Uracil-producing strain, construction method and application thereof

Abstract

This invention provides a uracil-producing strain, its construction method, and its application. The strain utilizes CRIPSR / Cas9 gene editing technology to produce... E. coli On the UR14 genome, firstly by knocking out psuG Gene, black Gene, rutA Genes and upp Genes were introduced to block the breakdown of uracil; then, the uridine phosphorylase gene was overexpressed. udp and pyrimidine-5'-nucleotide nuclease gene ppnN This synergistically enhances the synthesis and accumulation of uracil; finally, overexpression of the phosphoribosyltransferase gene... pgm, Further enhancing the conversion of the byproduct 1-phosphate ribose into the precursor 5-phosphate ribose-1-pyrophosphate improves carbon utilization. The resulting strain exhibits good genetic stability and high fermentation yield, enabling stable production of uracil and demonstrating broad application prospects.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering and genetic engineering, and in particular to a uracil-producing strain, its construction method, and its application. Background Technology

[0002] Uracil (CAS No.: 66-22-8) is an important pyrimidine base and one of the bases unique to RNA. Uracil participates in RNA synthesis and metabolism in organisms, playing a crucial role in gene expression regulation, protein synthesis, and other vital life activities. In practical applications, uracil is an important raw material for the synthesis of many drugs. Furthermore, uracil can be used to synthesize pyrimidine herbicides, achieving weed control by inhibiting plant photosynthesis.

[0003] Currently, the main methods for producing uracil include chemical synthesis and microbial fermentation. Chemical synthesis involves multiple organic reactions to produce uracil; the process is mature but costly. In contrast, microbial fermentation offers advantages such as simple process, readily available raw materials, low cost, and high safety. Therefore, developing a uracil-producing strain with high acid production efficiency and good genetic stability for uracil production via microbial fermentation is a pressing technical challenge. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a uracil-producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned uracil-producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned uracil-producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] A uracil-producing strain, named strain E.coli Ura9 is a strain that was created using a targeted modification method. E.coli It was obtained through further modifications based on UR14, specifically: E.coli UR14 genome knockout psuG Gene (Gene ID: 946699) preTA Gene( preT Gene ID: 949049 preA Gene ID: 949037) rutA Gene (Gene ID: 945643) and upp Gene (Gene ID: 946979), overexpressing uridine phosphorylase gene udp(Gene ID:948987) ， Pyrimidine-5'-nucleotide nuclease gene ppnN (Gene ID: 947266) and phosphoribosyltransferase gene pgm (Gene ID:945271).

[0009] Preferably, the above-mentioned uracil-producing strain, the starting strain E.coli UR14 is the strain described in CN 202411087442.4 (A uridine-producing strain and its construction method and application). E.coli UR14.

[0010] Preferably, in the above-mentioned uracil-producing strain, the targeted modification method utilizes CRISPR / Cas9 gene editing technology to completely modify the starting strain. E.coli The UR14 chromosome genome was modified.

[0011] Preferably, the above-mentioned uracil-producing strain, in yjiK , ychG , yncK The uridine phosphorylase gene is integrated at each of the three sites. udp and controlled by the TRC promoter; in yeeL Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN and controlled by the TRC promoter; in mbhA Integrating phosphoribosyltransferase gene at the site pgm, It is regulated by the BBa_J23107 promoter.

[0012] Preferably, in the above-mentioned uracil-producing strain, the uridine phosphorylase gene... udp The nucleotide sequence of the trc promoter is shown in SEQ ID NO.1, and the nucleotide sequence of the trc promoter is shown in SEQ ID NO.2; the pyrimidine-5'-nucleotide nuclease gene ppnN The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing; the phosphoribosyltransferase gene pgm The nucleotide sequence of the BBa_J23107 promoter is shown in SEQ ID NO.4 of the sequence listing, and the nucleotide sequence of the BBa_J23107 promoter is shown in SEQ ID NO.5 of the sequence listing.

[0013] Preferably, the above-mentioned uracil-producing strain, wherein psuG The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing; preTA The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing; rutA The nucleotide sequence of the gene is shown in SEQ ID NO.8 of the sequence listing;upp The nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing.

[0014] The specific steps for constructing the above-mentioned uracil-producing strain are as follows:

[0015] (1) Knockout psuG Gene, preTA Gene, rutA Genes and upp Genes that block the breakdown pathway of uracil;

[0016] (2) yjiK , ychG , yncK The uridine phosphorylase gene is integrated at each of the three sites. udp Furthermore, the conversion of uridine to uracil is enhanced by regulation using the TRC promoter;

[0017] (3) In yeeL Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN Furthermore, the conversion of UMP to uracil is enhanced by regulation using the trc promoter;

[0018] (4) In mbhA Integrating phosphoribosyltransferase gene at the site pgm, Furthermore, the BBa_J23107 promoter was used to regulate the conversion of the byproduct 1-phosphoribose to the precursor 5-phosphoribose-1-pyrophosphate, thereby improving carbon utilization.

[0019] The application of the above-mentioned uracil-producing strains in the fermentation production of uracil.

[0020] Preferably, the above-mentioned uracil-producing strain is used to produce uracil via shake-flask fermentation, and the specific steps are as follows:

[0021] (1) Seed activation and culture: The strain was evenly spread on the activation slant and cultured at 37℃ for 12h. It was then transferred to the activation slant and cultured for another 10h. Finally, it was transferred to a shaker containing seed culture medium for seed culture.

[0022] (2) Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 10-15%, and culture at 36℃ and 220r / min with shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; glucose solution is added to maintain the fermentation (fermentation cycle 30-32h).

[0023] Preferably, in the application of the above-mentioned uracil-producing strain, the slant culture medium used in step (1) is: glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.2 g / L, agar powder 25%, the remainder being water, pH 7.0-7.2.

[0024] Preferably, in the application of the above-mentioned uracil-producing strain, the seed culture medium used in step (1) is: yeast extract 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0025] Preferably, in the application of the above-mentioned uracil-producing strain, the fermentation medium used in step (2) is: yeast extract 10 g / L, (NaPO3)6 3.0 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 4.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, phenol red 2%, the remainder being water.

[0026] Preferably, the above-mentioned uracil-producing strain is used to produce uracil via fermentation in a fermenter, and the specific steps are as follows:

[0027] (A) Seed activation: The inoculum is evenly spread on the activation slant, incubated at 37℃ for 12h, and then transferred to a flask for further incubation for 12h.

[0028] (B) Seed culture: Inoculate the bacterial suspension into the seed culture medium, maintain pH 7.0, keep the temperature constant at 36℃, and keep the dissolved oxygen at 30%-60% for 6.5-7 hours;

[0029] (C) Fermentation culture: waiting for the seed cell OD 600 At 20-25°C, inoculate the fermentation medium at a rate of 20% to start fermentation. During fermentation, control the pH at 7.0, the temperature at 36°C, and the dissolved oxygen at 30%-60%. After the glucose in the medium is consumed, add glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L.

[0030] Preferably, in the application of the above-mentioned uracil-producing strain, the slant culture medium used in step (A) is: glucose 2 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.2 g / L, agar powder 25%, the remainder being water, pH 7.0-7.2.

[0031] Preferably, in the application of the above-mentioned uracil-producing strain, the seed culture medium used in step (B) is: glucose 10 g / L, yeast extract 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0032] Preferably, in the application of the above-mentioned uracil-producing strain, the fermentation medium used in step (C) is: glucose 10 g / L, yeast extract 10 g / L, (NaPO3)6 3.0 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 4.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0033] All of the above-mentioned culture media can be prepared using standard methods.

[0034] Beneficial effects:

[0035] The aforementioned uracil-producing strain is plasmid-free, defect-free, requires no induction, and possesses advantages such as good genetic stability and high fermentation yield. It is an excellent strain capable of stably producing uracil. The strain efficiently synthesizes uracil de novo using glucose as a substrate. After 32 hours of shake-flask fermentation, the uracil yield can reach up to 9.8 g / L, and after 48 hours of fermentation in a 5L fermenter, the uracil yield can reach 45.2 g / L, demonstrating broad application prospects. Attached Figure Description

[0036] Figure 1 A diagram illustrating the method for targeted modification of uracil-producing strains.

[0037] Figure 2 For the production strain E.coli Fermentation process curve of Ura9 in a 5L fermenter. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0039] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage, and the percentage of a solution refers to the number of grams of solute contained in 100 mL.

[0040] The starting strain used in the examples was a high-yield uridine strain. E.coli UR14, this strain is the strain described in Example 1 of CN202411087442.4 (A uridine-producing strain and its construction method and application). E.coli UR14; the corresponding promoter and gene are listed in the sequence listing.

[0041] 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 engineered plasmid pGRB involved in this method uses pUC18 as its backbone and includes the promoter J23100, the gRNA-Cas9 binding region sequence, the terminator sequence, and ampicillin resistance (working concentration: 100 mg / L). The technical terms related to gene integration and plasmid construction used in the following examples are explained in the article. Primers used in the strain construction process are shown in Table 1.

[0042] Table 1 Primers used in strain construction

[0043] Primer Sequence number Sequence (5' to 3') psuG-QC-1 SEQ ID NO.10 GTTCTGTGCATCCCGTCACAA psuG-QC-2 SEQ ID NO.11 AGTCGCACTCATCAGGTTGGATGTGAATATCCGGCGACATCAAT psuG-QC-3 SEQ ID NO.12 ATTGATGTCGCCGGATATTCACATCCAACCTGATGAGTGCGACT psuG-QC-4 SEQ ID NO.13 GTGAATCGCCTGGCAGCA pGRB-psuG-S SEQ ID NO.14 AGTCCTAGGTATAATACTAGTCTAACGCAAACCAATCAATCGTTTTAGAGCTAGAA pGRB-psuG-A SEQ ID NO.15 TTCTAGCTCTAAAACGATTGATTGGTTTGCGTTAGACTAGTATTATACCTAGGACT preTA-QC-1 SEQ ID NO.16 GGTGTATGTCTGGCGAGCAA preTA-QC-2 SEQ ID NO.17 GGGTGTTCTTTCTCGCCTTTCAGCGTCGTGACATAATAAACAGC preTA-QC-3 SEQ ID NO.18 \nGCTGTTTATTATGTCACGACGCTGAAAGGCGAGAAAGAACACCC preTA-QC-4 SEQ ID NO.19 ATTCACTGAAATACGCGCGTAAGAA pGRB-preTA-S SEQ ID NO.20 AGTCCTAGGTATAATACTAGTTATGAGAAAGAAGCGCACCCGTTTTAGAGCTAGAA pGRB-preTA-A SEQ ID NO.21 TTCTAGCTCTAAAACGATTGATTGGTTTGCGTTAGACTAGTATTATACCTAGGACT rutA-QC-1 SEQ ID NO.22 TGAAACACCCGCCAACTCTG rutA-QC-2 SEQ ID NO.23 TGCATCAGTGGTTGAATGCGCGTAAAGTGAAAGTCAGACGGGGC rutA-QC-3 SEQ ID NO.24 GCCCCGTCTGACTTTCACTTTACGCGCATTCAACCACTGATGCA rutA-QC-4 SEQ ID NO.25 TCACGGCGGTTTGAATGTTGG pGRB-rutA-S SEQ ID NO.26 AGTCCTAGGTATAATACTAGTCTGTCGATGATGATCAAACTGCGGTTTTAGAGCTAGAA pGRB-rutA-A SEQ ID NO.27 TTCTAGCTCTAAAACCGCAGTTTGATCATCGACAGACTAGTATTATACCTAGGACT upp-QC-1 SEQ ID NO.28 AAGAAGACTTGTGCCAGGGTAA upp-QC-2 SEQ ID NO.29 CCGAGGCCCGGAATAATGTTTGCTCACGCATCAGTCCCA upp-QC-3 SEQ ID NO.30 TGGGACTGATGCGGTGAGCAAACATTATTCCGGCCTCGG upp-QC-4 SEQ ID NO.31 ATGAAATAAGACGGGCACCAGG pGRB-upp-S SEQ ID NO.32 AGTCCTAGGTATAATACTAGTTCCGAAGTGGGTAGCCTGCTGTTTTAGAGCTAGAA pGRB-upp-A SEQ ID NO.33 TTCTAGCTCTAAAACAGCAGGCTACCCACTTCGGAACTAGTATTATACCTAGGACT yjiK-1 SEQ ID NO.34 GAATCTCACCTGCGAATGCC yjiK-trc-2 SEQ ID NO.35 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTTAATTGTCAAACACCATCCTGCTGGCAATAAA yjiK-3 SEQ ID NO.36 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCGATGGATGCCTCTGGT yjiK-4 SEQ ID NO.37 CCTAAGGTCAGAACCAGCATC pGRB-yjiK-S SEQ ID NO.38 AGTCCTAGGTATAATACTAGTTACGGGGTTTCTCCGCGTTAAGGGTTTTAGAGCTAGAA pGRB-yjiK-A SEQ ID NO.39 TTCTAGCTCTAAAACCCTTAACGCGGAAACCCCGTAACTAGTATTATACCTAGGACT udp-trc-S SEQ ID NO.40 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGTCCAAGTCTGATGTTTTTCATCTCG udp-trc-A SEQ ID NO.41 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCAGACGACGCGCCGCTT ychG-1 SEQ ID NO.42 GGGAGCCGTTTTCTTATGCCAC ychG-trc-2 SEQ ID NO.43 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGCGTTCTGCCGCTTAGTG ychG-3 SEQ ID NO.44 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATCAAAAACCCGTTATCCTGTGAAAC ychG-4 SEQ ID NO.45 CTGCGTCTTGATCAAGCAGG pGRB-ychG-S SEQ ID NO.46 AGTCCTAGGTATACTAGTAGGGACCATCCCGAAACGACGTTTTAGAGCTAGAA pGRB-ychG-A SEQ ID NO.47 TTCTAGCTCTAAAACGTCGTTTCGGGATGGTCCCTACTAGTATTATACCTAGGACT yncK-1 SEQ ID NO.48 CGCAGCGTAAACCACTGGTTAC yncK-trc-2 SEQ ID NO.49 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCCCTGAAGGATGGGGTTTTAC yncK-3 SEQ ID NO.50 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCATCAGGAAAACACAATGCCTTC yncK-4 SEQ ID NO.51 CATTTTTCACAACACCCGTGACCT pGRB-yncK-S SEQ ID NO.52 AGTCCTAGGTATAATACTAGTATCGACCAGATATTGCATTCGTTTTAGAGCTAGAA pGRB-yncK-A SEQ ID NO.53 TTCTAGCTCTAAAACGAATGCAATATCTGGTCGATACTAGTATTATACCTAGGACT yeeL-1 SEQ ID NO.54 TTCATCGGGACGAGTGGAGA yeeL-trc-2 SEQ ID NO.55 TATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCACCATAGCATCGCCAATCTGA yeeL-3 SEQ ID NO.56 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATAAAGC yeeL-4 SEQ ID NO.57 CATTCCCTCTACAGAACTAGCCCT pGRB-yeeL-A SEQ ID NO.58 AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA pGRB-yeeL-S SEQ ID NO.59 TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT ppnN-trc-S SEQ ID NO.60 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGATATGTTGTCGCAGCTGG ppnN-trc-A SEQ ID NO.61 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACGTGCAGATTTCGTAGCAAGGG mbhA-1 SEQ ID NO.62 GCCAGCACGAACATAATCCC mbhA-J23107-2 SEQ ID NO.63 GCTAGCATAATACCTAGGGCTGAGCTAGCCGTAAACACGGTGGCAGGTTTTGG mbhA-3 SEQ ID NO.64 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-4 SEQ ID NO.65 CGGCGTAATCACAAACTGGC pGRB-mbhA-S SEQ ID NO.66 AGTCCTAGGTATAATACTAGTTACCGGGCATACCGATGCGAGTTTTAGAGCTAGAA pGRB-mbhA-A SEQ ID NO.67 TTCTAGCTCTAAAACTCGCATCGGTATGCCCGGTAACTAGTATTATACCTAGGACT pgm-J23107-S SEQ ID NO.68 TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCATGGCAATCCACAATCGTGCAG pgm-J23107-A SEQ ID NO.69 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACGCGTTTTTCAGAACTTCGCTAA

[0044] Example 1

[0045] like Figure 1 As shown, the specific process for constructing genetically engineered strains is as follows:

[0046] E. coli UR14 / pRed-Cas9 electrocompetent cells were prepared using the starting strain E. coli UR14 according to the procedures mentioned in CN202411087442.4.

[0047] 1.1 Genes psuG Knockout

[0048] Using the *E. coli* W3110 genome as a template, PCR amplification was performed using primers psuG-QC-1 and psuG-QC-2, psuG-QC-3 and psuG-QC-4, respectively, to obtain the upstream homologous arm psuG-QC-UP and the downstream homologous arm psuG-QC-DW. Using the recovered upstream and downstream homologous arms as templates, overlap PCR was performed using primers psuG-QC-1 and psuG-QC-4 to obtain the complement fragment ΔpsuG required for the psuG gene knockout. Subsequently, the DNA fragment obtained by annealing primers pGRB-psuG-S and pGRB-psuG-A was ligated to plasmid pGRB to construct the pGRB-psuG plasmid. Finally, plasmid pGRB-psuG and the overlap fragment ΔpsuG were electroporated to… E.coli UR14 / pRed-Cas9 electrocompetent cells were used; positive transformants were screened using psuG-QC-1 and psuG-QC-4 primers to obtain the strain. E.coli Ura1.

[0049] 1.2 Genes preTA Knockout

[0050] Using the *E. coli* W3110 genome as a template, PCR amplification was performed using primers preTA-QC-1 and preTA-QC-2, preTA-QC-3 and preTA-QC-4, respectively, to obtain the upstream homologous arm preTA-QC-UP and the downstream homologous arm preTA-QC-DW. Using the recovered upstream and downstream homologous arms as templates, overlap PCR was performed using primers preTA-QC-1 and preTA-QC-4 to obtain the complement fragment ΔpreTA required for the knockout gene preTA. Subsequently, the DNA fragment obtained by annealing primers pGRB-preTA-S and pGRB-preTA-A was ligated to plasmid pGRB to construct the pGRB-preTA plasmid. Finally, the pGRB-preTA plasmid and the overlap fragment ΔpreTA were electroporated to…E.coli The strain was obtained by electrocompetentizing Ura1 / pRed-Cas9 cells; positive transformants were then screened using preTA-QC-1 and preTA-QC-4 primers. E.coli Ura2.

[0051] 1.3 Genes rutA Knockout

[0052] Using the *E. coli* W3110 genome as a template, PCR amplification was performed using primers rutA-QC-1 and rutA-QC-2, rutA-QC-3 and rutA-QC-4, respectively, to obtain the upstream homologous arm rutA-QC-UP and the downstream homologous arm rutA-QC-DW. Using the recovered upstream and downstream homologous arms as templates, overlap PCR was performed using primers rutA-QC-1 and rutA-QC-4 to obtain the complement fragment ΔrutA required for the rutA gene knockout. Subsequently, the DNA fragment obtained by annealing primers pGRB-rutA-S and pGRB-rutA-A was ligated to plasmid pGRB to construct the pGRB-rutA plasmid. Finally, the pGRB-rutA plasmid and the overlap fragment ΔrutA were electroporated into… E.coli The strain was obtained by electrocompetentizing Ura2 / pRed-Cas9 cells; positive transformants were screened using rutA-QC-1 and rutA-QC-4 primers. E.coli Ura3.

[0053] 1.4 Genes upp Knockout

[0054] Using the *E. coli* W3110 genome as a template, PCR amplification was performed using primers upp-QC-1 and upp-QC-2, upp-QC-3 and upp-QC-4, respectively, to obtain the upstream homologous arm upp-QC-UP and the downstream homologous arm upp-QC-DW. Using the recovered upstream and downstream homologous arms as templates, overlap PCR was performed using primers upp-QC-1 and upp-QC-4 to obtain the complement fragment Δupp required for the knockout gene upp. Subsequently, the DNA fragment obtained by annealing primers pGRB-upp-S and pGRB-upp-A was ligated to plasmid pGRB to construct the pGRB-upp plasmid. Finally, the pGRB-upp plasmid and the overlap fragment Δupp were electroporated to… E.coli The strain was obtained by electrocompetentizing Ura3 / pRed-Cas9 cells; positive transformants were then screened using upp-QC-1 and upp-QC-4 primers. E.coli Ura4.

[0055] 1.5 genes udp Integration ( yjiK::Ptrc- udp (integration)

[0056] Using *E. coli* W3110 as a template, PCR amplification was performed using primers yjiK-1 and yjiK-trc-2, yjiK-3 and yjiK-4, and udp-trc-S and udp-trc-A to obtain the upper homologous arm yjiK-trc-UP, the lower homologous arm yjiK-DW, and the intermediate target fragment trc-udp. Using the recovered upper and lower homologous arms and the intermediate target fragment as templates, overlap PCR was performed using primers yjiK-1 and yjiK-4 to obtain the target fragment yjiK-Ptrc-udp for integration. Then, the DNA fragment obtained by annealing primers pGRB-yjiK-S and pGRB-yjiK-A was ligated to plasmid pGRB to construct the pGRB-yjiK plasmid. Finally, the purified yjiK-Ptrc-udp integration fragment and plasmid pGRB-yjiK were simultaneously transformed into the target fragment via electroporation. E.coli In Ura4 / pRed-Cas9 competent cells, positive transformants were screened using primers yjiK-1 and yjiK-4 for identification, and the strain was finally obtained. E.coli Ura5.

[0057] 1.6 Genes udp Second copy ( ychG ::Ptrc- udp (integration)

[0058] Using *E. coli* W3110 as a template, PCR amplification was performed using primers ychG-1 and ychG-trc-2, ychG-3 and ychG-4 to obtain the upper homologous arm ychG-trc-UP and the lower homologous arm ychG-DW. Using the recovered upper and lower homologous arms and trc-udp as templates, overlap PCR was performed using primers ychG-1 and ychG-4 to obtain the target fragment ychG-Ptrc-udp for integration. Then, the DNA fragment obtained by annealing primers pGRB-ychG-S and pGRB-ychG-A was ligated to plasmid pGRB to construct the pGRB-ychG plasmid. Finally, the purified ychG-Ptrc-udp integration fragment and plasmid pGRB-ychG were simultaneously transformed into the target fragment via electroporation. E.coli In Ura5 / pRed-Cas9 competent cells, positive transformants were screened using primers ychG-1 and ychG-4 for identification, and the strain was finally obtained. E.coli Ura6.

[0059] 1.7 Genes udp Three copies ( yncK ::Ptrc- udp (integration)

[0060] Using *E. coli* W3110 as a template, PCR amplification was performed using primers yncK-1 and yncK-trc-2, yncK-3 and yncK-4 to obtain the upper homologous arm yncK-trc-UP and the lower homologous arm yncK-DW. Using the recovered upper and lower homologous arms and trc-udp as templates, overlap PCR was performed using primers yncK-1 and yncK-4 to obtain the target fragment yncK-Ptrc-udp for integration. Then, the DNA fragment obtained by annealing primers pGRB-yncK-S and pGRB-yncK-A was ligated to plasmid pGRB to construct the pGRB-yncK plasmid. Finally, the purified yncK-Ptrc-udp integration fragment and plasmid pGRB-yncK were simultaneously transformed into the target fragment via electroporation. E.coli In Ura6 / pRed-Cas9 competent cells, positive transformants were screened using primers yncK-1 and yncK-4 for identification, and the strain was finally obtained. E.coli Ura7.

[0061] 1.8 Genes ppnN Integration ( yeeL ::P trc - ppnN (integration)

[0062] Using the Escherichia coli W3110 genome as a template, primers yeeL-1 and yeeL-trc-2, yeeL-3 and yeeL-4 were used. ppnN -trc-S and ppnN PCR amplification was performed using -trc-A to obtain the upper homologous arm yeeL-trc-UP, the lower homologous arm yeeL-DW, and the intermediate target fragment trc- ppnN Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, the target fragment yeeL-Ptrc- required for integration was obtained by overlap PCR with primers yeeL-1 and yeeL-4. ppnN Next, the DNA fragments obtained by annealing primers pGRB-yeeL-S and pGRB-yeeL-A were ligated to plasmid pGRB to construct the pGRB-yeeL plasmid. Finally, the purified yeeL-P trc - ppnN The integrated fragment and plasmid pGRB-yeeL were simultaneously transferred via electroporation. E.coli In Ura7 / pRed-Cas9 competent cells, positive transformants were screened using primers yeeL-1 and yeeL-4 as identification primers, and the strain was finally obtained. E.coli Ura8.

[0063] 1.9 Genes pgmIntegration ( mbhA ::P J23107 - pgm (integration)

[0064] Using the Escherichia coli W3110 genome as a template, primers mbhA-1 and mbhA-J23107-2, mbhA-3 and mbhA-4 were used. pgm -J23107-S and pgm PCR amplification of J23107-A yielded the upper homologous arm mbhA-J23107-UP, the lower homologous arm mbhA-DW, and the intermediate target fragment J23107-. ppnN Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, the target fragment mbhA-PJ23107- was obtained by overlap PCR with primers mbhA-1 and mbhA-4. ppnN Next, the DNA fragments obtained by annealing primers pGRB-mbhA-S and pGRB-mbhA-A were ligated to plasmid pGRB to construct the pGRB-mbhA plasmid. Finally, the purified mbhA-P... J23107 - ppnN The integrated fragment and plasmid pGRB-mbhA were simultaneously transferred via electroconversion. E.coli In Ura8 / pRed-Cas9 competent cells, positive transformants were screened using primers mbhA-1 and mbhA-4 as identification primers, and the strain was finally obtained. E.coli Ura9.

[0065] The strains involved in the above construction process are shown in Table 2.

[0066] Table 2 lists the strains involved.

[0067]

[0068] Example 2

[0069] Using the uracil-producing strain described in Example 1 E.coli Ura9 is used in shake-flask fermentation to produce uracil.

[0070] 2.1 Culture medium

[0071] 2.1.1 Slant Culture Medium

[0072] Glucose 2g / L, peptone 10g / L, yeast powder 5g / L, sodium chloride 2.5g / L, KH2PO4 1.0g / L, MgSO4·7H2O 0.2g / L, agar powder 25%, dissolved in water, then adjusted to pH 7.0-7.2 with sodium hydroxide, and brought to a final volume of 500ml. The solution was dispensed into test tubes (9ml / tube) and flasks (45ml / flask), and sterilized in a high-pressure steam autoclave at 121℃ for 20min.

[0073] 2.1.2 Seed Culture Medium

[0074] Yeast powder 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0075] 2.1.3 Fermentation medium

[0076] Yeast powder 10 g / L, (NaPO3)6 3.0 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 4.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, phenol red 2%, the remainder being water.

[0077] 2.2 Cultivation Methods

[0078] 2.2.1 Seed activation and culture:

[0079] The bacterial strain was evenly spread on the activation slant and incubated at 37°C for 12 h. The strain was then transferred to the activation slant and incubated for another 10 h. Finally, the strain was transferred to a shaker containing 5 ml of seed culture medium for seed culture.

[0080] 2.2.2 Fermentation culture:

[0081] Inoculate the seed culture at a rate of 15% into a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL). Seal the flask with nine layers of gauze and incubate at 36°C with shaking at 220 rpm. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia. Add 60% (m / v) glucose solution to maintain fermentation (using phenol red as an indicator; when the fermentation broth color no longer changes, it indicates a sugar deficiency; add 1-2 mL of 60% (m / v) glucose solution if a sugar deficiency is detected). The fermentation cycle is 32 h. No antibiotics or inducers are added during fermentation.

[0082] After 32 hours of shake-flask fermentation, the yield of uridine can reach as high as 9.8 g / L.

[0083] Example 3

[0084] Using the uracil-producing strain described in Example 1 E.coli Ura9 was used to produce uracil via fermentation in a 5 L fermenter.

[0085] 3.1 Culture medium

[0086] 3.1.1 Slant Culture Medium

[0087] Glucose 2g / L, peptone 10g / L, yeast powder 5g / L, sodium chloride 2.5g / L, KH2PO4 1.0g / L, MgSO4·7H2O 0.2g / L, agar powder 25%, dissolved in water, then adjusted to pH 7.0-7.2 with sodium hydroxide, and brought to a final volume of 500ml. The solution was dispensed into test tubes (9ml / tube) and flasks (45ml / flask), and sterilized in a high-pressure steam autoclave at 121℃ for 20min.

[0088] 3.1.2 Seed Culture Medium

[0089] Glucose 10 g / L, yeast extract 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0090] 3.1.3 Fermentation medium

[0091] Glucose 10 g / L, yeast powder 10 g / L, (NaPO3)6 3.0 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 4.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0092] 3.2 Cultivation Methods

[0093] 3.2.1 Seed activation: The inoculum was evenly spread on the activation slant and incubated at 37℃ for 12 h, then transferred to a flask and incubated for another 12 h;

[0094] 3.2.2 Seed culture: The bacterial suspension was inoculated into the seed culture medium, pH 7.0, temperature constant at 36℃, dissolved oxygen at 30%-60%, and cultured for 7 h;

[0095] 3.2.3 Fermentation culture: Wait until the seed cell count reaches OD600 to 22, then inoculate the fermentation medium at a 20% inoculum rate to begin fermentation. During fermentation, maintain pH 7.0, temperature 36℃, and dissolved oxygen at 30%-60%. After the glucose in the medium is consumed, add an 80% glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L. Do not add antibiotics or inducers during fermentation.

[0096] like Figure 2 As shown, after 48 hours of fermentation, the yield of uracil can reach as high as 45.2 g / L.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A uracil-producing strain, characterized in that: It was obtained using a targeted modification method, specifically: in E. coli UR14 genome knockout psuG Gene, preTA Gene, rutA Genes and upp Gene, overexpression of uridine phosphorylase gene UDP And regulated by the TRC promoter; overexpression of the pyrimidine-5'-nucleotide nuclease gene ppnN And regulated by the trc promoter; overexpression of the phosphoribosyltransferase gene pgm And it is controlled by the BBa_J23107 starter, wherein the... psuG The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing; preTA The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing; rutA The nucleotide sequence of the gene is shown in SEQ ID NO.8 of the sequence listing; upp The nucleotide sequence of the gene is shown in SEQ ID NO. 9 of the sequence listing, uridine phosphorylase gene. UDP The nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing, pyrimidine-5'-nucleotide nuclease gene. ppnN The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing, phosphoribosyltransferase gene. pgm The nucleotide sequence of the trc promoter is shown in SEQ ID NO.4; the nucleotide sequence of the BBa_J23107 promoter is shown in SEQ ID NO.2; and the nucleotide sequence of the BBa_J23107 promoter is shown in SEQ ID NO.

5.

2. The uracil-producing strain according to claim 1, characterized in that: exist yjiK , ychG , yncK The uridine phosphorylase gene is integrated at each of the three sites. UDP and controlled by the TRC promoter; in yeeL Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN and controlled by the TRC promoter; in mbhA Integrating phosphoribosyltransferase gene at the site pgm, It is regulated by the BBa_J23107 promoter.

3. The method for constructing the uracil-producing strain according to claim 1 or 2, characterized in that: The specific steps are as follows: (1) Knockout psuG Gene, preTA Gene, rutA Genes and upp Gene; (2) yjiK , ychG , yncK The uridine phosphorylase gene is integrated at each of the three sites. UDP And it is regulated by the TRC promoter; (3) In yeeL Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN And it is regulated by the TRC promoter; (4) In mbhA Integrating phosphoribosyltransferase gene at the site pgm, It is regulated by the BBa_J23107 promoter.

4. The use of the uracil-producing strain according to claim 1 or 2 in the fermentation production of uracil.

5. The application according to claim 4, characterized in that: The specific steps for producing uracil using shake-flask fermentation are as follows: (1) Seed activation and culture: After the strain is evenly spread on the activation slant for culture, it is transferred to the activation slant for further culture, and then transferred to a shaker containing seed culture medium for seed culture. (2) Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 10-15% and shake culture. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; glucose solution is added to maintain the fermentation.

6. The application according to claim 5, characterized in that: The slant culture medium used in step (1) is: glucose 2 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.2 g / L, agar powder 25%, the remainder being water, pH 7.0-7.2; the seed culture medium used in step (1) is: yeast extract 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water; the fermentation medium used in step (2) is: yeast powder 10 g / L, (NaPO3)6 3.0 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 4.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, phenol red 2%, the remainder being water.

7. The application according to claim 4, characterized in that: The specific steps for producing uracil using a fermenter are as follows: (A) Seed activation: After the inoculum is evenly spread on the activation slant and cultured, it is transferred to a flask for further culture; (B) Seed culture: The bacterial suspension was inoculated into the seed culture medium and cultured at pH 7.0, constant temperature of 36℃, and dissolved oxygen of 30%-60%. (C) Fermentation culture: waiting for the seed cell OD 600 At 20-25°C, inoculate with fermentation medium to start fermentation. During fermentation, control the pH at 7.0, the temperature at 36°C, and the dissolved oxygen at 30%-60%. After the glucose in the medium is consumed, add glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L.

8. The application according to claim 7, characterized in that: The slant culture medium used in step (A) is as follows: glucose 2 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.2 g / L, agar powder 25%, the remainder being water, pH 7.0-7.2; the seed culture medium used in step (B) is as follows: glucose 10 g / L, yeast extract 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water; the fermentation medium used in step (C) is: glucose 10 g / L, yeast extract 10 g / L, (NaPO3)6 3.0 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 4.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

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