Beta-alanine producing strain as well as construction method and application thereof

Abstract

The invention provides a beta-alanine producing strain as well as a construction method and application thereof. According to the bacterial strain, an acetaldehyde dehydrogenase gene adhE, a lactic dehydrogenase gene ldhA, an acetokinase gene ackA, a pyruvate dehydrogenase gene poxB, a DNA binding transcription inhibition factor coding gene lacI, an aspartate kinase thrA, a pantothenic acid synthase gene panC, an alanine synthesis transaminase gene cycA and an alanine synthesis transaminase gene yfbQ are knocked out from an E.coli W3110 genome, and the bacterial strain is obtained. A phosphoenolpyruvate carboxylase gene ppc, a pyridine nucleotide transhydrogenase coding gene pntAB, an aspartic acid transaminase gene aspC, an aspartic acid decarboxylase gene panD derived from pseudomonas aeruginosa and a beta-alanine transporter gene NCgl0580 derived from corynebacterium glutamicum are integrated at the same time, and the bacillus subtilis is used for producing beta-alanine and has the advantage of high fermentation yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology and fermentation engineering, and particularly relates to a beta-alanine production strain and a construction method and application thereof. BACKGROUND

[0002] Beta-alanine is one of the few naturally occurring beta-amino acids, and is widely used in the fields of pharmaceuticals, food, cosmetics, environmental science and material science. According to statistics, the global beta-alanine market sales reached 520 million yuan in 2023, and is expected to reach 711 million yuan in 2030, with a broad market prospect.

[0003] At present, chemical synthesis, enzyme method and fermentation method are commonly used in the industry to produce beta-alanine. Although the chemical method has the advantages of high efficiency and short cycle, the reaction conditions are extreme, and it needs to be carried out under the conditions of high temperature and high pressure, strong acid and strong base, and has many by-products and high energy consumption. The enzyme method has the problems of low enzyme activity and easy mechanism inactivation. Compared with the above two methods, the fermentation method has the characteristics of low cost, environmental friendliness and easy cultivation, and has become the preferred method for producing beta-alanine.

[0004] However, the beta-alanine production strain currently used basically carries a plasmid and the synthetic metabolic pathway is not clear, so it has the problems of genetic instability, difficult control of the production process and low acid production efficiency. Therefore, it is an urgent technical problem to construct a beta-alanine production strain with genetic stability and high acid production efficiency. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a beta-alanine production strain.

[0006] Another technical problem to be solved by the present application is to provide a construction method of the above-mentioned beta-alanine production strain.

[0007] Another technical problem to be solved by the present application is to provide an application of the above-mentioned beta-alanine production strain.

[0008] To solve the above technical problems, the technical scheme of the present application is as follows: A beta-alanine production strain, named E. coli Al1-18, is constructed by knocking out the acetaldehyde dehydrogenase gene E. coli W3110 , the lactate dehydrogenase gene adhE , the acetate kinase gene ldhA , the pyruvate dehydrogenase gene ackA , the DNA binding transcriptional repressor encoding gene poxB , the aspartate kinase lacI and the DNA binding transcriptional repressor encoding gene on the genome of the wild-type Escherichia coli thrApyruvate carboxylase gene panC alanine synthesis transaminase gene cycA alanine synthesis transaminase gene yfbQ phosphoenolpyruvate carboxylase gene ppc pyridine nucleotide transhydrogenase encoding gene pntAB, aspartate transaminase gene aspC aspartate decarboxylase gene from Pseudomonas aeruginosa panD β-alanine transporter gene from Corynebacterium glutamicum NCgl0580 .

[0009] Preferably, the above β-alanine production strain has codon-optimized phosphoenolpyruvate carboxylase gene mbhA integrated at the genomic ycdN site and ppc promoted by Ptrc promoter; pyridine nucleotide transhydrogenase encoding gene pntAB integrated at the genomic ygaY site and regulated by the same Ptrc promoter; aspartate transaminase gene ybiC integrated at the genomic yeeP site and aspC regulated by the same Ptrc promoter; aspartate decarboxylase gene from Pseudomonas aeruginosa ylbE integrated at the genomic ilvG site, yjiT site and regulated by Ptrc promoter; β-alanine transporter gene from Corynebacterium glutamicum panD integrated at the genomic rph site and regulated by the same Ptrc promoter. NCgl0580

[0010] Preferably, the above β-alanine production strain is completely reformed on the wild-type Escherichia coli E. coli W3110 chromosomal genome of the starting strain by using CRISPR / Cas9 gene editing technology.

[0011] Preferably, the above β-alanine production strain has wild-type Escherichia coli E. coli W3110 as Escherichia coli E. coli W3110 ATCC 27325.

[0012] Preferably, the above β-alanine production strain has nucleotide sequence of adhE as shown in the sequence listing SEQ ID NO. 1; ldhA as shown in the sequence listing SEQ ID NO. 2; ackA as shown in the sequence listing SEQ ID NO. 3; poxB ​The nucleotide sequence is shown in SEQ ID NO.4 of the sequence listing; thrA The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing; panC The nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing; cycA The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing; yfbQ The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing; lacI The nucleotide sequence is shown in the sequence listing SEQ ID NO.9.

[0013] Preferably, the above-mentioned β-alanine-producing strain, wherein ppc The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing; aspC The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing; panD The nucleotide sequence is shown in SEQ ID NO. 12 of the sequence listing; NCgl0580 The nucleotide sequence of pntAB is shown in SEQ ID NO.13 of the sequence listing; the nucleotide sequence of pntAB is shown in SEQ ID NO.14 of the sequence listing.

[0014] Preferably, in the above-mentioned β-alanine producing strain, the nucleotide sequence of the Ptrc promoter is shown in the sequence listing SEQ ID NO. 15.

[0015] The above-mentioned method for constructing β-alanine-producing strains utilizes CRISPR / Cas9-mediated gene editing technology. E. coli W3110 The specific steps for targeted modification of the chromosome genome are as follows: (1) In order to further enhance the supply of pyruvate and block the branched acid synthesis pathway, knock out the pyruvate. adhE , ldhA , ackA , poxB Gene; (2) In order to weaken the L-aspartate degradation pathway, the gene encoding aspartate kinase was targeted. thrA Knock it out; (3) In order to weaken the β-alanine endocytosis pathway and block the intracellular transport of β-alanine, the gene encoding the β-alanine transporter was targeted. cycA Knock it out; (4) In order to reduce the repression of the Ptrc promoter and enhance its expression effect, the gene encoding the DNA-binding transcription repressor was targeted. lacI Knock it out; (5) In order to weaken the pantothenic acid branch of metabolism that further breaks down β-alanine into pantothenic acid, the gene encoding pantothenic acid synthase was targeted. panC Knock it out; (6) In order to weaken the L-alanine synthesis pathway, the alanine synthesis transaminase gene is knocked out yfbQ ; (7) In order to further strengthen the oxaloacetate synthesis pathway, the codon-optimized phosphoenolpyruvate carboxylase gene ppc is integrated into the mbhA site and ycdN site of the E. coli genome, and is controlled by the Ptrc promoter for transcription; (8) In order to further strengthen the L-aspartate synthesis pathway, the codon-optimized aspartate transaminase gene aspC is integrated into the ybiC site, yeeP site of the E. coli genome, and is controlled by the Ptrc promoter for transcription; (9) In order to strengthen the β-alanine synthesis pathway, the codon-optimized aspartate decarboxylase gene from Pseudomonas aeruginosa panD is integrated into the ylbE site, ilvG site and yjiT site of the E. coli genome, and is controlled by the same Ptrc promoter for transcription; (10) In order to strengthen the supply of NADPH in the cell, the pyridine nucleotide transhydrogenase encoding gene pntAB is integrated into the ygaY site of the E. coli genome, and is controlled by the same Ptrc promoter for transcription; (11) In order to strengthen the transport of β-alanine out of the cell, the β-alanine transporter gene NCgl0580 is integrated into the rph site of the genome, and is controlled by the same Ptrc promoter for transcription.

[0016] The above β-alanine producing strain is applied in the production of β-alanine.

[0017] Preferably, the above application uses a fermentation method to produce β-alanine.

[0018] Preferably, the above application has the following specific steps for shake flask fermentation: (1) Seed activation and culture: The strain is evenly coated on the activation slope, cultured at 37°C for 12h, and then transferred to the activation slope for further culture for 10h, and then transferred to a shake flask containing 5ml of seed culture medium for seed culture; (2) Fermentation culture: Inoculate 10%-15% of the culture medium into Erlenmeyer flasks containing fermentation medium, seal the flasks with nine layers of gauze, and incubate at 37℃ with shaking at 220 rpm. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia water; add glucose solution to maintain fermentation. The fermentation cycle is 24-32 hours.

[0019] Preferably, the above-mentioned application uses the following slant culture medium: glucose 2g / L, peptone 10g / L, yeast powder 5g / L, sodium chloride 2.5g / L, KH2PO4 1.0g / L, MgSO4 0.2g / L, agar powder 25%. After dissolving in water, the pH is adjusted to 7.0-7.2 with sodium hydroxide, and the volume is brought to 500 ml. The medium is then dispensed into test tubes (9 ml / tube) and flasks (45 ml / flask), and sterilized in an autoclave at 121℃ for 20 min.

[0020] Preferably, in the above application, the seed culture medium used is: yeast extract 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, the remainder being water.

[0021] Preferably, the fermentation medium used in the above application is: glucose 30.0 g / L, yeast powder 4.0 g / L, peptone 2.0 g / L, ammonium sulfate 2.0 g / L, citric acid 2.0 g / L, monosodium glutamate 2.0 g / L, MgSO4·7H2O 1.2 g / L, KH2PO4 3.0 g / L, threonine 0.5 g / L, MnSO4·H2O 5.0 mg / L, FeSO4·7H2O 15 mg / L, phenol red 2.5%, and the remainder is water.

[0022] All of the above-mentioned culture media can be prepared using standard methods.

[0023] Beneficial effects: The aforementioned β-alanine-producing strain is a plasmid-free strain that produces Escherichia coli. W3110As a chassis strain, the genetically engineered strain for de novo synthesis of high-yield beta-alanine has the advantages of easy operation, good genetic stability, high fermentation yield, etc. The beta-alanine engineering strain efficiently synthesizes beta-alanine from cheap carbon sources such as glucose, and the yield of beta-alanine can reach 6 g / L after 24 h of shake flask fermentation, which has a good industrial application prospect. The construction method of the genetically engineered strain is simple. Firstly, the beta-alanine synthesis pathway in Escherichia coli is analyzed and reconstructed to strengthen the synthesis and accumulation of beta-alanine. Then the codon-optimized aspartate decarboxylase gene of Pseudomonas aeruginosa is introduced to further strengthen the synthesis of beta-alanine, which is suitable for the needs of large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Schematic diagram of beta-alanine terminal metabolic pathway and its modification. DETAILED DESCRIPTION

[0025] In order to make those skilled in the art better understand the technical solutions of the present application, the technical solutions of the present application will be further described in detail below in combination with specific embodiments.

[0026] The percentage sign "%" involved in the embodiments refers to volume percentage, unless otherwise specified; the percentage of the solution "% (m / v)" refers to the number of grams of solute contained in 100 ml of solution.

[0027] The starting strain used in the embodiments is E. coli W3110 ATCC 27325; the codon-optimized aspartate decarboxylase gene of Pseudomonas aeruginosa panD Synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.; the corresponding promoter and gene are shown in the sequence listing. The primers used in the strain construction process are shown in Table 1.

[0028] Table 1 Primers involved in the strain construction process Primer name SEQ ID NO. Primer sequence (5'-3') adhE-US SEQ ID NO. 16 CTTAACGCACTCGTAGAGCG adhE-UA SEQ ID NO. 17 GTCTAACGCACTGATGCACCGCCGCATACCTATCGGTGTAG adhE-DS SEQ ID NO. 18 CTACACCGATAGGTATGCGGCGGTGCATCAGTGCGTTAGAC adhE-DA SEQ ID NO. 19 GGAACCCAGTTTGTGCGCC pGRB-adhE-S SEQ ID NO. 20 AGTCCTAGGTATAATACTAGTATCAACCTGATCCTCGCGACGTTTTAGAGCTAGAA pGRB-adhE-A SEQ ID NO. 21 TTCTAGCTCTAAAACGTCGCGAGGATCAGGTTGATACTAGTATTATACCTAGGACT ldhA-US SEQ ID NO. 22 CTCGCCGTTTATAGCACAAAACAG ldhA-UA SEQ ID NO. 23 GTTAGCATCACGGGTACGCTGCAGCCCCAGTTCTTTTGCCG ldhA-DS SEQ ID NO. 24 CGGCAAAAGAACTGGGGCTGCAGCGTACCCGTGATGCTAAC ldhA-DA SEQ ID NO. 25 GGTATTGACGATCATCACGCC pGRB-ldhA-S SEQ ID NO. 26 AGTCCTAGGTATAATACTAGTGCCGTTGCTGAACACGCCATGTTTTAGAGCTAGAA pGRB-ldhA-A SEQ ID NO. 27 TTCTAGCTCTAAAACATGGCGTGTTCAGCAACGGCACTAGTATTATACCTAGGACT ackA-US SEQ ID NO. 28 GTACTGGTTCTGAACTGCGGTAG ackA-UA SEQ ID NO. 29 GAAGCTGCATCTTTGATACCCTGCCTGCACCTAAAGCCGCTTC ackA-DS SEQ ID NO. 30 GAAGCGGCTTTAGGTGCAGGCAGGGTATCAAAGATGCAGCTTC ackA-DA SEQ ID NO. 31 CCGGATCGATATCACCAGAACG pGRB-ackA-S SEQ ID NO. 32 AGTCCTAGGTATAATACTAGTTCTGCGCAGCTGACTGCTATGTTTTAGAGCTAGAA pGRB-ackA-A SEQ ID NO. 33 TTCTAGCTCTAAAACATAGCAGTCAGCTGCGCAGAACTAGTATTATACCTAGGACT poxB-US SEQ ID NO. 34 CGGTTGCAGCTTATATCGCC poxB-UA SEQ ID NO. 35 ACCTGCACTTAATCAACGGCCCCCTCCAGCGAAATTGGCAG poxB-DS SEQ ID NO. 36 CTGCCAATTTCGCTGGAGGGGGCCGTTGATTAAGTGCAGGT poxB-DA SEQ ID NO. 37 GGTTTAGCTAAATCGTCCAGCCC pGRB-poxB-S SEQ ID NO. 38 AGTCCTAGGTATAATACTAGTTTGCCACCGCAATCACGTTCGTTTTAGAGCTAGAA pGRB-poxB-A SEQ ID NO. 39 TTCTAGCTCTAAAACGAACGTGATTGCGGTGGCAAACTAGTATTATACCTAGGACT thrA-US SEQ ID NO. 40 ACAGAGCACAAAATGCTGCCT thrA-UA SEQ ID NO. 41 GCATATCCAGCAAGTTCACGAAGTCAACACGAAGTTGATTTGGCCAC thrA-DS SEQ ID NO. 42 GTGGCCAAATCAACTTCGTGTTGACTTCGTGAACTTGCTGGATATGC thrA-DA SEQ ID NO. 43 GATAAGTGTGAGCACAACCAGGC pGRB-thrA-S SEQ ID NO. 44 AGTCCTAGGTATAATACTAGTCAGCGGCGAACCGCTTTCTGGTTTTAGAGCTAGAA pGRB-thrA-A SEQ ID NO. 45 TTCTAGCTCTAAAACCAGAAAGCGGTTCGCCGCTGACTAGTATTATACCTAGGACT cycA-DA SEQ ID NO. 46 ttatttccgcagttcagcagcc cycA-DS SEQ ID NO. 47 ctggttccccgatctctccgcgaaagggctgacgttctcg cycA-UA SEQ ID NO. 48 cggagagatcggggaaccagcgagaacgtcagccctttcg cycA-US SEQ ID NO. 49 atggtagatcaggtaaaagtcgttgc pGRB-cycA-S SEQ ID NO. 50 AGTCCTAGGTATAATACTAGTTTCGCGCATTTGTGGAATGACGGGTTTTAGAGCTAGAA pGRB-cycA-A SEQ ID NO. 51 TTCTAGCTCTAAAACCCGTCATTCCACAAATGCGCGAAACTAGTATTATACCTAGGACT lacI-US SEQ ID NO. 52 ATGTCGCAGAGTATGCCGG lacI-UA SEQ ID NO. 53 GTTAATGATCAGCCCACTGACGCCATCGACACCACCACGCTG lacI-DS SEQ ID NO. 54 CAGCGTGGTGGTGTCGATGGCGTCAGTGGGCTGATCATTAAC lacI-DA SEQ ID NO. 55 TCGTGCCAGCTGCATTAATGAAT pGRB-lacI-S SEQ ID NO. 56 AGTCCTAGGTATAATACTAGTGCCGTTGCTGAACACGCCATGTTTTAGAGCTAGAA pGRB-lacI-A SEQ ID NO. 57 TTCTAGCTCTAAAACATGGCGTGTTCAGCAACGGCACTAGTATTATACCTAGGACT panC-DA SEQ ID NO. 58 GGAGTCTATGATGACGCTGGCG panC-DS SEQ ID NO. 59 TTGCAGGAGGACTGCGAGAACTGGGGAACGGGATCTCGAT panC-UA SEQ ID NO. 60 ATCGAGATCCCGTTCCCCAGTTCTCGCAGTCCTCCTGCAA panC-US SEQ ID NO. 61 CAGCTGCTGGTGCTGGAATG pGRB-panC-A SEQ ID NO. 62 GGACGGCTGGCACCTTCCAGACTAGTATTATACCTAGGACTGAGC pGRB-panC-S SEQ ID NO. 63 CTGGAAGGTGCCAGCCGTCCGTTTTAGAGCTAGAAATAGCAAGTTAA yfbQ-DA SEQ ID NO. 64 TTACAGCTGATGATAACCAGAAAGGAAAC yfbQ-DS SEQ ID NO. 65 TTACCCACTCTGGACCGCGTTTATGAGCAGCGTAACCGCG yfbQ-UA SEQ ID NO. 66 CGCGGTCCAGAGTGGGTAACGCGGTTACGCTGCTCATAAA yfbQ-US SEQ ID NO. 67 ATGTCCCCCATTGAAAAATCCAGCAA pGRB-yfbQ-S SEQ ID NO. 68 AGTCCTAGGTATAATACTAGTATCAACCCAAATAACCCAACCGGGTTTTAGAGCTAGAA pGRB-yfbQ-A SEQ ID NO. 69 TTCTAGCTCTAAAACCCGGTTGGGTTATTTGGGTTGATACTAGTATTATACCTAGGACT ppc-trc-S SEQ ID NO. 70 TAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAACGAACAATATTCCGCATTGCG ppc-trc-A SEQ ID NO. 71 ACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGCCGGTATTACGCATACCTGC mbhA-US SEQ ID NO. 72 GCCAGCACGAACATAATCCCG mbhA-UA SEQ ID NO. 73 CCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACACGGTGGCAGGTTTTGGC mbhA-DS SEQ ID NO. 74 CTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-DA SEQ ID NO. 75 CGGCGTAATCACAAACTGGCC ycdN-US SEQ ID NO. 76 GATTTTGACGCCACCAACACC ycdN-UA SEQ ID NO. 77 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAATCCACATCACACAATCCATC ycdN-DS SEQ ID NO. 78 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAAGGGATTTTTGGCTATCAGG ycdN-DA SEQ ID NO. 79 GTATTCGCCAGGCTGTAAATTC pGRB-ycdN-S SEQ ID NO. 80 AGTCCTAGGTATAATACTAGTGCGTGGAAATCATCATGGCTGTTTTAGAGCTAGAA pGRB-ycdN-A SEQ ID NO. 81 TTCTAGCTCTAAAACAGCCATGATGATTTCCACGCACTAGTATTATACCTAGGACT aspC-trc-A SEQ ID NO. 82 AGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCACTGCCACAATCGC aspC-trc-S SEQ ID NO. 83 ATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTTGAGAACATTACCGCCGC ybiC-US SEQ ID NO. 84 GCGCGTTAATCAGGCATTTTAATTCT ybiC-UA SEQ ID NO. 85 TCCTGCTAGCACTGTACCTAGGACTGAGCTAGCCGTCAACCATGTGAATCATGCCCTGC ybiC-DS SEQ ID NO. 86 GAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGATGATAAGCCGATTTTGCTACCGG YbiC-DA SEQ ID NO. 87 GCAACAGATGCCTGATGCGAC pGRB-ybiC-S SEQ ID NO. 88 AGTCCTAGGTATAATACTAGTTATGTACGCTCCTGGAGTCAGTTTTAGAGCTAGAA pGRB-ybiC-A SEQ ID NO. 89 TTCTAGCTCTAAAACTGACTCCAGGAGCGTACATAACTAGTATTATACCTAGGACT yeeP-US SEQ ID NO. 90 GGTCAGGAGGTAACTTATCAGCG yeeP-UA SEQ ID NO. 91 GGTATATCTCCTTGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAGAAAACGGAGCCCTGCCAT yeeP-DS SEQ ID NO. 92 TTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAACTGGATTTTCTTCTGAACCTGT yeeP-DA SEQ ID NO. 93 ACGATGTCAGCAGCCAGCA pGRB-yeeP-S SEQ ID NO. 94 AGTCCTAGGTATAATACTAGTACAGAATATTCGCGAAAAAACGGGTTTTAGAGCTAGAA pGRB-yeeP-A SEQ ID NO. 95 TTCTAGCTCTAAAACCCGTTTTTTCGCGAATATTCTGTACTAGTATTATACCTAGGACT panD-trc-A SEQ ID NO. 96 GCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCAGGCGACCTGTATCGGAATC panD-trc-S SEQ ID NO. 97 ATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCACGCCATAATGCTCAAAG ylbE-US SEQ ID NO. 98 ACCCAACCTTACGCAACCAG ylbE-UA SEQ ID NO. 99 GGTATATCTCCTTGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAGTTGTTCGATAACCGCAGCAT ylbE-DS SEQ ID NO. 100 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA ylbE-DA SEQ ID NO. 101 GGCGTAACTCAGCAGGCAG pGRB-yibE-S SEQ ID NO. 102 AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA pGRB-yibE-A SEQ ID NO. 103 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT ilvG-US SEQ ID NO. 104 ACCGAGGAGCAGACAATGAATAA ilvG-UA SEQ ID NO. 105 TTGCTAGCACTGTACCTAGGACTGAGCTAGCCGTCAAGGTGATGGCAACAACAGGGA ilvG-DS SEQ ID NO. 106 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGT ilvG-DA SEQ ID NO. 107 GCGCTGGCTAACATGAGGAA pGRB-ilvG-S SEQ ID NO. 108 AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA pGRB-ilvG-A SEQ ID NO. 109 TTCTAGCTCTAAAACTGATGCGCGGCAACTCTTCCACTAGTATTATACCTAGGACT yjiT-US SEQ ID NO. 110 AATAGTTGTTGCCGCCTGAGT yjiT-UA SEQ ID NO. 111 GGTATATCTCCTTGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAGAAAACAGGCAGCAAAGTCCC yjiT-DS SEQ ID NO. 112 ATCTGGAAAACAAAGGCCAGTAAAAGCACTACCTGTGAAGGGATGT yjiT-DA SEQ ID NO. 113 CAGGGCTTCCACAGTCACAAT pGRB-yjiT-S SEQ ID NO. 114 AGTCCTAGGTATAATACTAGTAGGGATTATGAACGGCAATGGTTTTAGAGCTAGAA pGRB-yjiT-A SEQ ID NO. 115 TTCTAGCTCTAAAACCATTGCCGTTCATAATCCCTACTAGTATTATACCTAGGACT pntAB-trc-S SEQ ID NO. 116 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAATTGGCATACCAAGAG pntAB-trc-A SEQ ID NO. 117 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGAGCTTTCAGGATTGCATCCAC ygaY-US SEQ ID NO. 118 CCTACAAACCACATCGCACATT ygaY-UA SEQ ID NO. 119 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAACACCGAAGCAACCCAAAAG ygaY-DS SEQ ID NO. 120 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACC ygaY-DA SEQ ID NO. 121 GGAGTAGGGCTTTCCATAGAGTGT pGRB-ygaY-S SEQ ID NO. 122 AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA pGRB-ygaY-A SEQ ID NO. 123 TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT NCgl0580-trc-S SEQ ID NO. 124 ACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAACTAGGTGTGTGTACTCGCCTCT NCgl0580-trc-A SEQ ID NO. 125 GTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAATAAACAGTCCGCTGCAGTG rph-US SEQ ID NO. 126 GCTGTTTAGCCAGTTTTTCGTTGAG rph-UA SEQ ID NO. 127 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACACA rph-DS SEQ ID NO. 128 CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAGAAAAGTAAGATGAGGAGCGAAGG rph-DA SEQ ID NO. 129 ATTGGCCTGAATAATCTCCATCGAC pGRB-rph-S SEQ ID NO. 130 AGTCCTAGGTATAATACTAGTGGCTGGATCACCGCAGAGTAGTTTTAGAGCTAGAA pGRB-rph-A SEQ ID NO. 131 TTCTAGCTCTAAAACTACTCTGCGGTGATCCAGCCACTAGTATTATACCTAGGACT pGRB-mbhA-S SEQ ID NO. 132 AGTCCTAGGTATAATACTAGTGCGTGATGTGAATGAGAAAAGTTTTAGAGCTAGAA pGRB-mbhA-A SEQ ID NO. 133 TTCTAGCTCTAAAACTTTTCTCATTCACATCACGCACTAGTATTATACCTAGGACT The gene editing method used refers to 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.), and the professional terms involved in the present application can be explained in the article, unless otherwise specified. The "knockout" referred to in the present application means inactivating the target gene, and the "integration" means inserting the exogenous gene connected with the promoter and terminator into the genome of the engineering strain.

[0029] Example 1 Starting strain E. coli W3110 ATCC 27325 prepared according to the operation mentioned in CN 202411087442.4 E. coli UR12 / pRed-Cas9 electrocompetent cells, such as Figure 1 As shown, constructing genetically engineered strains E. coli Al1-18, the specific steps are as follows: 1.1 Knockout thrA Genes: W3110 Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using thrA-US, thrA-UA, thrA-DS, and thrA-DA primers, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were amplified by overlapping PCR using thrA-US and thrA-DA primers. Annealing was then performed using pGRB-thrA-S and pGRB-thrA-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain thrA-pGRB. Electroporation of competent cells of the desired strain was then performed, and the overlapping fragments and thrA-pGRB were co-electroporated into the competent cells. Positive transformants were then screened for. thrA Gene knockout complete, finally obtaining the strain. E. coli Al1-1.

[0030] 1.2 Knockout adhE Gene: The procedure is the same as in step 1.1, except that the primers used are adhE-US, adhE-UA, adhE-DS, adhE-DA, pGRB-adhE-S, and pGRB-adhE-A. Electroporation is performed to... E. coli The strain was finally obtained in Al1-1 / pRed-Cas9 electrocompetent cells. E. coli Al1-2.

[0031] 1.3 Knockout ldhA Gene: The procedure is the same as in step 1.1, except that the primers used are ldhA-US, ldhA-UA, ldhA-DS, ldhA-DA, pGRB-ldhA-S, and pGRB-ldhA-A. Electroporation is performed to... E. coli The strain was finally obtained from the electrocompetent cells of Al1-2 / pRed-Cas9. E. coli Al1-3.

[0032] 1.4 Knockout ackAGene: same procedure as in step 1.1, except that primers used were ackA-US, ackA-UA, ackA-DS, ackA-DA, pGRB-ackA-S, pGRB-ackA-A, electrotransformed into E. coli Al1-3 / pRed-Cas9 electrocompetent cells, resulting in the final strain E. coli Al1-4.

[0033] 1.5 Knockout poxB Gene: same procedure as in step 1.1, except that primers used were poxB-US, poxB-UA, poxB-DS, poxB-DA, pGRB-poxB-S, pGRB-poxB-A, electrotransformed into E. coli Al1-4 / pRed-Cas9 electrocompetent cells, resulting in the final strain E. coli Al1-5.

[0034] 1.6 Knockout cycA Gene: same procedure as in step 1.1, except that primers used were cycA-US, cycA-UA, cycA-DS, cycA-DA, pGRB-cycA-S, pGRB-cycA-A, electrotransformed into E. coli Al1-5 / pRed-Cas9 electrocompetent cells, resulting in the final strain E. coli Al1-6.

[0035] 1.7 Knockout lacI Gene: same procedure as in step 1.1, except that primers used were lacI-US, lacI-UA, lacI-DS, lacI-DA, pGRB-lacI-S, pGRB-lacI-A, electrotransformed into E. coli Al1-6 / pRed-Cas9 electrocompetent cells, resulting in the final strain E. coli Al1-7.

[0036] 1.8 Knockout panC Gene: same procedure as in step 1.1, except that primers used were panC-US, panC-UA, panC-DS, panC-DA, pGRB-panC-S, pGRB-panC-A, electrotransformed into E. coli Al1-7 / pRed-Cas9 electrocompetent cells, resulting in the final strain E. coli Al1-8.

[0037] 1.9 Knockout yfbQGene: The procedure is the same as in step 1.1, except that the primers used are yfbQ-US, yfbQ-UA, yfbQ-DS, yfbQ-DA, pGRB-yfbQ-S, and pGRB-yfbQ-A, and the gene is electroporated to... E. coli The strain was finally obtained in Al1-8 / pRed-Cas9 electrocompetent cells. E. coli Al1-9.

[0038] 1.10 in mbhA Site integration ppc Genes: W3110 Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using primers mbhA-US, mbhA-UA, mbhA-DS, and mbhA-DA, respectively. Using the *E. coli* genome as a template, the target gene fragment was amplified by PCR using primers ppc-trc-S and ppc-trc-A. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, overlapping fragments were amplified by PCR using primers mbhA-US and mbhA-DA to obtain overlapping fragments. Using primers pGRB-mbhA-S and pGRB-mbhA-A, gRNA fragments were annealed and ligated into the pGRB vector to obtain mbhA-pGRB. Electroporation competent cells of the strain to be modified were prepared, and the overlapping fragments and mbhA-pGRB were electroporated together into the competent cells. E. coli Competent cells of Al1-9 / pRed-Cas9 were selected and positive transformants were obtained. Thus, in mbhA Site integration originates from E. coli ppc Genetic modification completed, strain finally obtained E. coli Al1-10.

[0039] 1.11 In ycdN Site integration ppc Gene: The procedure is the same as in step 1.10, except that the primers used are ycdN-US, ycdN-UA, ycdN-DS, ycdN-DA, pGRB-ycdN-S, and pGRB-ycdN-A, and the gene is electroporated to... E. coli The strain was finally obtained from the electrocompetent cells of Al1-10 / pRed-Cas9. E. coli Al1-11.

[0040] 1.12 In ybiC Site integration aspCGene: same procedure as in step 1.10, except that the primers used were ybiC-US, ybiC-UA, ybiC-DS, ybiC-DA, pGRB-ybiC-S, pGRB-ybiC-A, aspC-trc-S, aspC-trc-A, and the electrocompetent cells were transformed with E. coli Al1-11 / pRed-Cas9, and the final strain was obtained E. coli Al1-12.

[0041] 1.13 In the strain Al1-12, the gene yeeP was integrated at the aspC Gene: same procedure as in step 1.10, except that the primers used were yeeP-US, yeeP-UA, yeeP-DS, yeeP-DA, pGRB-yeeP-S, pGRB-yeeP-A, aspC-trc-S, aspC-trc-A, and the electrocompetent cells were transformed with E. coli Al1-12 / pRed-Cas9, and the final strain was obtained E. coli Al1-13.

[0042] 1.14 In the strain Al1-13, the gene ylbE of Pseudomonas aeruginosa origin was integrated at the panD Gene: same procedure as in step 1.10, except that the primers used were ylbE -US, ylbE -UA, ylbE -DS, ylbE -DA, pGRB- ylbE -S, pGRB- ylbE -A, panD-trc-S, panD-trc-A, and the electrocompetent cells were transformed with E. coli Al1-13 / pRed-Cas9, and the final strain was obtained E. coli Al1-14.

[0043] 1.15 In the strain Al1-14, the gene ilvG of Pseudomonas aeruginosa origin was integrated at the panD Gene: same procedure as in step 1.10, except that the primers used were ilvG-US, ilvG-UA, ilvG-DS, ilvG-DA, pGRB-ilvG-S, pGRB-ilvG-A, panD-trc-S, panD-trc-A, and the electrocompetent cells were transformed with E. coli Al1-14 / pRed-Cas9, and the final strain was obtained E. coli Al1-15.

[0044] 1.16 In the yjiT site of the Pseudomonas aeruginosa-derived panD gene: the same operation as in step 1.10, except that the primers used are yjiT-US, yjiT-UA, yjiT-DS, yjiT-DA, pGRB-yjiT-S, pGRB-yjiT-A, panD-trc-S, panD-trc-A, and the electrotransformation is performed on the electrotransformation competent cells of E. coli Al1-15 / pRed-Cas9, to obtain the strain E. coli Al1-16.

[0045] 1.17 In the ygaY site of the pntAB gene: the same operation as in step 1.10, except that the primers used are ygaY-US, ygaY-UA, ygaY-DS, ygaY-DA, pGRB-ygaY-S, pGRB-ygaY-A, pntAB-trc-S, pntAB-trc-A, and the electrotransformation is performed on the electrotransformation competent cells of E. coli Al1-16 / pRed-Cas9, to obtain the strain E. coli Al1-17.

[0046] 1.18 In the rph site of the NCgl0580 gene: the same operation as in step 1.10, except that the primers used are rph-US, rph-UA, rph-DS, rph-DA, pGRB-rph-S, pGRB-rph-A, NCgl0580-trc-S, NCgl0580-trc-A, and the electrotransformation is performed on the electrotransformation competent cells of E. coli Al1-17 / pRed-Cas9, to obtain the strain E. coli Al1-18.

[0047] Example 2 This example aims to illustrate the application method of the strain in the fermentation production of beta-alanine, and to verify the production performance of the strain E. coli Al1-18.

[0048] The beta-alanine production strain E. coli E. coli Al1-18 in Example 1 is used to produce beta-alanine in a shake flask, and the specific steps are as follows: (1) Seed activation and culture: The seed is uniformly coated on the activation slope, and cultured at 37°C for 12 h. The activation slope is further cultured for 10 h, and then transferred to a shake flask containing 5 ml of seed culture medium for seed culture.

[0049] (2) Fermentation culture: Inoculate the seed culture at a rate of 15% into a 500mL Erlenmeyer flask containing fermentation medium (final volume 30mL). Seal the flask with nine layers of gauze and incubate at 37℃ with shaking at 220rpm. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia. Add 60% (m / v) glucose solution to maintain fermentation (using phenol red as an indicator; when the fermentation broth color no longer changes, it indicates a sugar deficiency; add 1-2 mL of 60% (m / v) glucose solution if a sugar deficiency is detected). The fermentation cycle is 24 hours. No antibiotics or inducers are added during fermentation.

[0050] The slant culture medium used in the above fermentation process consisted of: glucose 2 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 2.5 g / L, KH₂PO₄ 1.0 g / L, MgSO₄ 0.2 g / L, and agar powder 25%. After dissolving in water, the pH was adjusted to 7.0-7.2 with sodium hydroxide, and the volume was brought to 500 ml. The solution was dispensed into test tubes (9 ml / tube) and flasks (45 ml / flask), and sterilized in an autoclave at 121℃ for 20 min. The seed culture medium used consisted of: yeast extract 8.0 g / L, peptone 2.0 g / L, (NH₄)₂SO₄ 2.0 g / L, KH₂PO₄ 3.0 g / L, and V₂SO₄. B1 V B2 V B3 V B5 V B12 2 mg / L each, V H The following ingredients were added: 1 mg / L glucose, 0.5 g / L yeast extract, 0.32 mg / L ammonium molybdate, 4.5 mg / L boric acid, 1.6 mg / L CoCl2·6H2O, and the remainder was water. The fermentation medium used was: 30.0 g / L glucose, 4.0 g / L yeast extract, 2.0 g / L peptone, 2.0 g / L ammonium sulfate, 2.0 g / L citric acid, 2.0 g / L monosodium glutamate, 1.2 g / L MgSO4·7H2O, 3.0 g / L KH2PO4, 0.5 g / L threonine, 5.0 mg / L MnSO4·H2O, 15 mg / L FeSO4·7H2O, and 2.5% phenol red. The solid components were weighed and dissolved in 1 L of water and stored at 4℃.

[0051] After 24 hours of shake-flask fermentation, the yield of β-alanine can reach up to 6 g / L.

[0052] The above merely describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application. Any improvements and refinements made by those skilled in the art based on the method of the present application or based on the method are considered to be within the protection scope of the present application.

Claims

1. A β-alanine-producing strain, characterized in that: In wild-type Escherichia coli E. coli W3110 Knockout of acetaldehyde dehydrogenase gene in the genome adhE lactate dehydrogenase gene ldhA Acetylkinase gene ackA pyruvate dehydrogenase gene poxB DNA-binding transcription repressor gene lacI, Aspartate kinase thrA pantothenic acid synthase gene panC Alanine transaminase gene cycA Genes that synthesize transaminases with alanine yfbQ Simultaneously integrating the phosphoenolpyruvate carboxylase gene PPC pyridine nucleotide transhydrogenase encoding gene pntAB, aspartate transaminase gene aspC Aspartate decarboxylase gene derived from Pseudomonas aeruginosa panD and the β-alanine transporter gene from Corynebacterium glutamicum NCgl0580 .

2. The β-alanine-producing strain according to claim 1, characterized in that: In the genome mbhA site and ycdN The codon-optimized phosphoenolpyruvate carboxylase gene was integrated at the site. PPC And is initiated by the Ptrc promoter; integrates the pyridine nucleotide transhydrogenase encoding gene pntAB at the ygaY site in the genome, and is regulated by the same Ptrc promoter; in the genome ybiC site and yeeP Aspartate transaminase gene integrated at the site aspC And are regulated by the same Ptrc promoter; exist ylbE site, ilvG site, yjiT The site sequentially integrates codon-optimized aspartate decarboxylase genes derived from Pseudomonas aeruginosa. panD And is regulated by the Ptrc promoter; In the genome rph Integrating the β-alanine transporter gene from Corynebacterium glutamicum at the site NCgl0580 Both are regulated by the same Ptrc promoter.

3. The β-alanine-producing strain according to claim 1, characterized in that: The wild-type Escherichia coli E. coli W3110 Escherichia coli E. coli W3110 ATCC 27325.

4. The β-alanine-producing strain according to claim 1, characterized in that: The adhE The nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing; ldhA The nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing; ackA The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing; poxB The nucleotide sequence is shown in SEQ ID NO.4 of the sequence listing; thrA The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing; panC The nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing; cycA The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing; yfbQ The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing; lacI The nucleotide sequence is shown in the sequence listing SEQ ID NO.

9.

5. The β-alanine-producing strain according to claim 1 or 2, characterized in that: The PPC The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing; aspC The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing; panD The nucleotide sequence is shown in SEQ ID NO.12 of the sequence listing; NCgl0580 The nucleotide sequence of pntAB is shown in SEQ ID NO.13 of the sequence listing; the nucleotide sequence of pntAB is shown in SEQ ID NO.14 of the sequence listing.

6. The β-alanine-producing strain according to claim 2, characterized in that: The nucleotide sequence of the Ptrc promoter is shown in the sequence listing SEQ ID NO.

15.

7. The method for constructing the β-alanine-producing strain according to any one of claims 1-6, characterized in that: Using CRISPR / Cas9-mediated gene editing technology E. coli W3110 The specific steps for targeted modification of the chromosome genome are as follows: (1) Knock adhE , ldhA , ackA , poxB Gene; (2) The gene encoding aspartate kinase thrA Knock it out; (3) Genes encoding β-alanine transporters cycA Knock it out; (4) Genes encoding DNA-binding transcriptional repressor factors lacI Knock it out; (5) The gene encoding pantothenic acid synthase panC Knock it out; (6) Knockout of the alanine transaminase gene yfbQ ; (7) The codon-optimized phosphoenolpyruvate carboxylase gene PPC Integration into the E. coli genome mbhA site and ycdN The site is used, and transcription is controlled by the Ptrc promoter; (8) The codon-optimized aspartate transaminase gene aspC It was subsequently integrated into the Escherichia coli genome. ybiC site, yeeP The site is used, and transcription is controlled by the Ptrc promoter; (9) The codon-optimized Pseudomonas aeruginosa-derived aspartate decarboxylase gene panD It was subsequently integrated into the Escherichia coli genome. ylbE site, ilvG site and yjiT The same Ptrc promoter controls transcription at the same site; (10) The gene encoding pyridine nucleotide transhydrogenase pntAB Integration into the E. coli genome ygaY The same Ptrc promoter controls transcription at the same site; (11) The β-alanine transporter gene NCgl0580 Integration into the genome rph The same Ptrc promoter is used to control transcription at the same site.

8. The use of the β-alanine-producing strain according to any one of claims 1-6 in the production of β-alanine.

9. The application according to claim 8, characterized in that: The specific steps for producing β-alanine using shake-flask fermentation are as follows: (1) Seed activation and culture: The strain was evenly spread on the activation slant and cultured at 37°C. The culture was then transferred to the activation slant for further culture and then transferred to the shaker tube of the seed culture medium for seed culture. (2) Fermentation culture: Inoculate 10%-15% into Erlenmeyer flasks containing fermentation culture medium, and culture at 37℃ and 220 r / min with shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; glucose solution is added to maintain the fermentation.

10. The application according to claim 9, characterized in that: The slant culture medium used contained: glucose 2 g / L, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 2.5 g / L, KH₂PO₄ 1.0 g / L, MgSO₄ 0.2 g / L, and agar powder 25%; the seed culture medium used contained: yeast extract 8.0 g / L, peptone 2.0 g / L, (NH₄)₂SO₄ 2.0 g / L, KH₂PO₄ 3.0 g / L, and vitamin C. B1 V B2 V B3 V B5 V B12 2 mg / L each, V H The following were the main components of the fermentation medium: 1 mg / L glucose, 0.5 g / L yeast extract, 2.0 g / L peptone, 2.0 g / L ammonium sulfate, 2.0 g / L citric acid, 2.0 g / L monosodium glutamate, 1.2 g / L MgSO4·7H2O, 3.0 g / L KH2PO4, 0.5 g / L threonine, 5.0 mg / L MnSO4·H2O, 15 mg / L FeSO4·7H2O, 2.5% phenol red, and the remainder water.

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