Bacillus subtilis for expressing bovine-derived beta-lactoglobulin and construction method thereof

Abstract

The invention relates to bacillus subtilis for expressing bovine-derived beta-lactoglobulin and a construction method of the bacillus subtilis, and belongs to the technical field of microorganisms. According to the bacillus subtilis provided by the invention, bacillus subtilis BSF01 is used as a host, a constitutive promoter P566 is used for heterologous expression of a bovine-derived beta-lactoglobulin coding gene optimized by a codon, the yield of beta-lactoglobulin obtained by shake flask fermentation for 30 hours reaches 0.37 g / L, the yield of beta-lactoglobulin obtained by 3L fermentation tank fermentation for 44 hours reaches 3.06 g / L, the production intensity is 0.07 g / L / h, and the production cost is low. The bovine-derived beta-lactoglobulin is efficiently and insolubly expressed in a food safety level strain, the food industrial application of the beta-lactoglobulin is promoted, and the problems of food safety and excessive glycosylation possibly existing in heterologous expression of the beta-lactoglobulin in the prior art are solved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a Bacillus subtilis expressing bovine beta-lactoglobulin and a construction method thereof. Background Art

[0002] The proteins in milk are mainly composed of two major categories: casein and whey protein. Among them, β-lactoglobulin (β-Lg) accounts for 52% of the total whey protein and is the most abundant whey protein component. β-Lg is a high-quality dietary protein, rich in essential amino acids and branched-chain amino acids, and has immunomodulatory functions. At the same time, as a lipid transport protein, β-Lg can bind to a variety of hydrophobic ligands and is regarded as a potential drug delivery carrier in the biopharmaceutical field. Currently, membrane separation, isoelectric precipitation, affinity chromatography, and aqueous two-phase extraction have been developed to separate and purify β-Lg from whey protein. However, these methods not only require a large amount of raw milk, but also have complex extraction processes, and also have problems such as long time consumption and high cost.

[0003] Currently, heterologous expression of β-Lg primarily utilizes Escherichia coli and yeast strains as hosts. Previous studies have achieved insoluble intracellular expression of β-Lg in E. coli, with inclusion bodies constituting approximately 15% of the total intracellular protein. To achieve soluble expression of β-Lg, researchers engineered a fusion expression system with a bacterial disulfide isomerase and achieved proper folding of β-Lg in E. coli origami. Loch et al. introduced L1A / I2S mutations to ensure proper cleavage of the N-terminal methionine, resulting in properly folded, soluble β-Lg with a yield of 40 mg / L. In addition to E. coli, yeast expression systems have also been widely used for β-Lg expression. Researchers have achieved secretory expression of β-Lg in Pichia pastoris, but the presence of three glycosylation sites is inconsistent with native bovine β-Lg. Denton et al. fused the α-mating factor leader peptide of Saccharomyces cerevisiae to the cDNA coding sequence for β-Lg to construct an engineered Pichia pastoris strain expressing β-Lg. After 72 hours of methanol induction, the secreted β-Lg production reached 1.5 g / L.

[0004] Although β-Lg can be heterologously expressed in Escherichia coli and Pichia pastoris, neither strain is food-safe, and the methanol-induced system also poses certain food safety risks. Compared to natural bovine β-Lg, which lacks glycosylation, β-Lg expressed in Pichia pastoris exhibits over-glycosylation, and protein expression still needs to be further improved.

[0005] With the rapid development of food synthetic biology, Bacillus subtilis, a generally recognized as safe (GRAS) microorganism, has become a research hotspot in the field of food protein synthesis. Its efficient protein synthesis capacity and lack of a protein glycosylation modification system make it suitable for the microbial synthesis of bovine β-Lg. Furthermore, the use of Bacillus subtilis in food protein synthesis is expected to increase public acceptance of recombinant proteins. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a Bacillus subtilis expressing bovine β-lactoglobulin, using Bacillus subtilis BSF01 as the chassis strain, and confirming that the constitutive promoter P 566 Heterologous expression of the codon-optimized bovine β-lactoglobulin encoding gene can significantly increase the yield of the target protein. Subsequently, by adjusting the culture medium formulation and fermentation parameters, efficient fermentation production of β-lactoglobulin was achieved.

[0007] The first object of the present invention is to provide a recombinant Bacillus subtilis, wherein the recombinant Bacillus subtilis uses a constitutive promoter P 566 The bovine β-lactoglobulin is heterologously expressed, wherein the nucleotide sequence of the bovine β-lactoglobulin is codon-optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.2.

[0008] The present invention has been found through screening to use constitutive promoter P 566 The yield of bovine β-lactoglobulin was significantly higher than that of the constitutive promoter P odhA or constitutive promoter P 43 .

[0009] Furthermore, the recombinant Bacillus subtilis uses Bacillus subtilis BSF01 as a chassis strain.

[0010] The second object of the present invention is to provide a method for constructing a recombinant Bacillus subtilis, which comprises constructing a recombinant Bacillus subtilis comprising P 566 The promoter and the expression vector of the sequence shown in SEQ ID NO. 2 are introduced into Bacillus subtilis competent cells to obtain the recombinant Bacillus subtilis.

[0011] Furthermore, the expression vector uses pHT01 as a backbone.

[0012] In one embodiment of the present invention, the P 566 The nucleotide sequence of the promoter is shown in SEQ ID NO.5.

[0013] The third object of the present invention is to provide a microbial agent comprising the above-mentioned recombinant Bacillus subtilis.

[0014] The fourth object of the present invention is to provide the use of the above-mentioned recombinant Bacillus subtilis or the above-mentioned microbial agent in the production of β-lactoglobulin.

[0015] A fifth object of the present invention is to provide a method for producing β-lactoglobulin, comprising the following steps:

[0016] Step S1, inoculating the above-mentioned recombinant Bacillus subtilis into a seed culture medium to obtain a primary seed solution;

[0017] Step S2, transferring the primary seed solution to a shake flask containing fermentation medium to obtain a secondary seed solution;

[0018] Step S3: centrifuging the secondary seed liquid to collect the bacterial cells, transferring them to a fermentation tank containing a fermentation medium, and fermenting to obtain β-lactoglobulin.

[0019] Furthermore, in step S2, the OD of the secondary seed solution 600 The value is 15-20.

[0020] Preferably, in step S2, the OD of the secondary seed solution is 600 The value is 15.

[0021] Furthermore, the fermentation medium includes 40-45 g / L glucose, 12-15 g / L tryptone, 24-30 g / L yeast powder, 2.3-2.5 g / L KH2PO4 and 12.54-13 g / L K2HPO4.

[0022] Preferably, the fermentation medium comprises 40 g / L glucose, 12 g / L tryptone, 24 g / L yeast extract, 2.3 g / L KH2PO4 and 12.54 g / L K2HPO4.

[0023] Furthermore, in step S3, the fermentation time is 32-44 hours.

[0024] Preferably, in step S3, the fermentation time is 44 hours.

[0025] Furthermore, in step S3, the glucose concentration in the fermentation tank is maintained at 10-20 g / L.

[0026] Beneficial effects of the present invention:

[0027] The present invention optimizes the codon of the bovine β-lactoglobulin coding gene sequence and combines the constitutive promoter P 566The application of this technology solves the problem of low expression efficiency of bovine β-lactoglobulin in food-safe Bacillus subtilis, achieving efficient and sustained expression without the need for inducers. This addresses the food safety and excessive glycosylation issues that may exist in heterologous β-lactoglobulin expression in existing technologies. By optimizing the fermentation process (including culture medium formulation, fermentation time, and feeding strategy), β-lactoglobulin production has been significantly increased, making it suitable for large-scale production. This provides a low-cost, high-purity production method for the food industry application of β-lactoglobulin, which has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0029] Figure 1 The results of intracellular expression of recombinant Bacillus subtilis in Example 1 of the present invention are shown in Figure a, wherein a is an SDS-PAGE result showing the effect of different promoters on the intracellular expression level of β-lactoglobulin, and b is the expression yield of β-lactoglobulin under the influence of different promoters;

[0030] Figure 2 The results of β-lactoglobulin synthesis by recombinant Bacillus subtilis in a 3 L fermenter in Example 2 of the present invention are shown in FIG. 1 , wherein a is the fermentation process curve of the recombinant Bacillus subtilis and b is the yield and production intensity of β-lactoglobulin. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0032] Bacillus subtilis BSF01 is disclosed in the document Synergistic regulation of chassis cell growth and screening of promoters, signal peptides and fusion protein linkers for enhanced recombinant protein expression in Bacillus subtilis.

[0033] DNA polymerase was purchased from Takara, restriction endonucleases and T4 ligase were purchased from New England Biolabs, plasmid extraction kits were purchased from Shanghai Biotech Co., Ltd., and PCR product nucleic acid purification kits were purchased from Thermo Scientific.

[0034] Seed culture was performed using Luria-Bertani (LB) medium consisting of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0035] Shake flask fermentation was carried out using Terrific Broth (TB) medium: 5 g / L glycerol, 12 g / L tryptone, 24 g / L yeast powder, 2.3 g / L KH2PO4 and 12.54 g / L K2HPO4.

[0036] The medium formula for fed-batch fermentation in a 3 L fermentor was as follows: 40 g / L glucose, 12 g / L tryptone, 24 g / L yeast extract, 2.3 g / L KH2PO4, and 12.54 g / L K2HPO4.

[0037] Feed medium: 750 g / L glucose and 200 g / L yeast powder.

[0038] SDS-PAGE analysis and yield determination of protein: First, the harvested bacteria were resuspended in PBS and the bacteria were broken by ultrasonic disruptor under ice bath conditions. The intracellular soluble supernatant and the lysed cell fragments were obtained by centrifugation. The cell fragments were resuspended in an equal volume of PBS. For SDS-PAGE analysis, the protein sample was first treated with 4× sample buffer, boiled and centrifuged. The sample was separated by 10% SDS-PAGE gel, and MESSDS buffer (Thermo Scientific, USA) was used as the electrophoresis buffer. After the electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 staining solution (Biyuntian Biotechnology Co., Ltd., Shanghai). The β-Lg yield of the intracellular insoluble component was determined using a modified Bradford protein assay kit (Shanghai Sangon Biotechnology Co., Ltd.).

[0039] Example 1: Construction of a recombinant Bacillus subtilis strain expressing bovine β-Lg

[0040] The gene sequence encoding bovine β-Lg (amino acid sequence shown in SEQ ID NO. 1) was codon-optimized, and the optimized gene sequence is shown in SEQ ID NO. 2. The optimized bovine β-Lg gene sequence lgb was obtained by PCR amplification using primers lgb-F1 and lgb-R1.

[0041] Use primers P 43 -F1 and P 43 -R1, P odhA -F1 and P odhA -R1, P 566 -F1 and P 566 -R1 promoter P was obtained by PCR amplification 43(as shown in SEQ ID NO.3), P odhA (as shown in SEQ ID NO.4) and P 566 (as shown in SEQ ID NO.5).

[0042] The pHT01 vector backbone was obtained by PCR amplification using primers pHT-F1 and pHT-R1. The promoter P 43 、P odhA and P 566 The gene fragment lgb was connected to the pHT01 vector backbone and transformed into Escherichia coli DH5α competent cells. After sequencing verification, the recombinant plasmid pHT01-P was obtained. 43 -lgb、pHT01-P odhA -lgb and pHT01-P 566 -lgb. The primer sequences involved are shown in Table 1.

[0043] The target plasmid pHT01-P 43 -lgb、pHT01-P odhA -lgb and pHT01-P 566 -lgb was transformed into Bacillus subtilis BSF01, and the recombinant strain BSF01-pHT01-P was obtained. 43 、BSF01-pHT01-P odhA and BSF01-pHT01-P 566 After 30 h of shake flask fermentation, the expression of intracellular β-Lg was analyzed by SDS-PAGE and the protein yield was determined. Figure 1 As shown, using promoter P 566 The expression of bovine β-lactoglobulin can significantly increase the yield, with the yield reaching 0.37 g / L after 30 hours of shake flask fermentation.

[0044] Table 1 Primer sequences

[0045]

[0046] Example 2: Recombinant strain BSF01-pHT01-P 566 -lgb's 3L fermenter fed-batch fermentation

[0047] Pick up recombinant Bacillus subtilis BSF01-pHT01-P 566 A single colony of -lgb was inoculated into a 250 mL shake flask containing 10 mL of LB medium and cultured at 37 °C and 220 rpm for 10 h to obtain the primary seed solution.

[0048] The first-stage seed solution was transferred to a 500 mL shake flask containing 25 mL of fermentation medium at a transfer volume of 1%, and cultured at 37 °C and 220 rpm until the OD 600 Reach 15 and obtain the secondary seed liquid.

[0049] The secondary seed liquid was centrifuged to collect the cells, resuspended in 100 mL of fermentation medium, and inoculated into a 3-L fermentor (T&J-Atype, T&J Bioengineering Co., Ltd., Shanghai) containing 0.9 L of fermentation medium. Throughout the fermentation process, the pH was adjusted to 6.5 using aqueous ammonia and 8 M HCl. The glucose concentration was maintained between 10 g / L and 20 g / L. The fermentation temperature was maintained at 30°C, the aeration rate was set at 1.5 vvm, and the DO was maintained at approximately 35% by controlling the agitation rate.

[0050] The fermentation process curve is as follows Figure 2 As shown in a, the strain grew rapidly in the early stage of fermentation, and feeding was started after 10 hours of fermentation. After 32 hours, OD 600 Stable at around 115. The yield and production intensity of β-Lg are as follows Figure 2 As shown in Figure (b), after 44 hours of fermentation, the intracellular insoluble yield of β-Lg reached 3.06 g / L, an 8.27-fold increase compared to the yield in shake flask fermentation, with a maximum production intensity of 0.070 g / L / h. Therefore, efficient expression of β-Lg was achieved using Bacillus subtilis as the expression host.

[0051] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A recombinant Bacillus subtilis, characterized in that: The recombinant Bacillus subtilis uses the constitutive promoter P 566 The bovine β-lactoglobulin is heterologously expressed, wherein the nucleotide sequence of the bovine β-lactoglobulin is codon-optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.

2.

2. The recombinant Bacillus subtilis according to claim 1, characterized in that: The recombinant Bacillus subtilis uses Bacillus subtilis BSF01 as a chassis strain.

3. The method for constructing the recombinant Bacillus subtilis according to claim 1 or 2, characterized in that: The construction method includes constructing a P 566 The promoter and the expression vector of the sequence shown in SEQ ID NO. 2 are introduced into Bacillus subtilis competent cells to obtain the recombinant Bacillus subtilis.

4. A microbial agent comprising the recombinant Bacillus subtilis according to claim 1 or 2.

5. Use of the recombinant Bacillus subtilis according to claim 1 or 2 or the microbial agent according to claim 4 in producing β-lactoglobulin.

6. A method for producing β-lactoglobulin, characterized in that: The following steps are involved: Step S1, inoculating the recombinant Bacillus subtilis according to claim 1 or 2 into a seed culture medium to obtain a primary seed solution; Step S2, transferring the primary seed solution to a shake flask containing fermentation medium to obtain a secondary seed solution; Step S3: centrifuging the secondary seed liquid to collect the bacterial cells, transferring them to a fermentation tank containing a fermentation medium, and fermenting to obtain β-lactoglobulin.

7. The method according to claim 6, characterized in that: In step S2, the OD of the secondary seed solution 600 The value is 15-20.

8. The method according to claim 6, wherein: The fermentation medium includes 40-45 g / L of glucose, 12-15 g / L of tryptone, 24-30 g / L of yeast powder, 2.3-2.5 g / L of KH2PO4 and 12.54-13 g / L of K2HPO4.

9. The method according to claim 6, wherein: In step S3, the fermentation time is 32-44 hours.

10. The method according to claim 6, wherein: In step S3, the glucose concentration in the fermentation tank is maintained at 10-20 g / L.

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