Uracil production strain as well as construction method and application thereof

Abstract

The invention relates to the technical production field of metabolic engineering and genetic engineering, and particularly provides a uracil production strain as well as a construction method and application thereof. According to the strain, a uridine kinase gene udk is knocked out on an E.coli W3110 genome, and meanwhile, a T7RNA polymerase gene is integrated; the uridine monophosphate kinase gene pyrH is weakened; the method comprises the following steps: overexpressing a carbamyl phosphate synthetase gene carAB, a ribose phosphate pyrophosphate kinase gene prsA, a pyrimidine-5 '-nucleoside enzyme gene ppnN, UMP phosphohydrolase genes umpH and umpG, a ribonucleoside hydrolase 1 gene rihA and a glutamine synthetase gene glnA; an orotic acid nucleoside monophosphate decarboxylase gene pyrF and an orotic acid ribosyltransferase gene pyrE of wild type bacillus subtilis B.subtilis 168 are introduced in a heterologous manner, so that an orotate nucleoside monophosphate decarboxylase gene pyrF and an orotate ribosyltransferase gene pyrE are obtained. The safe, efficient and low-cost production of the uracil 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 uracil production strain and a construction method and application thereof. Background Art

[0002] Uracil is a base unique to RNA and one of the basic building blocks for the formation of RNA and vitamin B3 (an important cofactor in the metabolism of terrestrial life). In addition, in the medical field, drugs synthesized from it as raw materials are widely used. For example, synthetic 5-fluorouracil and its derivatives have good clinical effects on a variety of cancers. In the agricultural field, uracil can be used to synthesize certain pyrimidine herbicides, such as cypermethrin, herbicide, and terbacil, which achieve weed control effects by inhibiting photosynthesis.

[0003] The current industrial production method of uracil is mainly chemical, but the chemical method of preparing uracil has the disadvantages of using dangerous reagents, cumbersome operation and high raw material cost, while microbial fermentation has the characteristics of low pollution, high production efficiency and low cost. Coupled with the rapid development of synthetic biology, more and more researchers have gradually replaced the chemical method by constructing microbial cell factories to produce many natural products and chemicals. Therefore, constructing a uracil-producing strain with high uracil production efficiency and good genetic stability is an urgent problem to be solved.

[0004] In view of this, this application is hereby filed. Summary of the invention

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

[0006] In order to achieve the above purpose, this application adopts the following technical solutions: A uracil production strain The uracil production strain is based on Escherichia coli Escherichia coli W3110 was constructed using a directed transformation method as the starting strain: exist E.coli Knockout of uridine kinase gene in the W3110 genome udk At the same time, the T7 RNA polymerase gene was integrated; the uridylate kinase gene was weakened pyrH ; Overexpression of carbamoyl phosphate synthetase gene carAB ribose phosphate pyrophosphokinase gene prsA , pyrimidine-5'-nucleotidase gene ppnN , UMP phosphohydrolase gene umpH and umpG , ribonucleoside hydrolase 1 gene rihA and glutamine synthetase gene glnA; Heterologous introduction of wild-type Bacillus subtilis B.subtilis Orotidine monophosphate decarboxylase gene pyrF and orotate ribosyltransferase gene pyrE .

[0007] In some embodiments, the targeted modification method is to use CRISPR / Cas9 gene editing technology to completely transform Escherichia coli Escherichia coli The W3110 chromosome genome was modified.

[0008] In some embodiments, upon knocking out the uridine kinase gene udk The T7 RNA polymerase gene was integrated into the xylF Regulation; udk The nucleotide sequence of the 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.

[0009] In some embodiments, an artificial promoter P j23109 Replacement of uridylate kinase gene pyrH The natural promoter of pyrH The nucleotide sequence of the promoter is shown in SEQ ID NO.4. j23109 The nucleotide sequence of is shown in SEQ ID NO.5, the uridylate kinase gene pyrH The nucleotide sequence of the natural promoter is shown in SEQ ID NO.6.

[0010] In some embodiments, yciQ carbamoyl phosphate synthetase gene carAB , and with promoter P trc Regulation; the carbamyl phosphate synthetase gene carAB The nucleotide sequence of the promoter is shown in SEQ ID NO.13; trc The nucleotide sequence is shown in SEQ ID NO.11.

[0011] In some embodiments, ylbE ribose phosphate pyrophosphokinase gene prsA , and with promoter P trc Regulation; the ribose phosphate pyrophosphokinase gene prsA The nucleotide sequence is shown in SEQ ID NO.14.

[0012] In some embodiments, yghXpyrimidine-5'-nucleotidase gene ppnN , and with promoter P T7 Regulation; the pyrimidine-5'-nucleotide nucleosidase 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.

[0013] In some embodiments, ygaY UMP phosphohydrolase gene umpH , and with promoter P T7 Regulation; The UMP phosphohydrolase gene umpH The nucleotide sequence is shown in SEQ ID NO.9.

[0014] In some embodiments, yeeP UMP phosphohydrolase gene umpG , and with promoter P trc Regulation; The UMP phosphohydrolase gene umpG The nucleotide sequence is shown in SEQ ID NO.10.

[0015] In some embodiments, yjiT Ribonucleoside hydrolase 1 gene rihA , and with promoter P T7 Regulation; the ribonucleoside hydrolase 1 gene rihA The nucleotide sequence is shown in SEQ ID NO.12.

[0016] In some embodiments, gapC Glutamine synthetase gene glnA , and with promoter P trc Regulation; The glutamine synthetase gene glnA The nucleotide sequence is shown in SEQ ID NO.15.

[0017] In some embodiments, ilvG Bacillus subtilis B.subtilis Orotidine monophosphate decarboxylase gene pyrF and orotate ribosyltransferase gene pyrE , and with promoter P trc Regulation; the orotidine monophosphate decarboxylase gene pyrF The nucleotide sequence is shown in SEQ ID NO.16, and the orotate ribosyltransferase gene pyrE The nucleotide sequence is shown in SEQ ID NO.17.

[0018] The method for constructing a uracil-producing strain specifically comprises the following steps: (a) In Escherichia coli Escherichia coli Knockout of uridine kinase gene in the W3110 genome udk At the same time, the T7 RNA polymerase gene was integrated and the promoter P xylF Regulation; (b) Using artificial promoter P j23109 Replace the natural promoter of the uridylate kinase gene pyrH; (c) In yciQ carbamoyl phosphate synthetase gene carAB , by the promoter P trc Regulation; (d) In ilvG Bacillus subtilis B.subtilis 168 Orotidine monophosphate decarboxylase gene pyrF and orotate ribosyltransferase gene pyrE , by the promoter P trc Regulation; (e) In ylbE ribose phosphate pyrophosphokinase gene prsA , by the promoter P trc Regulation; (f) In yghX pyrimidine-5'-nucleotidase gene ppnN , by the promoter P T7 Regulation; (g) In ygaY UMP phosphohydrolase gene umpH , by the promoter P T7 Regulation; (h) yeeP UMP phosphohydrolase gene umpG , by the promoter P trc Regulation; (i) in yjiT Ribonucleoside hydrolase 1 gene rihA , by the promoter P T7 Regulation; (j) in gapC Glutamine synthetase gene glnA , by the promoter P trc Regulation.

[0019] Application of uracil-producing strains in fermentation production of uracil.

[0020] A method for producing uracil, the method comprising: fermenting the above-mentioned uracil-producing strain to obtain uracil.

[0021] In some embodiments, uracil is produced by fermentation in a fermenter, and the specific steps are as follows: (1) Seed activation: Spread the strain evenly on the activation slope, culture at 36-38℃ for 10-14 h, transfer to eggplant-shaped bottle and continue culture for 10-14 h; (2) Seed culture: inoculate the bacterial suspension into the seed culture medium, pH 6.9-7.1, constant temperature at 35-37°C, dissolved oxygen at 30%-60%, and culture for 6.5-7 hours; (3) Fermentation culture: Wait for the seed bacteria volume OD 600 To 15~20, access the fermentation medium according to the inoculation amount of 30%, start fermentation, control the pH at 6.9~7.1, temperature at 35~37℃, dissolved oxygen at 30%~60% during the fermentation process; when the glucose in the medium is consumed, add 78%~82% glucose solution to maintain the glucose concentration in the fermentation medium at 0.1~1 g / L, when the bacterial cell OD600 reaches 20~25 and 40~45, pour in 9~11 g / (L fermentation liquid) of sterilized xylose for induction.

[0022] In some embodiments, the slant culture medium used in the seed activation is: glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, NaCl 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, the rest is water, pH 7.0-7.2; the seed culture medium used in the seed culture is: glucose 30 g / L, yeast powder 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, and the rest is water; the fermentation medium used in the fermentation culture is: glucose 10 g / L, 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 H0.1 mg / L, ammonium molybdate 0.35 mg / L, boric acid 3.5 mg / L, CoCl2·6H2O1.5 mg / L, and the rest is water.

[0023] The above culture media can be prepared using standard methods.

[0024] Compared with the prior art, the technical effects of this application are: The uracil production strain of the present application is produced by knocking out the uridine kinase gene udk , weakening the uridylate kinase gene pyrH , while maintaining bacterial growth, reducing carbon loss; and then overexpressing the carbamoyl phosphate synthetase gene carAB , pyrimidine-5'-nucleotidase gene ppnN , UMP phosphohydrolase gene umpH and umpG and ribonucleoside hydrolase 1 gene rihA Bacillus subtilis B.subtilis 168 Orotidine monophosphate decarboxylase and orotidine ribosyltransferase genes pyrFE , unblocking the uracil de novo synthesis pathway; finally overexpressing the ribose phosphate pyrophosphokinase gene prsA and glutamine synthetase gene glnA , strengthen the supply of precursors ribose phosphate pyrophosphate PRPP and glutamine, and synergistically achieve rapid accumulation of uracil. The strain does not contain plasmids, has good genetic stability and high fermentation yield, and does not require the addition of antibiotics and defective substances during the production process, effectively reducing production costs; the strain uses cheap carbon source glucose as a substrate to efficiently synthesize uracil from scratch. After 48 hours of fermentation in a 5 L fermenter, the uracil yield can be as high as 35.8 g / L, which has a high prospect for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a diagram of the metabolic pathway of the directed transformation method of uracil-producing strains; Figure 2 Genetically engineered strains E.coli Illustration of ura10 genome modification; Figure 3 Genetically engineered strains E.coli ura10 fermentation process curve. DETAILED DESCRIPTION

[0026] 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.

[0027] The uracil production strain provided in this application is a strain produced by direct transformation 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.

[0028] The specific construction content includes: knocking out the uridine kinase gene udk At the same time, the T7 RNA polymerase gene was integrated; the uridylate kinase gene was weakened pyrH ; Overexpression of carbamoyl phosphate synthetase gene carAB ribose phosphate pyrophosphokinase gene prsA , pyrimidine-5'-nucleotidase gene ppnN , UMP phosphohydrolase gene umpH and umpG , ribonucleoside hydrolase 1 gene rihA and glutamine synthetase gene glnA ; Heterologous introduction of wild-type Bacillus subtilis B.subtilis Orotidine monophosphate decarboxylase gene pyrF and orotate ribosyltransferase gene pyrE .

[0029] It should be noted that the present invention knocks out the uridine kinase gene udk , weakening the uridylate kinase gene pyrH , while maintaining bacterial growth, reducing carbon loss; and then overexpressing the carbamoyl phosphate synthetase gene carAB , pyrimidine-5'-nucleotidase gene ppnN , UMP phosphohydrolase gene umpH and umpG and ribonucleoside hydrolase 1 gene rihA Bacillus subtilis B.subtilis 168 Orotidine monophosphate decarboxylase and orotidine ribosyltransferase genes pyrFE , unblocking the uracil de novo synthesis pathway; finally overexpressing the ribose phosphate pyrophosphokinase gene prsA and glutamine synthetase gene glnA , strengthen the supply of precursors ribose phosphate pyrophosphate PRPP and glutamine, and synergistically achieve rapid accumulation of uracil. Therefore, as long as the strains constructed can achieve the above functions, they are all uracil 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 uracil production strains.

[0030] In some embodiments, the specific technical means used in the directed transformation method is CRISPR / Cas9 gene editing technology. This method is completely and directly performed on the genome of the starting strain, and the obtained strain does not contain plasmids, has good genetic stability, high fermentation yield, and does not require the addition of antibiotics and defective substances during the production process, effectively reducing production costs.

[0031] In some embodiments, the knocked-out uridine kinase gene udk The specific sequence of is shown in SEQ ID NO.1, and the integrated T7 RNA polymerase gene is shown in SEQ ID NO.2. In addition, the promoter P shown in SEQ ID NO.3 is used. xylF Regulation.

[0032] In some embodiments, to attenuate the uridylate kinase gene pyrH , the artificial promoter P j23109 Replacement of uridylate kinase gene pyrH More specifically, the uridylate kinase gene pyrH The nucleotide sequence of the promoter is shown in SEQ ID NO.4. j23109 The nucleotide sequence of SEQ ID NO.5 is shown, uridylate kinase gene pyrH The nucleotide sequence of the natural promoter is shown in SEQ ID NO.6.

[0033] In some embodiments, to achieve overexpression of the carbamoyl phosphate synthetase gene carAB ribose phosphate pyrophosphokinase gene prsA , pyrimidine-5'-nucleotidase gene ppnN , UMP phosphohydrolase gene umpH and umpG , ribonucleoside hydrolase 1 gene rihA and glutamine synthetase gene glnA , the following solutions can be adopted: In one embodiment, yciQ carbamoyl phosphate synthetase gene carAB , and with promoter P trc Regulation. Carbamoyl phosphate synthetase gene carAB The nucleotide sequence of the promoter is shown in SEQ ID NO.13; trc The nucleotide sequence is shown in SEQ ID NO.11.

[0034] In one embodiment, ylbE ribose phosphate pyrophosphokinase gene prsA , and with promoter P trc Regulation. Ribose phosphate pyrophosphokinase geneprsA The nucleotide sequence is shown in SEQ ID NO.14.

[0035] In one embodiment, yghX pyrimidine-5'-nucleotidase gene ppnN , and with promoter P T7 Regulation. Pyrimidine-5'-nucleotide nucleosidase 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.

[0036] In one embodiment, ygaY UMP phosphohydrolase gene umpH , and with promoter P T7 Regulation. UMP phosphohydrolase gene umpH The nucleotide sequence is shown in SEQ ID NO.9.

[0037] In one embodiment, yeeP UMP phosphohydrolase gene umpG , and with promoter P trc Regulation. UMP phosphohydrolase gene umpG The nucleotide sequence is shown in SEQ ID NO.10.

[0038] In one embodiment, yjiT Ribonucleoside hydrolase 1 gene rihA , and with promoter P T7 Regulation. Ribonucleoside hydrolase 1 gene rihA The nucleotide sequence is shown in SEQ ID NO.12.

[0039] In one embodiment, gapC Glutamine synthetase gene glnA , and with promoter P trc Regulation. Glutamine synthetase gene glnA The nucleotide sequence is shown in SEQ ID NO.15.

[0040] In some embodiments, to heterologously introduce wild-type Bacillus subtilis B.subtilis Orotidine monophosphate decarboxylase gene pyrF and orotate ribosyltransferase gene pyrE , the following technical solutions can be adopted: In one embodiment, wild-type Bacillus subtilis is integrated at the ilvG locus. B.subtilis Orotidine monophosphate decarboxylase genepyrF and orotate ribosyltransferase gene pyrE and regulated by the promoter Ptrc. pyrF The nucleotide sequence of the orotate ribosyltransferase gene is shown in SEQ ID NO.16. pyrE The nucleotide sequence is shown in SEQ ID NO.17.

[0041] The present application provides a method for constructing a uracil-producing strain, which specifically comprises the following steps: Escherichia coli On the W3110 genome: (a) Knockout of uridine kinase gene udk At the same time, the T7 RNA polymerase gene was integrated and the promoter P xylF Regulation; (b) Using artificial promoter P j23109 Replacement of uridylate kinase gene pyrH The natural promoter of (c) In yciQ carbamoyl phosphate synthetase gene carAB , by the promoter P trc Regulation; (d) In ilvG Bacillus subtilis B.subtilis 168 Orotidine monophosphate decarboxylase gene pyrF and orotate ribosyltransferase gene pyrE , by the promoter P trc Regulation; (e) ylbE ribose phosphate pyrophosphokinase gene prsA , by the promoter P trc Regulation; (f) In yghX pyrimidine-5'-nucleotidase gene ppnN , by the promoter P T7 Regulation; (g) In ygaY UMP phosphohydrolase gene umpH , by the promoter P T7 Regulation; (h) yeeP UMP phosphohydrolase gene umpG , by the promoter P trc Regulation; (i) in yjiT Ribonucleoside hydrolase 1 gene rihA , by the promoter P T7 Regulation; (j) ingapC Glutamine synthetase gene glnA , by the promoter P trc Regulation.

[0042] 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.

[0043] The transformation method of uracil production strain, the specific steps are as follows: (1) In wild type E.coli Knockout of uridine kinase gene in W3110 genome udk (Gene ID: 946597) was integrated with the T7 RNA polymerase gene (Protein ID: QJZ11468.1), and the promoter P xylF Regulated to block uridine replenishment to the UMP pathway and introduce T7 RNA polymerase to induce T7 promoter expression.

[0044] (2) Using artificial promoter P j23109 Replacement of uridylate kinase gene pyrH (Gene ID: 944989)'s natural promoter, weakening the UMP to UDP branch pathway and reducing carbon loss.

[0045] (3) yciQ carbamoyl phosphate synthetase gene carA (Gene ID: 949025) and carB (Gene ID: 944775), driven by the promoter P trc regulation; ilvG Bacillus subtilis B.subtilis 168 Orotidine monophosphate decarboxylase gene pyrF (Gene ID: 935960) and orotate ribosyltransferase gene pyrE (Gene ID: 936714), driven by the promoter P trc Regulate and strengthen the UMP de novo synthesis pathway.

[0046] (4) ylbE ribose phosphate pyrophosphokinase gene prsA (Gene ID: 945772), driven by the promoter P trc Regulate and strengthen the supply of precursor PRPP.

[0047] (5) yghX pyrimidine-5'-nucleotidase gene ppnN (Gene ID: 947266), driven by the promoter P T7Regulate and strengthen the UMP direct synthesis pathway to uracil.

[0048] (6) ygaY UMP phosphohydrolase gene umpH (Gene ID: 945283), driven by the promoter P T7 regulation; yeeP UMP phosphohydrolase gene umpG (Gene ID: 947211), driven by the promoter P trc regulation; yjiT Ribonucleoside hydrolase 1 gene rihA (Gene ID: 945503), driven by the promoter P T7 Regulate and strengthen the UMP synthesis pathway from uridine to uracil.

[0049] (7) gapC Glutamine synthetase gene glnA (Gene ID: 948370), driven by the promoter P trc Regulate and strengthen the supply of precursor glutamine.

[0050] The uracil-producing strain constructed in the present application can realize the production of uracil and is suitable for industrial production.

[0051] The present application also provides a method for producing uracil. The uracil-producing strain provided in the present application can produce uracil by fermentation. The fermentation process can be carried out with reference to conventional fermentation methods in the art.

[0052] The specific steps of producing uracil by fermentation in a fermenter are as follows: (1) Seed activation: Spread the strain evenly on the activation slope, culture at 37°C for 12 h, transfer to an eggplant-shaped bottle and continue culture for 12 h; (2) Seed culture: Inoculate the bacterial suspension into the seed culture medium, pH 7.0, constant temperature at 36°C, dissolved oxygen at 30%-60%, and culture for 6.5-7 hours; (3) Fermentation culture: Wait until the OD600 of the seed bacteria reaches 15-20, inoculate the fermentation medium at a rate of 30%, and start fermentation. During the fermentation process, control the pH at 7.0, the temperature at 36°C, and the dissolved oxygen at 30%-60%. When 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 OD600 of the bacteria reaches 20-25 and 40-45, pour in 10 g / (L fermentation liquid) of sterilized xylose for induction.

[0053] The percentage sign "%" in the examples, unless otherwise specified, refers to volume percentage; the percentage of a solution "% (m / v)" refers to the number of grams of solute contained in 100 ml of the solution.

[0054] The starting strain used in the embodiment is wild-type Escherichia coli E.coli W3110, deposit number ATCC 27325; wild-type Bacillus subtilis B. subtilis 168, accession number ATCC 23857; Escherichia coli E.coli The corresponding promoter and gene of BL21 (DE3) SHBCC D24923 genome are shown in the sequence listing.

[0055] The gene editing method used was 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.

[0056] Table 1 Primers involved in strain construction Primer Sequence (5' to 3') SEQ ID NO. -1 GTTGGGTGCCAAACTGCG SEQ ID NO.18 -xyl-2 GATTTATGACCGAGATCTTACTTTTGTTGCGCAATTGTACTTATTGCATTTTTCTCTTCGAGGAATTACCCAGTTTCATCATTCCATTTTATTTTGCGAGCGAGCGCACACTTGTGAATTATCTCCCGATAATGACGCACTGATGAGAC SEQ IDNO.19 -3 CGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCATCGCGATCGATATATTGAAAGCG SEQ IDNO.20 -4 CTGGGTGAAGATAGAACGCCTCG SEQ IDNO.21 T7RNA-A CGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGGGAGCAGTTATAGCTCAGGCC SEQ IDNO.22 T7RNA-S CAAAAGTAAGATCTCGGTCATAAATCAAGAAATAAACCAAAAATCGTAATCGAAAGATAAAAATCTGTAATTGTTTTCCCCTGTTTAGTTGCTAAAAATTGGTTACGTTTATCGCGGTGATTGTTACTTATTAAAACTGTCCTCTAACTACAGAAGGCCCTACACCATGAACACGATTAACATCGCTAAGAACG SEQ IDNO.23 pGRB--S AGTCCTAGGTATAATACTAGTTAACGAGCGTGGGCGTTCAAGTTTTAGAGCTAGAA SEQ IDNO.24 pGRB--A TTCTAGCTCTAAAACTTGAACGCCCACGCTCGTTAACTAGTATTATACCTAGGACT SEQ IDNO.25 DW-PpyrH-A TAGCTTCTGCCCAGCTGTAGC SEQ IDNO.26 DW-PpyrH-js09-S TTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGCAGGAAACAGACCATGGCTACCAATGCAAAACCC SEQ IDNO.27 UP-PpyrH-js09-A GTCCCTAGGACTGAGCTAGCTGTAAAAATTAAGACTGCTTGGACATCGCAG SEQ IDNO.28 UP-PpyrH-S CTATGCACGTTGCTGCAAGC SEQ IDNO.29 Pjs09-JD-S CAGCTAGCTCAGTCCTAGGGAC SEQ IDNO.30 pGRB-PpyrH-S AGTCCTAGGTATAATACTAGTTAAATTCAGCTAACCCTTGTGTTTTAGAGCTAGAA SEQ IDNO.31 pGRB-PpyrH-A TTCTAGCTCTAAAACACAAGGGTTAGCTGAATTTAACTAGTATTATACCTAGGACT SEQ IDNO.32 yciQ-1 TTACTTGAAGCATTGGGCGAAC SEQ IDNO.33 yciQ-trc-2 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAGTCAAGATGCCAGGGTTC SEQ IDNO.34 trc-carAB-S TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGATTAAGTCAGCGCTATTGGTTC SEQ IDNO.35 carAB-①-A AGTTGCTGGATTACTATGACCCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCATCCATCACTTCGCCAACC SEQ IDNO.36 yciQ-4#-3 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACTTATAAGACCCGTTTTTCTCTCCAGT SEQ IDNO.37 yciQ-4 CATTATTTCTGGTCGCGCCT SEQ IDNO.38 carAB-②-S AAATTCCTCGCTTCAACTTCG SEQ IDNO.39 trc-carAB-②-A CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTG TTATTTGATCTGTGCGTGCATTT SEQ IDNO.40 yciQ-trc-3 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTATAAGACCCGTTTTTCTCTCCAGT SEQ IDNO.41 pGRB-yciQ-S AGTCCTAGGTATAATACTAGTAAACAACGTTTCTTGCCTCAGTTTTAGAGCTAGAA SEQ IDNO.42 pGRB-yciQ-A TTCTAGCTCTAAAACTGAGGCAAGAAACGTTGTTTACTAGTATTATACCTAGGACT SEQ IDNO.43 pGRB-4#-S ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACT SEQ IDNO.44 pGRB-4#-A AGTTGCTGGATTACTATGACCCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCAT SEQ IDNO.45 ilvG-1 ACCGAGGAGCAGACAATGAATAA SEQ IDNO.46 ilvG-trc-2 CCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGATGGA SEQ IDNO.47 ilvG-3 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGT SEQ IDNO.48 ilvG-4 GCGCTGGCTAACATGAGGAA SEQ IDNO.49 pGRB-ilvG-S AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA SEQ IDNO.50 pGRB-ilvG-A TTCTAGCTCTAAAACTGATGCGCGGCAACTCTTCCACTAGTATTATACCTAGGACT SEQ IDNO.51 pyrFE-S GTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAAACAACCTGCCCATCATCG SEQ IDNO.52 pyrFE-A CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATTTTTTAAACCAATCTTTTGACTCG SEQ IDNO.53 ylbE-1 ACCCAACCTTACGCAACCAG SEQ IDNO.54 ylbE-trc-2 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT SEQ IDNO.55 ylbE-3 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA SEQ IDNO.56 ylbE-4 GGCGTAACTCAGCAGGCAG SEQ IDNO.57 pGRB-ylbE-S AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA SEQ IDNO.58 pGRB-ylbE-A TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT SEQ IDNO.59 prsA-S TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCCTGATATGAAGCTTTTTGC SEQ IDNO.60 prsA-A CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGTGTTCGAACATGGCAGAGAT SEQ IDNO.61 yghX-1 GCGCAACGTAGAACAGGAATT SEQ IDNO.62 yghX-T7-2 AATTATTTCTAGACCCTATAGTGAGTCGTATTAGATTGAAGCGCCTTTACTACTCC SEQ IDNO.63 yghX-T7-3 AACGGGTCTTGAGGGGTTTTTTGGTCATAGTAATCCAGCAACTCTTGTG SEQ IDNO.64 yghX-4 GAGCAGGTATTTACGTGAACCG SEQ IDNO.65 pGRB-yghX-S AGTCCTAGGTATAATACTAGTCCCACTTTGCCTGTCGCTTGGTTTTAGAGCTAGAA SEQ IDNO.66 pGRB-yghX-A TTCTAGCTCTAAAACCAAGCGACAGGCAAAGTGGGACTAGTATTATACCTAGGACT SEQ IDNO.67 ppnN-T7-A ACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTACGTGCAGATTTCGTAGCAAGGG SEQ IDNO.68 ppnN-T7-S AGGGTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGATATGTTGTCGCAGCTGGAA SEQ IDNO.69 ygaY-1 CCTACAAACCACATCGCACATT SEQ IDNO.70 ygaY-T7-2 TAAAGTTAAACAAAATTATTTCTAGACCCTATAGTGAGTCGTATTAACACCGAAGCAACCCAAAAG SEQ IDNO.71 ygaY-T7-3 TGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTTGCTTGCCGCTCCACC SEQ IDNO.72 ygaY-4 GGAGTAGGGCTTTCCATAGAGTGT SEQ IDNO.73 pGRB-ygaY-UP AGTCCTAGGTATAATACTAGTCTCAACTACCCACAGTTGTTGTTTTAGAGCTAGAA SEQ IDNO.74 pGRB-ygaY-DW TTCTAGCTCTAAAACAACAACTGTGGGTAGTTGAGACTAGTATTATACCTAGGACT SEQ IDNO.75 umpH-T7-A GACCCGTTTAGAGGCCCCATCAGATAACGTCGATTTCAGCGACT SEQ IDNO.76 umpH-T7-S GGTCTAGAAATAATTTTGTTTAACTTTAATGACCATTAAAAATGTAATTTGCGATATCGAC SEQ IDNO.77 yeeP-1 GGTCAGGAGGTAACTTATCAGCG SEQ IDNO.78 yeeP-trc-2 AAAACGGAGCCCTGCCATTAATACGACTCACTATAGGGTCTAGAAATAATTTTG SEQ IDNO.79 yeeP-3 GGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGAACTGGATTTTCTTCTGAACCTGT SEQ IDNO.80 yeeP-4 ACGATGTCAGCAGCCAGCA SEQ IDNO.81 pGRB-yeeP-S AGTCCTAGGTATAATACTAGTACAGAATATTCGCGAAAAAACGGGTTTTAGAGCTAGAA SEQ IDNO.82 pGRB-yeeP-A TTCTAGCTCTAAAACCCGTTTTTTCGCGAATATTCTGTACTAGTATTATACCTAGGACT SEQ IDNO.83 umpG-S GGTCTAGAAATAATTTTGTTTAACTTTAATGCGCATATTGCTGAGTAATGATGAC SEQ IDNO.84 umpG-A GACCCGTTTAGAGGCCCCATTACCATTGCGTGCCAACTCC SEQ IDNO.85 yjiT-1 AATAGTTGTTGCCGCCTGAGT SEQ IDNO.86 yjiT-T7-2 AATTATTTCTAGACCCTATAGTGAGTCGTATTAAAACAGGCAGCAAAGTCCC SEQ IDNO.87 yjiT-T7-3 TGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCACTACCTGTGAAGGGATGT SEQ IDNO.88 yjiT-4 CAGGGCTTCCACAGTCACAAT SEQ IDNO.89 pGRB-yjiT-S AGTCCTAGGTATAATACTAGTAGGGATTATGAACGGCAATGGTTTTAGAGCTAGAA SEQ IDNO.90 pGRB-yjiT-A TTCTAGCTCTAAAACCATTGCCGTTCATAATCCCTACTAGTATTATACCTAGGACT SEQ IDNO.91 rihA-T7-S TAGAAATAATTTTGTTTAACTTTAATGGCACTGCCAATTCTGTTAGATT SEQ IDNO.92 rihA-T7-A AATCTAACAGAATTGGCAGTGCCATTAAAGTTAAACAAAATTATTTCTA SEQ IDNO.93 gapC-1 TGGGAAGAAACCACGAAACTC SEQ IDNO.94 gapC-trc-2 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGTTTCAGCAGGTAGGCGAGA SEQ IDNO.95 gapC-trc-3 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAAACGGTCGCCTGGTACG SEQ IDNO.96 gapC-4 TTATCCGCCGACATTGCTG SEQ IDNO.97 glnA-S TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTCCGCTGAACACGTACTGA SEQ IDNO.98 glnA-A AGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGACGCTGTAGTACAGCTCAAACTC SEQ IDNO.99 Example 1 like Figure 1 As shown, the specific process of constructing genetically engineered strains is as follows: 1.1 Knockout of udk gene and integration of T7 RNA polymerase gene The E. coli W3110 genome extracted and diluted to an acceptable concentration was used as a template and primers udk -1 and udk -xyl-2, udk -3 and udk -4; Using the available concentration of Escherichia coli BL21 (DE3) genome as a template, primers T7RNA-S and T7RNA-A were used to perform PCR amplification to obtain the upstream homology arm udk -xyl-UP and downstream homology arms udk -DW, target fragment xyl-T7RNA uses the recovered upper, middle and downstream homology arms as templates and primers udk -1 and udk-4 Perform overlapping PCR to obtain the target fragment required for integration udk- Then, primer pGRB- udk -S and pGRB- udk -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- udk Finally, pGRB- udk Plasmids and overlapping fragments udk- xyl-T7RNA was electroporated into E.coli W3110 / pRed-Cas9 was electroporated into competent cells; udk -1 and udk -4 is the identification primer, screen the positive transformants and obtain the strain E.coli ura1.

[0057] 1.2 Gene pyrH promoter (P pyrH ::P j23109 ) Using E. coli W3110 as a template, primers UP-PpyrH-S and UP-PpyrH-js09-A, DW-PpyrH-A and DW-PpyrH-js09-S were used for PCR amplification to obtain the upper homology arm UP-PpyrH-js09 and the lower homology arm DW-PpyrH. Using the recovered upstream and downstream homology arms as templates, primers UP-PpyrH-S and DW-PpyrH-A were used for overlapping PCR to obtain the target fragment PpyrH-js09 required for integration. Afterwards, the DNA fragment obtained by annealing primers pGRB-PpyrH-S and pGRB-PpyrH-A was connected to the plasmid pGRB line to construct the pGRB-PpyrH plasmid. Finally, the purified PpyrH-js09 integration fragment and plasmid pGRB-PpyrH were simultaneously transferred into E.coli ura1 / pRed-Cas9 competent cells, and then use primers Pjs09-JD-S and DW-PpyrH-A as identification primers to screen positive transformants, and finally obtain the strain E.coli ura2.

[0058] 1.3 Genes carAB Segmental integration of yciQ ::Ptrc- carAB ) Using E. coli W3110 as template, primers 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 amplified by PCR to obtain the upper homology arms yciQ -up, lower homology arm yciQ -trc-dw and yciQ -4#-dw and intermediate destination fragment carAB -① and carAB -②, with the recycled upper homology arm yciQ -up, downstream homology arm yciQ -4#-dw and intermediate destination fragment carAB -① is the template, using primers yciQ -1 and yciQ -4 Obtain the target fragment Ptrc- required for integration by overlapping PCR carAB -①, then, primer pGRB- yciQ -S and pGRB- yciQ -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- yciQ Plasmid. carAB -①Integration fragment and plasmid pGRB- yciQ At the same time, the strain was transformed into E.coli ura2 / pRed-Cas9 competent cells, and then yciQ -1 and yciQ -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura3-①. carAB -② and yciQ -trc-dw as template, with primers carAB -②-S and yciQ -4 Obtain the target fragment Ptrc- required for integration by overlapping PCR carAB -②, then, the DNA fragment obtained by annealing primers pGRB-4#-S and pGRB-4#-A was connected to the plasmid pGRB line to construct pGRB-4# plasmid. carAB -① The integration fragment and plasmid pGRB-4# were simultaneously transferred into the strain by electroporation E.coli ura3-① / pRed-Cas9 competent cells, and then yciQ -1 and yciQ -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura3.

[0059] 1.4 Genes pyrFE Integration of ilvG::P trc - pyrFE Integration of Using E. coli W3110 as template, primers ilvG -1 and ilvG -trc-2, ilvG -3 and ilvG -4; Bacillus subtilis B.subtilis 168 genome as template, with primers pyrFE -S and pyrFE -A was amplified by PCR to obtain the upper homology arm ilvG -trc-UP, lower homology arm ilvG -DW and intermediate destination fragments pyrFE , using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, and using primers ilvG -1 and ilvG -4 Obtain the target fragment P required for integration by overlapping PCR trc - pyrFE , then, primer pGRB- ilvG -S and pGRB- ilvG -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- ilvG Finally, the purified P trc - pyrFE Integration fragment and plasmid pGRB- ilvG At the same time, electroporation E.coli ura3 / pRed-Cas9 competent cells, and then ilvG -1 and ilvG -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura4.

[0060] 1.5 Gene prsA Integration of ylbE ::P trc - prsA Integration of Using E. coli W3110 as template, primers ylbE -1 and ylbE -trc-2, ylbE -3 and ylbE -4, prsA -S and prsA -A was amplified by PCR to obtain the upper homology arm ylbE -trc-UP, lower homology arm ylbE -DW and intermediate destination fragments prsA , using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, and using primers ylbE -1 and ylbE-4 Obtain the target fragment required for integration by overlapping PCR ylbE -P trc - prsA , then, primer pGRB- ylbE -S and pGRB- ylbE -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- ylbE Finally, the purified ylbE -P trc - prsA Integration fragment and plasmid pGRB- ylbE At the same time, electroporation E.coli ura4 / pRed-Cas9 competent cells, and then ylbE -1 and ylbE -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura5.

[0061] 1.6 Genes ppnN Integration of yghX ::P T7 - ppnN Integration of Using E. coli W3110 as template, primers yghX -1 and yghX -T7-2, yghX -T7-3 and yghX -4, ppnN -T7-S and ppnN -T7-A for PCR amplification to obtain the upper homology arm yghX -T7-UP, lower homology arm yghX -DW and intermediate target fragment T7- ppnN , using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, and using primers yghX -1 and yghX -4 Obtain the target fragment required for integration by overlapping PCR yghX -P T7 - ppnN , then, primer pGRB- yghX -S and pGRB- yghX -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- yghX Finally, the purified yghX -P T7 - ppnN Integration fragment and plasmid pGRB- yghX At the same time, electroporation E.coli ura5 / pRed-Cas9 competent cells, and then yghX-1 and yghX -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura6.

[0062] 1.7 Genes umpH Integration of ygaY ::P T7 - umpH Integration of Using E. coli W3110 as template, primers ygaY -1 and ygaY -T7-2, ygaY -T7-3 and ygaY -4, umpH -T7-S and umpH -T7-A for PCR amplification to obtain the upper homology arm ygaY -T7-UP, lower homology arm ygaY -DW and intermediate target fragment T7- umpH , using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, and using primers ygaY -1 and ygaY -4 Obtain the target fragment required for integration by overlapping PCR ygaY -P T7 - umpH , then, primer pGRB- ygaY -S and pGRB- ygaY -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- ygaY Finally, the purified ygaY -P T7 - umpH Integration fragment and plasmid pGRB- ygaY At the same time, electroporation E.coli ura6 / pRed-Cas9 competent cells, and then ygaY -1 and ygaY -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura7.

[0063] 1.8 Gene umpG Integration of yeeP ::P trc - umpG Integration of Using E. coli W3110 as template, primers yeeP -1 and yeeP -trc-2, yeeP -3 and yeeP -4, umpG -S and umpG-A was amplified by PCR to obtain the upper homology arm yeeP -trc-UP, lower homology arm yeeP -DW and intermediate destination fragments umpG , using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, and using primers yeeP -1 and yeeP -4 Obtain the target fragment required for integration by overlapping PCR yeeP -P trc - umpG , then, primer pGRB- yeeP -S and pGRB- yeeP -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- yeeP Finally, the purified yeeP -P trc - umpG Integration fragment and plasmid pGRB- yeeP At the same time, electroporation E.coli ura7 / pRed-Cas9 competent cells, and then yeeP -1 and yeeP -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura8.

[0064] 1.9 Gene rihA Integration of yjiT ::P T7 - rihA Integration of Using E. coli W3110 as template, primers yjiT -1 and yjiT -T7-2, yjiT -T7-3 and yjiT -4, rihA -T7-S and rihA -T7-A for PCR amplification to obtain the upper homology arm yjiT -T7-UP, lower homology arm yjiT -DW and intermediate target fragment T7- rihA , using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, and using primers yjiT -1 and yjiT -4 Obtain the target fragment required for integration by overlapping PCR yjiT -P T7 - rihA , then, primer pGRB- yjiT -S and pGRB- yjiT -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- yjiTFinally, the purified yjiT -P T7 - rihA Integration fragment and plasmid pGRB- yjiT At the same time, electroporation E.coli ura8 / pRed-Cas9 competent cells, and then yjiT -1 and yjiT -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura9.

[0065] 1.10 Genes glnA Integration of gapC ::P trc - glnA Integration of Using E. coli W3110 as template, primers gapC -1 and gapC -trc-2, gapC -3 and gapC -4, glnA -S and glnA -A was amplified by PCR to obtain the upper homology arm gapC -trc-UP, lower homology arm gapC -DW and intermediate destination fragments glnA , using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, and using primers gapC -1 and gapC -4 Obtain the target fragment required for integration by overlapping PCR gapC -P trc - glnA , then, primer pGRB- gapC -S and pGRB- gapC -A annealed DNA fragment was connected with plasmid pGRB to construct pGRB- gapC Finally, the purified gapC -P trc - glnA Integration fragment and plasmid pGRB- gapC At the same time, electroporation E.coli ura9 / pRed-Cas9 competent cells, and then gapC -1 and gapC -4 is the identification primer to screen positive transformants and finally obtain the strain E.coli ura10.

[0066] The strains involved in the above construction process are shown in Table 2.

[0067] Table 2 Involved strains 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 <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> E.coli <h2 style=";text-align:left;direction:ltr"> ura3,<h2 style=";text-align:left;direction:ltr"> ilvG <h2 style=";text-align:left;direction:ltr"> ::P<h2 style=";text-align:left;direction:ltr"> trc <h2 style=";text-align:left;direction:ltr"> -<h2 style=";text-align:left;direction:ltr"> pyrFE <h2 style=";text-align:left;direction:ltr"> (bsu) <h2 style=";text-align:left;direction:ltr"> ]]><h2 style=";text-align:left;direction:ltr"> 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 <h2 style=";text-align:left;direction:ltr"><![CDATA[ <h2 style=";text-align:left;direction:ltr"> E.coli <h2 style=";text-align:left;direction:ltr"> ura7,<h2 style=";text-align:left;direction:ltr"> yeeP <h2 style=";text-align:left;direction:ltr"> ::P<h2 style=";text-align:left;direction:ltr"> trc <h2 style=";text-align:left;direction:ltr"> -<h2 style=";text-align:left;direction:ltr"> umpG <h2 style=";text-align:left;direction:ltr"> ]]><h2 style=";text-align:left;direction:ltr"> ura9 <![CDATA[ E.coli ura8, yjiT:: P T7 -rihA ]]> ura10 <![CDATA[ E.coli ura9, gapC ::P trc - glnA ]]> Example 2 The uracil producing strain E. coli ura10 described in Example 1 was used to produce uracil in a 5 L fermenter.

[0068] 2.1 Culture medium 2.1.1 Seed activation and slant culture medium Glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, NaCl 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, dissolve in water, adjust pH to 7.0-7.2 with sodium hydroxide, make up to 500 ml, dispense into test tubes (9 ml / tube) and eggplant-shaped bottles (45 ml / bottle), and sterilize in a high-pressure steam cooker at 121°C for 20 min.

[0069] 2.1.2 Seed culture medium Glucose 30 g / L (digested), yeast powder 8 g / L, peptone 2.0 g / L, (NH4)2SO4 4.0 g / L, KH2PO43.0 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, and the rest is water.

[0070] 2.1.3 Fermentation medium Glucose 10 g / L (digested), 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, and the rest is water.

[0071] 2.2 Cultivation methods 2.2.1 Slant activation culture Scrape a loop of bacteria from the -80℃ freezer tube, spread evenly on the activated slant, culture at 37℃ for 12 h, transfer to an eggplant-shaped bottle and continue culture for 12 h; 2.2.2 Seed culture Take an appropriate amount of sterile water in an eggplant-shaped bottle, inoculate the bacterial suspension into the seed culture medium, stabilize the pH at around 7.0, keep the temperature constant at 36°C, and keep the dissolved oxygen between 30% and 60%, and culture for 7 hours; 2.2.3 Fermentation culture Waiting for the seed cell volume OD 600 When the temperature reaches about 20, fresh fermentation medium is added at a 30% inoculation rate to start fermentation. During the fermentation process, the pH is kept stable at about 7.0, the temperature is maintained at 36°C, and the dissolved oxygen is between 30% and 60%. When the glucose in the medium is consumed, 80% glucose solution is added to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L. When the bacterial cell OD600 reaches 25 and 43, 10 g / (L fermentation liquid) of sterilized xylose is poured in for induction, and the fermentation cycle is 48 hours. No antibiotics or inducers are added during the fermentation process.

[0072] like Figure 3 As shown in the figure, the uracil production in the 5 L fermenter reached 35.8 g / L after 48 h of fermentation.

[0073] The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present application or on the basis of the method are deemed to be within the scope of protection of the present application.

[0074] 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.

Claims

1. A uracil-producing strain, characterized in that: The uracil production strain is Escherichia coli Escherichia coli W3110 was constructed using a directed transformation method as the starting strain: exist E. coli Knockout of uridine kinase gene in the W3110 genome udk At the same time, the T7 RNA polymerase gene was integrated; the uridylate kinase gene was weakened pyr ; Overexpression of carbamoyl phosphate synthetase gene carAB ribose phosphate pyrophosphokinase gene prsA , pyrimidine-5'-nucleotidase gene N , UMP phosphohydrolase gene umpH and umpG , ribonucleoside hydrolase 1 gene AH and glutamine synthetase gene gLn ; Heterologous introduction of wild-type Bacillus subtilis B. subtilis Orotidine monophosphate decarboxylase gene pyr and orotate ribosyltransferase gene pyrE .

2. The uracil-producing strain according to claim 1, characterized in that The directed transformation method is to use CRISPR / Cas9 gene editing technology to completely Escherichia coli The W3110 chromosome genome was modified.

3. The uracil-producing strain according to claim 1, characterized in that Knockout of uridine kinase gene udk The T7 RNA polymerase gene was integrated into the xylF Regulation; udk The nucleotide sequence of the 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.

4. The uracil-producing strain according to claim 1, characterized in that With artificial promoter P j23109 Replacement of uridylate kinase gene pyr The natural promoter of pyr The nucleotide sequence of the promoter is shown in SEQ ID NO.

4. j23109 The nucleotide sequence of is shown in SEQ ID NO.5, the uridylate kinase gene pyr The nucleotide sequence of the natural promoter is shown in SEQ ID NO.

6.

5. The uracil-producing strain according to claim 1, characterized in that exist QUR carbamoyl phosphate synthetase gene carAB , and with promoter P trc Regulation; the carbamyl phosphate synthetase gene carAB The nucleotide sequence of the promoter is shown in SEQ ID NO.13; trc The nucleotide sequence is shown in SEQ ID NO.11; Preferably, YlB ribose phosphate pyrophosphokinase gene prsA , and with promoter P trc Regulation; the ribose phosphate pyrophosphokinase gene prsA The nucleotide sequence is shown in SEQ ID NO.14; Preferably, yx pyrimidine-5'-nucleotidase gene N , and with promoter P T7 Regulation; the pyrimidine-5'-nucleotide nucleosidase gene N The nucleotide sequence is shown in SEQ ID NO.7; promoter P T7 The nucleotide sequence is shown in SEQ ID NO.8; Preferably, Y UMP phosphohydrolase gene umpH , and with promoter P T7 Regulation; The UMP phosphohydrolase gene umpH The nucleotide sequence is shown in SEQ ID NO.9; Preferably, yeeP UMP phosphohydrolase gene umpG , and with promoter P trc Regulation; The UMP phosphohydrolase gene umpG The nucleotide sequence is shown in SEQ ID NO.10; Preferably, Yj Ribonucleoside hydrolase 1 gene AH , and with promoter P T7 Regulation; the ribonucleoside hydrolase 1 gene AH The nucleotide sequence is shown in SEQ ID NO.12; Preferably, gapC Glutamine synthetase gene gLn , and with promoter P trc Regulation; The glutamine synthetase gene gLn The nucleotide sequence is shown in SEQ ID NO.

15.

6. The uracil-producing strain according to claim 1, characterized in that exist ilvG Bacillus subtilis B. subtilis Orotidine monophosphate decarboxylase gene pyr and orotate ribosyltransferase gene pyrE , and with promoter P trc Regulation; the orotidine monophosphate decarboxylase gene pyr The nucleotide sequence is shown in SEQ ID NO.16, and the orotate ribosyltransferase gene pyrE The nucleotide sequence is shown in SEQ ID NO.

17.

7. The method for constructing the uracil-producing strain according to any one of claims 1 to 6, characterized in that: The specific steps are as follows: (a) In Escherichia coli Escherichia coli Knockout of uridine kinase gene in the W3110 genome udk At the same time, the T7 RNA polymerase gene was integrated and the promoter P xylF Regulation; (b) Using artificial promoter P j23109 Replace the natural promoter of the uridylate kinase gene pyrH; (c) In QUR carbamoyl phosphate synthetase gene carAB , by the promoter P trc Regulation; (d) In ilvG Bacillus subtilis B. subtilis 168 Orotidine monophosphate decarboxylase gene pyr and orotate ribosyltransferase gene pyrE , by the promoter P trc Regulation; (e) In YlB ribose phosphate pyrophosphokinase gene prsA , by the promoter P trc Regulation; (f) In yx pyrimidine-5'-nucleotidase gene N , by the promoter P T7 Regulation; (g) In Y UMP phosphohydrolase gene umpH , by the promoter P T7 Regulation; (h) in yeeP UMP phosphohydrolase gene umpG , by the promoter P trc Regulation; (i) in Yj Ribonucleoside hydrolase 1 gene AH , by the promoter P T7 Regulation; (j) in gapC Glutamine synthetase gene gLn , by the promoter P trc Regulation.

8. Use of the uracil-producing strain according to any one of claims 1 to 6 in fermentation production of uracil.

9. A method for producing uracil, characterized in that: The method comprises: fermenting the uracil-producing strain according to any one of claims 1 to 6 to obtain uracil.

10. The method according to claim 9, characterized in that The specific steps of producing uracil by fermentation in a fermenter are as follows: (1) Seed activation: Spread the strain evenly on the activation slope, culture at 36-38℃ for 10-14 h, transfer to eggplant-shaped bottle and continue culture for 10-14 h; (2) Seed culture: inoculate the bacterial suspension into the seed culture medium, pH 6.9-7.1, constant temperature at 35-37°C, dissolved oxygen at 30%-60%, and culture for 6.5-7 hours; (3) Fermentation culture: Wait for the seed bacteria volume OD 600 To 15-20, inoculate the fermentation medium at 30% inoculation volume, start fermentation, control pH 6.9-7.1, temperature 35-37℃, dissolved oxygen 30%-60% during fermentation; when the glucose in the medium is consumed, add 78%-82% glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L; when the bacterial cell OD600 reaches 20-25 and 40-45, pour 9-11 g / (L fermentation liquid) of sterilized xylose for induction; Preferably, the slant culture medium used in the seed activation is: glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, NaCl 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, the rest is water, pH 7.0-7.2; the seed culture medium used in the seed culture is: glucose 30 g / L, yeast powder 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, and the rest is water; the fermentation medium used in the fermentation culture is: glucose 10 g / L, 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 1mg / 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, and the rest is water.

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