph-dw-a were used as identification primers to screen positive transformants and obtain strain ur-2.

[0032] 1.3 Genes glnA Integration Using pGRB-ylbE-s and pGRB-ylbE-a as primers, the pGRB-ylbE plasmid was constructed. Then, using ylbE-up-s, ylbE-up-a, glnA-s, glnA-a, ylbE-dw-s, and ylbE-dw-a as primers, E.coli The genome of W3110 was used as a template to obtain the upstream homology arm ylbE-up, the downstream homology arm ylbE-dw, and the target gene glnA; then the fusion fragment ylbE::glnA was obtained. Finally, the pGRB-ylbE plasmid and the overlapping fragment ylbE::glnA were electroporated into the electroporation competent cells of ur-2; ylbE-up-s and ylbE-dw-a were used as identification primers to screen positive transformants and obtain strain ur-3.

[0033] 1.4 Genes pyrH Integration Using pGRB-ycdN-s and pGRB-ycdN-a as primers, pGRB-ycdN plasmid was constructed. pyrH -s, pyrH -a, ycdN-dw-s, ycdN-dw-a were used as primers, with E.coli The genome of W3110 was used as a template to obtain the upstream homology arm ycdN-up, the downstream homology arm ycdN-dw, and the target gene pyrH ; Then obtain the fusion fragment ycdN:: pyrH Finally, the pGRB-ycdN plasmid was combined with the overlapping fragment ycdN:: pyrH The strain was electroporated into the electroporation competent cells of ur-3. Then, ycdN-up-s and ycdN-dw-a were used as identification primers to screen positive transformants and obtain strain ur-4.

[0034] 1.5 Gene yfbR Integration pGRB-ilvG-s and pGRB-ilvG-a were used as primers to construct the pGRB-ilvG plasmid. Then ilvG-up-s, ilvG-up-a, yfbR-s, yfbR-a, ilvG-dw-s, and ilvG-dw-a were used as primers to construct the pGRB-ilvG plasmid. E.coli The genome of W3110 was used as a template to obtain the upstream homology arm ilvG-up, the downstream homology arm ilvG-dw, and the target gene yfbR; then the fusion fragment ilvG::yfbR was obtained. Finally, the pGRB-ilvG plasmid and the overlapping fragment ilvG::yfbR were electroporated into the electroporation competent cells of ur-4; ilvG-up-s and ilvG-dw-a were used as identification primers to screen positive transformants and obtain strain ur-5.

[0035] 1.6 Gene P trc -Segmental integration of T4 nrdBCA 1.6.1 Overlap of the first paragraph Using pGRB-yeeP-s and pGRB-yeeP-a as primers, the pGRB-yeeP plasmid was constructed. Using yeeP-up-s and yeeP-up-trc-a as primers, E.coli The genome of W3110 was used as a template to obtain the upstream homology arm yeeP-up; yeeP-dw-trc-s and yeeP-dw-a were used as primers to E.coli The genome of W3110 was used as a template to obtain the downstream homology arm yeeP-dw-trc; T4 nrdBCA -first-s and T4nrdBCA-first-4#-a were used as primers, and the gene fragment synthesized after codon optimization of the nrdBCA gene in T4 phage was used as a template to obtain the target gene T4 nrdBCA -first; then obtain the fusion fragment yeeP::T4 nrdBCA -first. Finally, the pGRB-yeeP plasmid was combined with the overlapping fragment yeeP::T4 nrdBCA -first electroporated into the electroporation competent cells of ur-5; then yeeP-up-s and yeeP-dw-a were used as identification primers to screen positive transformants to obtain strain ur-6-1.

[0036] 1.6.2 Overlap of the second paragraph Using pGRB-4#-s and pGRB-4#-a as primers, the pGRB-4# plasmid was constructed. Using yeeP-dw-4#-s and yeeP-dw-a as primers, E.coli The genome of W3110 was used as a template to obtain the downstream homology arm yeeP-dw-4#; nrdBCA -second-s and T4nrdBCA-second-1#-a were used as primers, and the gene fragment synthesized after codon optimization of the nrdBCA gene in T4 phage was used as a template to obtain the target gene T4 nrdBCA -second; then obtain the fused fragment T4 nrdBCA -second::yeeP-dw-4#. Finally, the pGRB-4# plasmid was combined with the overlapping fragment T4 nrdBCA -second::yeeP-dw-4# was electroporated into the electroporation competent cells of ur-6-1; then yeeP-up-s and yeeP-dw-a were used as identification primers to screen positive transformants and obtain strain ur-6-2.

[0037] 1.6.3 Overlap of the third paragraph Using pGRB-1#-s and pGRB-1#-a as primers, the pGRB-1# plasmid was constructed. Using yeeP-dw-1#-s and yeeP-dw-a as primers, E.coli The genome of W3110 was used as a template to obtain the downstream homology arm yeeP-dw-1#; nrdBCA -third-s and T4nrdBCA-third-a were used as primers, and the gene fragment synthesized after codon optimization of the nrdBCA gene in T4 phage was used as a template to obtain the target gene T4 nrdBCA -third; then obtain the fusion fragment T4 nrdBCA -third::yeeP-dw-1#. Finally, the pGRB-1# plasmid was combined with the overlapping fragment T4 nrdBCA -third::yeeP-dw-1# was electroporated into the electroporation competent cells of ur-6-2; then yeeP-up-s and yeeP-dw-a were used as identification primers to screen positive transformants and obtain strain ur-6-3.

[0038] 1.7 Genes pyrG * Integration Using pGRB-mbhA-s and pGRB-mbhA-a as primers, the pGRB-mbhA plasmid was constructed. Using mbhA-up-s, mbhA-up-a, mbhA-dw-s, and mbhA-dw-a as primers, E.coli The genome of W3110 was used as a template to obtain the upstream homology arm mbhA-up and the downstream homology arm mbhA-dw; then mbhA-pyrG-up-1, pyrG*-dw-2, pyrG*-up-3, and mbhA-pyrG-dw-4 were used as primers to Corynebacterium glutamicum The genome of pyrG was used as a template to obtain the fragments before and after the mutant gene * -up and pyrG * -dw; then obtain the fusion fragment mbhA::pyrG * Finally, the pGRB-mbhA plasmid was combined with the overlapping fragment mbhA::pyrG *The strain was electroporated into the electroporation competent cells of thy-6-3. Then, mbhA-up-s and mbhA-dw-a were used as identification primers to screen positive transformants and obtain strain ur-7.

[0039] Example 2 The strain ur7 obtained in Example 1 was used to ferment and produce 2'-deoxyuridine in a fermenter, and the specific steps were as follows: (1) Activation of bacterial strains: Inoculate the bacterial solution from the bacterial preservation tube into the slant culture medium for activation and culture for 12 hours; transfer the colonies from the slant culture medium to the eggplant flask culture medium and continue to activate and expand the culture for 10-12 hours at 32°C.

[0040] The slant and eggplant culture medium is: glucose 2g / L, yeast extract powder 5g / L, peptone 10g / L, NaCl 2.5g / L, KH2PO4 1g / L, MgSO4·7H2O 0.2g / L, agar powder 2.5g / L, and the rest is water.

[0041] (2) Seed culture: 25% ammonia solution is added automatically through the fermenter to maintain the pH value at 7.1 during the culture process; the dissolved oxygen is maintained at 30%-50% by adjusting the stirring speed or ventilation volume of the fermenter; the culture temperature is maintained at 34°C, and the inoculation requirement is met when the OD reaches 20; Seed culture medium: yeast extract powder 6g / L, peptone 4g / L, MgSO4·7H2O 1g / L, ammonium sulfate 2g / L, magnesium sulfate 1g / L, VH 1 mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 2 mg / L each, MSG 1 g / L, kanamycin sulfate 50 mg / L, and the rest is water.

[0042] (3) Fermentation culture: Inoculate at a 20% inoculation rate, control the pH at 7.1±0.1, temperature at 34°C, and dissolved oxygen at 30%-50% during the fermentation process; feed 80% glucose solution during the culture process to maintain the carbon source required by the fermentation process, and the fermentation cycle is 40 h; The fermentation medium is: yeast extract powder 6g / L, peptone 2g / L, citric acid 2g / L, MgSO4·7H2O 3g / L, KH2PO46g / L, FeSO4·7H2O 20mg / L, MnSO4 10mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 3 mg / L each, VH 1 mg / L, kanamycin sulfate 50 mg / L, sodium hexametaphosphate 3 g / L, glycine 2 g / L, and the rest is water.

[0043] The yield of 2'-deoxyuridine can reach 7.3 g / L in a fermenter after 40 h of fermentation, which has good industrial application value.

[0044] It can be seen that the genetically engineered bacteria for producing 2'-deoxyuridine provided in the present application can utilize glucose to synthesize 2'-deoxyuridine from scratch, and has the advantages of stable genetic performance, no defects, high production efficiency, etc.

[0045] Unless otherwise defined, all technical and scientific terms used in this application are the same as those generally understood by those skilled in the art to which this application belongs. If there is any inconsistency, the meaning described in the full text of this application or the meaning derived from the content recorded in the full text of this application shall prevail. In addition, the terms used in this description are only for the purpose of describing the embodiments of the present application and are not intended to limit the present application.

[0046] Note that the above are only preferred embodiments of the present application and the technical principles used. Those skilled in the art will understand that the present application is not limited to the specific embodiments described herein, and that various obvious changes, readjustments and substitutions can be made by those skilled in the art without departing from the protection scope of the present application. Therefore, although the present application is described in more detail through the above embodiments, the present application is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the technical concept of the present application, all of which belong to the protection scope of the present application.

Claims

1. A genetically engineered bacterium for producing 2'-deoxyuridine, characterized in that: The genetically engineered bacteria is Escherichia coli Escherichia coli W3110 is the starting strain constructed by genetic modification: exist E. coli Deoxyuridine kinase knockout in the W3110 genome tdk Integrate T7 RNA polymerase and overexpress aspartate aminotransferase gene aspC , glutamine synthetase gene gLn , 5'-deoxynucleotidase gene Yj Y and uridylate kinase gene pyr ; Heterologous tandem introduction from Escherichia phage T4 ribonucleoside diphosphate reductase gene nrdBCA ; Corynebacterium glutamicum Corynebacterium glutamicum CTP synthase gene pyr * .

2. The genetically engineered bacterium according to claim 1, characterized in that: Deoxyuridine kinase gene tdk The nucleotide sequence of the T7 RNA polymerase is shown in SEQ ID NO.1; the nucleotide sequence of the T7 RNA polymerase is shown in SEQ ID NO.

2.

3. The genetically engineered bacterium according to claim 1, characterized in that In the genome r Aspartate aminotransferase gene aspC , and with promoter P trc Regulation; the aspartate aminotransferase gene aspC The nucleotide sequence is shown in SEQ ID NO.3; Preferably, in the genome YlB Glutamine synthetase gene gLn , and with promoter P trc Regulation; The glutamine synthetase gene gLn The nucleotide sequence is shown in SEQ ID NO.4; Preferably, in the genome ilvG 5'-deoxynucleotidase gene Yj Y , and with promoter P trc Regulation; the 5'-deoxynucleotidase gene Yj Y The nucleotide sequence is shown in SEQ ID NO.5; Preferably, in the genome Yj Y Urydinate kinase gene pyr , and with promoter P T7 Regulation; The uridylate kinase gene pyr The nucleotide sequence is shown in SEQ ID NO.

6.

4. The genetically engineered bacterium according to claim 1, characterized in that: In the genome yeeP Tandem integration at the locus Escherichia phage T4 ribonucleoside diphosphate reductase gene nrdBCA , connected by RBS and driven by the same promoter P trc Regulation; Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strain nB The nucleotide sequence is shown in SEQ ID NO.7; Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strain nD The nucleotide sequence is shown in SEQ ID NO.8; Said Escherichia phage Ribonucleoside diphosphate reductase gene of T4 strain nD The nucleotide sequence is shown in SEQ ID NO.9; Preferably, in the genome mbA Corynebacterium glutamicum Corynebacterium glutamicum CTP synthase gene pyr * , using promoter P trc Regulation: The CTP synthase gene of the Corynebacterium glutamicum strain pyr * The nucleotide sequence is shown in SEQ ID NO.

10.

5. The genetically engineered bacterium according to claim 3 or 4, characterized in that: The P trc The nucleotide sequence of the promoter is shown in SEQ ID NO.11; T7 The nucleotide sequence of the promoter is shown in SEQ ID NO.12; the nucleotide sequence of the RBS is shown in SEQ ID NO.

13.

6. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 5, characterized in that: The steps include: (1) By using the starting strain Escherichia coli Deoxyuridine kinase knockout in W3110 genome tdk And integrate T7 RNA polymerase to block the degradation pathway of 2'-deoxyuridine; (2) By r , YlB , ilvG Overexpression at each site aspC , gLn and Yj Y , and use P trc The promoters are regulated respectively to enhance the expression of key enzyme genes in the 2'-deoxyuridine synthesis pathway; (3) Through the genome Yj Y Overexpression at the site pyr , and use P T7 Promoter regulation enhances the metabolic flow of UMP to the target pathway; (4) Through the genome yeeP , mbA ribonucleoside diphosphate reductase gene nrdBCA and CTP synthase gene pyrG* and regulated separately by the Ptrc promoter to strengthen the cytidine diphosphate deoxyreduction pathway, weaken CTP feedback inhibition and strengthen the cytidine triphosphate ribozyme.

7. Use of the genetically engineered bacteria for producing 2'-deoxyuridine according to any one of claims 1 to 5 in the fermentation production of 2'-deoxyuridine.

8. A method for producing 2'-deoxyuridine, characterized in that: The method comprises: fermenting the genetically engineered bacteria for producing 2'-deoxyuridine as claimed in any one of claims 1 to 5 to obtain 2'-deoxyuridine.

9. The method according to claim 8, characterized in that The specific steps for producing 2'-deoxyuridine using a fermentation tank are as follows: (1) Activation of bacterial strains: inoculate the bacterial solution from the bacterial preservation tube to the slant culture medium for activation and culture for 10-14 hours; transfer the colonies from the slant culture medium to the eggplant flask culture medium and continue activation and expansion culture for 10-12 hours at a culture temperature of 30-34°C; (2) Seed culture: 24%~26% ammonia solution is added automatically through the fermenter to maintain the pH value of the culture process at 7.0~7.2; the dissolved oxygen is maintained at 30%~50% by adjusting the stirring speed or ventilation volume of the fermenter; the culture temperature is maintained at 32~36℃, and the inoculation requirements are met when the OD is 18~22; (3) Fermentation culture: Inoculate at an inoculum rate of 18% to 22%. During the fermentation process, the pH was controlled at 7.1±0.1, the temperature was 32 to 36°C, and the dissolved oxygen was 30% to 50%. During the culture process, 80% glucose solution was added to maintain the carbon source required by the fermentation process. The fermentation cycle was 36 to 44 hours.

10. The method according to claim 9, characterized in that The seed culture medium used in step (2) is: yeast extract powder 6g / L, peptone 4g / L, MgSO4·7H2O 1g / L, ammonium sulfate 2g / L, magnesium sulfate 1g / L, VH 1 mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 2 mg / L each, 1 g / L monosodium glutamate, 50 mg / L kanamycin sulfate, and the rest water; the fermentation medium used in step (3) is: 6 g / L yeast extract powder, 2 g / L peptone, 2 g / L citric acid, 3 g / L MgSO4·7H2O, 6 g / L KH2PO4, 20 mg / L FeSO4·7H2O, 10 mg / L MnSO4, Vb 1、 Vb 3、 Vb 5、 Vb 12 3 mg / L each, VH 1 mg / L, kanamycin sulfate 50 mg / L, sodium hexametaphosphate 3 g / L, glycine 2 g / L, and the rest is water.

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