CN118272285A - Uracil nucleotide production strain, directional transformation method and application thereof - Google Patents

Abstract

The invention provides a uracil nucleotide production strain, a directional transformation method and application thereof, wherein the strain blocks UMP to uridine and uracil degradation paths by knocking out ushA, nagD, surE, yjjG and ygdH genes by using CRISPSR/Cas 9 gene editing technology; knocking out the pykF gene to reduce the carbon metabolism flow to the TCA cycle, and improving the conversion rate; heterologous introduction and overexpression of the ribophosphopyrophosphatase gene prs, the orotidine monophosphate decarboxylase gene pyrF and the orotic ribosyl transferase gene pyrE; overexpression of the phosphate ABC transporter gene pstC-pstA-pstB synergistically enhances UMP anabolic pathways; finally, dynamically regulating and controlling a uridylic acid kinase gene pyrH, and realizing UMP rapid accumulation under the condition of not affecting the growth of thalli; the obtained strain does not contain plasmids, does not need induction and defectives, and has good genetic stability.

Description

Uracil nucleotide production strain, directional transformation method and application thereof

Technical Field

The invention relates to the technical production fields of metabolic engineering and genetic engineering, in particular to a uracil nucleotide production strain, and a directional transformation method and application thereof.

Background

Uracil nucleotides (Uridine' monophosphate, UMP) have found wide application in agriculture, animal husbandry, and pharmaceutical fields. Today, with the gradual and deep research on uracil nucleotide functions, the market demand is continuously expanding.

The current industrialized production method of uracil nucleotide is mainly a nucleic acid hydrolysis method, which has the advantages of sufficient raw materials, low cost, mild production conditions and the like, but also has the problems of incomplete enzymolysis, low reaction efficiency, difficult separation, limited single nucleotide variety, low purity and the like. With the rapid development of synthetic biology, more and more researchers have gradually replaced chemical and hydrolytic processes by constructing microbial cell factories to ferment and produce many natural products and chemicals. The prior microbial fermentation method for synthesizing uracil nucleotide is mainly an enzyme catalysis method and a whole cell catalysis method, and a large amount of precursor orotic acid is required to be added into a substrate in the two methods, so that the production cost is high, and industrialization cannot be realized. Therefore, there is a need for a strain and process that can efficiently synthesize uracil nucleotides from scratch.

Disclosure of Invention

The invention aims to provide a uracil nucleotide production strain.

Another technical problem to be solved by the present invention is to provide a method for directional transformation of the uracil nucleotide production strain.

Another technical problem to be solved by the present invention is to provide an application of the uracil nucleotide production strain.

In order to solve the technical problems, the technical scheme of the invention is as follows:

The uracil nucleotide producing strain is strain E.colummp 11, which is obtained by further modifying an original strain E.columora 12 by a directional modification method, and specifically comprises the following steps: the ushA gene, nagD gene, surE gene, yjjG gene, ygdH gene and pykF gene were knocked out on E.coli Ora12 genome, and the ribophosphopyrophosphatase gene prs, orotidine monophosphate decarboxylase gene pyrF and orotic ribosyltransferase gene pyrE were introduced heterologous into B.subtilis168 of Bacillus subtilis, over-expressed in phosphate ABC transporter gene pstC, pstA, pstB and regulated dynamically in the uridylate kinase gene pyrH.

Preferably, the uracil nucleotide producing strain is a strain E.coli Ora12 in CN202410290893.1 (a orotic acid producing strain, a directional modification method and application thereof).

Preferably, the uracil nucleotide production strain is modified on the genome of the original strain E.collOra12 completely by using CRISPR/Cas9 gene editing technology.

Preferably, the nucleotide sequence of the ushA gene of the uracil nucleotide producing strain is shown in a sequence table SEQ ID NO. 1; the nucleotide sequence of nagD gene is shown in sequence table SEQ ID NO. 2; the nucleotide sequence of surE gene is shown in sequence table SEQ ID NO. 3; the nucleotide sequence of yjjG gene is shown in sequence table SEQ ID NO. 4; the nucleotide sequence of ygdH gene is shown in sequence table SEQ ID NO. 5; the nucleotide sequence of the pykF gene is shown in a sequence table SEQ ID NO. 6.

Preferably, the uracil nucleotide producing strain integrates a codon-optimized wild type B.subtilis 168 ribophosphopyrophosphatase gene prs at ylbE site, is regulated by a promoter P _{BBa_J23108} and is integrated again at yeeP site; the nucleotide sequence of the prs gene after codon optimization is shown in a sequence table SEQ ID NO.7, and the nucleotide sequence of the promoter P _{BBa_J23108} is shown in a sequence table SEQ ID NO. 8.

Preferably, the uracil nucleotide production strain integrates a wild type B.subtilis168 orotidine monophosphate decarboxylase gene pyrF and a orotidine transferase gene pyrE at the ilvG locus and is regulated by a promoter P _{BBa_J23100}; the nucleotide sequences of the orotidine monophosphate decarboxylase gene and the orotidine ribosyl transferase gene pyrF-pyrE are shown in the sequence table SEQ ID NO. 9; the nucleotide sequence of the promoter P _{BBa_J23100} is shown in a sequence table SEQ ID NO. 10.

Preferably, the uracil nucleotide production strain integrates the phosphate ABC transporter gene pstC, pstA, pstB in series at yjiT locus and is regulated by the same promoter P _{BBa_J23108}; the nucleotide sequence of the phosphoric acid ABC transporter gene pstC-pstA-pstB is shown in a sequence table SEQ ID NO. 11.

Preferably, the uracil nucleotide producing strain replaces the natural promoter of the uridylic kinase gene pyrH with the promoter P _fliC of the E.coli flagellum filaggrin gene fliC; the nucleotide sequence of the uridylic acid kinase gene pyrH is shown in a sequence table SEQ ID NO.12, the nucleotide sequence of the promoter P _fliC is shown in a sequence table SEQ ID NO.13, and the nucleotide sequence of the natural promoter of the uridylic acid kinase gene pyrH is shown in a sequence table SEQ ID NO. 14.

The directional transformation method of the uracil nucleotide production strain comprises the following specific steps:

(1) The ushA Gene (Gene ID: 947331), nagD Gene (Gene ID: 945283), surE Gene (Gene ID: 947211), yjjG Gene (Gene ID: 948899) and ygdH Gene (Gene ID: 947266) were knocked out successively on the E.coli Ora12 genome to block the UMP degradation pathway to uridine and uracil; knocking out the pykF Gene (Gene ID: 946179) to reduce the carbon metabolism flow to the TCA cycle, improving the conversion;

(2) Sequentially integrating the wild type bacillus subtilis B.subilis 168 ribophosphopyrophosphatase Gene prs (Gene ID: 936985) subjected to codon optimization at ylbE and yeeP sites, regulating and controlling by using a promoter P _{BBa_J23108}, introducing heterologously and double-copying ribophosphopyrophosphatase Gene prs, and enhancing PRPP synthesis;

(3) The wild type B.subtilis168 orotidine monophosphate decarboxylase Gene pyrF (Gene ID: 935960) and the orotidine transferase Gene pyrE (Gene ID: 936714) were integrated at the ilvG locus and regulated by the promoter P _{BBa_J23100}; heterologous introduction and overexpression of orotidine monophosphate decarboxylase gene pyrF and orotidine ribosyltransferase gene pyrE, enhancing UMP synthesis pathway;

(4) The phosphate ABC transporter genes pstC (Gene ID: 948238), pstA (Gene ID: 948239), pstB (Gene ID: 948240) were integrated in tandem at the yjiT position and regulated with the same promoter P _{BBa_J23108}; over-expressing the phosphoric acid ABC transporter gene pstC-pstA-pstB, enhancing the utilization efficiency of the strain on phosphate, and facilitating the synthesis of PRPP;

(5) The natural promoter of the uridylic acid kinase Gene pyrH (Gene ID: 944989) is replaced by the promoter P _fliC of the E.coli flagellum filaggrin Gene fliC, and the uridylic acid kinase Gene pyrH is regulated dynamically, so that UMP accumulation can be enhanced under the condition of ensuring the growth of thalli.

The uracil nucleotide production strain is applied to fermentation production of uracil nucleotide.

Preferably, the uracil nucleotide is produced by shake flask fermentation by using the uracil nucleotide production strain, and the specific steps are as follows:

(1) Seed activation and culture: uniformly coating the strain on an activation inclined plane, culturing at 37 ℃ for 12 h, transferring the activation inclined plane to continue culturing for 10h, and transferring the strain into a shaking tube containing a seed culture medium for seed culture;

(2) Fermentation culture: inoculating seed solution into triangular flask containing fermentation medium according to 10-15% inoculum size, sealing nine layers of gauze, shake culturing at 36deg.C at 200 r/min, and maintaining pH at 7.0-7.2 by adding ammonia water during fermentation; adding 60% (m/v) glucose solution to maintain fermentation (taking phenol red as indicator, treating as sugar deficiency when color of fermentation liquid is no longer changed, and adding 1-2 mL 60% (m/v) glucose solution when sugar deficiency occurs); the fermentation period is 30-32 h.

Preferably, the application of the uracil nucleotide production strain, wherein the slant culture medium adopted in the seed activation and culture is: glucose 2 g/L, peptone 10 g/L, yeast extract 5 g/L, sodium chloride 2.5 g/L, KH ₂PO₄1.0 g/L,MgSO₄ 0.2 g/L, agar powder 25% and water for the rest, and pH 7.0-7.2; the seed culture medium is as follows: yeast powder 8 g/L, peptone 2.0 g/L,(NH₄)₂SO₄2.0 g/L, KH₂PO₄3.0 g/L,V_B1 、V_B3、V_B5、V_B12 each 2 mg/L, V _H1 mg/L,MgSO₄·7H₂ O0.5 g/L, ammonium molybdate 0.32 mg/L, boric acid 4.5 mg/L, coCl ₂·6H₂ O1.6 mg/L, and water in balance;

Preferably, the uracil nucleotide producing strain is applied, and a fermentation medium adopted in the fermentation culture is: yeast powder 10 g/L, (NaPO ₃)₆, g/L, citric acid 2 g/L,(NH₄)₂SO₄2.5 g/L,KH₂PO₄10.0 g/L,MgSO₄·7H₂O 2.0 g/L,FeSO₄·7H₂O 40 mg/L,V_B1 1 mg/L,V_B3 1 mg/L,V_B5 1 mg/L,V_B12 1 mg/L,V_H0.1 mg/L,, ammonium molybdate 0.32, mg/L, boric acid 4.5, mg/L, coCl ₂·6H₂ O1.6, mg/L, phenol red 2% (v/v), balance water.

The above culture medium can be prepared by standard method.

The beneficial effects are that:

The uracil nucleotide production strain blocks UMP to uridine and uracil degradation paths by knocking out ushA, nagD, surE, yjjG and ygdH genes by using CRISPSR/Cas 9 gene editing technology; knocking out the pykF gene to reduce the carbon metabolism flow to the TCA cycle, and improving the conversion rate; heterologous introduction and overexpression of the ribophosphopyrophosphatase gene prs, the orotidine monophosphate decarboxylase gene pyrF and the orotic ribosyl transferase gene pyrE; overexpression of the phosphate ABC transporter gene pstC-pstA-pstB synergistically enhances UMP anabolic pathways; finally, dynamically regulating and controlling a uridylic acid kinase gene pyrH, and realizing UMP rapid accumulation under the condition of not affecting the growth of thalli; the strain does not contain plasmid, does not need induction, has the advantages of good genetic stability and the like, and can efficiently synthesize uracil nucleotide from the head by taking glucose as a substrate, and the uracil nucleotide yield can be up to 2.3 g/L after 32 h shake flask fermentation.

Drawings

FIG. 1 is a schematic representation of a directed engineering process for uracil nucleotide production strains.

Detailed Description

In order to enable those skilled in the art to better understand the technical scheme of the present invention, the technical scheme of the present invention will be further described in detail below with reference to the specific embodiments.

The percentage "%" referred to in the examples refers to volume percentage unless otherwise specified; the percentage "% (m/v)" of the solution refers to the grams of solute contained in the 100 ml solution.

The starting strain used in the examples was orotic acid high-yielding strain E.coli Ora12 as strain E.coli Ora12 in CN 202410290893.1; wild type bacillus subtilis b.subtilis 168 is b.subtilisatcc 23857 (commercially available); the corresponding promoter and gene are shown in the sequence table.

The specific operation steps of the adopted gene editing method refer to the patent (ZL 202210417923.1) of a genetic engineering bacterium for producing orotic acid, a construction method and application thereof, and the adopted genetic editing method refers to the patent (the specific operation steps refer to the patent) of the engineering plasmid pGRB which takes pUC18 as a framework and comprises a promoter J23100, a gRNA-Cas9 binding region sequence, a terminator sequence and ampicillin resistance (the working concentration is 100 mg/L). The terms of gene integration, construction of plasmids, etc., referred to in the examples below, are explained in this article. The primers used in the strain construction are shown in Table 1.

TABLE 1 primers involved in the construction of strains

Primer(s)	Sequence number	Sequence (5 'end to 3' end)
			ushA-QC-1	SEQ ID NO.15	ACTGAAGCAGCGGATAAATCGC
ushA-QC-2	SEQ ID NO.16	GCTCACCTCACCTTTCGGTTCTGGTTAACAGCGCTAACGCCA
			ushA-QC-3	SEQ ID NO.17	TGGCGTTAGCGCTGTTAACCAGAACCGAAAGGTGAGGTGAGC
ushA-QC-4	SEQ ID NO.18	GGTGGCAGTGAACGGTGATATG
			pGRB-ushA-S	SEQ ID NO.19	AGTCCTAGGTATAATACTAGTTGAATATGGCGAATATGGTCGTTTTAGAGCTAGAA
pGRB-ushA-A	SEQ ID NO.20	TTCTAGCTCTAAAACGACCATATTCGCCATATTCAACTAGTATTATACCTAGGACT
			nagD-QC-1	SEQ ID NO.21	TGCCGGTGAAATCACCGAAG
nagD-QC-2	SEQ ID NO.22	AGCGACTGACGGGTAAATCCGGCGACGTTATCGTGCATCAG
			nagD-QC-3	SEQ ID NO.23	CTGATGCACGATAACGTCGCCGGATTTACCCGTCAGTCGCT
nagD-QC-4	SEQ ID NO.24	CGTAATGGTGATTGTCGATTGGTGAA
			pGRB-nagD-S	SEQ ID NO.25	AGTCCTAGGTATAATACTAGTCGCAGCATTAAACAAAATGCGTTTTAGAGCTAGAA
pGRB-nagD-A	SEQ ID NO.26	TTCTAGCTCTAAAACGCATTTTGTTTAATGCTGCGACTAGTATTATACCTAGGACT
			surE-QC-1	SEQ ID NO.27	TCGAACAAGCAGCTGTCGC
surE-QC-2	SEQ ID NO.28	CAGCGGCGTGATGGAGACATAACGTCAGCAAACTCACGCAA
			surE-QC-3	SEQ ID NO.29	TTGCGTGAGTTTGCTGACGTTATGTCTCCATCACGCCGCTG
surE-QC-4	SEQ ID NO.30	CAGGATTGCCGTTTGATATCCCGAA
			pGRB-surE-S	SEQ ID NO.31	AGTCCTAGGTATAATACTAGTCGCCCGGACATTGTTGTGTCGTTTTAGAGCTAGAA
pGRB-surE-A	SEQ ID NO.32	TTCTAGCTCTAAAACGACACAACAATGTCCGGGCGACTAGTATTATACCTAGGACT
			yjjG-QC-1	SEQ ID NO.33	CTGACTATCAGCGTCAGGGATTGG
yjjG-QC-2	SEQ ID NO.34	CTCCAGTTCGTGCAACGAAGAACCGTTTTGATAATCCACCCACAGTG
			yjjG-QC-3	SEQ ID NO.35	CACTGTGGGTGGATTATCAAAACGGTTCTTCGTTGCACGAACTGGAG
yjjG-QC-4	SEQ ID NO.36	TCTCCATCCAGTCCGACTTAGC
			pGRB-yjjG-S	SEQ ID NO.37	AGTCCTAGGTATAATACTAGTCGAAGCCTTTATTAATGCGAGTTTTAGAGCTAGAA
pGRB-yjjG-A	SEQ ID NO.38	TTCTAGCTCTAAAACTCGCATTAATAAAGGCTTCGACTAGTATTATACCTAGGACT
			ygdH-QC-1	SEQ ID NO.39	TACCTGGTCTCATTCCGTCATCAC
ygdH-QC-2	SEQ ID NO.40	CCTGGCAACTTCATACGATGCTGCAGTTGATAGAGGTCGCTGCTG
			ygdH-QC-3	SEQ ID NO.41	CAGCAGCGACCTCTATCAACTGCAGCATCGTATGAAGTTGCCAGG
ygdH-QC-4	SEQ ID NO.42	GACATCTGACTTCTTCCGGGGC
			pGRB-ygdH-S	SEQ ID NO.43	AGTCCTAGGTATAATACTAGTCGCCAAATATGGTGGTCTGCGTTTTAGAGCTAGAA
pGRB-ygdH-A	SEQ ID NO.44	TTCTAGCTCTAAAACGCAGACCACCATATTTGGCGACTAGTATTATACCTAGGACT
			pykF-QC-1	SEQ ID NO.45	TCAGCACTTTGGACTGTAGAACTCA
pykF-QC-2	SEQ ID NO.46	TTCGTTGGTGGTCAGTGCCCGGCGGTTTTACCAGTTTTG
			pykF-QC-3	SEQ ID NO.47	CAAAACTGGTAAAACCGCCGGGCACTGACCACCAACGAA
pykF-QC-4	SEQ ID NO.48	TACAGGACGTGAACAGATGCG
			pGRB-pykF-S	SEQ ID NO.49	AGTCCTAGGTATAATACTAGTCGCACCATGAAACTGGAAGGGTTTTAGAGCTAGAA
pGRB-pykF-A	SEQ ID NO.50	TTCTAGCTCTAAAACCCTTCCAGTTTCATGGTGCGACTAGTATTATACCTAGGACT
			ylbE-1	SEQ ID NO.51	ACCCAACCTTACGCAACCAG
ylbE-J23108-2	SEQ ID NO.52	GGTATATCTCCTTGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAGTTGTTCGATAACCGCAGCAT
			ylbE-3	SEQ ID NO.53	AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA
ylbE-4	SEQ ID NO.54	GGCGTAACTCAGCAGGCAG
			pGRB-ylbE-S	SEQ ID NO.55	AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA
pGRB-ylbE-A	SEQ ID NO.56	TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT
			bsu-prs-A	SEQ ID NO.57	AGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGGCTGAACAGGTAAGAGACGCTTTG
bsu-prs-S	SEQ ID NO.58	CTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAGGAAACAGACCATGAGCAACCAGTACGGCGATA
			yeeP-1	SEQ ID NO.59	GGTCAGGAGGTAACTTATCAGCG
yeeP-J23108-2	SEQ ID NO.60	GGTATATCTCCTTGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAGAAAACGGAGCCCTGCCAT
			yeeP-3	SEQ ID NO.61	TTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAACTGGATTTTCTTCTGAACCTGT
yeeP-4	SEQ ID NO.62	ACGATGTCAGCAGCCAGCA
			pGRB-yeeP-S	SEQ ID NO.63	AGTCCTAGGTATAATACTAGTACAGAATATTCGCGAAAAAACGGGTTTTAGAGCTAGAA
pGRB-yeeP-A	SEQ ID NO.64	TTCTAGCTCTAAAACCCGTTTTTTCGCGAATATTCTGTACTAGTATTATACCTAGGACT
			ilvG-1	SEQ ID NO.65	ACCGAGGAGCAGACAATGAATAA
ilvG-J23100-2	SEQ ID NO.66	TTGCTAGCACTGTACCTAGGACTGAGCTAGCCGTCAAGGTGATGGCAACAACAGGGA
			ilvG-3	SEQ ID NO.67	AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGT
ilvG-4	SEQ ID NO.68	GCGCTGGCTAACATGAGGAA
			pGRB-ilvG-S	SEQ ID NO.69	AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA
pGRB-ilvG-A	SEQ ID NO.70	TTCTAGCTCTAAAACTGATGCGCGGCAACTCTTCCACTAGTATTATACCTAGGACT
			pyrFE-J23100-S	SEQ ID NO.71	TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAAGGAGATATACCATGAAAAACAACCTGCCCATCATCG
pyrFE-A	SEQ ID NO.72	CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATTTTTTAAACCAATCTTTTGACTCG
			yjiT-1	SEQ ID NO.73	AATAGTTGTTGCCGCCTGAGT
yjiT-J23108-2	SEQ ID NO.74	GGTATATCTCCTTGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAGAAAACAGGCAGCAAAGTCCC
			yjiT-3	SEQ ID NO.75	ATCTGGAAAACAAAGGCCAGTAAAAGCACTACCTGTGAAGGGATGT
yjiT-4	SEQ ID NO.76	CAGGGCTTCCACAGTCACAAT
			pGRB-yjiT-S	SEQ ID NO.77	AGTCCTAGGTATAATACTAGTAGGGATTATGAACGGCAATGGTTTTAGAGCTAGAA
pGRB-yjiT-A	SEQ ID NO.78	TTCTAGCTCTAAAACCATTGCCGTTCATAATCCCTACTAGTATTATACCTAGGACT
			pstC-J23108-S	SEQ ID NO.79	CTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAGGAGATATACCATGGCTGCAACCAAGCCTG
pstA-RBS-A	SEQ ID NO.80	GGTCTGTTTCCTTCAACCGTGTTTATTCTTCGCAAAAACA
			pstB-RBS-S	SEQ ID NO.81	AGGAAACAGACCATGAGTATGGTTGAAACTGCCCC
pstB-A	SEQ ID NO.82	TCAACCGTAACGACCGGTGAT
			DW-PpyrH-A	SEQ ID NO.83	TAGCTTCTGCCCAGCTGTAGC
pyrH-fliC-DW-S	SEQ ID NO.84	ACAGGGTTGACGGCGATTGAGCCGACGGGTGGAAACCCAATACGTAATCAACGACTTGCAATATAGGATAACGAATCATGGCTACCAATGCAAAACCC
			UP-PpyrH-fliC-A	SEQ ID NO.85	TCAATCGCCGTCAACCCTGTTATCGTCTGTCGTAAAACAACCTTTAGAATTTTTTTCAAAAACAGCCATTTTTTAATTAAGACTGCTTGGACATCGCAG
UP-PpyrH-S	SEQ ID NO.86	CTATGCACGTTGCTGCAAGC
			PfliC-JD-S	SEQ ID NO.87	ACGACAGACGATAACAGGGTTGAC
pGRB-PpyrH-S	SEQ ID NO.88	AGTCCTAGGTATAATACTAGTTAAATTCAGCTAACCCTTGTGTTTTAGAGCTAGAA
			pGRB-PpyrH-A	SEQ ID NO.89	TTCTAGCTCTAAAACACAAGGGTTAGCTGAATTTAACTAGTATTATACCTAGGACT

Example 1

As shown in fig. 1, the specific process of constructing the genetically engineered strain is as follows:

1.1 knockout of ushA Gene

E.coli Ora12/pRed-Cas9 electrotransformation competent cells were prepared from the starting strain E.coli Ora12 by the procedure described in patent ZL 202210417923.1.

The genome of Escherichia coli W3110 (wild-type Escherichia coli) extracted and diluted to a usable concentration is used as a template, PCR amplification is performed with primers ushA-QC-1 and ushA-QC-2, ushA-QC-3 and ushA-QC-4, respectively, to obtain an upstream homology arm ushA-QC-UP and a downstream homology arm ushA-QC-DW, and overlapping PCR is performed with the recovered upstream and downstream homology arms as a template with primers ushA-QC-1 and ushA-QC-4 to obtain a copy patch segment DeltaushA required for knocking out the gene ushA. Then, DNA fragments obtained by annealing the primers pGRB-ushA-S and pGRB-ushA-a were ligated with the plasmid pGRB in a linear manner to construct pGRB-ushA. Finally, carrying out electrotransformation on pGRB-ushA plasmid and an overlapped fragment delta ushA into E.coliOra12/pRed-Cas9 electrotransformation competent cells; then using ushA-QC-1 and ushA-QC-4 as identification primers, screening positive transformants to obtain the strain E.

1.2 Knockout of nagD Gene

The E.coli W3110 genome with available concentration is used as a template, primers nagD-QC-1 and nagD-QC-2, nagD-QC-3 and nagD-QC-4 are used for PCR amplification respectively to obtain an upstream homology arm nagD-QC-UP and a downstream homology arm nagD-QC-DW, the recovered upstream homology arm and the recovered downstream homology arm are used as templates, and the primers nagD-QC-1 and nagD-QC-4 are used for overlap PCR to obtain a patch returning segment ΔnagD required by knockout of the gene nagD. Then, the DNA fragments obtained by annealing the primers pGRB-nagD-S and pGRB-nagD-A were ligated with the plasmid pGRB in a line, thereby constructing pGRB-nagD. Finally, carrying out electrotransformation on pGRB-nagD plasmid and the overlapped fragment delta nagD into electrotransformation competent cells of E.colummp 1/pRed-Cas 9; and then, using nagD-QC-1 and nagD-QC-4 as identification primers, screening positive transformants to obtain the strain E.

1.3 Knockout of Gene surE

The E.coli W3110 genome with the available concentration is used as a template, primers surE-QC-1 and surE-QC-2, survivin-QC-3 and surE-QC-4 are used for PCR amplification respectively to obtain an upstream homology arm surE-QC-UP and a downstream homology arm surE-QC-DW, and the recovered upstream homology arm and the recovered downstream homology arm are used as templates for overlap PCR by using the primers surE-QC-1 and surE-QC-4 to obtain a copy patch segment delta surE required by the knocked-out gene surE. Then, the DNA fragments obtained by annealing the primers pGRB-surE-S and pGRB-surE-A were ligated with plasmid pGRB in a linear manner to construct pGRB-surE. Finally, carrying out electrotransformation on pGRB-surE plasmid and an overlapped fragment delta surE into electrotransformation competent cells of E.coli 2/pRed-Cas 9; then surE-QC-1 and surE-QC-4 are used as identification primers, positive transformants are screened, and the strain E.colump3 is obtained.

1.4 Knockout of Gene yjjG

The E.coli W3110 genome with the available concentration is used as a template, primers yjjG-QC-1 and yjjG-QC-2, yjjG-QC-3 and yjjG-QC-4 are used for PCR amplification to obtain an upstream homology arm yjjG-QC-UP and a downstream homology arm yjjG-QC-DW, and the recovered upstream homology arm and the recovered downstream homology arm are used as templates, and the primers yjjG-QC-1 and yjjG-QC-4 are used for overlap PCR to obtain a copy patch segment delta yjjG required by the knocked-out gene yjjG. Then, the DNA fragments obtained by annealing the primers pGRB-yjjG-S and pGRB-yjjG-A were ligated with plasmid pGRB in a linear manner to construct pGRB-yjjG. Finally, carrying out electrotransformation on pGRB-yjjG plasmid and an overlapped fragment delta yjjG into electrotransformation competent cells of E.coli 3/pRed-Cas 9; then yjjG-QC-1 and yjjG-QC-4 are used as identification primers, positive transformants are screened, and the strain E.colump4 is obtained.

1.5 Knockout of Gene ygdH

The E.coli W3110 genome with the available concentration is used as a template, primers ygdH-QC-1 and ygdH-QC-2, ygdH-QC-3 and ygdH-QC-4 are used for PCR amplification respectively to obtain an upstream homology arm ygdH-QC-UP and a downstream homology arm ygdH-QC-DW, and the recovered upstream homology arm and the recovered downstream homology arm are used as templates, and the primers ygdH-QC-1 and ygdH-QC-4 are used for overlap PCR to obtain a copy patch segment delta ygdH required by the knocked-out gene ygdH. Then, the DNA fragments obtained by annealing the primers pGRB-ygdH-S and pGRB-ygdH-A were ligated with plasmid pGRB in a linear manner to construct pGRB-ygdH. Finally, carrying out electrotransformation on pGRB-ygdH plasmid and an overlapped fragment delta ygdH into electrotransformation competent cells of E.coli 4/pRed-Cas 9; then ygdH-QC-1 and ygdH-QC-4 are used as identification primers, positive transformants are screened, and the strain E.colump5 is obtained.

1.6 Knockout of the Gene pykF

The E.coli W3110 genome with the available concentration is used as a template, primers pykF-QC-1 and pykF-QC-2, pykF-QC-3 and pykF-QC-4 are used for PCR amplification respectively to obtain an upstream homology arm pykF-QC-UP and a downstream homology arm pykF-QC-DW, the recovered upstream homology arm is used as a template, and the primers pykF-QC-1 and pykF-QC-4 are used for overlap PCR to obtain a patch returning segment delta pykF required by knocking out the gene pykF. Thereafter, the DNA fragments obtained by annealing the primers pGRB-pykF-S and pGRB-pykF-A were ligated with plasmid pGRB in line to construct pGRB-pykF. Finally electrotransferring pGRB-pykF plasmid and overlapping fragment Δpykf into electrotransferring competent cells of e.colump5/pRed-Cas 9; then, positive transformants were selected with pykF-QC-1 and pykF-QC-4 as identification primers to obtain strain E.colump6.

1.7 Integration of the gene prs (ylbE:: integration of P _J23108 -prs)

Coli W3110 was used as a template with primers ylbE-1 and ylbE-J23108-2, ylbE-3 and ylbE-4; PCR amplification is carried out by using synthesized gene prs after codon optimization as a template and primers bsu-prs-S and bsu-prs-A to obtain an upper homology arm ylbE-J23108-UP, a lower homology arm ylbE-DW and an intermediate target fragment bsu-prs, overlapping PCR is carried out by using recovered upstream and downstream homology arms and the intermediate target fragment as templates and primers ylbE-1 and ylbE-4 to obtain target fragment ylbE-P _J23108 -bsu-prs required for integration, and then DNA fragments obtained by annealing primers pGRB-ylbE-S and pGRB-ylbE-A are connected with plasmid pGRB in a line to construct pGRB-ylbE plasmid. Finally, the purified ylbE-P _J23108 -bsu-prs integrated fragment and the plasmid pGRB-ylbE are simultaneously transferred into competent cells of E.coli 6/pRed-Cas9 in an electrotransformation mode, and positive transformants are screened by using primers ylbE-1 and ylbE-4 as identification primers, so that the strain E.coli 7 is finally obtained.

1.8 Double copy of the prs gene (yeeP:: integration of P _J23108 -prs)

The E.coli W3110 was used as a template, primers yeeP-1 and yeeP-J23108-2, yeeP-3 and yeeP-4 were used for PCR amplification to obtain upper homology arm yeeP-J23108-UP, lower homology arm yeeP-DW was used as a template, the recovered upstream and downstream homology arms and intermediate target fragment bsu-prs were used as templates, primers yeeP-1 and yeeP-4 were used for overlap PCR to obtain target fragment yeeP-P _J23108 -bsu-prs required for integration, and then the DNA fragment obtained by annealing primers pGRB-yeeP-S and pGRB-yeeP-A was ligated on-line with plasmid pGRB to construct plasmid pGRB-yeeP. Finally, the purified yeeP-P _J23108 -bsu-prs integrated fragment and the plasmid pGRB-yeeP are simultaneously transferred into competent cells of E.coli 7/pRed-Cas9 in an electrotransformation mode, and positive transformants are screened by using primers yeeP-1 and yeeP-4 as identification primers, so that the strain E.coli 8 is finally obtained.

1.9 Integration of Gene pyrFE (ilvG:: integration of P _J23100 -pyrFE)

Using Escherichia coli W3110 as a template, and using primers ilvG-1 and ilvG-J23100-2, ilvG-3 and ilvG-4; ext> theext> bacillusext> subtilisext> B.subtilis168ext> genomeext> isext> usedext> asext> aext> templateext>,ext> primersext> pyrFEext> -ext> Jext> 23100ext> -ext> Sext> andext> pyrFEext> -ext> Aext> areext> usedext> forext> carryingext> outext> PCRext> amplificationext> toext> obtainext> anext> upperext> homologyext> armext> ilvGext> -ext> Jext> 23100ext> -ext> UPext>,ext> aext> lowerext> homologyext> armext> ilvGext> -ext> DWext> andext> anext> intermediateext> targetext> fragmentext> pyrFEext>,ext> theext> recoveredext> upstreamext> andext> downstreamext> homologyext> armsext> andext> theext> intermediateext> targetext> fragmentext> areext> usedext> asext> templatesext>,ext> primersext> ilvGext> -ext> 1ext> andext> ilvGext> -ext> 4ext> areext> usedext> forext> carryingext> outext> overlappingext> PCRext> toext> obtainext> aext> targetext> fragmentext> Pext> _J23100ext> -ext> pyrFEext> requiredext> forext> integrationext>,ext> andext> thenext> DNAext> fragmentsext> obtainedext> byext> annealingext> theext> primersext> pGRBext> -ext> ilvGext> -ext> Sext> andext> pGRBext> -ext> ilvGext> -ext> Aext> areext> connectedext> withext> aext> plasmidext> pGRBext> inext> aext> lineext> modeext> toext> constructext> aext> pGRBext> -ext> ilvGext> plasmidext>.ext> Finally, the purified P _J23100 -pyrFE integrated fragment and the plasmid pGRB-ilvG are simultaneously transferred into competent cells of E.colump 8/pRed-Cas9 in an electrotransformation mode, and positive transformants are screened by taking primers ilvG-1 and ilvG-4 as identification primers, so that the strain E.colump 9 is finally obtained.

1.10 Integration of Gene pstCAB (yjiT:: integration of P _J23108 -pstCAB)

The E.coli W3110 was used as se:Sup>A template, primers yjiT-1 and yjiT-J23108-2, yjiT-3 and yjiT-4, pstC-J23108-S and pstA-RBS-A, pstB-RBS-S and pstB-A were used for PCR amplification to obtain upper homology arms yjiT-J23108-UP, lower homology arms yjiT-DW and intermediate target fragments pstCAB-1 and pstCAB-2, the recovered upstream and downstream homology arms and intermediate target fragments were used as templates, primers yjiT-1 and yjiT-4 were used for overlap PCR to obtain the target fragment yjiT-P _J23108 -pstCAB for integration, and then DNA fragments obtained by annealing primers pGRB-yjiT-S and pGRB-yjiT-A were ligated with plasmid pGRB in se:Sup>A line, to construct plasmid pGRB-yjiT. Finally, the purified yjiT-P _J23108 -pstCAB integrated fragment and the plasmid pGRB-yjiT are simultaneously transferred into competent cells of E.coli 9/pRed-Cas9 in an electrotransformation mode, and positive transformants are screened by taking primers yjiT-1 and yjiT-4 as identification primers, so that the strain E.coli 10 is finally obtained.

1.11 Gene pyrH promoter (P _pyrH::P_fliC)

The E.coli W3110 was used as se:Sub>A template, primers UP-PpyrH-S and UP-PpyrH-fliC-A, DW-PpyrH-A and pyrH-fliC-DW-S were used for PCR amplification to obtain an upper homology arm UP-PpyrH-fliC, se:Sub>A lower homology arm DW-PpyrH-A was used as se:Sub>A template, the primers UP-PpyrH-S and DW-PpyrH-A were used for overlapping PCR to obtain se:Sub>A target fragment PpyrH-fliC required for integration, and then DNA fragments obtained by annealing primers pGRB-PpyrH-S and pGRB-PpyrH-A were ligated with plasmid pGRB line to construct plasmid pGRB-PpyrH. Finally, the purified PpyrH-fliC integration fragment and plasmid pGRB-PpyrH are simultaneously transferred into competent cells of E.colummp 10/pRed-Cas9 in an electrotransformation mode, and positive transformants are screened by using primers PfliC-JD-S and DW-PpyrH-A as identification primers, so that the strain E.colummp 11 is finally obtained.

The strains involved in the above construction are shown in Table 2.

Strains according to Table 2

Strain name	Genotype ("x" represents codon optimized)
		E.coliump1	E.coliOra12,ΔushA
E.coliump2	E.coliump1,ΔnagD
		E.coliump3	E.coliump2,ΔsurE
E.coliump4	E.coliump3,ΔyjjG
		E.coliump5	E.coliump4,ΔygdH
E.coliump6	E.coliump5,ΔpykF
		E.coliump7	E.coliump6,ylbE::PJ23108-prs*
E.coliump8	E.coliump7,yeeP::PJ23108-prs*
		E.coliump9	E.coliump8,ilvG::PJ23100-pyrFE
E.coliump10	E.coliump9,yjiT::PJ23108-pstCAB
		E.coliump11	E.coliump10,PpyrH::PfliC

Example 2

Uracil nucleotides were produced by shake flask fermentation using uracil nucleotide producing strain E.colummpl 11 as described in example 1.

2.1 Medium

2.1.1 Inclined plane culture medium

Glucose 2 g/L, peptone 10 g/L, yeast extract 5 g/L, sodium chloride 2.5 g/L, KH ₂PO₄1.0 g/L,MgSO₄ 0.2 g/L, agar 25% and water, adjusting pH to 7.0-7.2 with sodium hydroxide, constant volume to 500: 500 ml, packaging into test tube (9 ml/tube) and eggplant-shaped bottle (45 ml/bottle), and sterilizing in 121 deg.C autoclave for 20 min.

2.1.2 Seed Medium

Yeast powder 8 g/L, peptone 2.0 g/L,(NH₄)₂SO₄2.0 g/L, KH₂PO₄3.0 g/L,V_B1 、V_B3、V_B5、V_B12 each 2mg/L, V _H1 mg/L,MgSO₄·7H₂ O0.5 g/L, ammonium molybdate 0.32 mg/L, boric acid 4.5 mg/L, coCl ₂·6H₂ O1.6 mg/L, and the balance water.

2.1.3 Fermentation Medium

Yeast powder 10 g/L, (NaPO ₃)₆, g/L, citric acid 2 g/L,(NH₄)₂SO₄2.5 g/L,KH₂PO₄10.0 g/L,MgSO₄·7H₂O 2.0 g/L,FeSO₄·7H₂O 40 mg/L,V_B1 1 mg/L,V_B3 1 mg/L,V_B51 mg/L,V_B12 1 mg/L,V_H0.1 mg/L,, ammonium molybdate 0.32, mg/L, boric acid 4.5, mg/L, coCl ₂·6H₂ O1.6, mg/L, phenol red 2% (v/v), balance water.

2.2 Culture method

2.2.1 Seed activation and cultivation:

Uniformly coating the strain on an activation inclined plane, culturing at 37 ℃ for 12 h, transferring the activation inclined plane to continue culturing for 10 h, and transferring the strain into a shaking tube containing 5 ml seed culture medium for seed culture;

2.2.2 fermentation culture:

Inoculating the seed solution into 500 mL triangular flask (final volume of 30 mL) filled with fermentation medium according to 15% inoculum size, sealing nine layers of gauze, shake culturing at 36deg.C at 200 r/min, and maintaining pH at 7.0-7.2 by adding ammonia water during fermentation; the fermentation is maintained by adding 60% (m/v) glucose solution (phenol red is used as an indicator, the color of the fermentation liquor is regarded as sugar deficiency when no change occurs, and 1-2 mL of 60% (m/v) glucose solution is added when sugar deficiency occurs). The fermentation period is 32 h, and no antibiotics or inducers are added in the fermentation process.

After 32 h shake flask fermentation, uracil nucleotide yield is up to 2.3 g/L.

The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be appreciated by those skilled in the art that variations and modifications of the invention and strain changes, which are carried out by or based on the methods of this invention, may be made without departing from the spirit of this invention.

Claims (10)

1. A uracil nucleotide producing strain, characterized by: is obtained by further reforming on the basis of an original strain E.coliOra12 by using a directional reforming method, and specifically comprises the following steps: the ushA gene, nagD gene, surE gene, yjjG gene, ygdH gene and pykF gene were knocked out on E.coli Ora12 genome, and the ribophosphopyrophosphatase gene prs, orotidine monophosphate decarboxylase gene pyrF and orotic ribosyltransferase gene pyrE were introduced heterologous into B.subtilis 168 of Bacillus subtilis, over-expressed in phosphate ABC transporter gene pstC, pstA, pstB and regulated dynamically in the uridylate kinase gene pyrH.

2. The uracil nucleotide producing strain according to claim 1, wherein: the directional transformation method is to completely transform the original strain E.coli Ora12 genome by using CRISPR/Cas9 gene editing technology.

3. The uracil nucleotide producing strain according to claim 1, wherein: the nucleotide sequence of the ushA gene is shown in a sequence table SEQ ID NO. 1; the nucleotide sequence of nagD gene is shown in sequence table SEQ ID NO. 2; the nucleotide sequence of surE gene is shown in sequence table SEQ ID NO. 3; the nucleotide sequence of yjjG gene is shown in sequence table SEQ ID NO. 4; the nucleotide sequence of ygdH gene is shown in sequence table SEQ ID NO. 5; the nucleotide sequence of the pykF gene is shown in a sequence table SEQ ID NO. 6.

4. The uracil nucleotide producing strain according to claim 1, wherein: integrating a wild type bacillus subtilis 168 ribophosphopyrophosphatase gene prs after codon optimization at ylbE site, regulating and controlling by using a promoter P _{BBa_J23108}, and integrating again at yeeP site; the nucleotide sequence of the prs gene after codon optimization is shown in a sequence table SEQ ID NO.7, and the nucleotide sequence of the promoter P _{BBa_J23108} is shown in a sequence table SEQ ID NO. 8.

5. The uracil nucleotide producing strain according to claim 1, wherein: integrating a wild type bacillus subtilis 168 orotidine monophosphate decarboxylase gene pyrF and a orotidine transferase gene pyrE at the ilvG locus and regulated by a promoter P _{BBa_J23100}; the nucleotide sequences of the orotidine monophosphate decarboxylase gene and the orotidine ribosyl transferase gene pyrF-pyrE are shown in the sequence table SEQ ID NO. 9; the nucleotide sequence of the promoter P _{BBa_J23100} is shown in a sequence table SEQ ID NO. 10; the phosphate ABC transporter gene pstC, pstA, pstB is integrated in series at yjiT site and regulated by the same promoter P _{BBa_J23108}; the nucleotide sequence of the phosphoric acid ABC transporter gene pstC-pstA-pstB is shown in a sequence table SEQ ID NO. 11.

6. The uracil nucleotide producing strain according to claim 1, wherein: replacing the natural promoter of the uridylic acid kinase gene pyrH with the promoter P _fliC of the E.coli flagellum filaggrin gene fliC; the nucleotide sequence of the uridylic acid kinase gene pyrH is shown in a sequence table SEQ ID NO.12, and the nucleotide sequence of the promoter P _fliC is shown in a sequence table SEQ ID NO. 13; the nucleotide sequence of the natural promoter of the uridylic acid kinase gene pyrH is shown in a sequence table SEQ ID NO. 14.

7. A method for the directed engineering of uracil nucleotide producing strains according to any of claims 1-6, characterized in that: the method comprises the following specific steps:

(1) The ushA gene, nagD gene, surE gene, yjjG gene and ygdH gene are knocked out on the E.coli Ora12 genome; knocking out the pykF gene;

(2) Sequentially integrating the codon-optimized wild bacillus subtilis B.subilis 168 ribophosphopyrophosphatase gene prs at ylbE and yeeP sites, regulating and controlling by using a promoter P _{BBa_J23108}, and introducing heterologously and double-copying the ribophosphopyrophosphatase gene prs;

(3) Integrating a wild type bacillus subtilis 168 orotidine monophosphate decarboxylase gene pyrF and a orotidine transferase gene pyrE at the ilvG locus and regulated by a promoter P _{BBa_J23100}; heterologous introduction and overexpression of the orotidine monophosphate decarboxylase gene pyrF and the orotidine ribosyltransferase gene pyrE;

(4) The phosphate ABC transporter gene pstC, pstA, pstB is integrated in series at yjiT site and regulated by the same promoter P _{BBa_J23108}; overexpression of the phosphate ABC transporter gene pstC-pstA-pstB;

(5) The natural promoter of the uridylic acid kinase gene pyrH is replaced by the promoter P _fliC of the E.coli flagellum filaggrin gene fliC.

8. Use of the uracil nucleotide production strain according to any of claims 1-6 for fermentative production of uracil nucleotides.

9. The use of uracil nucleotide producing strain according to claim 8, wherein: uracil nucleotides are produced by shake flask fermentation, and the specific steps are as follows:

(1) Seed activation and culture: uniformly coating the strain on an activation inclined plane, culturing for 12 hours at 37 ℃, transferring the activation inclined plane to continue culturing for 10 hours, and transferring the strain into a shaking tube containing a seed culture medium to perform seed culture;

(2) Fermentation culture: inoculating seed solution into triangular flask containing fermentation medium according to 10-15% inoculum size, sealing nine layers of gauze, shake culturing at 36deg.C at 200r/min, and maintaining pH at 7.0-7.2 by adding ammonia water during fermentation; adding 60% glucose solution to maintain fermentation; the fermentation period is 30-32h.

10. The use of uracil nucleotide producing strain according to claim 9, wherein: the slant culture medium adopted in the seed activation and culture is as follows: glucose 2 g/L, peptone 10 g/L, yeast extract 5 g/L, sodium chloride 2.5 g/L, KH ₂PO₄ 1.0 g/L,MgSO₄ 0.2 g/L, agar powder 25% and water for the rest, and pH 7.0-7.2; the seed culture medium is as follows: yeast powder 8 g/L, peptone 2.0 g/L,(NH₄)₂SO₄ 2.0 g/L, KH₂PO₄3.0 g/L,V_B1 、V_B3、V_B5、V_B12 each 2 mg/L, V _H1 mg/L,MgSO₄·7H₂ O0.5 g/L, ammonium molybdate 0.32 mg/L, boric acid 4.5 mg/L, coCl ₂·6H₂ O1.6 mg/L, and water in balance; the fermentation culture medium adopted in the fermentation culture is as follows: yeast powder 10 g/L, (NaPO ₃)₆, g/L, citric acid 2 g/L,(NH₄)₂SO₄ 2.5 g/L,KH₂PO₄10.0 g/L,MgSO₄·7H₂O 2.0 g/L,FeSO₄·7H₂O 40 mg/L,V_B1 1 mg/L,V_B3 1 mg/L,V_B5 1 mg/L,V_B12 1 mg/L,V_H 0.1 mg/L,, ammonium molybdate 0.32, mg/L, boric acid 4.5, mg/L, coCl ₂·6H₂ O1.6, mg/L, phenol red 2%, balance water.