Uracil-producing strain, construction method and application thereof

Abstract

This application relates to the field of metabolic engineering and genetic engineering technology for production, specifically providing a uracil-producing strain, its construction method, and its application. This strain is... E. coli Knockout of the uridine kinase gene in the W3110 genome udk Simultaneously integrates the T7 RNA polymerase gene; weakens the uridine kinase gene. pyrH Overexpression of carbamoyl phosphate synthase gene carAB ribose-phosphokinase gene prsA pyrimidine-5'-nucleotide nuclease gene ppnN UMP phosphohydrolase gene umpH and umpG Ribonucleotide hydrolase 1 gene rihA and glutamine synthase gene glnA Heterogeneous introduction of wild-type Bacillus subtilis B. subtilis 168 orotate nucleoside monophosphate decarboxylase gene pyrF and orotic acid ribosyltransferase gene pyrE To achieve safe, efficient, and low-cost production of uracil.

Description

Technical Field

[0001] This application relates to the field of metabolic engineering and genetic engineering technology production, specifically providing a uracil-producing strain, its construction method, and its application. Background Technology

[0002] Uracil is a base unique to RNA and is one of the basic building blocks for the formation of RNA and vitamin B3 (an important cofactor in the metabolism of terrestrial life). Moreover, in the pharmaceutical field, drugs synthesized from uracil have wide applications. For example, synthesized 5-fluorouracil and its derivatives have shown good clinical efficacy against various cancers. In agriculture, uracil can be used to synthesize certain pyrimidine herbicides, such as cyclohexidine, chlorpyrifos, and terbufos, which achieve weed control by inhibiting photosynthesis.

[0003] Currently, the main method for industrial production of uracil is the chemical method. However, the chemical method suffers from drawbacks such as the use of hazardous reagents, cumbersome operations, and high raw material costs. Microbial fermentation, on the other hand, offers advantages such as low pollution levels, high production efficiency, and low cost. Furthermore, with the rapid development of synthetic biology, more and more researchers are gradually replacing chemical methods by constructing microbial cell factories to ferment and produce many natural products and chemicals. Therefore, constructing a uracil-producing strain with high efficiency and good genetic stability is a pressing issue that needs to be addressed.

[0004] In view of the above, this application is hereby submitted. Summary of the Invention

[0005] One of the purposes of this application is to provide a uracil-producing strain, its construction method, and its application, so as to realize the industrial production of uracil by microbial fermentation.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] A uracil-producing strain, the uracil-producing strain being *Escherichia coli* Escherichia coli W3110 was constructed using a directional modification method from the starting strain.

[0008] exist E.coli On the W3110 genome, knocking out the uridine kinase gene udk Simultaneously integrates the T7 RNA polymerase gene; weakens the uridine kinase gene. pyrH Overexpression of carbamoyl phosphate synthase gene carAB ribose-phosphokinase gene prsA pyrimidine-5'-nucleotide nuclease gene ppnN UMP phosphohydrolase gene umpH and umpG Ribonucleotide hydrolase 1 gene rihAand glutamine synthase gene glnA Heterogeneous introduction of wild-type Bacillus subtilis B.subtilis 168 orotate nucleoside monophosphate decarboxylase gene pyrF and orotic acid ribosyltransferase gene pyrE .

[0009] In some embodiments, the targeted modification method utilizes CRISPR / Cas9 gene editing technology to completely modify *E. coli*. Escherichia coli Modifications were made to the genome of chromosome W3110.

[0010] In some implementations, the uridine kinase gene is knocked out. udk Based on this, the T7 RNA polymerase gene is integrated and promoted using the promoter P xylF Regulation; the aforementioned udk The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the nucleotide sequence of the T7 RNA polymerase gene is shown in SEQ ID NO.2. The promoter P xylF The nucleotide sequence is shown in SEQ ID NO.3.

[0011] In some implementations, an artificial promoter P is used. j23109 Replacement of uridine kinase gene pyrH The natural promoter of the uridine kinase gene; pyrH The nucleotide sequence is shown in SEQ ID NO.4, and the promoter P... j23109 The nucleotide sequence of the uridine kinase gene is shown in SEQ ID NO.5. pyrH The nucleotide sequence of the natural promoter is shown in SEQ ID NO. 6.

[0012] In some implementations, in yciQ Integrating carbamoyl phosphate synthase gene at the site carAB and with promoter P trc Regulation; the carbamoyl phosphate synthase gene carAB The nucleotide sequence is shown in SEQ ID NO.13; the promoter P trc The nucleotide sequence is shown in SEQ ID NO.11.

[0013] In some implementations, in ylbE Integrating ribose-phosphokinase gene at the site prsA and with promoter P trc Regulation; the ribose-phospho-pyrophosphate kinase gene prsA The nucleotide sequence is shown in SEQ ID NO.14.

[0014] In some implementations, inyghX Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN and with promoter P T7 Regulation; the pyrimidine-5'-nucleotide nuclease gene ppnN The nucleotide sequence is shown in SEQ ID NO.7; promoter P T7 The nucleotide sequence is shown in SEQ ID NO.8.

[0015] In some implementations, in ygaY UMP phosphatase gene integrated at the site umpH and with promoter P T7 Regulation; the UMP phosphohydrolase gene umpH The nucleotide sequence is shown in SEQ ID NO.9.

[0016] In some implementations, in yeeP UMP phosphatase gene integrated at the site umpG and with promoter P trc Regulation; the UMP phosphohydrolase gene umpG The nucleotide sequence is shown in SEQ ID NO.10.

[0017] In some implementations, in yjiT Ribonucleotide hydrolase 1 gene integrated at the site rihA and with promoter P T7 Regulation; the ribonucleoside hydrolase 1 gene rihA The nucleotide sequence is shown in SEQ ID NO.12.

[0018] In some implementations, in gapC Integrating glutamine synthase gene at the site glnA and with promoter P trc Regulation; the glutamine synthase gene glnA The nucleotide sequence is shown in SEQ ID NO.15.

[0019] In some implementations, in ilvG wild-type Bacillus subtilis integrated at the site B.subtilis 168 orotate nucleoside monophosphate decarboxylase gene pyrF and orotic acid ribosyltransferase gene pyrE and with promoter P trc Regulation; the orotate nucleoside monophosphate decarboxylase gene pyrF The nucleotide sequence is shown in SEQ ID NO.16, and the orotic acid ribosyltransferase gene is described. pyrE The nucleotide sequence is shown in SEQ ID NO.17.

[0020] The method for constructing uracil-producing strains includes the following steps:

[0021] (a) in Escherichia coli Escherichia coli On the W3110 genome, knocking out the uridine kinase gene udk Simultaneously integrates the T7 RNA polymerase gene, via promoter P xylF Regulation;

[0022] (b) Using an artificial promoter P j23109 Replace the natural promoter of the uridine kinase gene pyrH;

[0023] (c) in yciQ Integrating carbamoyl phosphate synthase gene at the site carAB By promoter P trc Regulation;

[0024] (d) in ilvG wild-type Bacillus subtilis integrated at the site B.subtilis 168 orotic acid nucleoside monophosphate decarboxylase gene pyrF and orotic acid ribosyltransferase gene pyrE By promoter P trc Regulation;

[0025] (e) in ylbE Integrating ribose-phosphokinase gene at the site prsA By promoter P trc Regulation;

[0026] (f) in yghX Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN By promoter P T7 Regulation;

[0027] (g) in ygaY UMP phosphatase gene integrated at the site umpH By promoter P T7 Regulation;

[0028] (h) in yeeP UMP phosphatase gene integrated at the site umpG By promoter P trc Regulation;

[0029] (i) in yjiT Ribonucleotide hydrolase 1 gene integrated at the site rihA By promoter P T7 Regulation;

[0030] (j) in gapC Integrating glutamine synthase gene at the site glnABy promoter P trc Regulation.

[0031] Application of uracil-producing strains in the fermentation production of uracil.

[0032] A method for producing uracil, the method comprising: fermenting the above-mentioned uracil-producing strain to obtain uracil.

[0033] In some implementations, uracil is produced by fermentation in a fermenter, with the following specific steps:

[0034] (1) Seed activation: The strain is evenly spread on the activation slant and incubated at 36~38℃ for 10~14 h, then transferred to a flask and incubated for another 10~14 h;

[0035] (2) Seed culture: Inoculate the bacterial suspension into the seed culture medium, pH 6.9~7.1, temperature constant at 35~37℃, dissolved oxygen at 30%-60%, and culture for 6.5~7h;

[0036] (3) Fermentation culture: wait for the OD of the seed cell mass 600 At 15-20°C, inoculate the fermentation medium at a 30% inoculation rate to begin fermentation. During fermentation, control the pH at 6.9-7.1, the temperature at 35-37°C, and the dissolved oxygen at 30%-60%. After the glucose in the medium is consumed, add a 78%-82% glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L. When the OD600 of the cell count reaches 20-25 and 40-45, add 9-11 g / (L fermentation broth) of sterilized xylose for induction.

[0037] In some embodiments, the slant culture medium used in the seed activation is: glucose 2 g / L, peptone 10 g / L, yeast extract 5 g / L, NaCl 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, the remainder being water, pH 7.0-7.2; the seed culture medium used in the seed culture is: glucose 30 g / L, yeast extract 8 g / L, peptone 2.0 g / L, (NH4)2SO4 4.0 g / L, KH2PO4 3.0 g / L, V B1 V B3 V B5 V B12 1 mg / L each, V H1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.35 mg / L, boric acid 3.5 mg / L, CoCl2·6H2O 1.5 mg / L, the remainder being water; the fermentation medium used in the fermentation culture was: glucose 10 g / L, yeast extract 10 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 6.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 60 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.35 mg / L, boric acid 3.5 mg / L, CoCl2·6H2O 1.5 mg / L, the remainder being water.

[0038] All of the above-mentioned culture media can be prepared using standard methods.

[0039] Compared with the prior art, the technical effects of this application are as follows:

[0040] The uracil-producing strain described in this application, by knocking out the uridine kinase gene... udk Weakened uridine kinase gene pyrH This reduces carbon loss while maintaining bacterial growth; further, it involves overexpressing the carbamoyl phosphate synthase gene. carAB pyrimidine-5'-nucleotide nuclease gene ppnN UMP phosphohydrolase gene umpH and umpG and ribonucleoside hydrolase 1 gene rihA and heterologous introduction of wild-type Bacillus subtilis B.subtilis 168 orotic acid nucleoside monophosphate decarboxylase and orotic acid ribosyltransferase genes pyrFE Unblock the de novo synthesis pathway of uracil; finally, overexpress the ribose-phosphoric acid pyrophosphate kinase gene. prsA and glutamine synthase gene glnA This strain enhances the supply of precursors ribophosphate pyrophosphate (PRPP) and glutamine, synergistically achieving rapid accumulation of uracil. Lacking plasmids, this strain exhibits good genetic stability and high fermentation yield. No antibiotics or defective substances are required during production, effectively reducing production costs. Using inexpensive glucose as a substrate, this strain efficiently synthesizes uracil de novo. After 48 hours of fermentation in a 5 L fermenter, the uracil yield can reach as high as 35.8 g / L, demonstrating significant potential for industrial application. Attached Figure Description

[0041] Figure 1This is a diagram illustrating the metabolic pathway of a directional modification method for uracil-producing strains;

[0042] Figure 2 Genetically engineered strain E.coli Diagram of ura10 genome modification;

[0043] Figure 3 Genetically engineered strain E.coli ura10 fermentation process curve. Detailed Implementation

[0044] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0045] The uracil-producing strain provided in this application is an Escherichia coli strain produced using a targeted modification method. Escherichia coli This *E. coli* was obtained through further modification based on W3110. Escherichia coli W3110 (Escherichia coli) E.coli W3110 or wild-type Escherichia coli E.coli W3110) Collection number ATCC 27325.

[0046] The specific construction content includes: knocking out the uridine kinase gene. udk Simultaneously integrates the T7 RNA polymerase gene; weakens the uridine kinase gene. pyrH Overexpression of carbamoyl phosphate synthase gene carAB ribose-phosphokinase gene prsA pyrimidine-5'-nucleotide nuclease gene ppnN UMP phosphohydrolase gene umpH and umpG Ribonucleotide hydrolase 1 gene rihA and glutamine synthase gene glnA Heterogeneous introduction of wild-type Bacillus subtilis B.subtilis 168 orotate nucleoside monophosphate decarboxylase gene pyrF and orotic acid ribosyltransferase gene pyrE .

[0047] It should be noted that this application involves knocking out the uridine kinase gene. udk Weakened uridine kinase gene pyrH This reduces carbon loss while maintaining bacterial growth; further, it involves overexpressing the carbamoyl phosphate synthase gene. carAB pyrimidine-5'-nucleotide nuclease gene ppnN UMP phosphohydrolase gene umpH and umpGand ribonucleoside hydrolase 1 gene rihA and heterologous introduction of wild-type Bacillus subtilis B.subtilis 168 orotic acid nucleoside monophosphate decarboxylase and orotic acid ribosyltransferase genes pyrFE Unblock the de novo synthesis pathway of uracil; finally, overexpress the ribose-phosphoric acid pyrophosphate kinase gene. prsA and glutamine synthase gene glnA This process enhances the supply of precursors ribophosphate pyrophosphate (PRPP) and glutamine, synergistically achieving rapid accumulation of uracil. Therefore, any strain that can achieve the above-mentioned functions can be considered a uracil-producing strain as described in this application. Furthermore, those skilled in the art can utilize conventional experimental design and techniques to achieve the above functions and obtain uracil-producing strains.

[0048] In some implementations, the targeted modification method employs CRISPR / Cas9 gene editing technology. This method is performed entirely and directly on the genome of the starting strain, resulting in a strain that is plasmid-free, exhibits good genetic stability, high fermentation yield, and eliminates the need for antibiotics and defective substances during production, effectively reducing production costs.

[0049] In some implementations, the uridine kinase gene is knocked out. udk The specific sequence is shown in SEQ ID NO.1, and the integrated T7 RNA polymerase gene is shown in SEQ ID NO.2. Furthermore, the promoter P shown in SEQ ID NO.3 is used. xylF Regulation.

[0050] In some implementations, in order to weaken the uridine kinase gene pyrH It can be achieved using the artificial promoter P. j23109 Replacement of uridine kinase gene pyrH The natural promoter. More specifically, the uridine kinase gene. pyrH The nucleotide sequence is shown in SEQ ID NO.4, and the promoter P j23109 The nucleotide sequence is shown in SEQ ID NO.5, uridine kinase gene. pyrH The nucleotide sequence of the natural promoter is shown in SEQ ID NO.6.

[0051] In some implementations, in order to achieve overexpression of the carbamoyl phosphate synthase gene carAB ribose-phosphokinase gene prsA pyrimidine-5'-nucleotide nuclease gene ppnN UMP phosphohydrolase gene umpH and umpG Ribonucleotide hydrolase 1 gene rihA and glutamine synthase geneglnA The following solutions can be adopted:

[0052] In one implementation, yciQ Integrating carbamoyl phosphate synthase gene at the site carAB and with promoter P trc Regulation. Carbamoyl phosphate synthase gene carAB The nucleotide sequence is shown in SEQ ID NO.13; promoter P trc The nucleotide sequence is shown in SEQ ID NO.11.

[0053] In one implementation, ylbE Integrating ribose-phosphokinase gene at the site prsA and with promoter P trc Regulation. Ribophosphokinase gene prsA The nucleotide sequence is shown in SEQ ID NO.14.

[0054] In one implementation, yghX Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN and with promoter P T7 Regulation. Pyrimidine-5'-nucleotide nuclease gene ppnN The nucleotide sequence is shown in SEQ ID NO.7; promoter P T7 The nucleotide sequence is shown in SEQ ID NO.8.

[0055] In one implementation, ygaY UMP phosphatase gene integrated at the site umpH and with promoter P T7 Regulation. UMP phosphohydrolase gene umpH The nucleotide sequence is shown in SEQ ID NO.9.

[0056] In one implementation, yeeP UMP phosphatase gene integrated at the site umpG and with promoter P trc Regulation. UMP phosphohydrolase gene umpG The nucleotide sequence is shown in SEQ ID NO.10.

[0057] In one implementation, yjiT Ribonucleotide hydrolase 1 gene integrated at the site rihA and with promoter P T7 Regulation. Ribonucleotide hydrolase 1 gene rihA The nucleotide sequence is shown in SEQ ID NO.12.

[0058] In one implementation, gapC Integrating glutamine synthase gene at the site glnA and with promoter P trc Regulation. Glutamine synthase gene glnA The nucleotide sequence is shown in SEQ ID NO.15.

[0059] In some implementations, wild-type Bacillus subtilis is introduced heterologously. B.subtilis 168 orotate nucleoside monophosphate decarboxylase gene pyrF and orotic acid ribosyltransferase gene pyrE The following technical solutions can be adopted:

[0060] In one implementation, wild-type Bacillus subtilis is integrated at the ilvG site. B.subtilis 168 orotate nucleoside monophosphate decarboxylase gene pyrF and orotic acid ribosyltransferase gene pyrE And regulated by the promoter Ptrc. Orotic nucleoside monophosphate decarboxylase gene pyrF The nucleotide sequence is shown in SEQ ID NO.16, orotate ribosyltransferase gene. pyrE The nucleotide sequence is shown in SEQ ID NO.17.

[0061] This application provides a method for constructing a uracil-producing strain, specifically including the following steps: in Escherichia coli... Escherichia coli On the W3110 genome:

[0062] (a) Knockout of uridine kinase gene udk Simultaneously integrates the T7 RNA polymerase gene, via promoter P xylF Regulation;

[0063] (b) Using an artificial promoter P j23109 Replacement of uridine kinase gene pyrH The natural promoter;

[0064] (c) in yciQ Integrating carbamoyl phosphate synthase gene at the site carAB By promoter P trc Regulation;

[0065] (d) in ilvG wild-type Bacillus subtilis integrated at the site B.subtilis 168 orotic acid nucleoside monophosphate decarboxylase gene pyrF and orotic acid ribosyltransferase gene pyrE By promoter P trc Regulation;

[0066] (e) inylbE Integrating ribose-phosphokinase gene at the site prsA By promoter P trc Regulation;

[0067] (f) in yghX Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN By promoter P T7 Regulation;

[0068] (g) in ygaY UMP phosphatase gene integrated at the site umpH By promoter P T7 Regulation;

[0069] (h) in yeeP UMP phosphatase gene integrated at the site umpG By promoter P trc Regulation;

[0070] (i) in yjiT Ribonucleotide hydrolase 1 gene integrated at the site rihA By promoter P T7 Regulation;

[0071] (j) in gapC Integrating glutamine synthase gene at the site glnA By promoter P trc Regulation.

[0072] It should be noted that the order of the above steps is not strictly required, as long as the above purpose and function are achieved.

[0073] The specific steps for modifying uracil-producing strains are as follows:

[0074] (1) In the wild type E.coli Knockout of the uridine kinase gene in the W3110 genome udk Based on (Gene ID: 946597), the T7 RNA polymerase gene (Protein ID: QJZ11468.1) is integrated, driven by the promoter P. xylF Regulation was employed to block uridine complementation to the UMP pathway and to introduce T7 RNA polymerase in order to induce T7 promoter expression.

[0075] (2) Using the artificial promoter P j23109 Replacement of uridine kinase gene pyrH The natural promoter of (Gene ID: 944989) weakens the UMP-to-UDP branching pathway, reducing carbon loss.

[0076] (3) In yciQIntegrating carbamoyl phosphate synthase gene at the site carA (Gene ID: 949025) and carB (Gene ID: 944775), generated by promoter P trc Regulation; in ilvG wild-type Bacillus subtilis integrated at the site B.subtilis 168 orotic acid nucleoside monophosphate decarboxylase gene pyrF (Gene ID: 935960) and orotic acid ribosyltransferase gene pyrE (Gene ID: 936714), generated by promoter P trc Regulation and enhancement of the de novo UMP synthesis pathway.

[0077] (4) In ylbE Integrating ribose-phosphokinase gene at the site prsA (Gene ID: 945772), generated by promoter P trc Regulation and strengthening of the supply of precursor PRPP.

[0078] (5) In yghX Integrating pyrimidine-5'-nucleotide nuclease gene at the site ppnN (Gene ID: 947266), generated by promoter P T7 Regulation and enhancement of the UMP-to-uracil synthesis pathway.

[0079] (6) In ygaY UMP phosphatase gene integrated at the site umpH (Gene ID: 945283), generated by promoter P T7 Regulation; in yeeP UMP phosphatase gene integrated at the site umpG (Gene ID: 947211), generated by promoter P trc Regulation; in yjiT Ribonucleotide hydrolase 1 gene integrated at the site rihA (Gene ID: 945503), generated by promoter P T7 Regulation enhances the UMP synthesis pathway via uridine to uracil.

[0080] (7) In gapC Integrating glutamine synthase gene at the site glnA (Gene ID: 948370), generated by promoter P trc Regulation and strengthening of the supply of the precursor glutamine.

[0081] The uracil-producing strain constructed in this application can produce uracil and is suitable for industrial production.

[0082] This application also provides a method for producing uracil, wherein the uracil-producing strain provided in this application can be fermented to obtain uracil. This fermentation process can be carried out with reference to conventional fermentation methods in the art.

[0083] The specific steps for producing uracil using a fermenter are as follows:

[0084] (1) Seed activation: The strain 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.

[0085] (2) Seed culture: Inoculate the bacterial suspension into the seed culture medium, pH 7.0, constant temperature 36℃, dissolved oxygen 30%-60%, and culture for 6.5-7h;

[0086] (3) Fermentation culture: Wait until the seed cell count OD600 reaches 15-20, inoculate the fermentation medium at a rate of 30%, and start fermentation. During fermentation, control the pH at 7.0, the temperature at 36℃, and the dissolved oxygen at 30%-60%. After the glucose in the medium is consumed, add 80% glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L. When the cell count OD600 reaches 20-25 and 40-45, add 10 g / (L fermentation broth) of sterilized xylose for induction.

[0087] Unless otherwise specified, the percentage sign "%" in the examples refers to volume percentage; the percentage of a solution "% (m / v)" refers to the number of grams of solute contained in 100 ml of solution.

[0088] The starting strain used in the examples was wild-type Escherichia coli. E.coli W3110, accession number ATCC 27325; wild-type Bacillus subtilis B. subtilis 168, Accession No. ATCC 23857; Escherichia coli E.coli The corresponding promoter and genes of the BL21(DE3)SHBCC D24923 genome are shown in the sequence listing.

[0089] The gene editing method used is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coliUsing 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.

[0090] Table 1 Primers used in strain construction

[0091] Primer Sequence (5' to 3') Sequence number -1 GTTGGGTGCCAAACTGCG SEQ ID NO.18 -xyl-2 GATTTATGACCGAGATCTTACTTTTGTTGCGCAATTGTACTTATTGCATTTTTCTCTTCGAGGAATTACCCAGTTTCATCATTCCATTTTATTTTGCGAGCGAGCGCACACTTGTGAATTATCTCCCGATAATGACGCACTGATGAGAC SEQ ID NO.19 -3 CGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCATCGCGATCGATATATTGAAAGCG SEQ ID NO.20 -4 CTGGGTGAAGATAGAACGCCTCG SEQ ID NO.21 T7RNA-A CGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGGGAGCAGTTATAGCTCAGGCC SEQ ID NO.22 T7RNA-S CAAAAGTAAGATCTCGGTCATAAATCAAGAAATAAACCAAAAATCGTAATCGAAAGATAAAAATCTGTAATTGTTTTCCCCTGTTTAGTTGCTAAAAATTGGTTACGTTTATCGCGGTGATTGTTACTTATTAAAACTGTCCTCTAACTACAGAAGGCCCTACACCATGAACACGATTAACATCGCTAAGAACG SEQ ID NO.23 pGRB--S AGTCCTAGGTATAATACTAGTTAACGAGCGTGGGCGTTCAAGTTTTAGAGCTAGAA SEQ ID NO.24 pGRB--A TTCTAGCTCTAAAACTTGAACGCCCACGCTCGTTAACTAGTATTATACCTAGGACT SEQ ID NO.25 DW-PpyrH-A TAGCTTCTGCCCAGCTGTAGC SEQ ID NO.26 DW-PpyrH-js09-S TTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGCAGGAAACAGACCATGGCTACCAATGCAAAACCC SEQ ID NO.27 UP-PpyrH-js09-A GTCCCTAGGACTGAGCTAGCTGTAAAAATTAAGACTGCTTGGACATCGCAG SEQ ID NO.28 UP-PpyrH-S CTATGCACGTTGCTGCAAGC SEQ ID NO.29 Pjs09-JD-S CAGCTAGCTCAGTCCTAGGGAC SEQ ID NO.30 pGRB-PpyrH-S AGTCCTAGGTATAATTACTAGTTAAATTCAGCTAACCCTTGTGTTTTAGAGCTAGAA SEQ ID NO.31 pGRB-PpyrH-A TTCTAGCTCTAAAACCAAGGGTTAGCTGAATTTAACTAGTATTATACCTAGGACT SEQ ID NO.32 yciQ-1 TTACTTGAAGCATTGGGCGAAC SEQ ID NO.33 yciQ-trc-2 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAGTCCAAGATGCCAGGGTTC SEQ ID NO.34 trc-carAB-S TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGATTAAGTCAGCGCTATTGGTTC SEQ ID NO.35 carAB-①-A AGTTGCTGGATTACTATGACCCTAGAAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCATCCATCACTTCGCCAACC SEQ ID NO.36 yciQ-4#-3 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTTAGGGTCATAGTAATCCAGCAACTTATAAGACCCGTTTTCTCTCCAGT SEQ ID NO.37 yciQ-4 CATTATTTCTGGTCGCGCCCT SEQ ID NO.38 carAB-②-S AAATTCCTCGCTTCAACTTCG SEQ ID NO.39 trc-carAB-②-A CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTG TTATTTGATCTTGTGCGTGCATTT SEQ ID NO.40 yciQ-trc-3 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTATAAGACCCGTTTTCTCTCCAGT SEQ ID NO.41 pGRB-yciQ-S AGTCCTAGGTATAATACTAGTAAACAACGTTTCTTGCCTCAGTTTTAGAGCTAGAA SEQ ID NO.42 pGRB-yciQ-A TTCTAGCTCTAAAACTGAGGCAAGAAACGTTGTTTACTAGTATTATACCTAGGACT SEQ ID NO.43 pGRB-4#-S ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACT SEQ ID NO.44 pGRB-4#-A AGTTGCTGGATTACTATGACCCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCAT SEQ ID NO.45 ilvG-1 ACCGAGGAGCAGACAATGAATAA SEQ ID NO.46 ilvG-trc-2 CCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGATGGA SEQ ID NO.47 ilvG-3 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGT SEQ ID NO.48 ilvG-4 GCGCTGGCTAACATGAGGAA SEQ ID NO.49 pGRB-ilvG-S AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA SEQ ID NO.50 pGRB-ilvG-A TTCTAGCTCTAAAACTGATGCGCGGCAACTCTTCCACTAGTATTATACCTAGGACT SEQ ID NO.51 pyrFE-S GTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAAACAACCTGCCCATCATCG SEQ ID NO.52 pyrFE-A CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATTTTTTAAACCAATCTTTTGACTCG SEQ ID NO.53 ylbE-1 ACCCAACCTTACGCAACCAG SEQ ID NO.54 ylbE-trc-2 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT SEQ ID NO.55 ylbE-3 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA SEQ ID NO.56 ylbE-4 GGCGTAACTCAGCAGGCAG SEQ ID NO.57 pGRB-ylbE-S AGTCCTAGGTATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA SEQ ID NO.58 pGRB-ylbE-A TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT SEQ ID NO.59 prsA-S TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGCCTGATATGAAGCTTTTTGC SEQ ID NO.60 prsA-A CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGTTCGAACATGGCAGAGAT SEQ ID NO.61 yghX-1 GCGCAACCGTAGAACAGGAATT SEQ ID NO.62 yghX-T7-2 AATTATTTCTAGACCCTATAGTGAGTCGTTAGATTGAAGCGCCTTTACTACTCC SEQ ID NO.63 yghX-T7-3 AACGGGTCTTGAGGGGTTTTTTGGTCATAGTAATCCAGCAACTCTTGTG SEQ ID NO.64 yghX-4 GAGCAGGTATTTACGTGAACCG SEQ ID NO.65 pGRB-yghX-S AGTCCTAGGTATACTAGTCCCACTTTGCCTGTCGCTTGGTTTTAGAGCTAGAA SEQ ID NO.66 pGRB-yghX-A TTCTAGCTCTAAAACCAAGCGACAGGCAAAGTGGGACTAGTATTATACCTAGGACT SEQ ID NO.67 ppnN-T7-A ACCCCTCAAGACCCGTTTAGGGCCCCAAGGGGTTATGCTAGTTACGTGCAGATTTCGTAGCAAGGG SEQ ID NO.68 ppnN-T7-S AGGGTCTAGAAATTTGTTTAACTTTAAGAAGGATATACCATGGATATGTTGTCGCAGCTGGAA SEQ ID NO.69 ygaY-1 CCTACAAACCACATCGCATT SEQ ID NO.70 ygaY-T7-2 TAAGTAAAAAATTATTTCTAGACCCTTAGTGAGTCGTATTAACCGAAGCACCCAAAAG SEQ ID NO.71 ygaY-T7-3 TGGGGGCCTCTAAAACGGGTCTTGAGGGGTTTTTTGTTGCTTGCCGCTCCACC SEQ ID NO.72 ygaY-4 GGAGTAGGGCTTTCCATAGAGTGT SEQ ID NO.73 pGRB-ygaY-UP AGTCCTAGGTAATACTAGTCTCAACTACCCACAGTTGTTGTTTTAGAGCTAGAA SEQ ID NO.74 pGRB-ygaY-DW TTCTAGCTCTAAAAEACTGTGGGTAGTTTACTAGTATTACCTAGCT SEQ ID NO.75 umpH-T7-A GACCCGTTTAGAGGCCCCATCAGATAACGTCGATTTCAGCGACT SEQ ID NO.76 umpH-T7-S GGTCTAGAAATTTGTTTAACTTATGACCTAAAATGTAATTTGCGATATCGAC SEQ ID NO.77 and P-1 GGTCAGGGTAACTTATCAGCG SEQ ID NO.78 YeP-trc-2 AAACGGAGCCCTGCCATTAATACGACTCACTATAGGGTCTAGAAATTATTG SEQ ID NO.79 and P-3 GGGGCCCCTTAAACGGGTCTTGAGGGGTTTTTTGGAACTGGATTTTCTCTCTGAACCTGT SEQ ID NO.80 and P-4 ACGATGTCAGCAGCCAGCA SEQ ID NO.81 pGRB-yeP-S AGTCCTAGGTATAATACTAGTACAGAATATTCGCGAAAAAACGGGTTTTAGAGCTAGAA SEQ ID NO.82 pGRB-yesP-A TTCTAGCTCTAAAACCCGTTTTTTCGCGAATATTCTGTACTAGTATTATACCTAGGACT SEQ ID NO.83 umpG-S GGTCTAGAAATAATTTTTGTTTAACTTTAATGCGCATATTGCTGAGTAATGATGAC SEQ ID NO.84 umpG-A GACCCGTTTAGAGGCCCCATTACCATTGCGTGCCAACTCC SEQ ID NO.85 yjiT-1 AATAGTTGTTGCCGCCTGAGT SEQ ID NO.86 yjiT-T7-2 AATTATTTCTAGACCCTATAGTGAGTCGTATTAAAACAGGCAGCAAAGTCCC SEQ ID NO.87 yjiT-T7-3 TGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCACTACCTGTGAAGGGATGT SEQ ID NO.88 yjiT-4 CAGGGCTTCCACAGTCACAAT SEQ ID NO.89 pGRB-yjiT-S AGTCCTAGGTATAATACTAGGGGATTATGAACGGCAATGGTTTTAGAGCTAGAA SEQ ID NO.90 pGRB-yjiT-A TTCTAGCTCTAAAACCATTGCCGTTCATAATCCCTACTAGTATTATACCTAGGACT SEQ ID NO.91 rihA-T7-S TAGAAATTTTTGTTTAACTTTAATGGCACTGCCAATTCTGTTAGATT SEQ ID NO.92 rihA-T7-A AATCTAACAGAATTGGCAGTGCCATTAAAGTTAAACAAAATTATTTCTA SEQ ID NO.93 gapC-1 TGGGAAGAAACCACGAAACTC SEQ ID NO.94 gapC-trc-2 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGTTTCAGCAGGTAGGCGAGA SEQ ID NO.95 gapC-trc-3 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAAACGGTCGCCTGGTACG SEQ ID NO.96 gapC-4 TTATCCGCCGACATTGCTG SEQ ID NO.97 glnA-S TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTCCGCTGAACACGTACTGA SEQ ID NO.98 glnA-A AGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGACGCTGTAGTACAGCTCAAACTC SEQ ID NO.99

[0092] Example 1

[0093] like Figure 1 As shown, the specific process for constructing genetically engineered strains is as follows:

[0094] 1.1 Knockout of gene udk and integration of T7 RNA polymerase gene

[0095] Using the extracted and diluted *E. coli* W3110 genome as a template, primers were used... udk -1 and udk -xyl-2, udk -3 and udk -4; Using a usable concentration of E. coli BL21(DE3) genome as a template, PCR amplification was performed using primers T7RNA-S and T7RNA-A, respectively, to obtain the upstream homologous arm. udk -xyl-UP and downstream homologous arms udk -DW, the target fragment xyl-T7 RNA was used as a template with the recovered upstream, midstream and downstream homologous arms, and primers were applied. udk -1 and udk -4 Perform overlap PCR to obtain the target fragment required for integration. udk- xyl-T7 RNA. Then, primer pGRB- udk -S and pGRB- udk The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- udk Finally, pGRB- udk Plasmids and overlapping fragments udk- xyl-T7 RNA electroporated to E.coli In W3110 / pRed-Cas9 electrocompetent cells; then with udk -1 and udk-4 is the identification primer; positive transformants are screened to obtain the strain. E.coli ura1.

[0096] 1.2 Gene pyrH promoter replacement (P pyrH ::P j23109 )

[0097] Using *E. coli* W3110 as a template, PCR amplification was performed using primers UP-PpyrH-S and UP-PpyrH-js09-A, and DW-PpyrH-A and DW-PpyrH-js09-S to obtain the upper homologous arm UP-PpyrH-js09 and the lower homologous arm DW-PpyrH. Using the recovered upper and lower homologous arms as templates, overlap PCR was performed using primers UP-PpyrH-S and DW-PpyrH-A to obtain the target fragment PpyrH-js09 for integration. Then, the DNA fragment obtained by annealing primers pGRB-PpyrH-S and pGRB-PpyrH-A was ligated to plasmid pGRB to construct the pGRB-PpyrH plasmid. Finally, the purified PpyrH-js09 integration fragment and plasmid pGRB-PpyrH were simultaneously transformed into the target fragment by electroporation. E.coli In competent cells of ura1 / pRed-Cas9, positive transformants were screened using primers Pjs09-JD-S and DW-PpyrH-A to finally obtain the strain. E.coli ura2.

[0098] 1.3 Genes carAB Segmented integration ( yciQ ::Ptrc- carAB )

[0099] Using Escherichia coli W3110 as a template, primers were used... yciQ -1 and yciQ -trc-2, yciQ -4 and yciQ -trc-3, yciQ -4 and yciQ -4#-3, carAB -trc-S and carAB -①-A, carAB -②-S and trc- carAB -②-A was subjected to PCR amplification, and the upper homologous arms were obtained successively. yciQ -up, lower homologous arm yciQ -trc-dw and yciQ -4#-dw and intermediate target fragments carAB -① and carAB -②, with the recovered upper homologous arm yciQ -up, downstream homologous arm yciQ-4#-dw and intermediate target fragments carAB -① serves as a template, using primers yciQ -1 and yciQ -4 The target fragment Ptrc required for integration was obtained through overlap PCR- carAB -①, then, primer pGRB- yciQ -S and pGRB- yciQ The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- yciQ Plasmid. The purified Ptrc- carAB -①Integrated fragment and plasmid pGRB- yciQ Simultaneously, the strain was transferred via electroporation. E.coli In competent cells of ura2 / pRed-Cas9, primers were then used. yciQ -1 and yciQ -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E.coli ura3-①. Again with carAB -② and yciQ -trc-dw is the template, and primers are used. carAB -②-S and yciQ -4 The target fragment Ptrc required for integration was obtained through overlap PCR- carAB -② Next, the DNA fragment obtained by annealing primers pGRB-4#-S and pGRB-4#-A was ligated to plasmid pGRB to construct plasmid pGRB-4#. Finally, the purified Ptrc- carAB -① The integrated fragment and plasmid pGRB-4# were simultaneously transformed into the strain via electroporation. E.coli In competent cells of ura3-① / pRed-Cas9, primers were then used... yciQ -1 and yciQ -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E.coli ura3.

[0100] 1.4 Genes pyrFE Integration ( ilvG ::P trc - pyrFE (integration)

[0101] Using Escherichia coli W3110 as a template, primers were used... ilvG -1 and ilvG -trc-2, ilvG -3 and ilvG -4; with Bacillus subtilis B.subtilis Using the 168 genome as a template, primers were used. pyrFE -S and pyrFE -A was used for PCR amplification to obtain the upper homologous arm.ilvG -trc-UP, lower homologous arm ilvG -DW and intermediate destination fragments pyrFE Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... ilvG -1 and ilvG -4 The target fragment P required for integration was obtained through overlap PCR. trc - pyrFE Then, the primer pGRB- ilvG -S and pGRB- ilvG The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- ilvG Plasmid. Finally, the purified P... trc - pyrFE Integration fragment and plasmid pGRB- ilvG Simultaneously transferred via electroconversion E.coli In competent cells of ura3 / pRed-Cas9, primers were then used. ilvG -1 and ilvG -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E.coli ura4.

[0102] 1.5 genes prsA Integration ( ylbE ::P trc - prsA (integration)

[0103] Using Escherichia coli W3110 as a template, primers were used... ylbE -1 and ylbE -trc-2, ylbE -3 and ylbE -4, prsA -S and prsA -A was used for PCR amplification to obtain the upper homologous arm. ylbE -trc-UP, lower homologous arm ylbE -DW and intermediate destination fragments prsA Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... ylbE -1 and ylbE -4 The target fragment required for integration was obtained through overlap PCR. ylbE -P trc - prsA Then, the primer pGRB- ylbE -S and pGRB- ylbE The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- ylbE Plasmid. Finally, the purified plasmid... ylbE -P trc - prsAIntegration fragment and plasmid pGRB- ylbE Simultaneously transferred via electroconversion E.coli In competent cells of ura4 / pRed-Cas9, primers were then used. ylbE -1 and ylbE -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E.coli ura5.

[0104] 1.6 genes ppnN Integration ( yghX ::P T7 - ppnN (integration)

[0105] Using Escherichia coli W3110 as a template, primers were used... yghX -1 and yghX -T7-2, yghX -T7-3 and yghX -4, ppnN -T7-S and ppnN -T7-A was used for PCR amplification to obtain the upper homologous arm. yghX -T7-UP, lower homologous arm yghX -DW and intermediate target fragment T7- ppnN Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... yghX -1 and yghX -4 The target fragment required for integration was obtained through overlap PCR. yghX -P T7 - ppnN Then, the primer pGRB- yghX -S and pGRB- yghX The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- [[ID=1 Plasmid. Finally, the purified plasmid... ​ -P T7 - ​ Integration fragment and plasmid pGRB- ​ Simultaneously transferred via electroconversion ​ In competent cells of ura5 / pRed-Cas9, primers were then used. ​ -1 and ​ -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. ​ ura6.

[0106] 1.7 genes ​ Integration ( ​ ::P T7 - ​ (integration)

[0107] Using Escherichia coli W3110 as a template, primers were used... ​ -1 and ​ -T7-2, ​ -T7-3 and ​ -4, ​ -T7-S and ​ -T7-A was used for PCR amplification to obtain the upper homologous arm. ​ -T7-UP, lower homologous arm ​ -DW and intermediate target fragment T7- ​ Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... ​ -1 and ​ -4 The target fragment required for integration was obtained through overlap PCR. ​ -P T7 - ​ Then, the primer pGRB- ​ -S and pGRB- ​ The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- ​ Plasmid. Finally, the purified plasmid... ​ -P T7 - ​ Integration fragment and plasmid pGRB- ygaY Simultaneously transferred via electroconversion E.coli In competent cells of ura6 / pRed-Cas9, primers were then used. ygaY -1 and ygaY -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E.coli ura7.

[0108] 1.8 genes umpG Integration ( yeeP ::P trc - umpG (integration)

[0109] Using Escherichia coli W3110 as a template, primers were used... yeeP -1 and yeeP -trc-2, yeeP -3 and yeeP -4, umpG -S and umpG -A was used for PCR amplification to obtain the upper homologous arm. yeeP -trc-UP, lower homologous arm yeeP -DW and intermediate destination fragments umpG Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... yeeP -1 and yeeP -4 The target fragment required for integration was obtained through overlap PCR. yeeP-P trc - umpG Then, the primer pGRB- yeeP -S and pGRB- yeeP The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- yeeP Plasmid. Finally, the purified plasmid... yeeP -P trc - umpG Integration fragment and plasmid pGRB- yeeP Simultaneously transferred via electroconversion E.coli In competent cells of ura7 / pRed-Cas9, primers were then used. yeeP -1 and yeeP -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E.coli ura8.

[0110] 1.9 genes rihA Integration ( yjiT ::P T7 - rihA (integration)

[0111] Using Escherichia coli W3110 as a template, primers were used... yjiT -1 and yjiT -T7-2, yjiT -T7-3 and yjiT -4, rihA -T7-S and rihA -T7-A was used for PCR amplification to obtain the upper homologous arm. yjiT -T7-UP, lower homologous arm yjiT -DW and intermediate target fragment T7- rihA Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... yjiT -1 and yjiT -4 The target fragment required for integration was obtained through overlap PCR. yjiT -P T7 - rihA Then, the primer pGRB- yjiT -S and pGRB- yjiT The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- yjiT Plasmid. Finally, the purified plasmid... yjiT -P T7 - rihA Integration fragment and plasmid pGRB- yjiT Simultaneously transferred via electroconversion E.coli In competent cells of ura8 / pRed-Cas9, primers were then used. yjiT -1 and yjiT-4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E.coli ura9.

[0112] 1.10 genes glnA Integration ( gapC ::P trc - glnA (integration)

[0113] Using Escherichia coli W3110 as a template, primers were used... gapC -1 and gapC -trc-2, gapC -3 and gapC -4, glnA -S and glnA -A was used for PCR amplification to obtain the upper homologous arm. gapC -trc-UP, lower homologous arm gapC -DW and intermediate destination fragments glnA Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primers were used... gapC -1 and gapC -4 The target fragment required for integration was obtained through overlap PCR. gapC -P trc - glnA Then, the primer pGRB- gapC -S and pGRB- gapC The DNA fragment obtained by -A annealing was ligated onto plasmid pGRB to construct pGRB- gapC Plasmid. Finally, the purified plasmid... gapC -P trc - glnA Integration fragment and plasmid pGRB- gapC Simultaneously transferred via electroconversion E.coli In competent cells of ura9 / pRed-Cas9, primers were then used. gapC -1 and gapC -4 indicates the primers used for identification to screen positive transformants, ultimately obtaining the strain. E.coli ura10.

[0114] The strains involved in the above construction process are shown in Table 2.

[0115] Table 2 lists the strains involved.

[0116] Strain Name Genotype ura1 <![CDATA[ E.coli W3110, udk ::P xylF -T7RNAP]]> ura2 <![CDATA[ E.coli hour1,P pyrH ::P j23109 ]]> ura3 <![CDATA[ E.coli ura2, yciQ ::P trc - carAB ]]> ura4 <![CDATA[ E.coli ura3, ilvG ::P trc - pyrFE (bsu) ]]> ura5 <![CDATA[ E.coli ura4, ylbE ::P trc - prsA ]]> ura6 <![CDATA[ E.coli day5, yghX ::P T7 - ppnN ]]> ura7 <![CDATA[ E.coli ura6, ygaY ::P T7 - umpH ]]> ura8 <![CDATA[ E.coli ura7, yeeP ::P trc - umpG ]]> ura9 <![CDATA[ E.coli ura8, yjiT:: P T7 -rihA ]]> ura10 <![CDATA[ E.coli ura9, gapC ::P trc - glnA ]]>

[0117] Example 2

[0118] Uracil was produced by fermentation in a 5 L fermenter using the uracil-producing strain E. coli ura10 described in Example 1.

[0119] 2.1 Culture medium

[0120] 2.1.1 Seed activation and slant culture medium

[0121] Glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride NaCl 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / 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 500 ml. The solution was dispensed into test tubes (9 ml / tube) and flasks (45 ml / flask), and sterilized in an autoclave at 121℃ for 20 min.

[0122] 2.1.2 Seed Culture Medium

[0123] Glucose 30 g / L (partially digested), yeast extract 8 g / L, peptone 2.0 g / L, (NH4)2SO4 4.0 g / L, KH2PO4 3.0 g / L, V B1 V B3 V B5 V B12 1 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.35 mg / L, boric acid 3.5 mg / L, CoCl2·6H2O 1.5 mg / L, the remainder being water.

[0124] 2.1.3 Fermentation medium

[0125] Glucose 10 g / L (digested separately), yeast powder 10 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 6.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 60 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.35 mg / L, boric acid 3.5 mg / L, CoCl2·6H2O 1.5 mg / L, the remainder being water.

[0126] 2.2 Cultivation Methods

[0127] 2.2.1 Slant activation culture

[0128] Scrape a loopful of bacterial culture from the -80℃ preservation tube, spread it evenly on the activation slant, incubate at 37℃ for 12 h, and then transfer to a flask to continue incubation for 12 h.

[0129] 2.2.2 Seed Culture

[0130] Take an appropriate amount of sterile water into an eggplant-shaped flask, inoculate the bacterial suspension into the seed culture medium, stabilize the pH at around 7.0, keep the temperature constant at 36℃, and keep the dissolved oxygen between 30% and 60%, and incubate for 7 h;

[0131] 2.2.3 Fermentation Culture

[0132] Waiting for seed cell count OD 600 At approximately 20°C, inoculate fresh fermentation medium at a 30% inoculation rate to begin fermentation. During fermentation, maintain a stable pH of around 7.0, a temperature of 36°C, and dissolved oxygen levels between 30% and 60%. Once the glucose in the medium is depleted, add an 80% glucose solution to maintain a glucose concentration of 0.1-1 g / L. When the OD600 of the cells reaches 25 and 43, respectively, add 10 g / (L fermentation broth) of sterilized xylose to induce fermentation. The fermentation cycle is 48 hours. No antibiotics or inducing agents are added during fermentation.

[0133] like Figure 3 As shown, the yield of uracil reached 35.8 g / L after 48 h of fermentation in a 5 L fermenter.

[0134] The above description is only a preferred embodiment of this application. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application. Improvements and modifications such as strain modification made by those skilled in the art based on the method of this application or based on the method are all considered to be within the scope of protection of this application.

[0135] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

Claims

1. A uracil-producing strain, characterized in that, The uracil-producing strain was constructed using a directed modification method based on Escherichia coli Escherichiacoli W3110. The construction process includes the following steps: (a) In the genome of Escherichia coli W3110, knocking out the uridine kinase gene udk and simultaneously integrating the T7 RNA polymerase gene, via promoter P xylF Regulation; the nucleotide sequence of the udk gene is shown in SEQ ID NO.1, the nucleotide sequence of the T7 RNA polymerase gene is shown in SEQ ID NO.2, and the promoter P xylF The nucleotide sequence is shown in SEQ ID NO.3; (b) Using an artificial promoter P j23109 Replace the natural promoter of the uridine kinase gene pyrH; the nucleotide sequence of the uridine kinase gene pyrH is shown in SEQ ID NO.4, and the promoter P j23109 The nucleotide sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the natural promoter of the uridine kinase gene pyrH is shown in SEQ ID NO.6; (c) The carbamoyl phosphate synthase gene carAB is integrated at the yciQ site, via promoter P trc Regulation; the nucleotide sequence of the carbamoyl phosphate synthase gene carAB is shown in SEQ ID NO.13; the promoter P trc The nucleotide sequence is shown in SEQ ID NO.11; (d) Integration of the wild-type Bacillus subtilis 168 orotic nucleotide monophosphate decarboxylase gene pyrF and orotic ribosyltransferase gene pyrE at the ilvG site, via promoter P trc Regulation; the nucleotide sequence of the orotate nucleoside monophosphate decarboxylase gene pyrF is shown in SEQ ID NO.16, and the nucleotide sequence of the orotate ribosyltransferase gene pyrE is shown in SEQ ID NO.17; (e) The ribose-phospho-pyrokinase gene prsA is integrated at the ylbE site, via promoter P trc Regulation; the nucleotide sequence of the ribophosphate pyrophosphate kinase gene prsA is shown in SEQ ID NO.14; (f) Integration of the pyrimidine-5'-nucleotide nuclease gene ppnN at the yghX site, via promoter P T7 Regulation; the nucleotide sequence of the pyrimidine-5'-nucleotide nuclease gene ppnN is shown in SEQ ID NO.7; promoter P T7 The nucleotide sequence is shown in SEQ ID NO. 8; (g) The UMP phosphatase gene umpH is integrated at the ygaY site, via the promoter P T7 Regulation; the umbilical sequence of the UMP phosphohydrolase gene umpH is shown in SEQ ID NO.9; (h) The UMP phosphatase gene umpG is integrated at the yeeP site, via promoter P. trc Regulation; the nucleotide sequence of the UMP phosphohydrolase gene umpG is shown in SEQ ID NO.10; (i) The ribonucleoside hydrolase 1 gene rihA is integrated at the yjiT site, via promoter P T7 Regulation; the ribonucleoside hydrolase 1 gene rihA nucleotide sequence is shown in SEQ ID NO.12; (j) The glutamine synthase gene glnA is integrated at the gapC site, via promoter P trc Regulation; the nucleotide sequence of the glutamine synthase gene glnA is shown in SEQ ID NO.

15.

2. The uracil-producing strain according to claim 1, characterized in that, The targeted modification method utilizes CRISPR / Cas9 gene editing technology to modify the genome of the Escherichia coli W3110 chromosome.

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

4. A method for producing uracil, characterized in that, The method includes: fermenting the uracil-producing strain according to claim 1 or 2 to obtain uracil.

5. The method according to claim 4, characterized in that, The specific steps for producing uracil using a fermenter are as follows: (1) Seed activation: The strain is evenly spread on the activation slant and incubated at 36~38℃ for 10~14 h, then transferred to a flask and incubated for another 10~14 h; (2) Seed culture: Inoculate the bacterial suspension into the seed culture medium, pH 6.9~7.1, temperature constant at 35~37℃, dissolved oxygen at 30%-60%, and culture for 6.5~7h; (3) Fermentation culture: wait for the OD of the seed cell mass 600 At 15-20°C, inoculate the fermentation medium at a rate of 30% to begin fermentation. During fermentation, maintain the pH at 6.9-7.1, the temperature at 35-37°C, and the dissolved oxygen at 30%-60%. After the glucose in the medium is consumed, add a 78%-82% glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L. When the OD600 of the bacterial cell count reaches 20-25 and 40-45, add 9-11 g of sterilized xylose per liter of fermentation broth to induce induction. The slant culture medium used for seed activation consisted of: glucose 2 g / L, peptone 10 g / L, yeast extract 5 g / L, NaCl 2.5 g / L, KH₂PO₄ 1.0 g / L, MgSO₄ 0.2 g / L, agar powder 25%, with the remainder being water, pH 7.0-7.

2. The seed culture medium used for seed culture consisted of: glucose 30 g / L, yeast extract 8 g / L, peptone 2.0 g / L, (NH₄)₂SO₄ 4.0 g / L, KH₂PO₄ 3.0 g / L, V B1 V B3 V B5 V B12 1 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.35 mg / L, boric acid 3.5 mg / L, CoCl2·6H2O 1.5 mg / L, the remainder being water; the fermentation medium used in the fermentation culture was: glucose 10 g / L, yeast extract 10 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 6.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 60 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.35 mg / L, boric acid 3.5 mg / L, CoCl2·6H2O 1.5 mg / L, the remainder being water.

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