2, 6-dipicolinic acid synthase PmDpa and application thereof

Abstract

The invention discloses 2, 6-dipicolinic acid synthase PmDpa and application thereof, and the amino acid sequence of the 2, 6-dipicolinic acid synthase PmDpa is shown as SEQ ID NO. 1. The invention also discloses a preparation method of the 2, 6-dipicolinic acid synthase PmDpa. The invention discloses the gene sequence of the DPA synthase PmDpa in paecilomyces megaterium for the first time, designs excavation, cloning, expression and purification of the DPA synthase in a paecilomyces megaterium genome to obtain the DPA synthase PmDpa, and further verifies the application of the DPA synthase PmDpa in catalytic synthesis of 2, 6-dipicolinic acid.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to 2,6-pyridinedicarboxylic acid synthase PmDpa and application thereof. Background Art

[0002] 2,6-pyridinedicarboxylic acid (DPA) is an intermediate metabolite of the lysine biosynthesis pathway in Bacillus or Clostridium. It is a specific marker component of bacterial spores, accounting for 5%-15% (w / w) of the dry weight of spores. The spore core has a very low free water content and is rich in divalent metal ions Ca 2+ , can form a chelate Ca-DPA with DPA in a 1:1 ratio. Ca-DPA is considered to be one of the main factors that make spores extremely resistant to heat, radiation, dryness, extreme pH and toxic chemicals, and plays a decisive role in spores' stress resistance.

[0003] In Bacillus, the biosynthesis of DPA depends on the DAP-lysine synthesis branch pathway. This branch pathway mainly consists of four steps. The first two steps catalyze the conversion of aspartate to aspartate-semialdehyde by aspartate kinase (DapG) and aspartate semialdehyde dehydrogenase (Asd); then, dihydropicolinate synthase (DapA) catalyzes the conversion of aspartate semialdehyde (L-ASA) and pyruvate (PYR) to 2,3-dihydropicolinate (DHD). Finally, DHD is oxidized to DPA by DPA synthase. Studies have shown that in Bacillus subtilis, the encoding of DPA synthase is regulated by the spoVF operon, and the transcription factor that regulates the spoVF operon is only expressed in spores. DPA is only synthesized in the sporulation stage, and DPA is almost undetectable in the fermentation broth of wild-type vegetative cells.

[0004] Like Bacillus, most of the fungi in the family Trichoderma can produce a large number of spores and have a high resistance to harsh environments. For example, the spores of Paecilomyces variabilis are the most heat-resistant spores reported so far, and Paecilomyces variabilis has a high tolerance to metals. Although there is also a lysine biosynthesis pathway in fungi, it mainly relies on the 2-aminoadipic acid (AAA) synthesis pathway, which is completely unrelated to the DAP pathway. Although there is still controversy over whether there is a DAP biosynthesis pathway in fungi, studies have found that dihydropicolinate synthase not only exists in bacteria and plants, but is also annotated in many fungal genomes. This enzyme is the first step of the DAP pathway, a class I aldolase that can catalyze aspartate semialdehyde (L-ASA) and pyruvate (PYR) to produce 4-hydroxy-2,3,4,5-tetrahydrodipyridine acid (HTPA). DPA has long been considered a secondary metabolite unique to bacterial spores until the 1960s when researchers discovered the presence of DPA in the fermentation broth of molds and the culture filtrate of some Penicillium molds, and then successfully used Penicillium molds to convert carbohydrates into DPA. Afterwards, researchers discovered that the divergent mycelium and conidia of Cordyceps sinensis and Paecilomyces fumosorum can produce DPA. These findings indicate that the DPA synthesis pathway may also exist in fungi. Summary of the invention

[0005] One of the purposes of the present invention is to provide a 2,6-pyridinedicarboxylic acid synthase PmDpa, the amino acid sequence of which is as follows:

[0006] MKLAVFSAKSYDREYFQSVQRSLYPSYQIEYHSFALSEETVPLASDCQAVCVFVNDVLDAKVLSALRTYGVGAVLLRCAGFDNVDLQKAEELGLFVARVPAYSPESVAEFAVALIQALNRKTHRAYTRVREGNFSLDGFTGFTLYGKTVGIVGIGKIGMALAKILKGFGCHLLAY DPYPNEEFEQYGRLVPFDELLAQSHIVSLHCPLSKATKYIINDQSLAKIKKGALLINTSRGGLVDTKAAITALKSRQLGGLALDVYEAESYLFYTDHSSEIIQDDDLMRLLTLPNALMCGHQAFLTEEALTQIAEITFRNLEDYAEQRKCENSLVQGRDILVSGDTQPVRI(SEQ ID NO.1).

[0007] The second object of the present invention is to provide the nucleotide sequence of the above-mentioned 2,6-pyridinedicarboxylic acid synthase PmDpa, as shown below:

[0008]

[0009] The third object of the present invention is to provide an expression vector of 2,6-pyridinedicarboxylic acid synthase PmDpa, wherein the expression vector comprises the above-mentioned nucleic acid molecule.

[0010] A fourth object of the present invention is to provide a recombinant bacterium comprising the above expression vector.

[0011] A fifth object of the present invention is to provide the use of the above-mentioned 2,6-pyridinedicarboxylic acid synthase PmDpa, the above-mentioned nucleic acid molecule, the above-mentioned expression vector or the above-mentioned recombinant bacteria in the biocatalytic synthesis of 2,6-pyridinedicarboxylic acid.

[0012] In one embodiment of the present invention, the process of using the 2,6-pyridinedicarboxylic acid synthase PmDpa to biocatalyze the synthesis of 2,6-pyridinedicarboxylic acid is as follows: first, L-2,3-dihydropyridinedicarboxylic acid synthase DapA is added to the substrate L-aspartate semialdehyde and sodium pyruvate for reaction, and then 2,6-pyridinedicarboxylic acid synthase PmDpa, Fe 2+ / Fe 3+ By reacting with auxiliary factors, 2,6-pyridinedicarboxylic acid can be obtained.

[0013] Furthermore, the cofactor is NAD + , NADP + , FMN or FAD.

[0014] In the previous study, the inventors detected 2,6-pyridinedicarboxylic acid (DPA) in the spores of Paecilomyces maximus NJC01, and based on this, the inventors inferred that Paecilomyces maximus NJC01 carries the expression gene of DPA synthase. The present invention designs the mining of DPA synthase in the Paecilomyces maximus genome, and its cloning, expression, and purification, obtains DPA synthase PmDpa, and further verifies the application of enzyme PmDpa in catalytic synthesis of 2,6-pyridinedicarboxylic acid.

[0015] The present invention discloses for the first time the gene sequence of DPA synthase PmDpa in Paecilomyces giganteus, and realizes the two-step enzymatic synthesis of DPA by using DPA synthase PmDpa and L-2,3-dihydropyridinedicarboxylic acid synthase DapA. In the method of the present invention, the catalyst preparation process is simple, the cost is low, the reaction conditions are mild, the process flow is short, and the method has high industrial application potential.

[0016] Beneficial effects:

[0017] Compared with the prior art, the present invention discloses for the first time the DPA synthase gene sequence in fungi and the kinetic parameters of the enzyme, and uses the enzyme to realize the in vitro enzymatic synthesis of DPA. Compared with the method of producing DPA by bacterial fermentation, the route is simple and the operation is convenient; compared with the chemical method, the enzymatic synthesis method disclosed in the present invention has mild conditions and is good for the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the TAIL-PCR chromosome walking principle in the present invention.

[0019] Figure 2 In the figure, A is the pmDpa gene fragment with a band size of 1041 bp amplified by PCR, and B is a single protein band of about 39.15 kD obtained after PmDpa was expressed and purified by BL21.

[0020] Figure 3 In the figure, A is the reaction formula of DPA synthesis reaction catalyzed by DPA synthase PmDpa, B is the result of LC-MS / MS detection of whether the reaction can generate DPA, and C is the result of LC-MS / MS detection of DPA generation under the reaction conditions catalyzed by different cofactors.

[0021] Figure 4 DPA synthase PmDpa catalyzes the reverse reaction of DPA synthesis. Figure 4 A is the reaction formula, Figure 4 B is the relative activity of PmDpa enzyme under different pH, temperature, metal ion and substrate conditions. DETAILED DESCRIPTION

[0022] The invention provides a method for mining a DPA synthase PmDpa in a Paecilomyces giganteus genome, as well as cloning, expression and purification thereof, and application of the enzyme in catalyzing the synthesis of DPA.

[0023] Specifically, the mining of DPA synthase PmDpa in the genome of Paecilomyces giganteus, and its cloning, expression, and purification include the following steps:

[0024] Step 1, using the DPA synthase gene sequence of Bacillus subtilis 168 as a template, designing degenerate primers to amplify the DPA synthase PmDpa gene in Paecilomyces maximus NJC01 (CCTCCM2021380);

[0025] Step 2, using the interlaced thermal asymmetric PCR (TAIL-PCR) method to amplify the full-length DPA synthase gene sequence (pmDpa);

[0026] Step 3, the pmDpa gene fragment is connected to the linearized pET30a expression plasmid to construct a recombinant plasmid, which is then transformed into Escherichia coli DH5α competent cells, and the fragment size is verified by PCR and sequenced; after sequencing is correct, the recombinant plasmid is transformed into Escherichia coli BL21 (DE3) competent cells, and the PmDpa protein is obtained by extraction and purification.

[0027] Specifically, the use of DPA synthase PmDpa in catalyzing the synthesis of DPA comprises the following steps:

[0028] Step 1, amplifying the L-2,3-dihydropicolinate synthase DapA gene sequence in the genome of Bacillus subtilis 168; connecting the dapA gene fragment with the linearized pET30a expression plasmid to construct a recombinant plasmid, and then transforming it into Escherichia coli DH5α competent cells, verifying the fragment size by PCR, and sequencing; after sequencing is correct, transforming the recombinant plasmid into Escherichia coli BL21 (DE3) competent cells, and extracting and purifying to obtain the DapA protein;

[0029] Step 2, preparing the substrate L-aspartate semialdehyde (L-ASA) in the catalytic reaction system, preparing substrate and cofactor solutions such as sodium pyruvate, and starting the DPA synthesis catalytic reaction.

[0030] The catalytic reaction process is shown below:

[0031]

[0032] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] Mining, cloning, expression and purification of pmDpa gene

[0037] (1) Design of degenerate primers for DPA synthase and cloning into vector for sequencing

[0038] Total RNA was extracted from Paecilomyces giganteus NJC01 (CCTCCM2021380) and reverse transcribed to obtain cDNA. Using the blast function of NCBI, the amino acid sequence of DPA synthase of Bacillus subtilis 168 strain (GenBank: Z22554) was used as a template to search for similar sequences in the genus Paecilomyces, and the retrieved sequences were compared to design degenerate primers to amplify DPA synthase in Paecilomyces giganteus NJC01.

[0039] The primer sequences are as follows:

[0040] Forward primer: 5'-GCCATATGAAGCTCGCTGTATTCAGTGCCAAG-3'

[0041] Reverse primer: 5′-ATAAGCTTGATTCTCACCGGCTGGGTG-3′.

[0042] The PCR reaction system is shown in Table 1.

[0043] Table 1 PmDpa gene cloning PCR reaction system

[0044]

[0045] PCR program: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 48℃ annealing for 5s, 72℃ extension for 1min 10s, 32 cycles of denaturation, annealing and extension, 72℃ full extension for 2min after the cycle, and finally reduced to 16℃ for insulation. The amplified product was detected by agarose gel electrophoresis, and the band closest to the expected size of 1000bp was recovered. The product was purified with a PCR product purification kit and stored at -20℃ for use.

[0046] The PCR purified product of the target fragment was ligated with the linearized PBBR1MCS5 plasmid. The ligation system is shown in Table 2.

[0047] Table 2 Fragment and linearized plasmid ligation system

[0048]

[0049] After the ligation system is placed in a 37°C water bath for 30 minutes, it is immediately placed on ice to terminate the reaction and can be stored at -20°C for later use or used immediately for transformation.

[0050] Recombinant fragment transformation steps:

[0051] ① Add all the recombinant fragments to the competent E. coli DH5α and let it stand on ice for 30 minutes. For the negative control, add 10 μL of sterile water;

[0052] ②Heat shock at 42℃ for 1 min, then immediately place on ice for 2 min;

[0053] ③ Add 1 mL of LB liquid medium and resuscitate on a shaker at 37°C and 200 r / min for 1 h;

[0054] ④ Centrifuge at 5000r / min for 2min at room temperature, remove the supernatant, spread the cells on LB plates containing 100μg / mL gentamicin, culture inverted at 37℃, and screen transformants;

[0055] ⑤ Select several single colonies and place them in 2 mL of LB liquid culture medium containing 100 μg / mL gentamicin. Send the bacterial solution to the company to sequence the sequence obtained by PCR using degenerate primers.

[0056] (2) Alternating Thermal Asymmetric PCR (TAIL-PCR) Steps

[0057] The sequencing results were BLASTed in NCBI to preliminarily determine the partial sequence. The TAIL-PCR method was used to perform chromosome walking to the unknown flanks at both ends of the known sequence to obtain the complete sequence of DPA synthase. Figure 1 shown.

[0058] According to the existing DNA sequence, nested specific primers were designed on both sides, as shown in Table 3, so that the annealing temperature of each primer was as high as possible above 68°C. The homology arms of some random primers were added to the 5' end of the second round primers (LB1 / RB1) (the bold part is the homology arm) to improve the amplification efficiency. At the same time, three random primers were designed, and two or three degenerate bases were added to the 5' end of the four random bases to increase the chance of binding to the template cDNA. The random primer design is shown in Table 4 (the bold part is the homology arm), and AC0 was added as an amplification primer to improve the amplification efficiency.

[0059] Table 3 Specific primer sequences

[0060]

[0061] Table 4 Random primer sequences

[0062]

[0063] The first round of PCR reaction was carried out according to the TAIL-PCR amplification reaction system and reaction procedure in Tables 5 and 6, and the TAIL-PCR first round product was verified by agarose gel electrophoresis. The first round TAIL-PCR product was diluted 10 times with sterile water and used as a second round TAIL-PCR template.

[0064] Table 5 The first round of TAIL-PCR amplification reaction system

[0065]

[0066] Table 6 The first round of TAIL-PCR amplification reaction program

[0067]

[0068] The second round of PCR was performed according to the TAIL-PCR amplification reaction system and reaction procedure in Tables 7 and 8. The second round of TAIL-PCR products were detected by agarose gel electrophoresis (1 μL was retained as a template for the third round of TAIL-PCR). If there were bands, a single large band was recovered as much as possible, and the recovered products were temporarily stored at -20°C.

[0069] Table 7 Second round TAIL-PCR amplification reaction system

[0070]

[0071]

[0072] Table 8 Second round TAIL-PCR amplification reaction program

[0073]

[0074] The second-round product diluted 10 times was used as a template for the third-round TAIL-PCR amplification. The third-round reaction was performed according to the TAIL-PCR amplification reaction system and reaction procedure in Tables 9 and 10. The three-round products of TAIL-PCR were detected by agarose gel electrophoresis, and a single large band was recovered.

[0075] Table 9 The third round of TAIL-PCR amplification reaction system

[0076]

[0077] Table 10 The third round of TAIL-PCR amplification reaction program

[0078]

[0079] The second round of recovered products (sequencing primers are LB1 / RB1) and the third round of recovered products (sequencing primers are LB2 / RB3) were sent to the company for sequencing; TAIL-PCR was repeated until the complete DPA synthase sequence (start codon to stop codon) was obtained. The results are as follows Figure 2 As shown in A, PCR amplification yielded a single band that matched the expected size of 1041 bp. This sequence was named pmdpa, and the encoded protein was named PmDpa. The band was recovered from the gel and sent to the company for sequencing.

[0080] (3) Construction of recombinant expression vector and heterologous expression

[0081] It was connected to the pET30a plasmid digested with NdeI and HindIII. The recombinant vector was introduced into E. coli DH5α competent cells, and the fragment size was verified by PCR. After sequencing, PmDpa was heterologously expressed using BL21 (DE3). The obtained protein was purified by Ni-NTA, and the purified protein obtained by SDS-PAGE was consistent with the predicted protein size, which was 39.15KDa ( Figure 2 The content of the purified PmDpa protein was detected by Coomassie Brilliant Blue colorimetry.

[0082] Example 2

[0083] PmDpa activity detection

[0084] (1) PCR to obtain the DapA gene fragment

[0085] Total RNA was extracted from Bacillus subtilis 168 strain and reverse transcribed to obtain cDNA. According to the reported dapA gene (GenBank: CP053102.1) sequence, the amplification primers dapA forward primer: 5'-CTCGAGTGCGGCCGCAAGCTTCAGTTCGCTGATCGTGC-3' and dapA reverse primer: 5'-GGATAACAATTCCCCTCTAGAATGAATTTCGGAAATGTGTCTACAG-3' were designed. The DapA sequence of Bacillus subtilis 168 strain was amplified using the above primers. The PCR system dosage is the same as Table 1.

[0086] PCR program: 98℃ pre-denaturation for 3min, 98℃ denaturation for 15s, 55℃ annealing for 10s, 72℃ extension for 1min, with 32 cycles of denaturation, annealing and extension. After the cycle, the temperature was fully extended at 72℃ for 5min, and finally reduced to 16℃ for insulation. The amplified product was detected by agarose gel electrophoresis. A single band was obtained by PCR amplification, which was consistent with the expected size of 870bp. The band was recovered by gel and sent to the company for sequencing.

[0087] (2) Construction of recombinant expression vector and heterologous expression

[0088] After the PCR band was verified by sequencing, it was connected to the pET30a plasmid digested with XbaI and HindIII. The recombinant plasmid was introduced into DH5α competent cells, the fragment size was verified by PCR, and after sequencing, DapA was heterologously expressed using BL21 (DE3). The obtained protein was purified by Ni-NTA, and the purified DapA protein obtained by SDS-PAGE was consistent with the predicted protein size, which was 31.87KDa.

[0089] (3) Preparation of L-aspartic acid semialdehyde (L-ASA)

[0090] 19 mg of homoserine and 46 mg of 2-iodobenzoic acid (IBX) were weighed and reacted in 1 mL of ethyl acetate at 78 °C for 6 h. 50% methanol and 50% ethyl acetate were used as chromatographic liquids. The formation of L-ASA was detected by silica gel. The supernatant was taken and dried with a fan. 300 μL of 2% DMSO ddH 2 O resuspend.

[0091] (4) DapA and PmDpa are purified proteins after column purification.

[0092] (5) Prepare sodium pyruvate at a concentration of 200 mM.

[0093] (6) Prepare 10 mM nicotinamide adenine dinucleotide (NAD + ), nicotinamide adenine dinucleotide phosphate (NADP + ), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN).

[0094] (7) Prepare Fe with a concentration of 10 mM 2+ / Fe 3+ .

[0095] The enzyme reaction system of L-ASA, sodium pyruvate, DapA, and PmDpa is shown in Table 11, and the reaction schematic diagram is shown in Figure 3 As shown in A. L-ASA and sodium pyruvate generate DHD under the catalysis of DapA, followed by DPD under the catalysis of PmDpa and cofactor NAD + and Fe 2+ In the control group, an equal volume of 0.9% sodium chloride was added to replace the purified DapA and PmDpa.

[0096] Table 11 DPA synthase catalyzed DPA synthesis reaction system

[0097]

[0098] Sodium pyruvate, L-ASA and purified DapA were reacted at 37°C for 30 min, and then PmDpa and NAD + / NADP + / FMN / FAD and Fe 2+ / Fe 3+After reacting at 37°C for 1 h, an equal volume of pre-cooled methanol was added to terminate the reaction, and the formation of the product DPA was detected by LC-MS / MS. MS / MS and LC-MS analysis were performed using a triple quadrupole / AB SCIEX 5500QTRAP system (Sciex, USA). The LC-MS / MS conditions were as follows: an SB-C18 column (2.1×75 mm, Agilent, USA) was used, the mobile phase was 10:90 acetonitrile:5 mM ammonium formate, the flow rate was 400 μL / min, the injection was 5 μL, the temperature was 30°C, DP: 98.0V, and CXP was 11.5V. From Figure 3 As shown in B, when DapA or PmDpa is missing, no reaction occurs, but when both DapA and PmDpa are present, DPA is produced.

[0099] Figure 3 Figure C shows that the conversion efficiency of the cofactor FMN is the highest, at 1.75%, followed by FAD, at 1.55%, and NAD + and NADP + The conversion efficiency is only 0.13% and 0.08%.

[0100] Example 3

[0101] Determination of PmDpa kinetic parameters

[0102] (1) preparing 2,6-pyridinedicarboxylic acid (DPA) at pH 7 and a concentration of 1 mM;

[0103] (2) preparing nicotinamide adenine dinucleotide (NADH) at a concentration of 1 mM;

[0104] (3) PmDpa is the purified protein after column purification, with a concentration of 0.13878 mg / mL.

[0105] (4) Prepare various metal ions with a concentration of 10 mM, such as Mn 2+ , Cu 2+ 、Zn 2+ , Fe 3+ , Fe 2+ , Ca 2+ Mg 2+ , and EDTA at a concentration of 10 mM.

[0106] The enzyme reaction system of DPA, NADH and PmDpa is shown in Table 12, and the schematic diagram of the reaction system is shown in A in 4. The control group is a control group in which an equal volume of 0.9% sodium chloride is added instead of purified PmDpa.

[0107] Table 12 Reverse reaction system of DPA synthesis reaction catalyzed by DPA synthase

[0108]

[0109] After the reaction started, use NanoDrop TM One micro-volume UV-Vis spectrophotometer detects the absorbance of the reaction solution at 340nm to confirm the optimal cofactor, optimal temperature, optimal pH and substrate specificity of the enzyme.

[0110] After adding the reaction at the optimal temperature and the optimal pH, the absorbance of the reaction solution was continuously detected at 340 nm every 1 min. The consumption rate of DPA was calculated using the NADH external standard method.

[0111] The results are as follows Figure 4 As shown in B, PmDpa has the characteristics of substrate specificity and requires the cofactor Fe 3+ or Fe 2+ The optimum reaction temperature is 25℃, the optimum pH is 8.0, the Km of PmDpa is 0.0131±0.00081mM, and V max 0.05723±0.00057mM / min, K cat 6.7276±0.6658s -1 .

Claims

1. A 2,6-pyridinedicarboxylic acid synthase PmDpa, whose amino acid sequence is shown in SEQ ID NO.

1. 2 . A nucleic acid molecule encoding the 2,6-pyridinedicarboxylic acid synthase PmDpa according to claim 1 .

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

4. An expression vector of 2,6-pyridinedicarboxylic acid synthase PmDpa, characterized in that: The expression vector comprises the nucleic acid molecule according to claim 2.

5. A recombinant bacterium comprising the expression vector according to claim 4.

6. Use of the 2,6-pyridinedicarboxylic acid synthase PmDpa according to claim 1, the nucleic acid molecule according to any one of claims 2 to 3, the expression vector according to claim 4 or the recombinant bacterium according to claim 5 in the biocatalytic synthesis of 2,6-pyridinedicarboxylic acid.

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