Recombinant genetic engineering bacteria for producing nattokinase and its application

Abstract

The present invention relates to a recombinant genetic engineering bacterium for producing nattokinase and its application, belonging to the field of genetic engineering technology. A signal peptide is modified and the modified signal peptide is screened to obtain a signal peptide, the sequence of which is shown in SEQ ID NO.4. A high-expression strain of Bacillus subtilis nattokinase is constructed to improve the activity of protease.

Description

[0001] Statement: This invention is a divisional case based on patent number 2022103268312 applied for on March 30, 2022, and patent name "A signal peptide for improving the secretion efficiency of nattokinase and its application". Technical Field

[0002] The invention relates to a recombinant genetic engineering bacterium for producing nattokinase and application thereof, belonging to the technical field of genetic engineering. Background Art

[0003] Nattokinase (NK, EC 3.4.21.62) is an alkaline serine protease produced by fermentation of Bacillus natto using nutrients in soybeans during the fermentation of natto. The enzyme has set off a research boom in the field of cardiovascular and cerebrovascular diseases due to its efficient and safe thrombolytic ability. Prior to this, commonly used clinical thrombolytic drugs mainly include streptokinase, urokinase and plasminogen activator, but such drugs have the disadvantages of high cost, low safety and short efficacy, and are not suitable for large-scale production. Nattokinase has significant advantages in terms of cost and safety, especially it can directly act on fibrin, greatly improving the thrombolytic efficiency, making it uniquely superior and having broad development prospects compared with the above-mentioned thrombolytic drugs. In addition, Nattokinase has been proven to have a significant effect in regulating blood pressure and blood lipids, and has research value in anti-fatigue, antibacterial, anti-oxidation, endocrine regulation, etc. It is a high-potential dietary supplement with great development significance in the food industry.

[0004] Nakamura et al. (1992) cloned the nattokinase gene for the first time by the shotgun method, which was more accurate at that time, and analyzed its primary structure in depth, and determined the complete gene sequence of the enzyme. Since then, researchers have begun to transform and optimize the nattokinase gene or its host by genetic engineering methods at the molecular level. Liu Beiyu et al. fished out the nattokinase progene from Bacillus natto, constructed a temperature-inducible expression plasmid pESX-1 in E.coli JF1125, and achieved the effective expression of nattokinase in E. coli. In addition, other hosts, such as Pichia pastoris, Bacillus licheniformis, and Spodoptera frugiperda, have achieved the effective expression of nattokinase.

[0005] In order to improve the exogenous expression level of nattokinase, researchers have also carried out a number of works on the optimization of expression elements. For example, Wu et al. tried to use the promoter (Paprv) to start the transcriptional expression of nattokinase, and transformed its core -35 and -10 sequences through point mutations, and found that the mutation transformation of the -10 region increased the enzyme activity by 1.36 times. Modifying the original gene and its key elements at the molecular level may significantly improve the enzyme activity of nattokinase. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a recombinant genetically engineered bacterium for producing nattokinase and its application, by modifying the signal peptide, screening the modified signal peptide, optimizing the nattokinase protein expression frame, constructing a Bacillus subtilis nattokinase high-expression strain, and improving the protease activity.

[0007] The present invention is achieved through the following technical solutions:

[0008] A recombinant genetically engineered bacterium for producing nattokinase, wherein the recombinant genetically engineered bacterium is obtained by transferring a recombinant plasmid into Bacillus subtilis WB800, wherein the recombinant plasmid comprises a gene of a signal peptide as shown in SEQ ID NO.4 and a nattokinase gene derived from Bacillus subtilis N2 as shown in SEQ ID NO.7, wherein the gene of the signal peptide is fused to the N-terminus of the nattokinase gene.

[0009] The invention also provides application of the recombinant genetic engineering bacteria in producing nattokinase.

[0010] The beneficial effects of the present invention compared with the prior art are as follows:

[0011] 1. The present invention constructs expression vectors combining various signal peptides with nattokinase encoding genes based on the nucleotide sequence of SEQ ID NO.6, i.e., the original nattokinase signal peptide gene, and combines a high-throughput screening method to screen out signal peptides that can increase the secretion of nattokinase. At the same time, an expression vector combining the signal peptide with the nattokinase proenzyme encoding gene is constructed to increase the secretion expression of the nattokinase proenzyme.

[0012] 2. The present invention provides a nucleotide fragment having a signal peptide with strong secretory expression activity, which can achieve high expression of nattokinase, and in particular provides an effective element for Bacillus subtilis to express the nattokinase gene.

[0013] 3. The present invention can increase the secretion of nattokinase and further improve the enzyme activity by fusing the signal peptide.

[0014] 4. The present invention improves the secretion expression of nattokinase proenzyme by fusing signal peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic diagram of the construction of plasmid PSPX in Example 1;

[0016] Figure 2 It is the electrophoresis diagram of the amplified signal peptide 1 fragment in Example 1; wherein lane M is marker DNA; lane 1 is the constructed signal peptide 1 fragment;

[0017] Figure 3 It is the electrophoresis diagram of the amplified signal peptide 2 fragment in Example 1; wherein lane M is marker DNA; lane 1 is the constructed signal peptide 2 fragment;

[0018] Figure 4 It is the electrophoresis diagram of the amplified signal peptide 3 fragment in Example 1; wherein lane M is marker DNA; lane 1 is the constructed signal peptide 3 fragment;

[0019] Figure 5 It is the electrophoresis diagram of the amplified signal peptide 4 fragment in Example 1; wherein lane M is marker DNA; lane 1 is the constructed signal peptide 4 fragment;

[0020] Figure 6 It is the electrophoresis diagram of the amplified signal peptide 5 fragment in Example 1; wherein lane M is marker DNA; lane 1 is the constructed signal peptide 5 fragment;

[0021] Figure 7 Comparison of the nattokinase enzyme activity between the constructed signal peptide 1 and the original signal peptide in Example 2;

[0022] Figure 8 Comparison of the nattokinase enzyme activity between the constructed signal peptide 2 and the original signal peptide in Example 2;

[0023] Fig. 9 Comparison of the nattokinase enzyme activity between the signal peptide 3 constructed in Example 2 and the original signal peptide;

[0024] Fig.10 Comparison of the nattokinase enzyme activity between the constructed signal peptide 4 and the original signal peptide in Example 2;

[0025] Fig.11 Comparison of the nattokinase enzyme activity between the signal peptide 5 constructed in Example 2 and the original signal peptide. DETAILED DESCRIPTION

[0026] The molecular biology experimental techniques used in the following examples include PCR amplification, plasmid extraction, DNA fragment ligation, gel electrophoresis, etc., which are specifically referred to in Molecular Cloning Experiment Guide (3rd Edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Translated by Huang Peitang et al., 2002, Beijing: Science Press). Bacillus subtilis WB800 can be purchased as a commercial strain. The Bacillus subtilis WB800 used in this example is preserved in the Applied Microbiology Laboratory of the School of Food Science and Engineering, Ocean University of China; the nucleotide sequence and amino acid sequence mentioned in this example are shown in Table 1.

[0027] Bacillus subtilis N2 was screened by the Applied Microbiology Laboratory of the School of Food Science and Engineering of Ocean University of China and deposited in the General Microbiology Center of China General Microbiological Culture Collection on March 30, 2020, with the deposit number CGMCC NO.19541, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0028] Example 1 Screening of signal peptides for efficient secretory expression of nattokinase

[0029] By optimizing the signal peptide of nattokinase, constructing expression vectors combining various signal peptides with the nattokinase coding gene, and combining high-throughput screening methods, signal peptides that improve the secretion effect of nattokinase to varying degrees are obtained from clones, specifically including the following steps:

[0030] (1) Construction of pP43NMK-NKF vector: Extract Bacillus subtilis N2 genomic DNA (Tian Gen, Bacterial Genome Extraction Kit), and use two artificial synthetic fragments SEQ ID NO.8 and SEQ ID NO.9 to PCR amplify the nattokinase gene from the Bacillus subtilis N2 genomic DNA; use two artificial synthetic fragments SEQ ID NO.10 and SEQ ID NO.11 to PCR amplify the linearized pP43NMK fragment from pP43NMK; use DNA seamless cloning (Clon Express II OneStep Cloning Kit) to recombinantly connect the nattokinase gene (SEQ ID NO.7) and the linearized pP43NMK fragment to construct the recombinant cloning vector pP43NMK-NKF.

[0031] (2) Construction of signal peptide-deficient linearized vector PSP0: Design primers SEQ ID NO.12 and SEQ ID NO.13 based on pP43NMK-NKF to amplify the linearized vector. The amplification system is 50 μl: 2×Phanta Max Buffer (Mg 2+Plus) 25μl, dNTP Mixture (2.5mM each) 1μl, upstream and downstream primers (10μM) 2μl each, Template 1μl, Phanta Max Super-Fidelity DNA Polymerase 1μl, ddH2O 28μl. The PCR amplification program is as follows: 95℃30sec, 95℃15sec (1cycle); 55℃15sec, 72℃8min (33cycles); 72℃10min (1cycle). The PSP0 linearized vector fragment was obtained. The linearized vector was digested with Fast Digest DpnI, and the treated fragment was purified using a purification kit (EZN Cycle Pure Kit D6492) and recovered for later use.

[0032] (3) Extraction and purification of signal peptide:

[0033] Primers were designed as follows: ①SEQ ID NO.14, SEQ ID NO.15 ②SEQ ID NO.16, SEQ ID NO.17 ③SEQ ID NO.18, SEQ ID NO.19 ④SEQ ID NO.20, SEQ ID NO.21 ⑤SEQ ID NO.22, SEQ ID NO.23; the first pair of primers (SEQ ID NO.14, SEQ ID NO.15) corresponded to signal peptide 1. The design concept was to convert the original signal peptide nucleotide sequence (SEQ ID NO.6), the first base of the original signal peptide nucleotide sequence is mutated from G to A, followed by adding three bases of ATG at the end of the nucleotide sequence; the second pair of primers corresponds to signal peptide 2, the fourth pair of primers corresponds to signal peptide 4, and the fifth pair of primers corresponds to signal peptide 5. The design concept is to increase the number of positive charges at the N end of the signal peptide by designing primers in addition to mutating the first base of the original signal peptide nucleotide sequence from G to A and adding three bases of ATG at the end of the nucleotide sequence; the third pair of primers corresponds to signal peptide 3. The design concept is to increase the length of the core H region of the signal peptide by designing primers in addition to mutating the first base of the original signal peptide nucleotide sequence from G to A and adding three bases of ATG at the end of the nucleotide sequence. The amino acid sequence of the original signal peptide is shown in SEQ ID NO.24.

[0034] Using the genome of Bacillus subtilis WB800 as a template, the target gene was obtained by gradient PCR. The amplification system was 50 μl: 5× PrimeSTAR Buffer (Mg 2+Plus) 10μl, dNTP Mixture (2.5mM each) 5μl, upstream and downstream primers (10μM) 2μl each, Template 1μl, PrimeSTAR HS DNA Polymerase (2.5U / μL) 1μl, ddH2O 29μl. The gradient PCR amplification program is as follows: 95℃30sec, 95℃15sec (1cycle); 62℃15sec, 72℃30sec, 95℃15sec (5cycles); 60℃15sec, 72℃30sec, 95℃15sec (5cycles); 58℃15sec, 72℃30sec (25cycles); 72℃7min (1cycle). The molecular weight was determined by nucleic acid electrophoresis analysis, and the correct band was purified and recovered (see Figure 2-6 ) to be used as the target fragment.

[0035] (4) Construction and verification of signal peptide-guided nattokinase cloning vector PSPX: According to the DNA concentration of the purified signal peptide fragment and PSP0 vector, dilution was performed to ensure that the molar ratio of the linearized vector and the target fragment was 1:2 for recombination ligation. The signal peptide gene was fused to the N-terminus of the nattokinase gene whose nucleotide sequence was the sequence shown in SEQ ID NO.7, so that the N-terminus encoded by the fused gene was fused with the corresponding signal peptide to form a recombinant gene. The recombinant gene was ligated to the expression plasmid to form a recombinant plasmid. The ligated system was transformed into E. coli DH5α by heat shock method, and then coated on LB (AMP) plates for screening of positive recombinants. The vector PSPX constructed with the universal primer pair of pP43NMK vector (see Figure 1 ) were sequenced for single clone verification.

[0036] (5) Construction and verification of signal peptide expression vector: After activating the correctly sequenced transformants, plasmids (EZN Plasmid DNA Mini Kit D6942 kit) to obtain nattokinase cloning vectors containing different signal peptides. The nattokinase cloning vectors were transformed into Bacillus subtilis WB800 by electroporation and verified by SDS-PAGE and fibrin plate enzyme production.

[0037] Example 2 Detection of signal peptide-mediated nattokinase expression level

[0038] (1) The positive transformants were inoculated into 5 mL of liquid LB (Kana). After incubation, the plasmids were extracted and sequenced. The transformants with the correct plasmids were inoculated into TB medium (Kana). The culture was shaken at 37°C and 180 rpm for 48 h. The fermentation broth was centrifuged at 8000 rpm for 10 min to obtain the enzyme solution.

[0039] (2) According to the UV spectrophotometric method developed by the Japan Nattokinase Association, the enzyme activity is defined as: 1 unit (1FU) is defined as the supernatant at OD 275 The amount of enzyme at which the absorbance increases by 0.01 per minute.

[0040] (3) Determination of nattokinase activity: Compared with the original signal peptide of nattokinase SEQ ID NO.6, five signal peptides that can improve the activity of nattokinase were screened out, namely, signal peptide 1 encoded by the amino acid sequence of SEQ ID NO.1, which can increase the enzyme activity of nattokinase from 22.3FU / mL to 171FU / mL (see Figure 7 ); The signal peptide 2 encoded by the amino acid sequence of SEQ ID NO.2 can increase the enzyme activity of nattokinase from 22.3FU / mL to 207.7FU / mL (see Figure 8 ); The signal peptide encoded by the amino acid sequence of SEQ ID NO.3 can increase the enzyme activity of nattokinase from 22.3FU / mL to 208.3FU / mL (see Fig. 9 ); The signal peptide encoded by the amino acid sequence of SEQ ID NO.4 can increase the enzyme activity of nattokinase from 22.3FU / mL to 225.4FU / mL (see Fig.10 ); The signal peptide encoded by the amino acid sequence of SEQ ID NO.5 can increase the enzyme activity of nattokinase from 22.3FU / mL to 235.2FU / mL (see Fig.11 ).

[0041] Table 1. All nucleotide and amino acid sequences used in this example

[0042]

[0043]

Claims

1. A recombinant genetically engineered bacterium for producing nattokinase, characterized in that: The recombinant genetically engineered bacteria is obtained by transferring a recombinant plasmid into Bacillus subtilis WB800, wherein the recombinant plasmid comprises a gene of a signal peptide as shown in SEQ ID NO.4 and a nattokinase gene derived from Bacillus subtilis N2 as shown in SEQ ID NO.7, wherein the gene of the signal peptide is fused to the N-terminus of the nattokinase gene.

2. Application of the recombinant genetically engineered bacteria according to claim 1 in the production of nattokinase.

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