L-aspartic acid alpha-decarboxylase with improved substrate tolerance

Abstract

The invention discloses L-aspartic acid alpha-decarboxylase with improved substrate tolerance. L-aspartic acid-alpha-decarboxylase from bacillus subtilis is subjected to site-specific mutagenesis, wherein phenylalanine at a No.4 site is mutated into tryptophan, phenylalanine at a No.6 site is mutated into tryptophan, and phenylalanine at a No.6 site is mutated into tryptophan; the isoleucine at the No.33 site is mutated into alanine; the isoleucine at the No.88th site is tryptophan; and transforming recombinant plasmids of the mutant into escherichia coli, carrying out induced expression, and then catalyzing a substrate L-aspartic acid to generate beta-alanine. When the substrate addition concentration of a whole-cell catalytic system is 60g / L, the conversion rates of the three enzyme mutants T4W, I33A and I88M can reach about 90%, the yields of the three enzyme mutants T4W, I33A and I88M are respectively 1.2, 1.3 and 1.2 times of those of non-mutated strains, and the substrate tolerance of the three enzyme mutants T4W, I33A and I88M is obviously improved; the tolerance of the combined mutation to the substrate is also improved to a certain extent, and the discovery has important research value for industrial preparation of beta-alanine.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to an L-aspartate α-decarboxylase with improved substrate tolerance. Background Art

[0002] L-aspartate α-decarboxylase (PanD) is the key to the synthesis of β-alanine by enzymatic and microbial fermentation methods. This enzyme can catalyze L-aspartate to produce β-alanine, which is crucial for the yield of β-alanine. β-alanine is widely used in fields such as medicine, food, chemical industry, and environment. Firstly, many important industrial compounds such as 3-hydroxypropionic acid, poly-3-hydroxypropionate, pantothenic acid, and carnosine are synthesized using β-alanine as an important precursor or intermediate. Secondly, in the food industry, β-alanine is both a food additive to improve the taste of food and a nutritional supplement for athletes to improve physical performance. In addition, it can be directly used to produce poly-β-alanine, which is widely used in fields such as cosmetics, water purification, and construction. In recent years, the global demand for β-alanine series products is approximately 50,000 tons and is still increasing continuously. It is known as one of the 12 most promising C3 chemical products in the world in the future.

[0003] β-alanine is mainly produced by chemical methods, biocatalytic conversion methods, and microbial fermentation methods. Although chemical methods are relatively more mature than biosynthesis, their waste treatment costs are high and environmental pollution is serious, which does not meet environmental protection standards. Therefore, the green and environmentally friendly enzymatic method with mild conditions has become a more promising method for the preparation of β-alanine at present. Currently, the L-aspartate α-decarboxylase used for the preparation of β-alanine mainly comes from Corynebacterium glutamicum, Bacillus subtilis, and Corynebacterium jeikeium. However, wild-type L-aspartate α-decarboxylase and its mutants generally have the problem of poor substrate tolerance. For example, Li Huanhuan used the L-aspartate α-decarboxylase from Corynebacterium glutamicum. After 20 h of biotransformation under whole-cell biocatalyst, the titer of β-alanine reached 24.8 g / L, and the yield of 40 g / L L-aspartate was 92.6%. However, when the concentration of L-aspartate was higher than 40 g / L, substrate inhibition occurred in the whole-cell biocatalyst system; Wang Jing performed site-directed mutagenesis of R12V on the L-aspartate-α-decarboxylase from Bacillus subtilis, but when the concentration of L-aspartate reached 100 g / L, the conversion rate was only 67%. Therefore, using site-directed mutagenesis technology to improve the substrate tolerance of this enzyme is of great significance for the industrial application of the biological method for the preparation of β-alanine. Summary of the Invention

[0004] To solve the above problems, the present invention provides an L-aspartate α-decarboxylase with improved substrate tolerance. By designing the L-aspartate α-decarboxylase derived from Bacillus subtilis, its tolerance and affinity for L-aspartate are improved, thereby achieving efficient production of β-alanine.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A mutant of L-aspartate α-decarboxylase with improved substrate tolerance according to the present invention. Compared with the L-aspartate α-decarboxylase encoded by the panD gene derived from Bacillus subtilis, it has mutations of T4W, I33A, I88W, T4W / I88W, and I33A / I88W.

[0007] The present invention provides a gene encoding the above L-aspartate α-decarboxylase mutant.

[0008] The nucleotide sequence encoding the L-aspartate α-decarboxylase mutant is shown in SEQ ID NO.1.

[0009] The present invention provides an expression vector carrying the above gene.

[0010] The expression vector is the pET-28a(+) vector.

[0011] The present invention provides a host cell carrying the above gene or the above expression vector.

[0012] The host cell is Escherichia coli BL21(DE3).

[0013] The present invention provides a method for preparing the above L-aspartate α-decarboxylase mutant. The method is to inoculate the above host cell into a fermentation medium for fermentation. After the fermentation is completed, the obtained fermentation broth is collected and centrifuged. After centrifugation, the above L-aspartate α-decarboxylase mutant is separated from the cell precipitate obtained by centrifugation.

[0014] The present invention provides the L-aspartate α-decarboxylase mutant prepared by the above method.

[0015] The present invention provides a method for preparing β-alanine. Using the mutant or the host cell as a catalyst, L-aspartate as a substrate, and adding 50 mM Fe 2+ to carry out a catalytic reaction to obtain β-alanine.

[0016] The catalytic reaction conditions are 37 °C, 200 rpm, and the reaction is carried out for 20 h.

[0017] Use of the mutant, or the gene, or the expression vector, or the host cell in the preparation of β-alanine.

[0018] The beneficial effects obtained by the present invention are as follows:

[0019] In the present invention, the L-aspartate α-decarboxylase encoded by the panD gene derived from Bacillus subtilis was subjected to single-site mutations or combined mutations at the following sites: (1) phenylalanine at the 4th site was mutated to tryptophan; (2) isoleucine at the 33rd site was mutated to alanine; (3) isoleucine at the 88th site was mutated to tryptophan; (4) phenylalanine at the 4th site was mutated to tryptophan, and isoleucine at the 88th site was mutated to tryptophan; (5) isoleucine at the 33rd site was mutated to alanine, and isoleucine at the 88th site was mutated to tryptophan; which greatly alleviated the substrate inhibition. When the substrate addition concentration was 60 g / L, the β-alanine yields were 1.2 times, 1.3 times, and 1.2 times that of the wild type respectively, and its substrate tolerance was significantly improved. The combined mutation also had a certain improvement in substrate tolerance. Description of the Drawings

[0020] Figure 1 It is a graph showing the influence results of adding substrates at different concentrations on the β-alanine yield and conversion rate. Detailed Embodiments

[0021] The present invention will be described below in conjunction with the drawings and embodiments.

[0022] In the present invention, the gene sequence of BspanD is as follows:

[0023] atgtatcgaacaatgatgagcggcaaacttcacagggcaactgttacggaagcaaacctgaactatgtgggaag

[0024] cattacaattgatgaagatctcattgatgctgtgggaatgcttcctaatgaaaaagtacaaattgtgaataataataatg

[0025] gagcacgtcttgaaacgtatattattcctggtaaacggggaagcggcgtcatatgcttaaacggtgcagccgcacgcctt

[0026] gtgcaggaaggagataaggtcattattatttcctacaaaatgatgtctgatcaagaagcggcaagccatgagccgaaa

[0027] gtggctgttctgaatgatcaaaacaaaattgaacaaatgctggggaacgaaccagcccgtacaattttgtag。

[0028] The amino acid sequence is:

[0029] MYRTMMSGKLHRATVTEANLNYVGSITIDEDLIDAVGMLPNEKVQIVNNNNGARLETYII PGKRGSGVICLNGAAARLVQEGDKVIIISYKMMSDQEAASHEPKVAVLNDQNKIEQMLGNEP ARTIL。

[0030] The culture media and measurement methods used are as follows:

[0031] (I) Culture media

[0032] LB medium (1 L): 10 g peptone, 5 g yeast extract, 10 g sodium chloride. 20 g / L agar powder is added to the solid medium.

[0033] TB medium (1 L): 12 g / L peptone, 24 g / L yeast extract, KH 2 PO 4 2.3 g / L, 5 g / L glycerol, K 2 HPO 4 ·3H 2 O 16.43 g / L; pH 7.0 - 7.2.

[0034] (II) Measurement methods

[0035] Measurement of cell concentration: Take a certain amount of cell suspension and dilute it appropriately with deionized water. Measure the OD value at 600 nm using a UV 7500 visible light spectrophotometer.

[0036] Measurement of amino acids: High performance liquid chromatography (HPLC). Instrument: Agilent high performance liquid chromatograph (equipped with a UV detector and an auto - sampler). Chromatographic conditions: Chromatographic column: C18 column; Mobile phase: Phase A (50% acetonitrile, filtered with a 0.45 μm filter membrane); Phase B (50 mM sodium acetate, pH 7.2, filtered with a 0.45 μm filter membrane); Derivatization is carried out with NaHCO3 solution and DNFB reagent before injection.

[0037] The present invention adopts the Gibson assembly method, specifically as follows: Use the Sangon rapid seamless cloning kit. The seamless cloning reaction system: Add 5 μL of 2X Seamless cloning Master Mix; for the linearized vector, add the volume according to the length (bp) × 0.02 and the concentration; for the insert fragment, add the volume according to the length (bp) × 0.02 and the concentration; add H 2 O to make up to 10 μL.

[0038] React at 50 °C for 30 min. After the reaction ends, immediately place the centrifuge tube on ice to cool for 2 min, waiting for transformation.

[0039] Transformation: Transfer 10 μL of the reaction solution into competent cells (JM109), flick gently several times, and incubate on ice for 30 minutes.

[0040] a. Heat shock in a 42 °C water bath for 90 s and then quickly place on ice for 5 min.

[0041] b. Add 800 - 900 μL of LB liquid medium and incubate at 37 °C for 45 - 60 min.

[0042] c. Centrifuge at 4000 rpm for 3 minutes to collect the bacteria, and spread a certain amount of the bacteria evenly on the plate containing the corresponding resistance as needed.

[0043] The strains involved are shown in Table 1, the primers used for assembly are shown in Table 2 specifically, and the plasmids used are shown in Table 3.

[0044] Table 1 Strains involved in the present invention

[0045] Strain Description E.coli JM109 Cloning host B.subtilis 168 Provide the panD gene B01 E.coli BL21(DE3), expression host B02 Strain B01, carrying plasmid pET-28a(+)-panD_Bs B03 <![CDATA[Strain B01, carrying plasmid pET-28a(+)-panD T4W _Bs]]> B04 <![CDATA[Strain B01, carrying plasmid pET-28a(+)-panD I33A _Bs]]> B05 <![CDATA[Strain B01, carrying plasmid pET-28a(+)-panD I88W _Bs]]> B06 <![CDATA[Strain B01, carrying plasmid pET28(a+)-panD T4W / I88W _Bs]]> B07 <![CDATA[Strain B01, carrying plasmid pET28(a+)-panD I33A / I88W _Bs]]>

[0046] Table 2 Primer table used

[0047] Primer Primer sequence (5'-3')* BspanD_F gtttaactttaagaaggagatataccatgtatcgaacaatgatgagcgg BspanD_R ctcagtggtggtggtggtggtgcaaaattgtacgggctggttcg pET-28a(+)_F GGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAG pET-28a(+)_R CACCACCACCACCACCACTGAG T4W_F taccatgtatcgaTGGatgatgagcgg T4W_R catcatCCAtcgatacatggtatatctccttct I33A_F agatctcGCAgatgctgtggga I33A_R gagatcttcatcaattgtaatgct I88W_F gaaggagataaggtcTGGattatttcctacaaaat I88W-R tgtaggaCCAaataatgaccttatctccttcctg

[0048] Table 3. Plasmids required

[0049] Plasmid Function pET-28a(+)-panD_Bs Used to express the panD gene derived from Bacillus subtilis <![CDATA[pET-28a(+)-panD T4W _Bs]]> <![CDATA[For expressing panD derived from Bacillus subtilis T4W gene]]> <![CDATA[pET-28a(+)-panD I33A _Bs]]> <![CDATA[For expressing panD derived from Bacillus subtilis I33A gene]]> <![CDATA[pET-28a(+)-panD I88W _Bs]]> <![CDATA[For expressing panD derived from Bacillus subtilis I88W gene]]> <![CDATA[pET-28a(+)-panD T4W / I88W _Bs]]> <![CDATA[For expressing panD derived from Bacillus subtilis T4W / I88W gene]]> <![CDATA[pET-28a(+)-panD I33A / I88W _Bs]]> <![CDATA[For expressing panD derived from Bacillus subtilis I33A / I88W gene]]>

[0050] Example 1: Construction of recombinant mutant strains

[0051] The L-aspartate α-decarboxylase in the embodiments of the present invention is derived from Bacillus subtilis, hereinafter abbreviated as BspanD. The BspanD gene fragment was amplified from the gene of Bacillus subtilis by BspanD_F / R. The plasmid pET-28a(+) was linearized using the primers pET-28a(+)_F / R to obtain a linearized fragment. The BspanD fragment was assembled with the linearized pET-28a(+) by Gibson assembly to obtain the recombinant plasmid pET-28a(+)-panD_Bs. The positive recombinant plasmid pET-28a(+)-panD_Bs was transformed into the expression host strain E. coli BL21(DE3), namely B01, to obtain the prokaryotic expression strain B02.

[0052] The construction processes of strains B03 - B07 are similar to that of B02. The plasmids carried by strains B02 - B07 are shown in Table 1.

[0053] Using the recombinant pET-28a(+)-panD_Bs as the DNA template and the sequences in Table 2 as primers, the plasmid in which the mutated gene was linked to the expression vector pET-28a(+) was obtained by whole plasmid PCR method. It was transformed into E. coli JM109 by heat shock of competent cells. Monoclonal colonies were selected, cultured overnight at 37°C with shaking at 220 rpm, and the plasmids were extracted and sent to a sequencing company (Shanghai Biological Company) for sequencing. The plasmids with correct sequencing results were transformed into E. coli BL21(DE3). The successfully constructed mutants were named T4W, I33A, and I88W respectively. Based on the above mutations, combined mutations were carried out to obtain mutants named T4W / I88W and I33A / I88W respectively.

[0054] Example 2: Induced expression of recombinant mutant L-aspartate-α-decarboxylase

[0055] The recombinant mutant expression strains B02 - B07 obtained in Example 1 were respectively inoculated into 5 mL of LB medium containing 50 μg / mL of kanamycin and cultured overnight at 37°C with shaking at 200 r / min. The above overnight cultures were inoculated into TB medium containing 50 μg / mL of kanamycin at an inoculation amount of 1% and cultured at 37°C with shaking at 200 r / min until the OD 600 of the bacterial solution reached 0.6 - 0.8. IPTG was added to a final concentration of 0.5 mmol / L, and the culture was induced at 30°C for 10 h. Then, it was centrifuged at 4°C and 8000 rpm for 5 min, and the supernatant was discarded to collect the precipitate, that is, the wet cells of recombinant Escherichia coli B02 - B07 containing the expressed recombinant mutant plasmids were obtained.

[0056] Example 3: Synthesis of β-alanine catalyzed by recombinant wild-type and mutant L-aspartate-α-decarboxylase

[0057] The wet cells of B02-B07 obtained in Example 2 were used as catalysts. Whole cell catalytic system: OD 600 =5 recombinant bacteria, the reaction was carried out in 10mL system. In the reaction system, the final concentration of substrate L-aspartic acid was 60g / L (one-time addition), Fe 2+ The concentration was 50mM, the buffer was 100mM, the pH was 7.0 phosphate, and the reaction conditions were 37°C, 200rpm, and 20h. The changes in substrate and product were determined by HPLC. The experiment was repeated three times and the average value was taken. After screening and verification, the conversion rate and yield of the wild type and mutants are shown in Table 4. When the substrate concentration was 60g / L, compared with the wild type strain, the conversion rate of the mutant strain was about 90%, and the maximum yield could reach 39.1g / L. The yields of the three mutant strains—T4W, I33A, and I88W were 1.2 times, 1.3 times, and 1.2 times that of the unmutated strain, respectively; the combined mutation also improved the conversion rate compared with the wild type.

[0058] Table 4 Whole cell catalysis results of wild type and mutant strains

[0059]

[0060] Example 4: Substrate tolerance test of recombinant mutant L-aspartate-α-decarboxylase

[0061] The effects of different concentrations of substrate addition on β-alanine production and conversion rate were determined by the whole-cell catalytic method of Example 4. The substrate concentration range was 40-100 g / L. The comparison group used B02 wet cells. The results are shown in Table 4. Figure 1 As shown, with the increase of substrate concentration, the conversion rate gradually decreased. Compared with the wild type, the overall conversion rate and yield of the mutant strain were improved, which improved the substrate tolerance to a certain extent.

[0062] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An L-aspartate-α-decarboxylase mutant, characterized in that: The L-aspartate-α-decarboxylase mutant is one of the following: (1) mutating the codon encoding the fourth amino acid in the nucleotide sequence of SEQ ID NO: 1 from a phenylalanine codon to a tryptophan codon, thereby replacing the fourth phenylalanine in the translated amino acid sequence with tryptophan; (2) mutating the codon encoding the amino acid at position 33 in the nucleotide sequence of SEQ ID NO: 1 from isoleucine to alanine, thereby replacing the isoleucine at position 33 in the translated amino acid sequence with alanine; (3) mutating the codon encoding the 88th amino acid in the nucleotide sequence of SEQ ID NO: 1 from an isoleucine codon to a tryptophan codon, thereby replacing the isoleucine at position 88 in the translated amino acid sequence with tryptophan; (4) mutating the codon encoding the amino acid at position 4 in the nucleotide sequence shown in SEQ ID NO: 1 from a codon for phenylalanine to a codon for tryptophan, thereby replacing the phenylalanine at position 4 in the translated amino acid sequence with tryptophan, and mutating the codon encoding the amino acid at position 88 in the nucleotide sequence shown in SEQ ID NO: 1 from a codon for isoleucine to a codon for tryptophan, thereby replacing the isoleucine at position 88 in the translated amino acid sequence with tryptophan; (5) The codon encoding the amino acid at position 33 in the nucleotide sequence shown in SEQ ID NO: 1 is mutated from isoleucine to alanine, thereby replacing the isoleucine at position 33 in the translated amino acid sequence with alanine, and the codon encoding the amino acid at position 88 in the nucleotide sequence shown in SEQ ID NO: 1 is mutated from isoleucine to tryptophan, thereby replacing the isoleucine at position 88 in the translated amino acid sequence with tryptophan.

2. The gene encoding the L-aspartate-α-decarboxylase mutant according to claim 1.

3. A recombinant expression vector containing the nucleic acid according to claim 2.

4. The recombinant expression vector of nucleic acid according to claim 3, characterized in that: The recombinant expression vector uses pET28-a(+) vector as the framework.

5. A genetically engineered bacterium containing the coding gene according to claim 2, or the recombinant expression vector according to claim 3 or 4.

6. The genetically engineered bacteria of the recombinant expression vector according to claim 5, characterized in that: The genetically engineered bacteria is Escherichia coli BL21 (DE3).

7. Use of the L-aspartate-α-decarboxylase mutant according to claim 1 in catalyzing the synthesis of β-alanine from L-aspartate.

8. The use according to claim 7, characterized in that: The method is implemented by the following method: L-aspartic acid is used as a substrate, the genetically engineered bacteria of the recombinant expression vector is used as a whole-cell catalyst, and beta-alanine is synthesized through a biocatalytic reaction.

9. The use according to claim 8, characterized in that: The catalytic reaction system is: 600 The wet bacteria with a value of 5 were used as the catalytic body to carry out the catalytic reaction in 100 mM phosphate buffer containing 60 g / L L-aspartic acid, 50 mM Fe2+, and pH 7.

0.

10. The use according to claim 8, characterized in that: The wet bacterial cells are prepared by the following method: centrifuging the fermented bacterial liquid, discarding the supernatant, and collecting the precipitate to obtain the wet bacterial cells.

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