Optimized bacillus subtilis expression vector, genetically engineered bacterium containing expression vector and application of genetically engineered bacterium

Abstract

The invention relates to the field of genetic engineering, in particular to an optimized bacillus subtilis expression vector, a genetically engineered bacterium containing the expression vector and application of the genetically engineered bacterium. The vector is based on pMA5 plasmids, and a shuttle type bacillus subtilis expression vector is optimized; an open reading framework with unknown functions in the bacillus subtilis expression vector is reduced, so that the size of the vector is reduced, the expression burden of cells is reduced, and an optimization effect is achieved; the optimized bacillus subtilis expression vector not only can be stably copied in escherichia coli, but also can be stably expressed in bacillus subtilis, and the expression level of heterologous gene coding protein is improved by utilizing the genetically engineered bacteria containing the optimized bacillus subtilis expression vector.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to an optimized Bacillus subtilis expression vector, a genetic engineering bacterium containing the expression vector and applications thereof. Background Art

[0002] Bacillus subtilis is an important industrial non-pathogenic microorganism belonging to the Gram-positive bacteria. Through the whole genome sequencing of this strain, we have a clear understanding of the genetic background and physiological characteristics of Bacillus subtilis. Using Bacillus subtilis to express foreign proteins has many advantages: (1) High safety, can be used in industrial production of food, medicine, etc.; (2) Can directly release secretory proteins into the culture medium, which is conducive to separation and purification; (3) Has the ability to efficiently secrete target proteins, has multiple secretion pathways, and the relevant secretory components in the pathways have been analyzed; The secreted recombinant protein can still maintain its natural conformation and biological activity in most cases; (4) Rapid growth, simple culture conditions, clear understanding of genetic background, and a good industrial production foundation.

[0003] The shuttle-type Bacillus subtilis expression vector is a recombinant vector that can be efficiently replicated and expressed in two different host cells (such as Bacillus subtilis and Escherichia coli). This type of vector is widely used in molecular biology and genetic engineering because of its ability to achieve gene transfer and expression between different types of cells. The key elements of this expression vector include a dual promoter system (regulating gene expression in Escherichia coli and Bacillus subtilis respectively), a multiple cloning site (MCS, used for flexible cloning and expression of foreign genes), a signal peptide sequence (such as AmyQ, used to guide secretory protein expression), a dual replicon system (adapting to the replication origin of the two hosts), a selection marker gene (facilitating the screening of successfully transformed strains), a terminator (such as rrnB, enhancing mRNA stability), and an optional replication enhancement element (increasing the number of plasmid copies). Through the synergistic effect of the above expression elements, the shuttle-type Bacillus subtilis expression vector can not only perform efficient plasmid cloning and amplification in Escherichia coli, but also achieve efficient expression of foreign genes in Bacillus subtilis.

[0004] Optimization of Bacillus subtilis expression vectors is crucial to improving the efficient expression of foreign genes, especially in terms of improving expression efficiency and stability. Further optimization of key elements of expression vectors will provide more possibilities for improving their expression efficiency and stability in different hosts, thereby promoting the progress of genetic engineering research. By streamlining open reading frames (ORFs) with unknown functions and reducing metabolic burden, it will help improve host growth rate and vector stability, achieve efficient expression of heterologous proteins, and provide cost-effective solutions for industry and scientific research.

[0005] Currently, there are few studies on optimizing key elements of expression vectors and streamlining open reading frames with unknown functions to reduce metabolic burden, which is of great significance for improving the growth rate of the host and the stability of the vector and achieving efficient expression of heterologous proteins.

[0006] Therefore, streamlining the unknown functional open reading frames of Bacillus subtilis expression vectors and optimizing the Bacillus subtilis expression vectors are of great value in promoting genetic engineering research, improving industrial production efficiency and reducing costs, and provide economical and effective solutions for industry and scientific research. Summary of the invention

[0007] The purpose of the present invention is to provide a Bacillus subtilis expression vector;

[0008] Another object of the present invention is to provide a genetically engineered bacterium containing a Bacillus subtilis expression vector and its application.

[0009] The technical solution adopted to achieve the purpose of the present invention is:

[0010] The purpose of the present invention is to significantly improve the expression level of heterologous proteins by reducing and optimizing the unknown functional sequences in the Bacillus subtilis expression vector.

[0011] Specifically, the optimized Bacillus subtilis expression vector is obtained by reducing the sequence of unknown function from the Bacillus subtilis expression vector having the nucleotide sequence of SEQ ID NO: 1, and SEQ ID NO: 1 is:

[0012]

[0013]

[0014]

[0015]

[0016] Furthermore, the Bacillus subtilis expression vector is a pMA5 plasmid.

[0017] Further, the sequence reducing unknown functions is selected from ORF sequences;

[0018] Furthermore, the reduced sequence is the 5213-5980 bp interval in SEQ ID NO: 1, and the nucleotide sequence is ORF-1 of SEQ ID NO: 2; SEQ ID NO: 2 is:

[0019]

[0020] Or the shortened sequence is the 2982-3510 bp interval in SEQ ID NO: 1, and the nucleotide sequence is ORF-2 of SEQ ID NO: 3; SEQ ID NO: 3 is:

[0021]

[0022] Furthermore, the present application also discloses a genetically engineered bacterium, which contains the optimized Bacillus subtilis expression vector.

[0023] Furthermore, the present application also discloses the use of the genetically engineered bacteria in the fermentation production of proteins encoded by heterologous genes;

[0024] Furthermore, the protein encoded by the heterologous gene is glycogen synthase.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present application provides a method for optimizing a Bacillus subtilis expression vector by reducing an open reading frame of unknown function in the Bacillus subtilis expression vector. The vector in the present application is based on the pMA5 plasmid, and the ORF sequence of unknown function in the vector is removed, thereby reducing the size of the vector and alleviating the expression burden of the cell. The optimized vector can be stably replicated in Escherichia coli. The fluorescence intensity of the recombinant Bacillus subtilis strain obtained after reducing the OFR-1 sequence is increased by 11% compared with that before the reduction, and the fluorescence intensity of the recombinant Bacillus subtilis strain obtained after reducing the OFR-2 sequence is increased by 17% compared with that before the reduction, which proves that both have a significant effect on improving the expression level of heterologous gene-encoded proteins in Bacillus subtilis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the genetic map of the plasmid vector pMATE-GFP-GlgA constructed in Example 1 of the present invention;

[0028] Figure 2 The OD value of the fermentation liquid obtained by fermenting Bacillus subtilis for 48 hours in Examples 1, 2 and 3 of the present invention is 600 Test chart;

[0029] Figure 3 This is a test graph of the fluorescence intensity of the fermentation sample obtained by Bacillus subtilis in Examples 1, 2 and 3 of the present invention when the fermentation was 48 hours. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0032] Example 1

[0033] The glycogen synthase gene used in this example is derived from Escherichia coli, and its nucleotide sequence is SEQ ID NO:4. SEQ ID NO:4 is:

[0034]

[0035]

[0036] (I) Construction of expression vector pMA5-GFP-GlgA

[0037] GFP is selected as the expression reporter gene, and the expression intensity of the target protein can be screened by the fluorescence intensity expressed by GFP. This method of screening directly through growth differences is an efficient screening solution.

[0038] Using the homologous recombination method, the glycogen synthase gene sequence and the pMA5 vector were first linearized by PCR, wherein the amplification primers used for the glycogen synthase gene sequence fragment and the GFP sequence fragment contained homologous sequences that were complementary to the two ends of the linearized vector fragment for subsequent homologous recombination connection reactions.

[0039] Linearized vector amplification: pMA5 was used as a template and the following primers were used for PCR. The components were mixed in a sterile thin-walled centrifuge tube in the following order. The reaction system was shown in Table 1:

[0040] Table 1

[0041] Reactants content Upstream primer 1 (10 μmol / L) 2.5μL Downstream primer 2 (10 μmol / L) 2.5μL DNA template 2μL PrimeSTARMaxPremix (2×) 25μL <![CDATA[ddH2O]]> 18μL Total volume 50μL

[0042] The amplification conditions were as follows: 98°C for 2 min, 1 cycle; 98°C for 30 s, 60°C for 15 s, 72°C for 3 min, 30 cycles; and the last cycle was 72°C for 5 min.

[0043] The glycogen synthase gene was obtained by gene synthesis, and the synthesized glycogen synthase gene was used as a template for PCR. The components were mixed in a sterilized thin-walled centrifuge tube in the following order. The reaction system is shown in Table 2:

[0044] Table 2

[0045] Reactants content Upstream primer 3 (10 μmol / L) 2.5μL Downstream primer 4 (10 μmol / L) 2.5μL DNA template 2μL PrimeSTARMaxPremix (2×) 25μL <![CDATA[ddH2O]]> 18μL Total volume 50μL

[0046] Amplification conditions were as follows: 98°C for 2 min, 1 cycle; 98°C for 30 s, 60°C for 15 s, 72°C for 2 min, 30 cycles; and the last cycle was 72°C for 5 min.

[0047] Using the GFP gene fragment as a template, PCR was performed. The components were mixed in a sterilized thin-walled centrifuge tube in the following order. The reaction system is shown in Table 3:

[0048] Table 3

[0049] Reactants content Upstream primer 5 (10 μmol / L) 2.5μL Downstream primer 6 (10 μmol / L) 2.5μL DNA template 2μL PrimeSTARMaxPremix (2×) 25μL <![CDATA[ddH2O]]> 18μL Total volume 50μL

[0050] The amplification conditions were as follows: 98°C for 2 min, 1 cycle; 98°C for 30 s, 60°C for 15 s, 72°C for 1 min, 30 cycles; and the last cycle was 72°C for 5 min.

[0051] The purified glycogen synthase gene fragment and the GFP gene fragment were used as templates at a molar ratio of about 1:2, and the two fragments were assembled in a two-step method:

[0052] In the first step, the components are mixed in a sterilized thin-walled centrifuge tube in the following order. The reaction system is shown in Table 4:

[0053] Table 4

[0054] Reactants content Glycogen synthase gene fragment 2.5μL GFP gene fragment 2.5μL PrimeSTARMaxPremix (2×) 10μL <![CDATA[ddH2O]]> 5μL Total volume 20μL

[0055] Amplification conditions were as follows: 98°C for 2 min, 1 cycle; 98°C for 30 s, 60°C for 15 s, 72°C for 2 min, 10 cycles; and the last cycle was 72°C for 5 min.

[0056] In the second step, the components were mixed in a sterilized thin-walled centrifuge tube in the following order. The reaction system was as shown in Table 5:

[0057] Table 5

[0058] Reactants content Upstream primer 5 (10 μmol / L) 2μL Downstream primer 4 (10 μmol / L) 2μL First step product 2μL PrimeSTARMaxPremix (2×) 25μL <![CDATA[ddH2O]]> 19μL Total volume 50μL

[0059] Amplification conditions were as follows: 98°C for 2 min, 1 cycle; 98°C for 30 s, 60°C for 15 s, 72°C for 2 min, 30 cycles; and the last cycle was 72°C for 5 min.

[0060] The linearized vector fragment and the Overlap fragment were mixed as templates at a molar ratio of about 1:2 for homologous recombination. The components were mixed in a sterilized thin-walled centrifuge tube in the following order. The reaction system is shown in Table 6:

[0061] Table 6

[0062] Reactants content Vector fragment 100ng Overlap Clip 200ng ExnaseⅡ 2μL 5×CEⅡBuffer 4μL <![CDATA[ddH2O]]> up to 20 μL Total volume 20μL

[0063] The reaction conditions were: 37°C for 30 min.

[0064] The ligation product was directly transformed into Escherichia coli DH5α, and spread on ampicillin plates (100 μg / mL), cultured at 37° C. overnight, and positive transformants were screened to obtain plasmid pMATE-GFP-GlgA.

[0065] The successfully constructed plasmid was transformed into Bacillus subtilis to obtain a recombinant strain.

[0066] (II) Fermentation of glycogen synthase by Bacillus subtilis strains

[0067] The recombinant strain of glycogen synthase was fermented. The strain was taken out of the -80℃ refrigerator and cultured overnight in LB solid medium. The next day, it was transferred to LB liquid medium and cultured at 37℃ and 220rpm for 12h as seed liquid. The fermentation medium was 2×SR medium, whose components were 1.5% (w / v) peptone, 2.5% (w / v) yeast extract and 0.3% (w / v) K2HPO4, pH 7.2. The volume of the culture medium in the 250mL triangular shake flask used for fermentation culture was 30mL, and the inoculation volume was 1:100 (v / v). The fermentation broth sample was obtained by fermentation at 37℃ and 220rpm for 48h; two parallels were set for each fermentation sample.

[0068] The fluorescence intensity was used to determine the expression intensity of glycogen synthase. The excitation wavelength of the fluorescent reporter protein was 488 nm, and there was an emission peak at about 507 nm. The fermentation broth sample was diluted twenty times and added to a 96-well black bottom transparent plate at a volume of 200 mL per well. 485 nm was selected as the excitation wavelength and 528 nm as the emission wavelength. The fluorescence intensity of the sample was detected using an ELISA reader.

[0069] Example 2 Reduction of ORF-1 for vector optimization and fermentation testing

[0070] (I) Construction of shortened plasmid pMATE-GFP-GlgA1

[0071] The sequence of the 5213-5980 region of the plasmid pMATE-GFP-GlgA was reduced by inverse PCR. The components were mixed in a sterilized thin-walled centrifuge tube in the following order. The reaction system is shown in Table 7:

[0072] Table 7

[0073] Reactants content Upstream primer 7 (10 μmol / L) 2.5μL Downstream primer 8 (10 μmol / L) 2.5μL DNA template 2μL PrimeSTARMaxPremix (2×) 25μL <![CDATA[ddH2O]]> 18μL Total volume 50μL

[0074] The amplification conditions were as follows: 98°C for 2 min, 1 cycle; 98°C for 30 s, 60°C for 15 s, 72°C for 3 min, 30 cycles; and the last cycle was 72°C for 5 min.

[0075] The 5' end of the linearized product obtained by inverse PCR was phosphorylated using PNK enzyme. The components were mixed in a sterile thin-walled centrifuge tube in the following order. The reaction system is shown in Table 8:

[0076] Table 8

[0077] Reactants content DNA 800ng PNK 1μL T4Buffer 2μL <![CDATA[ddH2O]]> up to 18 μL Total volume 18μL

[0078] The reaction conditions were as follows: phosphorylation at 37°C for 30 min, inactivation at 75°C for 10 min, cooling to room temperature, adding 2 μL of T4 ligase to the system to cyclize the ligation product at 16°C, and reacting overnight.

[0079] The cyclized product was directly transformed into Escherichia coli DH5α, and spread on ampicillin plates (100 μg / mL), cultured at 37°C overnight, and positive transformants were screened to obtain plasmid pMATE-GFP-GlgA1.

[0080] The successfully constructed plasmid was transformed into Bacillus subtilis to obtain a recombinant Bacillus subtilis strain containing the plasmid pMATE-GFP-GlgA1.

[0081] (ii) Fermentation production of glycogen synthase by recombinant Bacillus subtilis strain containing plasmid pMATE-GFP-GlgA1

[0082] The fermentation conditions were the same as those described in Example 1. The shake flask fermentation was carried out for 48 h. The OD600 value of the fermentation sample was measured and the fluorescence intensity of the sample was detected using an ELISA instrument. Figure 3 and Figure 2 As shown, after the OFR-1 sequence was reduced, the fluorescence intensity increased by 11% compared with that before reduction, proving that reducing ORF-1 has a significant optimization effect on the expression of glycogen synthase.

[0083] Example 3 Reduction of ORF-2 for vector optimization

[0084] (I) Construction of shortened plasmid pMATE-GFP-GlgA2

[0085] The sequence of the plasmid pMATE-GFP-GlgA in the range of 2982-3510 was reduced by inverse PCR. The components were mixed in a sterilized thin-walled centrifuge tube in the following order, and the reaction system was shown in Table 9:

[0086] Table 9

[0087] Reactants content Upstream primer 7 (10 μmol / L) 2.5μL Downstream primer 8 (10 μmol / L) 2.5μL DNA template 2μL PrimeSTARMaxPremix (2×) 25μL <![CDATA[ddH2O]]> 18μL Total volume 50μL

[0088] The amplification conditions were as follows: 98°C for 2 min, 1 cycle; 98°C for 30 s, 60°C for 15 s, 72°C for 3 min, 30 cycles; and the last cycle was 72°C for 5 min.

[0089] The 5' end of the linearized product obtained by inverse PCR was phosphorylated using PNK enzyme. The components were mixed in a sterilized thin-walled centrifuge tube in the following order. The reaction system is shown in Table 10:

[0090] Table 10

[0091]

[0092]

[0093] The reaction conditions were: phosphorylation at 37°C for 30 min, inactivation at 75°C for 10 min, and cooling to room temperature. 2 μL of T4 ligase was added to the system to circularize the ligation product at 16°C (overnight).

[0094] The cyclized product was directly transformed into Escherichia coli DH5α, and spread on ampicillin plates (100 μg / mL), cultured at 37°C overnight, and positive transformants were screened to obtain plasmid pMATE-GFP-GlgA2.

[0095] The successfully constructed plasmid was transformed into Bacillus subtilis to obtain a recombinant Bacillus subtilis strain containing the plasmid pMATE-GFP-GlgA2.

[0096] (ii) Fermentation production of glycogen synthase by recombinant Bacillus subtilis strain containing plasmid pMATE-GFP-GlgA2

[0097] The fermentation conditions were the same as those described in Example 1. The shake flask fermentation was performed for 48 h. The OD of the fermentation samples was measured. 600 The fluorescence intensity of the samples was measured using an enzyme-labeled instrument. Figure 3 and Figure 2 It can be seen that after the OFR-2 sequence was reduced, the fluorescence intensity increased by 17% compared with that before reduction, proving that reducing ORF-2 has a significant optimization effect on the expression of glycogen synthase.

[0098] Therefore, in combination with Example 2 and this example, high-level expression of heterologous proteins in Bacillus subtilis is achieved by reducing the ORF sequences of unknown functions in the vector.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An optimized Bacillus subtilis expression vector, characterized in that: It is obtained by reducing the sequence of unknown function from the Bacillus subtilis expression vector with the nucleotide sequence of SEQ ID NO: 1, The shortened sequence is the 5213-5980 bp interval in SEQ ID NO: 1, and the nucleotide sequence is ORF1 of SEQ ID NO: 2, or 2982-3510 bp interval, the nucleotide sequence is ORF2 of SEQ ID NO: 3, thereby obtaining the optimized Bacillus subtilis expression vector.

2. A genetically engineered bacterium comprising the optimized Bacillus subtilis expression vector according to claim 1.

3. Use of the genetically engineered bacteria as claimed in claim 2 in the fermentation production of proteins encoded by heterologous genes.

4. The use of the genetically engineered bacteria in the fermentation production of heterologous gene-encoded proteins as claimed in claim 3, characterized in that: The protein encoded by the heterologous gene is glycogen synthase.

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