Bacillus amyloliquefaciens expressing bovine lactoferricin and construction method and application thereof
By introducing a signal peptide into Bacillus amyloliquefaciens and optimizing fermentation conditions, efficient extracellular expression of lactoferrin was achieved, solving the problem of low efficiency in traditional methods and providing the possibility of domestic production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING TECH UNIV
- Filing Date
- 2023-08-03
- Publication Date
- 2026-07-03
AI Technical Summary
Existing technologies are insufficient for the efficient expression and production of lactoferrin. Traditional extraction methods are inefficient and costly, and heterologous proteins may cause antigen reactions. Lactoferrin production technology is monopolized by foreign countries, resulting in a high degree of import dependence in China.
Bacillus amyloliquefaciens or Bacillus subtilis were used with constitutive expression vector pMA5 to promote the extracellular secretion of lactoferrin by binding to a signal peptide, and the expression level was increased by optimizing the fermentation medium and fermentation system.
This study achieved highly efficient extracellular expression of lactoferrin, reaching an expression level of 105 mg/L, which solves the problem of low efficiency in traditional methods and provides the possibility for domestic production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a Bacillus amyloliquefaciens that efficiently expresses bovine lactoferrin, its construction method, and its application. Background Technology
[0002] Lactoferrin (LF) is an iron-binding protein belonging to the transferrin family, with a molecular weight of approximately 76-80 kDa and diverse biological activities. LF is mainly produced by the mammary epithelial cells of various mammals, including humans, cattle, goats, horses, camels, dogs, and several rodents. Depending on its origin, it can be classified into human lactoferrin (HLF), bovine lactoferrin (BLF), and ovine lactoferrin (OLF), with bovine lactoferrin being the most commonly used in applications.
[0003] Lactoferrin (LF) possesses a wide range of biological functions, including anti-cancer, antiviral, antibacterial, and anti-osteoporosis effects. Its diverse biological activities make it a promising candidate for applications in food additives, pharmaceuticals, infant formula, pet food, and daily chemical products. The most typical application of lactoferrin is as a nutritional fortifier added to infant formula, yogurt, and other dairy products. It is also used as a green feed additive in animal husbandry, playing a crucial role in the prevention and treatment of common diseases in domestic pigs and in feed preparation. Currently, it is a scarce and expensive nutritional protein. China is the world's second-largest dairy consumer market and the world's largest importer of dairy products. Data shows that in 2021, China imported a total of 3.8973 million tons of various dairy products, a year-on-year increase of 18.8%, of which dry dairy products, an important raw material for dairy products, reached 2.6012 million tons, a year-on-year increase of 17.7%. my country's import dependence on lactoferrin is as high as 98%. Furthermore, the global production technology for high-purity lactoferrin is monopolized by foreign countries. From 2017 to 2020, the price of lactoferrin rose from over 3,000 yuan to 30,000 yuan per kilogram. In May 2022, Feihe Dairy successfully obtained a production license for lactoferrin, and my country's first automated lactoferrin production line officially went into operation. Traditional extraction methods are inefficient, require large quantities of milk, and yield only 1 kilogram of purified lactoferrin per 10,000 liters of milk. The activity and stability of lactoferrin vary depending on the source of the milk, resulting in numerous byproducts, high equipment investment, complex extraction processes, and immature technology. Only a few companies can achieve a purity of over 95% for lactoferrin, with most achieving around 80%. Furthermore, as a heterologous protein for human use, lactoferrin extracted directly from cow's milk may cause certain antigenic reactions. Therefore, there is an urgent need to explore new and efficient methods for preparing LF (lactoferrin-free lactoferrin).
[0004] With the development of genetic engineering technology, the large-scale production of lactoferrin (LF) in reactors using genetically engineered bacteria has become a research hotspot, providing a new approach for the domestic production of LF. Currently, some attempts have been made to use genetic engineering combined with molecular biology methods to heterologously express LF in different microbial, plant, and animal systems, aiming to find a faster, more efficient, and environmentally friendly method for LF preparation. Most studies have achieved successful expression, but the expression levels are generally low. How to obtain large quantities of bioactive lactoferrin has been a persistent challenge for researchers.
[0005] Bacillus amyloliquefaciens is a Gram-positive bacterium that efficiently secretes a variety of proteins and metabolites without producing endotoxins. It is a generally recognized as safe (GRAS) food-grade microbial system with a clear genetic background, simple operation, and strong secretory expression capabilities. It is frequently used as an excellent chassis cell in the medical, health, and food industries, and is an ideal host for prokaryotic expression systems to secrete and express exogenous proteins. Currently, there are no reports on lactoferrin expression in Bacillus amyloliquefaciens. Using Bacillus amyloliquefaciens as an expression host for bovine lactoferrin provides a new approach for the production of lactoferrin using genetically engineered bacteria. Summary of the Invention
[0006] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a Bacillus amyloliquefaciens strain that efficiently expresses bovine lactoferrin, its construction method, and its application.
[0007] The present invention utilizes *Bacillus amyloliquefaciens* or *Bacillus subtilis* and the constitutive expression vector pMA5 to express bovine lactoferrin. Commonly used signal peptides are screened to promote the extracellular secretion of lactoferrin. Furthermore, through optimization of the fermentation medium and scale-up of the fermentation system, the expression level of lactoferrin is effectively increased.
[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0009] A recombinant Bacillus amyloliquefaciens expressing bovine lactoferrin was obtained by heterologously expressing the bovine lactoferrin-encoding gene BLF using Bacillus amyloliquefaciens as the host.
[0010] The recombinant Bacillus amyloliquefaciens also incorporates a signal peptide.
[0011] The *Bacillus amyloliquefaciens* mentioned above is *Bacillus amyloliquefaciens* NFΔSE, a modified strain of the food-grade host *Bacillus amyloliquefaciens* NF. Specifically, it is obtained by knocking out the gene cluster encoding extracellular polysaccharide synthesis (epsA-O), the lipopolysaccharide synthesis gene (lps), and the fructanase gene (sacB) from *Bacillus amyloliquefaciens* NF. (Detailed information on *Bacillus amyloliquefaciens* NFΔSE has been published in the article "Design and construction of a Bacillus amyloliquefaciens cell factory for hyaluronic acid synthesis from Jerusalemartichoke inulin".)
[0012] The bovine lactoferrin encoding gene BLF has the nucleotide sequence shown in SEQ ID NO.1.
[0013] The signal peptide is any one of DacB, AmyE, and SacB, with AmyE being the preferred signal peptide.
[0014] Specifically, the nucleotide sequence of the DacB signal peptide is shown in SEQ ID NO.2, the nucleotide sequence of the AmyE signal peptide is shown in SEQ ID NO.3, and the nucleotide sequence of the SacB signal peptide is shown in SEQ ID NO.4.
[0015] The recombinant Bacillus amyloliquefaciens used pMA5 as its expression vector (purchased from the BioVector NTCC plasmid vector strain cell protein antibody gene deposit center).
[0016] A method for constructing a recombinant Bacillus amyloliquefaciens expressing bovine lactoferrin includes the following steps:
[0017] (1) The bovine lactoferrin encoding gene BLF was cloned into the expression vector pMA5 and verified to obtain the recombinant plasmid pMA5-BLF;
[0018] (2) Using Bacillus amyloliquefaciens NFΔSE as a template, the signal peptide was amplified by PCR to obtain the signal peptide gene fragments. The obtained signal peptide gene fragments were cloned into the recombinant plasmid pMA5-BLF in step (1). After transformation, the recombinant plasmids pMA5-DacB-BLF, pMA5-AmyE-BLF, and pMA5-SacB-BLF were obtained by PCR verification.
[0019] (3) Transform the recombinant plasmid obtained in step (2) into the host Bacillus amyloliquefaciens competent cells to construct recombinant Bacillus amyloliquefaciens.
[0020] The host Bacillus amyloliquefaciens can also be Bacillus subtilis, specifically Bacillus subtilis 168. The preferred host is Bacillus amyloliquefaciens NFΔSE.
[0021] The application of the aforementioned recombinant Bacillus amyloliquefaciens in the fermentation preparation of lactoferrin is also within the scope of protection of this invention.
[0022] The fermentation conditions are as follows: fermentation temperature of 28-37℃; rotation speed of 300 r / min; fermentation time of 24-28 h.
[0023] Preferably, the fermentation conditions are: fermentation temperature of 32℃; rotation speed of 300 r / min; and fermentation time of 36 h.
[0024] The fermentation medium is formulated as follows: carbon source 20-40 g / L, peptone 12 g / L, K2HPO4 12.5 g / L, KH2PO4 2.3 g / L, glycerol 4 mL, (NH4)2Fe(SO4)2 0-1.5 mg / L.
[0025] The carbon source is any one or a combination of several of glucose, maltose, trehalose, sucrose, and corn starch, with glucose being preferred.
[0026] Preferably, the fermentation medium is formulated as follows: glucose 40 g / L, peptone 12 g / L, K2HPO4 12.5 g / L, KH2PO4 2.3 g / L, glycerol 4 mL, (NH4)2Fe(SO4)2 1.2 mg / L.
[0027] Beneficial effects:
[0028] (1) This invention is the first to use Bacillus amyloliquefaciens or Bacillus subtilis as the expression host of bovine lactoferrin, providing a new approach for the production of lactoferrin using genetically engineered bacteria.
[0029] (2) Commonly used signal peptides were screened to promote the extracellular secretion and expression of lactoferrin. Fermentation experiments showed that signal peptides could effectively promote the extracellular expression of lactoferrin. Among them, the secretion of lactoferrin in the recombinant strain corresponding to the AmyE signal peptide was significantly improved, and the extracellular lactoferrin expression level was as high as 39.4 mg / L.
[0030] (3) By optimizing the composition of the fermentation medium and scaling up the fermentation system, the expression level of lactoferrin can be effectively increased, and the extracellular lactoferrin expression level is 105 mg / L. Attached Figure Description
[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.
[0032] Figure 1 This is an SDS-PAGE electrophoresis image of the fermentation product of recombinant strain B. amyloliquefaciens SE / pMA5-BLF. Lane 1: SE / pMA5; Lane 2: SE / pMA5-BLF extracellular; Lane 3: SE / pMA5-BLF intracellular.
[0033] Figure 2 This is a graph showing the effect of different signal peptides on lactoferrin secretion and expression.
[0034] Figure 3 This is a diagram showing the expression of recombinant plasmids in different chassis strains.
[0035] Figure 4 This study examines the impact of fermentation medium composition optimization on lactoferrin yield.
[0036] Figure 5 These are the parameters for the fermentation process of the recombinant strain B. amyloliquefaciens SE / pMA5-AmyE / BLF. Detailed Implementation
[0037] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.
[0038] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, cell biology, recombinant DNA technology, and related fields, which have been well described in existing literature.
[0039] The reagents and culture media used in the following examples are as follows:
[0040] (1) Resuscitation medium: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, sorbitol 0.5 mol / L, mannitol 0.38 mol / L.
[0041] (2) Liquid LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.
[0042] (3) TB medium: yeast extract 24 g / L, peptone 12 g / L, K2HPO4 12.5 g / L, KH2PO4 2.3 g / L, glycerol 4 mL.
[0043] (4) Fermentation medium: carbon source (glucose, maltose, trehalose, sucrose or corn starch) 20-40 g / L, peptone 12 g / L, K2HPO4 12.5 g / L, KH2PO4 2.3 g / L, glycerol 4 mL, (NH4)2Fe(SO4)2 0-1.5 mg / L.
[0044] Example 1: Obtaining and expressing engineered Bacillus amyloliquefaciens strains for lactoferrin hydrolysis
[0045] Based on the gene sequence of bovine lactoferrin in NCBI GenBanK, it was artificially synthesized by a company and stored in the pMA5 plasmid to obtain the recombinant plasmid pMA5-BLF.
[0046] The plasmid pMA5-BLF was demethylated. 100 μL of Bacillus amyloliquefaciens NFΔSE competent cells were mixed with 10 μL of demethylated recombinant plasmid DNA and transferred to a pre-chilled 0.2 cm electroporation cuvette. The cuvette containing the mixture was then placed on ice for 5 min. The electroporation parameters were adjusted as follows: voltage 3.0 kV, capacitance 25 μf, resistance 200 Ω, and time approximately 4 ms. After electroporation, 1 mL of pre-chilled resuscitation medium was quickly added to the cuvette, and the mixture was gently aspirated and transferred to a centrifuge tube. The mixture was incubated at 37°C and 200 rpm for 2-3 h, centrifuged at 3000 rpm for 5 min, 900 μL of supernatant was discarded, and the remaining cells were spread evenly onto kanamycin medium (containing 25 μg / mL kanamycin) plates. The plates were incubated at 37°C overnight until colonies grew. Colony PCR was used to verify the correctness of the colony (SE / pMA5-BLF).
[0047] The correct colonies were inoculated into 5 mL of LB medium with the appropriate resistance liquid (containing 25 μg / mL kanamycin) and cultured at 37°C and 200 rpm for 12 h. The colonies were then transferred to TB medium at an inoculation rate of 1% v / v and fermented at 32°C and 200 rpm for 36 h. After fermentation, the cells were collected by centrifugation and then broken up to obtain the lactoferrin.
[0048] SDS-PAGE analysis of the fermentation broth supernatant and cell lysate showed that the recombinant strain SE / pMA5-BLF successfully expressed lactoferrin, and that it was expressed intracellularly. Figure 1To achieve the secretory expression of the target protein, a signal peptide screening process is subsequently performed.
[0049] Example 2: Amplification of signal peptide
[0050] Based on the bovine lactoferrin gene sequence (SEQ ID NO.1) from NCBI GenBanK, a lactoferrin DNA fragment was synthesized and stored in the pMA5 plasmid to obtain the recombinant plasmid pMA5-BLF. Using *Bacillus amyloliquefaciens* NFΔSE as a template, the corresponding signal peptides (DacB, AmyE, SacB) were amplified by PCR. The gene fragments of each signal peptide were then assembled with the linearized recombinant plasmid pMA5-BLF using a one-step cloning kit. The ligation product was transformed into DH5α competent cells, plated on LB agar plates containing 50 mg / mL Amp, and incubated at 37°C for 12 h. Single colonies were then picked for PCR verification. Recombinant strains with correct bands were sequenced to obtain the correct transformants (pMA5-DacB-BLF, pMA5-AmyE-BLF, pMA5-SacB-BLF).
[0051] The nucleotide sequence of the DacB signal peptide is shown in SEQ ID NO.2, the nucleotide sequence of the AmyE signal peptide is shown in SEQ ID NO.3, and the nucleotide sequence of the SacB signal peptide is shown in SEQ ID NO.4.
[0052] The PCR amplification system consisted of: forward primer F: 2 μL, reverse primer R: 2 μL, pMA5-BLF: 2 μL, 2×Phanta Max Master Mix: 25 μL, and ddH2O: 19 μL. The PCR reaction program was: 95℃ for 5 min, 95℃ for 30 s, 58℃ for 70 s, 72℃ for 30 s, for 30 cycles, followed by 72℃ for 5 min.
[0053] Table 1 Primer sequences for amplifying the signal peptide
[0054]
[0055] Example 3: Effects of different signal peptides on lactoferrin expression
[0056] The correctly sequenced recombinant plasmids were demethylated. 100 μL of *Bacillus amyloliquefaciens* NFΔSE competent cells from the chassis strain were mixed thoroughly with 10 μL of the demethylated recombinant plasmid DNA and transferred to a pre-chilled 0.2 cm electroporation cuvette. The cuvette containing the mixture was incubated on ice for 5 min. The electroporation parameters were adjusted: voltage 3.0 kV, capacitance 25 μf, resistance 200 Ω, and time approximately 4 ms. After electroporation, 1 mL of pre-chilled resuscitation medium was quickly added to the cuvette, and the mixture was gently aspirated and transferred to a centrifuge tube. The mixture was incubated at 37°C and 200 rpm for 2–3 h, centrifuged at 3000 rpm for 5 min, 900 μL of supernatant was discarded, and the remaining cells were reconstituted and spread onto kanamycin medium (containing 25%...). On a plate containing 1 μg / mL kanamycin, incubate at 37°C overnight until colonies grow. Verify the colonies (SE / pMA5-DacB-BLF, SE / pMA5-AmyE-BLF, SE / pMA5-SacB-BLF) by colony PCR.
[0057] The correct colonies were inoculated into 5 mL of LB medium with the appropriate resistance liquid (containing 25 μg / mL kanamycin) and cultured at 37°C and 200 rpm for 12 h. The colonies were then transferred to TB medium at an inoculation rate of 1% v / v and fermented at 32°C and 200 rpm for 36 h to obtain the lactoferrin.
[0058] The fermentation broth was centrifuged to obtain the fermentation supernatant. SDS-PAGE analysis was used to detect lactoferrin synthesis. The results showed that the signal peptide significantly affected lactoferrin synthesis. (See attached figures). Figure 2 The production of lactoferrin was detected using an ELISA kit (Caozhiyuan, Nanjing Caobenyuan Biotechnology Co., Ltd., catalog number: cby14070). The results showed that the signal peptide with the most significant impact was AmyE, and the lactoferrin production reached 39.4 mg / L.
[0059] Example 4: Effects of different chassis strains on lactoferrin expression
[0060] The chassis strain *Bacillus subtilis* NFΔSE from Example 2 was replaced with chassis strain *Bacillus subtilis* 168, with the AmyE signal peptide preferred, while the remaining experimental steps remained unchanged. The effects of different chassis strains on lactoferrin expression were investigated. The results showed that when *Bacillus subtilis* 168 was used as the chassis strain, the expression level of lactoferrin was significantly lower than when *Bacillus subtilis* NFΔSE was used as the chassis strain. Figure 3 ).
[0061] Example 5: Effect of culture medium composition on the expression of target protein in recombinant strains
[0062] The recombinant strain SE / pMA5-AmyE-BLF, screened in Example 2, was used as the preferred strain to study the effect of culture medium composition on the expression of the target protein in the recombinant strain. Using TB medium as a control, shake-flask fermentation was performed with glucose, maltose, trehalose, sucrose, and corn starch as carbon sources, respectively. After fermentation at 32℃ and 200 rpm for 36 h, lactoferrin under different carbon source fermentation conditions was obtained. The protein expression level was measured, and the results showed ( Figure 4 A) With the addition of different types of carbon sources, the recombinant strain SE / pMA5-AmyE-BLF could express the target protein. When glucose was used as the carbon source, the expression level of bovine lactoferrin was the highest, reaching 62 mg / L.
[0063] Using TB medium with optimized carbon source as a control, different concentrations (0.3 / 0.6 / 0.9 / 1.2 / 1.5 mg / L) of (NH4)2Fe(SO4)2 were added to the medium, and shake-flask fermentation was carried out at 32℃ and 200 rpm for 36 h to obtain lactoferrin. The protein expression level was measured, and the results showed ( Figure 4 (B) The addition of different concentrations of (NH4)2Fe(SO4)2 enhanced the expression of the target protein in the recombinant strain SE / pMA5-AmyE-BLF, and the supplementation of iron ions alleviated bacterial death caused by the iron-exclusion inhibitory effect of lactoferrin. When the concentration of (NH4)2Fe(SO4)2 was 1.2 mg / L, the recombinant strain expressed the highest amount of lactoferrin, reaching 80 mg / L.
[0064] Example 6: Cultivation in a 5L fermentation tank
[0065] To further promote the production of the target protein, the recombinant strain SE / pMA5-AmyE / BLF was fermented in a 5L tank. The fermentation medium formulation was: glucose 40 g / L, peptone 12 g / L, K₂HPO₄ 12.5 g / L, KH₂PO₄ 2.3 g / L, glycerol 4 mL, and (NH₄)₂Fe(SO₄)₂ 1.2 mg / L. The inoculum size was 6% v / v; the fermentation temperature was 32℃; the rotation speed was 300 rpm; and the fermentation time was 36 h. The results showed that fermentation in the tank increased the yield of the target protein by 0.3125 times, reaching 10⁵ mg / L. Figure 5 ).
[0066] This invention provides a method and approach for constructing and applying a Bacillus amyloliquefaciens expressing bovine lactoferrin. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.
Claims
1. A recombinant Bacillus amyloliquefaciens expressing bovine lactoferrin, characterized in that, Bacillus amyloliquefaciens B. amyloliquefaciens As a host, heterologous expression of the bovine lactoferrin encoding gene BLF Obtained; The recombinant Bacillus amyloliquefaciens also incorporates the signal peptide AmyE. B. amyloliquefaciens To decompose Bacillus amyloliquefaciens NFΔSE; Among them, the bovine lactoferrin encoding gene BLF Its nucleotide sequence is shown in SEQ ID NO.1; The signal peptide AmyE has the nucleotide sequence shown in SEQ ID NO.3; The method for constructing the recombinant Bacillus amyloliquefaciens expressing bovine lactoferrin includes the following steps: (1) The gene encoding bovine lactoferrin BLF The plasmid was cloned into the expression vector pMA5 and verified to obtain the recombinant plasmid pMA5-BLF. (2) Using Bacillus amyloliquefaciens NFΔSE as a template, the signal peptide AmyE was amplified by PCR to obtain the gene fragment of the signal peptide AmyE. The gene fragment of the signal peptide AmyE was cloned into the recombinant plasmid pMA5-BLF in step (1). After transformation, it was verified by PCR to obtain the recombinant plasmid pMA5-AmyE-BLF. (3) Transform the recombinant plasmid obtained in step (2) into the host Bacillus amyloliquefaciens. B. amyloliquefaciens Recombinant Bacillus amyloliquefaciens was constructed in competent cells.
2. The recombinant Bacillus amyloliquefaciens according to claim 1, characterized in that, The aforementioned *Bacillus amyloliquefaciens* NFΔSE is a modified strain of *Bacillus amyloliquefaciens* NF, specifically, *Bacillus amyloliquefaciens* NF with the gene cluster encoding extracellular polysaccharide synthesis knocked out. epsA-O lipopolysaccharide synthesis gene lps Fructanase gene sacB Obtained.
3. The use of the recombinant Bacillus amyloliquefaciens according to any one of claims 1-2 in the fermentation preparation of bovine lactoferrin.
4. The application according to claim 3, characterized in that, The fermentation conditions are as follows: fermentation temperature 28-37℃; rotation speed 300 r / min; fermentation time 24-48 h.