CN106834128A - Genetically engineered bacterium for producing beta-alanine by glucose fermentation and construction method and application thereof - Google Patents

Abstract

The invention discloses a strain of genetically engineered bacteria that utilizes glucose fermentation to produce β-alanine. The aceBA gene encoding malate synthase and isocitrate lyase in the strain whose preservation number is CGMCC NO: 2301 is knocked out and inactivated. , and insert the genes encoded by L-aspartase and L-aspartate-α-decarboxylase respectively into the position of aceBA gene to obtain Escherichia coli AL12; the nicotinic acid transfer phosphoribosyl kinase gene pncB clone On the expression plasmid, the recombinant plasmid is obtained, and the recombinant plasmid is transformed into Escherichia coli AL12 to obtain the genetically engineered bacterium AL13 which utilizes glucose fermentation to produce β-alanine. The present invention realizes the constitutive high-activity expression of L-aspartase and L-aspartic acid-α-decarboxylase, and completely uses renewable biomass resource glucose as raw material to ferment and prepare β-alanine , the route is green and environmentally friendly.

Description

A genetically engineered bacterium that uses glucose to ferment and produce β-alanine and its construction method and application

technical field

The invention belongs to the technical field of genetic engineering, and specifically relates to a genetically engineered bacterium for producing β-alanine by fermenting glucose, a construction method and application thereof.

Background technique

β-alanine, also known as 3-alanine, is the only β-type amino acid that exists in nature. The physiological function of β-alanine is mainly an intermediate product of metabolism, and it is widely used. Medicine has found that in the mammalian nervous system, it acts as a neurotransmitter in the brain. In medicine, it can be used as an important precursor for the synthesis of pantothenic acid and coenzymes. In chemical industry, it can be used as a catalyst for some chemical reactions. β-alanine has a wide range of applications and a good market prospect.

At present, the production of β-alanine mainly adopts chemical synthesis and enzymatic conversion. The chemical synthesis method is mainly to synthesize β-alanine through acrylonitrile method, β-aminopropionitrile method and succinimide interpretation method. The raw materials, intermediates and by-products of these methods are all poisonous, and the environment is extremely polluted. At present, there have been studies on enzymatic conversion methods in China, mainly using L-aspartic acid as raw material, and using bio-enzymatic synthesis. At present, L-aspartic acid is prepared from fumaric acid, and fumaric acid is mainly used It is prepared by chemical method, so from the analysis of the whole cycle, the preparation process of β-alanine is complicated and the cycle is long and still depends on fossil resources. Glucose is derived from renewable biomass resources, and its yield is abundant. It is of great significance to screen or construct a production strain that can directly use glucose to ferment β-alanine, but there is no related report yet.

Contents of the invention

The technical problem to be solved by the present invention is to provide a genetically engineered bacterium that uses glucose to ferment and produce β-alanine.

The technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned genetically engineered bacteria.

The final technical problem to be solved by the present invention is to provide the application of the above-mentioned genetically engineered bacteria.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

A genetically engineered bacterium that utilizes glucose fermentation to produce β-alanine. The preservation number is CGMCC NO: 2301. The gene aceBA encoding malate synthase and isocitrate lyase in the strain is knocked out to inactivate it, and L -The genes encoded by aspartase and L-aspartic acid-α-decarboxylase are inserted into the position of aceBA gene respectively to obtain Escherichia coli AL12, the encoding of said malate synthase and isocitrate lyase The GenBank accession number of gene aceBA is EU889415.1;

The nicotinic acid transphosphoriboskinase gene pncB is cloned into the expression plasmid to obtain the recombinant plasmid, and the recombinant plasmid is transformed into Escherichia coli AL12 to obtain the genetically engineered bacterium AL13 which utilizes glucose fermentation to produce β-alanine. The Escherichia coli CGMCC NO: 2301 is a genetically engineered bacterium with high fumaric acid production. The specific information of this strain has been disclosed in the patent application number 200810019216.7.

Nicotinic acid phosphoribosyltransferase is the rate-limiting enzyme of the NAD(H) synthesis system. Overexpression of pncB can increase the total amount of NAD(H) and maintain an appropriate NADH/NAD ⁺ , thereby promoting the growth and metabolism of cells using glucose. By knocking out the coding gene aceBA of malate synthase and isocitrate lyase, the glyoxylate cycle bypass path can be blocked, and the TCA cycle path can be enhanced. Through the co-expression of L-aspartase and L-aspartic acid-α-decarboxylase, the bacteria can directly use glucose to ferment and produce β-alanine.

Wherein, the nucleotide sequence of the L-aspartase gene AspC is shown in SEQ ID NO: 1, and the GenBank accession number of the L-aspartase gene is X03629.1.

Wherein, the nucleotide sequence of the L-aspartate-α-decarboxylase gene panD is shown in SEQ ID NO: 2, and the GenBank accession number of the L-aspartate-α-decarboxylase gene is NC_003450.3.

Wherein, the nucleotide sequence of the nicotinic acid phosphoribosyltransferase gene pncB is shown in SEQ ID NO: 3, and the EcoGene accession number of the nicotinic acid phosphoribosyltransferase gene pncB is EG10742.

Wherein, the expression plasmid is pTrc99a.

The above-mentioned method for constructing a genetically engineered bacterium that utilizes glucose fermentation to produce β-alanine comprises the following steps:

(1) Using the nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 as primers and plasmid pIJ773 as a template, linear fragment 1 was obtained by PCR amplification;

Using the nucleotide sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7 as primers, and the nucleotide sequence shown in SEQ ID NO: 1 as a template, linear fragment 2 was obtained by PCR amplification;

Using the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 as primers, and the nucleotide sequence shown in SEQ ID NO: 2 as a template, linear fragment 3 was obtained by PCR amplification;

Using the nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 9 as primers, linear fragment 1, linear fragment 2 and linear fragment 3 as templates to amplify to obtain gene knockout fragments;

(2) Transform the pKD46 plasmid into the CGMCC NO: 2301 strain, use L-arabinose to induce its expression of λ recombinase, and then make the strain competent;

(3) Transforming the knockout fragment in step (1) into the competence obtained in step (2), and screening out positive recombinants on a plate coated with apramycin;

(4) Transform pCP20 into the positive recombinants obtained in step (3), heat shock at 42°C to express FLP recombinase, use non-resistant plates and plates containing apramycin resistance for double picking, and the The strain that grows on non-resistant plates but cannot grow on apramycin-resistant plates is Escherichia coli AL12;

(5) Cloning the nucleotide sequence of the pncB gene shown in SEQ ID NO: 3 into the Nco I and Hind III restriction sites of the pTrc99a plasmid to obtain the pTrc99a-pncB recombinant plasmid, which was transformed into Escherichia coli AL12, That is, the genetically engineered bacteria Escherichia coli AL13 that utilizes glucose fermentation to produce β-alanine is obtained.

The application of the above-mentioned genetically engineered bacteria producing β-alanine by fermentation of glucose in the fermentative preparation of β-alanine is within the protection scope of the present invention.

Wherein, the seed liquid culture process is as follows:

(S1) Transfer from the cryopreservation tube to LB medium at a volume fraction of 1-2%, and culture it aerobically for 10-12 hours;

(S2) Transferring to the LB medium of the seed fermenter at a volume fraction of 1-2%;

(S3) When the cell OD ₆₀₀ reaches 2.5-4, the fermentation medium is inoculated with a volume ratio of 5-10%. The formulation of the fermentation medium is: JSP medium, nicotinic acid 0.1mM; citric acid 3.0g/ L; Na ₂ HPO ₄ ·7H ₂ O 3.00g/L; KH ₂ PO ₄ 8.00g/L; (NH ₄ ) ₂ HPO ₄ 20.00g/L; NH ₄ Cl 10g/L; (NH ₄ ) ₂ SO ₄ 5g/L; MgSO ₄ 7H ₂ O 1.00g/L; CaCl ₂ 2H ₂ O 10.0mg/L; ZnSO ₄ 7H ₂ O 0.5mg/L; CuCl ₂ 2H ₂ O 0.25mg/L; MnSO ₄ H ₂ O 2.5mg/L; CoCl ₂ 6H ₂ O 1.75mg/L; H ₃ BO ₃ 0.12mg/L; Al ₂ (SO ₄ ) ₃ xH ₂ O 1.77mg/L; Na ₂ MoO ₄ 2H ₂ O 0.5mg/L; Fe(III) citrate 16.1mg/L, the solvent is water, adjust the pH to 8.0 with ammonia water after sterilization, and glucose is added in 3 times after sterilization alone.

In steps (S1) and (S2), the culture temperature is 35-37°C.

In step (S3), a two-stage fermentation mode is adopted. When the OD ₆₀₀ of the bacteria is below 20, aerobic fermentation is carried out with oxygen, and the dissolved oxygen is 5-40%; when the OD ₆₀₀ of the bacteria is above 20, carbon dioxide gas is used Perform anaerobic fermentation.

Wherein, the temperature during the two-stage fermentation process is 30-32° C., and the pH during the cultivation process is adjusted to 7.8-8.1 with ammonia water.

Beneficial effect:

The present invention innovatively replaces the original method of enzymatic conversion of β-alanine, solves the problem of long production cycle of β-alanine, and provides a breeding purpose, high efficiency, and ability to synthesize β-alanine Strong engineering strains and completely use glucose as raw material to ferment and prepare β-alanine route, which is green and environment-friendly.

detailed description

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.

Example 1:

This example illustrates the use of overlapping PCR technology and homologous recombination technology to knock out the aceBA gene (SEQ ID NO: 3) in the parent Escherichia coli JM125 and simultaneously insert the L-aspartase gene AspC (SEQ ID NO: 1) and L - Aspartate-alpha-decarboxylase gene panD (SEQ ID NO: 2), and a process of eliminating apramycin-resistant strains was obtained.

(1) Using LB medium, culture Escherichia coli JM125 under aerobic conditions at 37°C to OD ₆₀₀ = 0.4-0.6, and make it competent for electroporation;

(2) Electrotransform the recombinant plasmid into competent Escherichia coli CGMCC NO: 2301. The electric shock conditions are: 200Ω, 25μF, electric shock voltage 2.3kv, electric shock time 4-5ms. Immediately after the electric shock, the bacteria were added to pre-cooled 1mL SOC medium, cultured at 150r/min, 30°C for 1h, and then spread on the LB medium plate with ampicillin (Amp) to screen out the positive transformant CGMCCNO: 2301 (pKD46);

(3) Adding 10 mM L-arabinose to LB medium, inducing plasmid pKD46 to express λ recombinase at 30°C to make electroporation competent;

(4) Apramycin resistance gene (pIJ773) with FRT sites on both sides, L-aspartase gene (GenBank: X03629.1) and L-aspartic acid-α-decarboxylation The enzyme gene is used as a template to design primers F1, R1, F2, R2 and F3, R3, and the specific sequence is:

F1 (SEQ ID NO: 4):

CCTTCGTTCACAGTGGGGAAGTTTTCGGATCCATGACGAGGAGCTGCACGTGTAGGCTGGAGCTGCTTCGAAG

R1 (SEQ ID NO: 5): ATTCCGGGGATCCGTCGACTACAAACTCTTGTAATGGCGGCGF2 (SEQ ID NO: 6): TAAGGCCCCTAGGCAGCTGATGTTTGAGAACATTACCGCCGCR2 (SEQ ID NO: 7):

TGCGGCGTGAACGCCTTATCCGGCCTACAGTCAGCAACGGTTGTTGTTGCCGGGCTTCATTGTTTTTAATGCTTACAGCA

F3 (SEQ ID NO: 8): ATGGGTCGCGGATCCGAATTCATGCTGCGCACCATCCTC

R3 (SEQ ID NO: 9):

CTCGAGTGCGGCCGCAAGCTTCTAAATGCTTCTCGACGTCAAAAGC

(5) Apramycin resistance gene (linear fragment 1), L-aspartase gene (linear fragment 2) and L-asparagus were amplified respectively with F1, R1, F2, R2 and F3, and R3 Amino acid-α-decarboxylase gene, and then use F1 and R3 as primers to amplify a DNA knockout fragment with homology arms of aceBA gene at both ends;

(6) Electrotransform the artificially synthesized linear DNA fragment into Escherichia coli CGMCCNO: 2301 (pKD46) competent to express lambda recombinase, and spread it on the LB plate with apramycin to select positive recombinants, and carry out PCR identification;

(7) Positive recombinants were prepared to be competent and poured into the plasmid pCP20 capable of inducing the expression of FLP recombinase, and the apramycin resistance could be eliminated after heat shock expression of FLP recombinase at 42°C. Use a pair of plates to spot samples in parallel, and the colony that can grow on the non-resistant plate but cannot grow on the resistant plate is the strain that has knocked out the resistance, named AL12(△aceBA-aspC-30- panD).

Example 2

This example illustrates the process of constructing an expression plasmid for overexpressing nicotinic acid phosphoribosyltransferase, increasing the consumption and regeneration rate of the coenzyme NAD ⁺ under anaerobic conditions, maintaining the balance of cofactors, and obtaining the strain AL13.

1. Construction of an expression plasmid for overexpressing niacin phosphoribosyltransferase, the process comprising:

(1) Artificially design and synthesize primers with Nco I and Hind III restriction sites:

Upstream primer (SEQ ID NO: 10): 5'-CGCCATGGATGACACAATTCGCTTCTCCTG-3'

Downstream primer (SEQ ID NO: 11): 5'-CCCAAGCTTCACTTGTCCACCCGTAAATGG-3'

(2) Escherichia coli K12 was used as a template for PCR amplification, and the reaction conditions were 94° C. for 45 seconds, 54° C. for 45 seconds, 72° C. for 1.2 minutes, and a total of 30 cycles. After purifying the amplified pncB gene, the expression plasmid pTrc99a was digested with Nco I and Hind III respectively, and ligated to obtain the recombinant plasmid pTrc99a-pncB.

2. The plasmid pTrc99a-pncB was introduced into Example 1 to eliminate the competence of AL12 (△aceBA-aspC-30-panD) of the apramycin-resistant strain. The obtained positive transformant is the newly constructed strain AL13 of the present invention.

Example 3

This example illustrates the comparison of the newly constructed recombinant Escherichia coli AL12, AL13 and the starting strain CGMCC NO: 2301 for producing β-alanine by fermentation.

1. Use LB medium to inoculate 1-2% (v/v) inoculum into the Erlenmeyer flask from the cryopreservation tube, cultivate aerobically for 10-12 hours, and further inoculate to 1-2% (v/v) inoculum Seed fermenter (the culture medium is also LB), after 4-6 hours of cultivation, when the OD ₆₀₀ of the bacteria is between 2.5-4, inoculate the fermentation medium (JSP medium, glucose as the carbon source) in batches at 5-10% );

2. The temperature of the seed cultivation process is controlled at 35-37°C, the pH does not need to be adjusted during the cultivation, and the dissolved oxygen is controlled at 5-40%. The fermentation process adopts a two-stage fermentation mode. When the cell OD is about ₆₀₀ to 20, carbon dioxide gas is used for anaerobic fermentation. The temperature of the fermentation process is controlled at 30-32 °C, and the pH of the cultivation process is controlled at 7.8-8.1 with ammonia water.

The results of the anaerobic fermentation of the three strains after 48h are shown in Table 1.

Table 1 Fermentation acid production of the starting strain and two recombinant strains

SEQUENCE LISTING

<110> Nanjing University of Technology

<120> A Genetically Engineered Bacteria Using Glucose Fermentation to Produce β-Alanine and Its Construction Method and Application

<130> 20151116002

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<170> PatentIn version 3.5

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<213> Nucleotide sequence of L-aspartase gene AspC

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ctgctgtttg gtaaaggtag cgccctgatc aatgacaaac gtgctcgcac ggcacagact 300

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tgccataacc caaccggtat cgaccctacg ctggaacaat ggcaaacact ggcacaactc 600

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ctggaagaag atgctgaagg actgcgcgct ttcgcggcta tgcataaaga gctgattgtt 720

gccagttcct actctaaaaa ctttggcctg tacaacgagc gtgttggcgc ttgtactctg 780

gttgctgccg acagtgaaac cgttgatcgc gcattcagcc aaatgaaagc ggcgattcgc 840

gctaactact ctaacccacc agcacacggc gcttctgttg ttgccaccat cctgagcaac 900

gatgcgttac gtgcgatttg ggaacaagag ctgactgata tgcgccagcg tattcagcgt 960

atgcgtcagt tgttcgtcaa tacgctgcag gaaaaaggcg caaaccgcga cttcagcttt 1020

atcatcaaac agaacggcat gttctccttc agtggcctga caaaagaaca agtgctgcgt 1080

ctgcgcgaag agtttggcgt atatgcggtt gcttctggtc gcgtaaatgt ggccgggatg 1140

acaccagata acatggctcc gctgtgcgaa gcgattgtgg cagtgctgta a 1191

<210> 2

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<212>DNA

<213> Nucleotide sequence of L-aspartate-α-decarboxylase gene panD

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atgctgcgcaccatcctcggaagtaagattcaccgagccactgtcactcaagctgatcta 60

gattatgttggctctgtaaccatcgacgccgacctggttcacgccgccggattgatcgaa 120

ggcgaaaaagttgccatcgtagacatcaccaacggcgctcgtctggaaacttatgtcatt 180

gtgggcgacgccggaacgggcaatatttgcatcaatggtgccgctgcacaccttattaat 240

cctggcgatcttgtgatcatcatgagctaccttcaggcaactgatgcggaagccaaggcg 300

tatgagccaaagattgtgcacgtggacgccgacaaccgcatcgttgcgctcggcaacgat 360

cttgcggaagcactacctggatccgggcttttgacgtcgagaagcatttag 411

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<212>DNA

<213> Nucleotide sequence of nicotinic acid phosphoribosyltransferase gene pncB

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atgacacaat tcgcttctcc tgttctgcac tcgttgctgg atacagatgc ttataagttg 60

catatgcagc aagccgtgtt tcatcactat tacgatgtgc atgtcgcggc ggagtttcgt 120

tgccgaggtg acgatctgct gggtatttat gccgatgcta ttcgtgaaca ggttcaggcg 180

atgcagcacc tgcgcctgca ggatgatgaa tatcagtggc tttctgccct gcctttcttt 240

aaggccgact atcttaactg gttacgcgag ttccgcttta acccggaaca agtcaccgtg 300

tccaacgata atggcaagct ggatattcgt ttaagcggcc cgtggcgtga agtcatcctc 360

tgggaagttc ctttgctggc ggttatcagt gaaatggtac atcgctatcg ctcaccgcag 420

gccgacgttg cgcaagccct cgacacgctg gaaagcaaat tagtcgactt ctcggcgtta 480

accgccggtc ttgatatgtc gcgcttccat ctgatggatt ttggcacccg tcgccgtttt 540

tctcgcgaag tacaagaaac catcgttaag cgtctgcaac aggaatcctg gtttgtgggc 600

accagcaact acgatctggc gcgtcggctt tccctcacgc cgatgggaac acaggcacac 660

gaatggttcc aggcacatca gcaaatcagc ccggatctag ccaacagcca gcgagctgca 720

cttgctgcct ggctggaaga gtatcccgac caacttggca ttgcattaac cgactgcatc 780

actatggatg ctttcctgcg tgatttcggt gtcgagttcg ctagtcggta tcagggcctg 840

cgtcatgact ctggcgaccc ggttgaatgg ggtgaaaaag ccattgcaca ttatgaaaag 900

ctgggaattg atccacagag taaaacgctg gttttctctg acaatctgga tttacgcaaa 960

gcggttgagc tataccgcca cttctcttcc cgcgtgcaat taagttttgg tattgggact 1020

cgcctgacct gcgatatccc ccaggtaaaa cccctgaata ttgtcattaa gttggtagag 1080

tgtaacggta aaccggtggc gaaactttct gacagccctg gcaaaactat ctgccatgat 1140

aaagcgtttg ttcgggcgct gcgcaaagcg ttcgaccttc cgcatattaa aaaagccagt 1200

taa 1203

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<212>DNA

<213> Artificial Sequence

<220>

<223> Upstream primer for amplifying linear fragment 1

<400> 4

ccttcgttca cagtggggaa gttttcggat ccatgacgag gagctgcacg tgtaggctgg 60

agctgcttcg aag 73

<210> 5

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<212>DNA

<213> Artificial Sequence

<220>

<223> Downstream primer for amplifying linear fragment 1

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attccgggga tccgtcgact acaaactctt gtaatggcgg cg 42

<210> 6

<211> 42

<212>DNA

<213> Artificial Sequence

<220>

<223> Upstream primer for amplifying linear fragment 2

<400> 6

taaggcccct aggcagctga tgtttgagaa cattaccgcc gc 42

<210> 7

<211> 80

<212>DNA

<213> Artificial Sequence

<220>

<223> Downstream primer for amplifying linear fragment 2

<400> 7

tgcggcgtga acgccttatc cggcctacag tcagcaacgg ttgttgttgc cgggcttcat 60

tgtttttaat gcttacagca 80

<210> 8

<211> 39

<212>DNA

<213> Artificial Sequence

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<223> Upstream primer for amplifying linear fragment 3

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atgggtcgcg gatccgaatt catgctgcgc accatcctc 39

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<212>DNA

<213> Artificial Sequence

<220>

<223> Downstream primer for amplifying linear fragment 3

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Ctcgcgtgcg gccgcaagct tctaaatgct tctcgacgtc aaaagc 46

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<213> Artificial Sequence

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<223> Upstream primer to amplify niacin-transphosphoriboskinase

<400> 10

cgccatggat gacacaattc gcttctcctg 30

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<211> 30

<212>DNA

<213> Artificial Sequence

<220>

<223> Downstream primers for amplifying the niacin-transphosphoriboskinase gene

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cccaagcttc acttgtccac ccgtaaatgg 30

Claims (10)

1. one plant utilizes the genetic engineering bacterium that glucose fermentation produces Beta-alanine, it is characterised in that knockout preserving number is CGMCC NO：The encoding gene aceBA of malate synthetase and isocitrate lyase, causes it to inactivate in 2301 bacterial strains, and by L- asparagus ferns The gene of the coding of the Gene A spC and L-Aspartic acid-α-decarboxylase PanD of propylhomoserin enzyme coding is inserted into aceBA genes jointly Position on, obtain Escherichia coli AL12；

Nicotinic acid phosphoribosyl transferase gene pncB is cloned on expression plasmid, recombinant plasmid is obtained, the recombinant plasmid is turned Change Escherichia coli AL12, that is, be utilized the genetic engineering bacterium AL13 that glucose fermentation produces Beta-alanine.

2. utilization glucose fermentation according to claim 1 produces the genetic engineering bacterium of Beta-alanine, it is characterised in that described Encode the gene order such as SEQ ID NO of L-Aspartic acid enzyme：Shown in 1, the L-Aspartic acid-α-decarboxylase gene sequence of coding Such as SEQ ID NO：Shown in 2.

3. utilization glucose fermentation according to claim 1 produces the genetic engineering bacterium of Beta-alanine, it is characterised in that nicotinic acid The nucleotide sequence of phosphoribosyl transferase gene pncB such as SEQ ID NO：Shown in 3.

4. utilization glucose fermentation according to claim 1 produces the genetic engineering bacterium of Beta-alanine, it is characterised in that described Expression plasmid be pTrc99a.

5. the utilization glucose fermentation described in claim 1 produces the construction method of the genetic engineering bacterium of Beta-alanine, and its feature exists In comprising the following steps：

(1) with SEQ ID NO：4 and SEQ ID NO：Nucleotides sequence shown in 5 is classified as primer, and plasmid pIJ773 is template, PCR Amplification obtains linear fragment 1；

With SEQ ID NO：6 and SEQ ID NO：Nucleotides sequence shown in 7 is classified as primer, SEQ ID NO：Nucleotides shown in 1 Sequence is template, and PCR amplifications obtain linear fragment 2；

With SEQ ID NO：8 and SEQ ID NO：Nucleotides sequence shown in 9 is classified as primer, SEQ ID NO：Nucleotides shown in 2 Sequence is template, and PCR amplifications obtain linear fragment 3；

With SEQ ID NO：4 and SEQ ID NO：Nucleotides sequence shown in 9 is classified as primer, linear fragment 1, linear fragment 2 and line Property fragment 3 obtains gene knockout fragment for template amplification；

(2) by pKD46 plasmids conversion CGMCC NO：2301 bacterial strains, its expression λ recombinase is induced using L-arabinose, then will The bacterial strain is prepared into competence；

(3) in the competence for obtaining gene knockout fragment step of converting (2) in step (1), it is coated with the flat screen of apramycin Select positive recombinant；

(4) pCP20 is transformed into the positive recombinant that step (3) is obtained, 42 DEG C of heat shocks make its expression FLP recombinase, utilize Non-resistant flat board and the flat board containing apramycin resistance carry out it is double choose, can grow on non-resistant flat board, but can not pacify The bacterial strain grown on general chloramphenicol resistance flat board is Escherichia coli AL12；

(5) by SEQ ID NO：The nucleotide sequence of pncB genes shown in 3 is cloned into the Nco I and Hind of pTrc99a plasmids III digestion site, obtains pTrc99a-pncB recombinant plasmids, by recombinant plasmid transformed Escherichia coli AL12, that is, obtains profit The genetic engineering bacterium Escherichia coli AL13 of Beta-alanine is produced with glucose fermentation.

6. the utilization glucose fermentation described in claim 1 produces the genetic engineering bacterium of Beta-alanine in fermentation prepares Beta-alanine Application.

7. application according to claim 6, it is characterised in that seed liquor incubation is as follows：

(S1) it is transferred in LB culture mediums from cryopreservation tube for 1~2% by volume fraction, 10~12h of aerobic culture；

(S2) it is 1~2% to be transferred in the LB culture mediums of seed fermentation tank by volume fraction；

(S3) thalline OD is treated₆₀₀It is by volume 5~10% inoculation fermentation culture mediums during to 2.5~4, the fermentation medium It is formulated and is：JSP culture mediums, nicotinic acid 0.1mM；Citric acid 3.0g/L；Na₂HPO₄·7H₂O 3.00g/L；KH₂PO₄8.00g/L； (NH₄)₂HPO₄20.00g/L；NH₄Cl 10g/L；(NH₄)₂SO₄5g/L；MgSO₄·7H₂O 1.00g/L；CaCl₂·2H₂O 10.0mg/L；ZnSO₄·7H₂O 0.5mg/L；CuCl₂·2H₂O 0.25mg/L；MnSO₄·H₂O 2.5mg/L；CoCl₂·6H₂O 1.75mg/L；H₃BO₃0.12mg/L；Al₂(SO₄)₃·xH₂O 1.77mg/L；Na₂MoO₄·2H₂O 0.5mg/L；Fe(III) Citrate 16.1mg/L, solvent is water, and it is 8.0 to adjust pH with ammoniacal liquor after sterilizing, and wherein glucose is divided into 3 times after individually sterilizing Add.

8. application according to claim 7, it is characterised in that in seed liquor incubation, in step (S1) and (S2), training It is 35~37 DEG C to support temperature.

9. application according to claim 7, it is characterised in that two benches fermentation pattern is used in step (S3), works as thalline OD₆₀₀For less than 20 when, logical oxygen carries out aerobic fermentation, and dissolved oxygen is 5%~40%；As thalline OD₆₀₀Change logical during to more than 20 Carbon dioxide carries out anaerobic fermentation.

10. application according to claim 9, it is characterised in that temperature is 30~32 DEG C in two benches fermentation process, culture Process pH ammoniacal liquor is adjusted to 7.8~8.1.