L-aspartate alpha-decarboxylase with improved substrate tolerance

Abstract

The application discloses an L-aspartate alpha-decarboxylase with improved substrate tolerance, and the L-aspartate alpha-decarboxylase is derived from Bacillus subtilis and is subjected to site-directed mutation, i.e., phenylalanine at the 4th site is changed into tryptophan, isoleucine at the 33rd site is changed into alanine, and isoleucine at the 88th site is changed into tryptophan, so as to obtain an enzyme mutant; the mutant is recombined into E. coli, and after expression is induced, the mutant catalyzes the substrate L-aspartate to generate beta-alanine. When the substrate addition concentration of the whole cell catalytic system is 60 g / L, the conversion rates of the three enzyme mutants T4W, I33A and I88M can reach about 90%, and the yields are 1.2, 1.3 and 1.2 times of that of the unmutated strain respectively, and the substrate tolerance is obviously improved; the combined mutation also has a certain improvement on the substrate tolerance, and the finding has important research value for industrial preparation of beta-alanine.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and specifically relates to an L-aspartic acid α-decarboxylase with improved substrate tolerance. Background Technology

[0002] L-Aspartate α-decarboxylase (PanD) is crucial for the enzymatic and microbial fermentation synthesis of β-alanine. This enzyme catalyzes the conversion of L-aspartate to β-alanine and is key to β-alanine yield. β-alanine has wide applications in medicine, food, chemicals, and the 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 used as a food additive to improve food flavor and as a nutritional supplement for athletes to improve physical function. Furthermore, it can be directly used to produce poly(β-alanine), which is widely used in cosmetics, water purification, and construction. In recent years, the global demand for β-alanine-related products has been approximately 50,000 tons and continues to increase, making it one of the 12 most promising C3 chemical products for future development.

[0003] β-Alanine is mainly produced through chemical methods, enzymatic conversion, and microbial fermentation. While chemical methods are relatively more mature than biosynthesis, they are costly to treat waste and cause severe environmental pollution, failing to meet environmental standards. Therefore, the green, environmentally friendly, and mild enzymatic method has become a more promising approach for β-alanine preparation. Currently, L-aspartate α-decarboxylases used to prepare β-alanine are mainly derived from *Corynebacterium glutamicum*, *Bacillus subtilis*, and *Corynebacterium jejuni*. However, wild-type L-aspartate α-decarboxylases and their mutants generally suffer from poor substrate tolerance. For example, Li Huanhuan used L-aspartate α-decarboxylase from Corynebacterium glutamicum. Under whole-cell biocatalysis, the β-alanine titer reached 24.8 g / L after 20 h of biotransformation, and the yield of L-aspartate at 40 g / L was 92.6%. However, when the L-aspartate concentration exceeded 40 g / L, substrate inhibition occurred in the whole-cell biocatalysis system. Wang Jing performed R12V site-directed mutagenesis on L-aspartate α-decarboxylase from Bacillus subtilis, but the conversion rate was only 67% when the L-aspartate concentration reached 100 g / L. Therefore, improving the substrate tolerance of this enzyme using site-directed mutagenesis technology is of great significance for the industrial application of biochemical preparation of β-alanine. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides an L-aspartate α-decarboxylase with enhanced substrate tolerance. By designing an 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 objectives, the present invention adopts the following technical solution:

[0006] The present invention discloses an L-aspartate α-decarboxylase mutant with improved substrate tolerance. Compared with the L-aspartate α-decarboxylase encoded by the panD gene from Bacillus subtilis, the mutant has the mutations T4W, I33A, I88W, T4W / I88W, and I33A / I88W.

[0007] This invention provides a gene encoding the above-mentioned L-aspartic acid 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-mentioned genes.

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

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

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

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

[0014] This invention provides an L-aspartic acid α-decarboxylase mutant prepared using the above method.

[0015] This invention provides a method for preparing β-alanine, using the mutant or the host cell as a catalyst, L-aspartic acid as a substrate, and simultaneously adding 50 mM Fe. 2+ β-alanine was prepared by catalytic reaction.

[0016] The catalytic reaction conditions were 37°C, 200 rpm, and 20 h.

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

[0018] The beneficial effects obtained by this invention are:

[0019] This invention utilizes single-point or combined mutations at the following sites to mutate the L-aspartate α-decarboxylase encoded by the panD gene derived from Bacillus subtilis: (1) phenylalanine at site 4 is mutated to tryptophan; (2) isoleucine at site 33 is mutated to alanine; (3) isoleucine at site 88 is mutated to tryptophan; (4) phenylalanine at site 4 is mutated to tryptophan, and isoleucine at site 88 is mutated to tryptophan; (5) isoleucine at site 33 is mutated to alanine, and isoleucine at site 88 is mutated to tryptophan. This significantly reduces substrate inhibition. At a substrate concentration of 60 g / L, the β-alanine yield is 1.2 times, 1.3 times, and 1.2 times that of the wild type, respectively, indicating a significant improvement in substrate tolerance. Combined mutations also improve substrate tolerance to some extent. Attached Figure Description

[0020] Figure 1 The graph shows the effect of different substrate concentrations on the yield and conversion of β-alanine. Detailed Implementation

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

[0022] In this 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 as follows:

[0029] MYRTMMSGKLHRATVTEANLNYVGSITIDEDLIDAVGMLPNEKVQIVNNNNGARLETYII PGKRGSGVICLNGAAARLVQEGDKVIIISYKMMSDQEAASHEPKVAVLNDQNKIEQMLGNEP ARTIL.

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

[0031] (I) Culture medium

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

[0033] TB medium (1L): peptone 12g / L, yeast extract 24g / L, KH2PO4 2.3 g / L, glycerol 5g / L, K2HPO4·3H2O 16.43g / L; pH 7.0-7.2.

[0034] (II) Measurement Method

[0035] Determination of bacterial concentration: A certain amount of bacterial suspension was diluted appropriately with deionized water, and the OD value was measured at 600 nm using a UV 7500 visible light spectrophotometer.

[0036] Determination of amino acids: High-performance liquid chromatography (HPLC). Instrument: Agilent HPLC system (equipped with UV detector and autosampler). Chromatographic conditions: Column: C18 column; Mobile phase: Phase A (50% acetonitrile, filtered through a 0.45 μm filter); Phase B (50 mM sodium acetate, pH 7.2, filtered through a 0.45 μm filter); Derivatization with DNFB reagent using NaHCO3 solution before injection.

[0037] This invention employs the Gibson assembly method, specifically as follows: using the Sangon Biotech Rapid Seamless Cloning Kit, the seamless cloning reaction system consists of: 5 μL of 2X Seamless cloning Master Mix; linearized vector, length (bp) × 0.02, added according to concentration; insert fragment, length (bp) × 0.02, added according to concentration; and H2O, to a final volume of 10 μL.

[0038] React at 50°C for 30 minutes. After the reaction is complete, immediately place the centrifuge tube on ice to cool for 2 minutes, and wait for the conversion.

[0039] Transformation: 10 μL of reaction solution was transferred to competent cells (JM109), gently tapped a few times, and incubated on ice for 30 minutes.

[0040] a. Heat shock in a 42℃ water bath for 90 seconds, then quickly place on ice for 5 minutes.

[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 bacterial cells, and spread a certain amount of bacterial cells evenly on a plate containing the corresponding resistance, as needed.

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

[0044] Table 1. Strains involved in this invention

[0045] strain describe E. coli JM109 Cloning host B.subtilis168 Provides panD gene B01 E. coli BL21(DE3), expressing host B02 Strain B01 carries 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 lists the primers used.

[0047] Primers 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. Required plasmids

[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 this embodiment of the invention is derived from Bacillus subtilis, hereinafter abbreviated as BspanD. The BspanD gene fragment was amplified from the Bacillus subtilis gene by BspanD_F / R. The plasmid pET-28a(+) was linearized using primer pET-28a(+)_F / R to obtain a linearized fragment. The BspanD fragment was assembled with the linearized pET-28a(+) using the Gibson assembly method to obtain the recombinant plasmid pET-28a(+)-panD_Bs. The positive recombinant plasmid pET-28a(+)-panD_Bs was transformed into the expression host bacterium E. coli BL21(DE3), i.e., B01, to obtain the prokaryotic expression strain B02.

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

[0053] Using recombinant pET-28a(+)-panD_Bs as a DNA template and the sequences in Table 2 as primers, a plasmid ligated with the expression vector pET-28a(+) was obtained by whole-plasmid PCR. The plasmid was then transformed into E. coli JM109 using competent cells with heat excitation. Single colonies were selected, and the cells were incubated overnight at 37°C and 220 rpm. The plasmid was extracted and sent to a sequencing company (Shanghai Biotechnology Co., Ltd.) for sequencing. Plasmids with correct sequencing results were transformed into E. coli BL21(DE3), and the successfully constructed mutants were named T4W, I33A, and I88W, respectively. Combinatorial mutations were performed based on the above mutations 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 strain B02-B07 obtained in Example 1 was inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 200 rpm. The overnight culture was then inoculated at a rate of 1% into TB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm until the bacterial culture reached OD500. 600 Add IPTG to a final concentration of 0.5 mmol / L, bring the concentration to 0.6–0.8, and induce culture at 30°C for 10 h. Centrifuge at 8000 rpm for 5 min at 4°C, discard the supernatant, and collect the precipitate to obtain wet cells of recombinant Escherichia coli B02-B07 containing the recombinant mutant plasmid.

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

[0057] The wet bacterial cells of B02-B07 obtained in Example 2 were used as catalysts. Whole-cell catalytic system: OD 600 The recombinant bacteria with a concentration of 5 were used in a 10 mL reaction system. The final concentration of the substrate L-aspartic acid in the reaction system was 60 g / L (added all at once), and Fe... 2+The concentration was 50 mM, the buffer was 100 mM, pH 7.0 phosphate, and the reaction conditions were 37℃, 200 rpm, 20 h. Changes in substrate and product were determined by HPLC. The experiment was performed in triplicate, and the average value was taken. After screening and verification, the conversion rates and yields of the wild-type and mutant strains are shown in Table 4. At a substrate concentration of 60 g / L, compared to the wild-type strain, the mutant strain achieved a conversion rate of approximately 90%, with a maximum yield of 39.1 g / 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 non-mutant strain, respectively. The combined mutant strain also showed an improved conversion rate compared to the wild-type strain.

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

[0059]

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

[0061] The effect of different substrate concentrations on β-alanine yield and conversion was determined using the whole-cell catalysis method of Example 4. The substrate concentration ranged from 40 to 100 g / L, and the control group used BO2 wet cells. The results are as follows: Figure 1 As shown, the conversion rate gradually decreased with increasing substrate concentration. Compared with the wild type, the overall conversion rate and yield of the mutant strain were improved, which improved substrate tolerance to a certain extent.

[0062] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An L-aspartate-α-decarboxylase mutant, characterized in that, The L-aspartate-α-decarboxylase mutant is specifically: The codon encoding the 33rd amino acid in the nucleotide sequence shown in SEQ ID NO: 1 is mutated from isoleucine to alanine, thereby replacing isoleucine with alanine in the translated amino acid sequence.

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

3. A recombinant expression vector containing the encoding gene as described in claim 2.

4. The recombinant expression vector encoding the gene according to claim 3, characterized in that, The recombinant expression vector uses the pET28-a(+) vector as its framework.

5. Genetically engineered bacteria containing the coding gene as described in claim 2, or the recombinant expression vector as described in claim 3 or 4.

6. The genetically engineered bacterium according to claim 5, characterized in that, The genetically engineered bacterium is Escherichia coli BL21(DE3).

7. The application of the L-aspartic acid-α-decarboxylase mutant according to claim 1 in the catalytic synthesis of β-alanine from L-aspartic acid.

8. The application according to claim 7, characterized in that, The process is carried out by using L-aspartic acid as a substrate and the genetically engineered bacteria described in claim 5 or 6 as a whole-cell catalyst to synthesize β-alanine through a biocatalytic reaction.

9. The application according to claim 8, characterized in that, The reaction system for the biocatalytic reaction is as follows: using OD 600 Wet bacterial cells with a pH of 5 were used as the catalytic host to carry out the catalytic reaction in a 100 mM phosphate buffer containing 60 g / L L-aspartic acid, 50 mM Fe²⁺, and pH 7.

0.

10. The application according to claim 9, characterized in that, The wet bacterial cells are prepared by the following method: centrifuging the fermented bacterial broth, discarding the supernatant, collecting the precipitate, and obtaining the wet bacterial cells.

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