CN117887748A

CN117887748A - Method for synthesizing selenoprotein by inserting selenocysteine into lactobacillus at fixed point and application

Info

Publication number: CN117887748A
Application number: CN202410127531.0A
Authority: CN
Inventors: 王成华; 刘辰星; 彭静静; 岳世阳; 方钰辉; 王玲钰; 陈格格
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2024-01-30
Filing date: 2024-01-30
Publication date: 2024-04-16

Abstract

The invention relates to a method for synthesizing selenoprotein by inserting selenocysteine into lactobacillus at fixed points and application thereof. Lactic acid bacteria lack regulatory genes for selenoprotein synthesis and do not have the capability of synthesizing selenoprotein by site-specific insertion of selenocysteine. The invention adopts the means of genetic engineering to integrate selenocysteine synthesized by escherichia coli into lactobacillus through an insertion way (SBIP), and the recombinant expression dual-function glutathione synthetase GshF increases selenium donor, thereby realizing the efficient fixed-point insertion of selenocysteine into lactobacillus to synthesize selenoprotein. The invention provides a new method for expression, engineering transformation and production of the exogenous functional active selenoprotein in the fields of food, biology, medicine and the like, and is suitable for development and utilization of novel selenium-enriched functional lactic acid bacteria.

Description

Method for synthesizing selenoprotein by inserting selenocysteine into lactobacillus at fixed point and application

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to a method for synthesizing selenoprotein by inserting selenocysteine into lactobacillus at fixed points and application thereof.

Background

Selenium in dietary selenium and selenium-enriched yeast mainly exists in a free form in the form of selenomethionine (SeMet), and can also be randomly inserted to replace methionine to enter protein, so that the selenium-enriched yeast can be well absorbed and stored. However, complicated selenium metabolic pathways and nonspecific doping cause the common problems of undefined selenium metabolic flow, unstable selenium spectrum composition and low selenoprotein content, which also causes the low yield, high cost and poor activity of the selenoprotein produced by the current extraction method, and limits the application of the selenoprotein in the fields of foods, medicines and nutritional products. The development of a selenium-enriched method for specifically guiding the selenium metabolic flow to directionally synthesize specific selenoprotein is helpful for fundamentally solving the bottleneck problem existing in the selenium supplementing mode.

Unlike the way SeMet inserts randomly into proteins, selenocysteine (Sec), which is regarded as the 21 st natural amino acid constituting the protein, is encoded by the stop codon UGA, and is specifically inserted into the protein in co-translated form, which is the main manifestation of selenium biological activity. Over 25 selenoproteins have been found so far, most of them being human, mouse, pig and other higher animal selenoproteins, except for a few prokaryotic selenoproteins such as E.coli formate dehydrogenase H (Formate dehydrogenase H, fdhH). Typically, glutathione peroxidase (glutathione peroxidase, GPx) and selenoprotein H and the like play an important role in regulating the redox state of cells, removing excessive active oxides generated in vivo and the like. Therefore, research on directional production of selenoprotein by means of biosynthesis and specific insertion mechanism of selenocysteine has important scientific significance and potential application prospect. The selenocysteine synthesis and insertion pathway (Sec biosynthesis and insertion pathway, SBIP) involves 1 cis-acting element and 4 regulatory protein factors, which has been resolved in E.coli at present, but E.coli is not a food grade safety strain (GRAS) and has limited its use in the fields of food, medicine and nutraceuticals to some extent.

Compared with escherichia coli, lactobacillus serving as probiotics may have a wider application prospect in selenium supplementation. The genome search and the analysis result by adopting the prokaryotic selenoprotein gene prediction program bSECIS show that the lactobacillus does not contain selenoprotein encoding genes and related regulatory factors for selenoprotein biosynthesis, and no report of the selenoprotein of the lactobacillus and the related regulatory factors for selenoprotein biosynthesis has been found so far. Researches on selenium-rich lactobacillus show that selenium-rich probiotics such as lactobacillus plantarum, enterococcus, streptococcus, bifidobacterium and the like and fermentation products thereof mainly have the forms of Sec, simple substances, nano selenium, methylated selenium and the like after inorganic selenium is converted, and the problems of complex selenium form composition and nonspecific insertion exist. If these problems could be solved, the use of selenium-enriched probiotics would be further.

Lactococcus lactis is the most widely used model of the food-grade gene expression system of lactic acid bacteria and has been successfully used as a cell factory for the efficient production of recombinant proteins and metabolites. Based on the important application potential of selenoprotein and Sec fixed-point insertion thereof in nutrition, health and metabolic engineering, the development of a method for directionally synthesizing probiotics selenoprotein has great significance. By means of pathway engineering, the synthesis of Sec and fixed-point insertion mechanism are introduced, and the introduction of lactic acid bacteria selenium metabolic flow into artificial design selenoprotein is an important direction for solving the problem, and has important application prospect for the production of selenoprotein with medical health efficacy and probiotics rich in specific selenoprotein.

Disclosure of Invention

The invention aims to solve the defects in the prior art, and enables the directional synthesis of selenocysteine by site-specific insertion of selenocysteine into lactobacillus which does not have selenocysteine synthesis and insertion ways and can not synthesize selenoprotein by genetic engineering means, thereby realizing the expression of selenocysteine-containing selenoprotein in probiotics.

In order to accomplish the above object, the present invention provides a method for synthesizing selenoprotein by site-directed insertion of selenocysteine into lactic acid bacteria, wherein the method comprises the steps of recombinantly expressing selenocysteine synthesis and insertion pathway (SBIP) element SelA, selB, selC, selD in lactic acid, adding selenium donor enzyme such as bifunctional glutathione synthetase GshF, and realizing efficient site-directed insertion expression of selenoprotein by the sequence shown as SEQ ID NO1, 2, 3 and 6, and comprising the following steps:

(1) Constructing a SelA, selB, selC, selD-containing plasmid as a vector for expressing the E.coli SBIP pathway in lactic acid bacteria;

(2) Constructing a plasmid containing GshF and a target selenoprotein gene;

(3) Introducing the plasmids constructed in the steps (1) and (2) into lactobacillus together by means of electric shock transformation and the like, and constructing a gene recombinant strain for expressing the target selenoprotein;

(4) And (3) fermenting and culturing the gene recombinant strain in the step (3) by adopting a selenium and Nisin induction expression mode to synthesize the target selenoprotein.

More preferably, the gene which is a key element of the selenocysteine synthesis and insertion pathway (SBIP) of E.coli is integrated in the form of a plasmid into lactococcus lactis NZ 9000; meanwhile, another compatible plasmid is used for loading difunctional glutathione peroxidase GshF capable of enhancing the generation of a selenium donor and target selenoprotein containing SECIS, and the difunctional glutathione peroxidase GshF and the target selenoprotein are introduced into lactococcus lactis NZ9000 containing a SBIP pathway to realize the expression of the selenoprotein in lactobacillus, and the specific steps are as follows:

1. Construction of helper plasmid pNZ8148-pC1ABD containing SBIP pathway element genes

2. Construction of lactic acid bacteria NZ9000/SBIP containing SBIP pathway

3. Construction of expression vector containing selenium protein of interest and bifunctional glutathione synthetase GshF required for enhancing selenium donor production

4. Construction of lactococcus lactis NZ9000/SBIP-NN with SBIP pathway genes and a target selenoprotein gene;

5. expression verification of selenoprotein of interest

Wherein:

Step 1 the construction of helper plasmid pNZ8148-pC1ABD containing SBIP pathway element genes comprises the steps of:

1) E, extracting the whole genome of the color MG 1655;

2) The SBIP pathway element gene SelA, selB, selC, selD, amplification of promoter P1;

3) Ligation of SBIP pathway element genes;

4) Verification of the pathway plasmid pNZ8148-pC1 ABD.

Step 2 the construction steps of lactic acid bacteria NZ9000/SBIP containing Sec synthesis and insertion pathways comprise:

1) Preparing competent cells of lactobacillus NZ 9000;

2) In the competence of lactococcus lactis NZ9000, the helper plasmid pNZ8148-pC1ABD was subjected to electric shock transformation;

3) Verification of lactococcus lactis NZ9000/SBIP

4) Preparation of lactococcus lactis NZ9000/SBIP competent.

Step 3 the construction of an expression vector containing a desired selenoprotein and a bifunctional glutathione synthetase GshF required for enhancing selenium donor production comprises the steps of:

1) Synthesizing a carrier containing GshF;

2) Obtaining a target selenoprotein gene sequence and preparing fragments;

3) Ligating the selenoprotein fragment of interest with a plasmid vector comprising GshF;

4) And (5) verifying the target selenoprotein expression vector.

Step 4 construction of lactococcus lactis NZ9000 having a SBIP pathway gene and a selenoprotein gene of interest comprising the steps of:

1) In the competence of lactococcus lactis NZ9000/SBIP, the electric shock transformation of the target selenoprotein expression vector plasmid;

2) Validation of lactococcus lactis NZ9000/SBIP-NN (NN represents the selenoprotein of interest).

The expression verification of the selenoprotein of step 5 comprises the following steps:

1) Preparing a target selenoprotein crude enzyme solution;

2) SDS-PAGE verification and enzyme activity determination of the selenium protein.

It is another object of the present invention to provide the use of the above method for the site-directed insertion of selenocysteine into lactic acid bacteria for the engineering of selenoprotein, preferably proteins whose active center comprises cysteine, by introducing the TGA codon encoding selenocysteine Sec, preferably simultaneously with the SECIS sequence, into the coding region of the selenocysteine gene to be engineered. The enzyme activity of the novel fdhF recombinant protein with the directed Sec introduced in one embodiment provided by the invention is 20.32U/g.

The invention also aims to provide a novel selenium-rich functional lactobacillus for recombinant expression of selenoprotein, which is constructed by the method for synthesizing selenocysteine by inserting selenocysteine into the lactobacillus at fixed points, and the recombinant lactobacillus has both the selenium-rich function and the activity function of the recombinant expression of selenoprotein.

The invention has the beneficial effects that:

1. The invention can realize the expression of selen protein containing Sec in lactobacillus, and lays a foundation for the production of selen protein with medical health efficacy and probiotics rich in specific selen protein.

2. The invention integrates SBIP approach derived from escherichia coli into lactobacillus by genetic engineering means, supplements selenoprotein anabolism approach for lactobacillus, successfully expresses recombinant selenoprotein fdhF with functional activity in lactobacillus, and the enzyme activity can reach 20.32U/g.

3. The method provided by the invention realizes efficient directional synthesis of selenoprotein in lactobacillus, and widens the application of lactobacillus in the fields of food, medicine, nutritional products and the like.

4. The method is characterized in that the selenoprotein lactobacillus GPX with one cysteine in the active center is engineered in the lactobacillus, the TGA codon of the Sec is used for replacing the cysteine codon of the active center, and a SECIS sequence is introduced, so that the Sec is inserted into the selenoprotein which does not contain the Sec at fixed points originally, and the TGA-SEICS mutant with the enzyme activity is successfully expressed.

5. According to the invention, through carrying out selenoprotein engineering transformation in lactobacillus, adverse effects of non-GRAS strains of escherichia coli are avoided, a new thought is provided for selenoprotein engineering transformation, and application potential of lactobacillus in industries such as food, medicine, nutrition and health is promoted.

6. The invention introduces the selenium metabolic flow of the lactobacillus into the artificial design selenoprotein by means of pathway engineering transformation, constructs a novel selenium-rich functional lactobacillus capable of directionally synthesizing selenoprotein fdhhF, has the functions of selenium enrichment and directionally synthesizing selenoprotein, provides a brand-new thought for solving the problem of complex selenium form caused by non-specific insertion in the selenium-rich lactobacillus, and provides a support for developing a new application scene of the selenium-rich lactobacillus.

Drawings

In order to more clearly illustrate the technical solution of the embodiments of the present invention, the drawings required to be used in the description of the embodiments will be briefly introduced. It is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art. Wherein:

FIG. 1 shows a plasmid map of the SBIP-way plasmid pNZ8148-pC1ABD constructed in accordance with the present invention.

FIG. 2 shows the results of the cleavage verification of the pathway plasmid pNZ8148-pC1 ABD.

FIG. 3 shows the results of the sequencing verification of the pathway plasmid pNZ8148-pC1 ABD.

FIG. 4 shows the results of the cleavage verification of the extracted plasmid in the pathway strain NZ 9000/SBIP.

FIG. 5 shows the results of sequencing verification of the extracted plasmid in the pathway strain NZ 9000/SBIP.

FIG. 6 shows the result of SDS-PAGE of pathway strain NZ9000/SBIP successfully expressing SelA, selB and SelD.

FIG. 7 shows a plasmid map of selenoprotein expression plasmid pTRKH-p 8G23FS constructed in a specific embodiment of the invention.

FIG. 8 shows the results of the cleavage assay for plasmid pTRKH2-p8G23 FS.

FIG. 9 shows the results of sequencing verification of plasmid pTRKH2-p8G23 FS.

FIG. 10 shows the results of the cleavage verification of two plasmids in lactic acid bacteria NZ 9000/SBIP-fdhF.

FIG. 11 shows the results of sequencing verification of two plasmids in lactic acid bacteria NZ 9000/SBIP-fdhF.

FIG. 12 shows the SDS-PAGE result of successful expression of fdhF by lactic acid bacteria NZ 9000/SBIP-fdhF.

FIG. 13 shows the SDS-PAGE results of crude enzyme solutions of various mutants of lactic acid bacteria GPX expressed by the method of the present invention.

FIG. 14 shows the results of enzyme activity detection of crude enzyme solutions of various mutants of lactic acid bacteria GPX expressed by the method of the present invention.

FIG. 15 shows the growth curves of recombinant selenium enriched functional lactic acid bacteria NZ9000/SBIP-fdhF and other control lactic acid bacteria.

FIG. 16 shows the selenium-enriched color difference results of recombinant selenium-enriched functional lactic acid bacteria NZ9000/SBIP-fdhF under different induction conditions.

FIG. 17 shows the SDS-PAGE results of recombinant selenium-enriched functional lactic acid bacteria NZ9000/SBIP-fdhF under different induction conditions.

FIG. 18 shows the result of fdhF enzyme activity of recombinant selenium-enriched functional lactic acid bacteria NZ9000/SBIP-fdhF under different induction conditions.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.

The primer used for PCR and other related sequences are listed in the sequence table; the relevant result graphs are all listed in the drawings of the specification.

The following examples are presented to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that many modifications may be made to the specific embodiments of the invention disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

Material

1 Strain, vector and plasmid

The strain vectors and plasmids used in the examples of the present invention are detailed in Table 1:

TABLE 1 plasmid of strains used in the present invention

2 Medium

LB broth: the components are 10g/L tryptone, 5g/L yeast extract powder and 10g/L sodium chloride; the pH value at 25 ℃ is 7.0+/-0.1; 25.0g of the extract is dissolved in 1000mL of distilled water and packaged; if a solid plate is prepared, 15g/L of agar powder is added on the basis.

M17 medium: 2.5g/L peptone; 2.5g/L casein peptone; 5g/L soytone; 5g/L beef powder; 2.5g/L yeast extract powder; 0.5g/L sodium ascorbate; 19g/L sodium beta-glycerophosphate; 0.25g/L anhydrous magnesium sulfate (MgSO ₄). The pH of the medium was adjusted to 7.0 to 7.4 with 1% NaOH and 1% HCl. If a solid plate is prepared, 15g/L of agar powder is added on the basis.

GM17 medium: to the sterilized and cooled M17 medium, sterilized and cooled glucose was added at a final concentration of 0.5% to obtain a GM17 medium.

G-SGM17B medium: 0.5M sucrose (171.15 g/L) was added to the sterilized cooled M17; 1.25% glycine (12.5 g/L); 0.5% glucose (5 g/L); sterilized CaCl ₂ and 20mM MgCl ₂ were then added to a final concentration of 2mM, respectively.

3, Reagent:

DNA electrophoresis solution-50×TAE buffer: 242g Tris,37.2g EDTA.N _a2·2H₂ O and 57.1mL glacial acetic acid were taken and mixed and then dissolved in a 1L volumetric flask and stored at room temperature. When used, the solution was diluted to 1×TAE Buffer.

Protein electrophoresis solution:

(1) 5 XTris-Glycine running buffer (pH 8.3): weighing 15.1g of Tris, 94g of glycine and 5g of SDS to a 1L beaker, adding 800mL of deionized water, stirring and dissolving, and adjusting the pH to 8.3; adding deionized water to fix the volume of the solution to 1L, and preserving at room temperature. When in use, deionized water is added for dilution.

(2) 5 Xloading buffer: 60mM Tris-HCl pH 6.8;2% SDS;0.1% bromophenol blue; 25% glycerol; 14.4mM beta-mercaptoethanol.

(3) Coomassie brilliant blue R250 staining solution: firstly, 1g of Coomassie brilliant blue R250 is taken in a 1000mL beaker, 250mL of isopropanol, 100mL of glacial acetic acid and 650mL of distilled water are added, and then the mixture is fully stirred and is subjected to ultrasonic treatment for 10 minutes to be uniformly mixed, and the mixture is preserved at room temperature for standby.

(4) Coomassie brilliant blue decolorized solution: 100mL of glacial acetic acid, 50mL of absolute ethyl alcohol and 850mL of distilled water are dissolved in a 1L volumetric flask, and the mixture is fully stirred and mixed for use.

Lactic acid bacteria competent solution:

(1) Solution I (600 mL formulation): 0.5M sucrose (102.69 g); 10% glycerol (60 mL glycerol+540 mL water).

(2) Solution II (200 mL formulation): 0.5M sucrose (34.23 g); 50mM EDTA. Na ₂·2H₂ O (3.722 g); 10% glycerol (20 mL glycerol+180 mL water).

Antibiotics:

(1) Preparation of 25mg/mL chloramphenicol resistant mother liquor: under aseptic conditions, 2.5g chloramphenicol was dissolved in 100mL absolute ethanol; filtering with 0.22 μm organic filter membrane, sterilizing, and storing at-20deg.C in dark place.

(2) Preparation of 25mg/mL erythromycin resistance mother solution: under aseptic condition, 2.5g of erythromycin is dissolved in 100mL of absolute ethyl alcohol, the volume is fixed by a volumetric flask, and the mother liquor is 25mg/mL; filtering with 0.22 μm organic filter membrane, sterilizing, and storing at-20deg.C in dark place.

(3) Preparation of 50mg/mL kanamycin resistant mother liquor: dissolving kanamycin in sterile water under sterile conditions, and fixing the volume by using a volumetric flask, wherein the mother solution is 50mg/mL; filtering with 0.22 μm water-based filter membrane, sterilizing, and standing at-20deg.C.

Preparation of 30. Mu.g/mL Nisin solution: 3mg of Nisin solid was dissolved in 100mL of 0.05% HAC, sterilized by filtration through a 0.22 μm organic filter under aseptic conditions and placed at-20℃for use.

8Mg/mL sodium selenite (Na ₂SeO₃) solution: 80mg of sodium selenite powder is weighed and dissolved in 10mL of sterile ultra-pure water; filtering with 0.22 μm water-based filter membrane, sterilizing, and standing at-20deg.C.

Preparation of GSH synthesis reaction solution:

(1) Preparation of 200mM Glu mother liquor, gly mother liquor and Cys mother liquor: 1.4713g of Glu, 0.7507g of Gly and 1.2116g of Cys are weighed and dissolved in 50mL of Tris-HCl buffer (100 mM, pH 8.0) respectively;

(2) Preparation of 100mM ATP mother liquor: 1g of ATP powder was completely dissolved in 9.86mL Tris-HCl buffer (100 mM, pH 8.0);

(3) 300mM MgCl ₂ stock: 0.57126g of MgCl ₂ were weighed and dissolved in 10mL Tris-HCl buffer (100 mM, pH 8.0).

Enzyme:

saira restriction endonuclease PstI, kpnI, apaL, xbaI, hindIII

Whole gold T4 ligase T4 DNA LIGASE

Lysozyme: 0.1g of lysozyme was dissolved in 1mL of 10mM Tris-HCl (pH 8.0) and stored at-20 ℃; when in use, the solution is diluted into 1mg/mL working solution.

The kit comprises:

full-type gold SeamLess Cloning and Assembly Kit seamless cloning kit;

Biyundian Beyotime glutathione peroxidase detection kit "

Kit for kang Cheng Ji BCA Protein Assay Kit

Kangji company bacterial genome DNA extraction reagent box (Bacteria Genomic DNA Kit)

OMEGA plasmid extraction kit (PLASMID MINI KIT I)

Reduced glutathione (reduced glutathione, GSH) content kit of Suzhou Grace biotechnology Co., ltd

Example 1

This example is an example of the method for synthesizing selenoprotein by site-directed insertion of selenocysteine into lactobacillus, and the method is proved to be feasible by verifying the expression of SEICS-containing escherichia coli formate dehydrogenase fdhF in lactobacillus, and the specific steps are as follows:

1 construction of helper plasmid pNZ8148-pC1ABD containing SBIP pathway element genes

1) E, extracting the whole genome of the color MG 1655;

In this example, E.coli MG1655 whole genome was extracted using the bacterial genome DNA extraction kit (Bacteria Genomic DNA Kit) from century Corp, and the procedure was carried out according to the instructions.

2) Amplification of SBIP pathway element gene SelA, selB, selC, selD and promoter p 1;

Using pBAD18-SelABC2-GPX-GW as template and "SelA-SelB-F (SEQ ID NO. 13)/SelA-SelB-R (SEQ ID NO. 14)" as primer, completing 30 cycles under 60-68 ℃ (15 s) annealing condition and 72 ℃ (3.3 min) extension condition to realize the clone amplification of SelAB (SEQ ID NO. 1); using pBAD18-SelABC2-GPX-GW as template and "SelC-F (SEQ ID NO. 11)/SelC-R (SEQ ID NO. 12)" as primer, completing 30 cycles under 50-60 ℃ (5 s) annealing condition and 72 ℃ (7 s) extension condition to realize SelC (SEQ ID NO. 2) cloning amplification; using E.coli MG1655 whole genome DNA as template and SelD-F (SEQ ID NO. 15)/SelD-R (SEQ ID NO. 16) as primer, completing 30 cycles under 55-65 deg.C (5 s) annealing condition and 72 deg.C (70 s) extension condition to realize SelD (SEQ ID NO. 3) clone amplification; the cloning and amplification of the promoter P1 (SEQ ID NO. 4) were achieved by 30 cycles of the primer "P1-F (SEQ ID NO. 21)/P1-R (SEQ ID NO. 22)" with pUC6P as the template under the conditions of annealing at 50-60℃for 5s and extension at 72℃for 13 s.

3) Ligation of SBIP pathway element genes;

t4 ligation of SelC-pNZ 8148:

The pNZ8148 circular plasmid was double digested with PstI and KpnI and gel recovered to give a linear fragment of pNZ8148, and then SelC was ligated at a position behind the promoter of pNZ8148 plasmid pNis (SEQ ID NO. 5) with reference to the full gold T4 ligase instructions.

The pNZ8148-SelC ligation products were parameterized to E.coli MC1061, and the verification and preservation was completed.

Information ligation of pC1 ABD:

the recombinant plasmid pNZ8148-SelC was first digested with Xbal and HindIII, the circular plasmid pNZ8148-SelC was digested with two enzymes and gel recovered to give linear fragments of pNZ8148-SelC, and then the ligation product was transferred to E.coli MC1061 for preservation using the full-size gold SeamLess Cloning and Assembly Kit kit for seamless cloning of SelAB, selD, P1 and vector pNZ8148-SelC according to the instructions.

4) Verification of pathway plasmid pNZ8148-pC1ABD

The E.coli MC1061-pNZ8148-pC1ABD obtained in the step 3 is extracted by using an OMEGA company plasmid extraction kit (PLASMID MINI KIT I), the enzyme digestion verification result is shown in figure 2, and the bright band appearing in the lane 1 corresponds to the closed circular plasmid pCABD of about 3800 bp; the single band of lane 2 corresponds to the 7527bp pCABD long fragment that is open after single cleavage with Xba I; lane 3 shows two bands of 4283bp and 3244bp after double digestion with Xba I and Hind III. All of the above bands are in agreement with theory.

The sequencing verification result is shown in FIG. 3, and the SnapGene comparison result after forward and reverse sequencing shows that SelAB and SelD are successfully connected to pNZ8148-SelC, and the construction of the pathway plasmid pNZ8148-pC1ABD is successful.

The plasmid map of the constructed pNZ8148-pC1ABD (SEQ ID NO. 27) is shown in FIG. 1.

2 Construction of lactic acid bacteria NZ9000/SBIP containing the SBIP pathway

1) Preparation of competent cells of lactic acid bacteria NZ 9000:

Culturing NZ9000 lactobacillus on a non-resistant GM17 plate by streaking, picking up single colony, amplifying and culturing in G-SGM17B until OD600 = 0.3-0.4, centrifugally collecting the colony, re-suspending by using a 4-degree precooled solution I, then collecting the colony, re-suspending by using a solution II, then collecting the colony, re-suspending by using the solution I for two times, re-suspending by using the solution I of 1/100 of a culture medium, and sub-packaging into 80 mu L of one tube. All operations except centrifugation were done in an ultra clean bench on ice.

2) In the competence of lactococcus lactis NZ9000, shock transformation of helper plasmid pNZ8148-pC1 ABD:

About 1. Mu.g of the plasmid pNZ8148-pC1ABD constructed in step 1 was introduced into the competence of lactococcus lactis NZ9000 by means of electric shock transformation. The whole procedure was performed in a sterile environment using a pre-chilled 2mM cuvette, and shock conversion was done at a voltage u=2000 v, pulse duration (pμ LSE LENGTH, PL) =150 μs, pulse number (mμ Ltiple P μ Lsing, MP) =30 times, total pulse duration=4.5 ms, pulse interval (pμ LSE INTERVAL, PI) =100 ms. Then the strain is coated on a GM17 flat plate containing chloramphenicol resistance after resuscitating culture, single colony is picked after stationary culture at 30 ℃, and the strain NZ9000/SBIP containing SBIP paths is obtained after liquid culture verification and preservation.

3) Verification of lactococcus lactis NZ9000/SBIP

NZ9000/SBIP performed plasmid restriction enzyme, sequencing, and SDS-PAGE. As shown in FIG. 4, lane 1 corresponds to a closed circular pC1ABD of about 4000bp without cleavage; lane 2 corresponds to a linear pC1ABD of 7730bp cut with a single enzyme; lane 3 corresponds to linear bands around 4500 and around 3300 of pC1ABD double digested with Hind iii, kpn I, all bands being expected.

The sequencing verification result is shown in FIG. 5, and the multi-primer sequencing comparison result accords with the expectation, which shows that the NZ9000/pC1ABD recombinant bacteria are successfully constructed.

SDS-PAGE results are shown in FIG. 6, and theoretical molecular weight sizes calculated by using detaibio on-line servers are 50.62kDa (about 51 kDa), 68.88kDa (about 69 kDa) and 36.69kDa (about 37 kDa), respectively, based on the protein molecular sequence of SelA, selB, selD. By contrast with background non-induced NZ9000/pNZ8148 (lane 1) and Nisin-induced NZ9000/pNZ8148 (lane 2), non-induced NZ9000/pC1ABD (lane 3) and Nisin-induced NZ9000/pC1ABD (lane 4) all appear as over-expressed bands at three positions in theory.

4) Preparation of lactococcus lactis NZ9000/SBIP competent.

NZ9000/SBIP was made competent using the same method as in 1).

3 Construction of expression vector containing E.coli formate dehydrogenase fdhF and bifunctional glutathione synthetase GshF required for enhancing selenium donor production

1) Synthesizing a carrier containing GshF;

Vector pTRKH-GshF containing GshF (SEQ ID NO. 6) was synthesized by Nanjing Jinsri Biotechnology Co.

2) Acquisition of fdhF Gene sequence and amplification of promoter fragment

Using E.coli MG1655 whole genome DNA as a template, using "fdhF-F (SEQ ID NO. 17)/fdhF-R (SEQ ID NO. 18)" as a primer, completing 30 cycles under 60-68 ℃ (15 s) annealing condition and 72 ℃ (130 s) extension condition to realize cloning amplification of fdhF (SEQ ID NO. 9); using pRHU-sfGFP as a template, using "sfGFP-F (SEQ ID NO. 19)/sfGFP-R (SEQ ID NO. 20)" as a primer, and completing 30 cycles under 60-68 ℃ (5 s) annealing condition and 72 ℃ (45 s) extension condition to realize cloning and amplification of sfGFP (SEQ ID NO. 10); taking P8-F (SEQ ID NO. 23)/P8-R (SEQ ID NO. 24) as a primer, taking pUC6P as a template, and completing 30 cycles under the annealing condition of 50-60 ℃ (5 s) and the extension condition of 72 ℃ (12 s) to realize the cloning and amplification of the promoter P8 (SEQ ID NO. 7); taking P23-F (SEQ ID NO. 25)/P23-R (SEQ ID NO. 26) as a primer, taking pUC6P as a template, and completing 30 cycles under the annealing condition of 55-65 ℃ (5 s) and the extension condition of 72 ℃ (12 s) to realize the cloning and amplification of the promoter P23 (SEQ ID NO. 8);

3) Ligation of fdhF and promoter and GshF-containing plasmid vector;

The pNZ8148-GshF circular plasmids were double digested with ApaLI and KpnI and gel recovered to give pNZ8148-GshF linear fragments, and then p8, p23, fdhF, sfGFP were subjected to seamless cloning using the full gold SeamLess Cloning and Assembly Kit kit to generate physical transformation into E.coli DH 5. Alpha.

4) Verification of fdhF expression vector:

The ligated plasmid was designated pTRKH-p 8G23FS (i.e., the plasmid contained three genes, gshF, fdhF, and sfGFP). As shown in FIG. 8, the bright band in lane 1 corresponds to a closed circular plasmid pGFS of about 6000 bp; the single band of lane 2 corresponds to the long chain 11533bp pGFS of Sal I after single cleavage. All of the above bands are in agreement with theory.

The sequencing verification results are shown in FIG. 9, and the SnapGene comparison results after forward and reverse sequencing show that FDH and sfGFP have been successfully connected to pTRKH2-GshF, and plasmid pTRKH2-p8G23FS is successfully constructed.

(SfGFP on vector was used for early construction for ease of verification, no actual effect in expression of fdhF.) plasmid map of the constructed expression vector pTRKH-p 8G23FS (SEQ ID NO. 28) is shown in FIG. 7.

4 Construction of lactococcus lactis NZ9000 having a SBIP pathway gene and an fdhF gene;

1) Shock transformation of fdhF expression vector plasmid in lactococcus lactis NZ9000/SBIP competence;

Plasmid pTRKH2-p8G23FS was introduced into recombinant lactic acid bacteria NZ9000/SBIP using the same conditions as for shock transformation of pNZ8148-pC1ABD, and positive selection was performed using plates with dual resistance to erythromycin and chloramphenicol; pTRKH2-GshF, which did not contain the fdhF gene, was used as a negative control for simultaneous introduction. Through screening of the resistance plate, a strain NZ9000/SBIP-fdhF capable of expressing selenoprotein fdhF is obtained.

2) Validation of lactococcus lactis NZ 9000/SBIP-fdhF.

The plasmid in the strain NZ9000/SBIP-fdhF was verified by digestion and sequencing. As shown in FIG. 10, the bright single band in lane 1 corresponds to a closed loop pC1ABD of about 3900bp and a closed loop p8G23FS of about 6000bp, which are not digested; lane 2 single band corresponds to 7730bp and 11882bp after single cleavage by Xho I unique restriction enzyme shared by pC1ABD and p8G23FS2 double plasmids; all bands are expected.

The sequencing results are shown in FIG. 11, and the sequencing results show that the strain NZ9000/SBIP-fdhF contains the plasmid pNZ8148-pC1ABD and the plasmid pTRKH-p 8G23FS at the same time and no mutation occurs.

Expression verification of 5fdhF

1) SDS-PAGE validation of fdhF

After the strain NZ9000/SBIP-fdhF was subjected to expanded culture to OD ₆₀₀ to 0.4 to 0.6 and then subjected to cell disruption and centrifugation after 30ng/mL Nisin and 8ng/mL Na ₂SeO₃ were added to induce expression for 16 hours, a crude enzyme solution was obtained, and SDS-PAGE was performed, as shown in FIG. 12, and the results were confirmed by using the crude enzyme solution (lane 1) of the strain NZ9000/p-p without empty plasmid pNZ8148, pTRKH2 as a control, and the genes SelA (about 51 kDa), selB (about 69 kDa), selD (about 37 kDa), gshF (about 86 kDa) and fdhF (about 79 kDa) in the element genes in the crude enzyme solution NZ9000/SBIP-fdhF (lane 2) were all expressed as bands at the expected positions in theory

2) Measurement of enzyme activity of fdhF.

The enzyme activity of fdhF was measured by using the NADH colorimetric method with reference to the reduced glutathione content kit instructions of Suzhou Granisi Biotechnology Co., ltd. And the final result was 20.32U/g of crude enzyme solution of NZ 9000/SBIP-fdhF.

The final results show that example 1 successfully constructs a method for synthesizing selenoprotein by site-specific insertion of selenocysteine into lactobacillus.

Example 2

The embodiment is the application of the method for synthesizing selenoprotein by inserting selenocysteine into lactobacillus at fixed point in engineering transformation of selenoprotein

Based on the method of example 1, the TGA site and SECIS sequence are introduced into the GPX gene of the glutathione photo-oxidase of the lactobacillus which does not contain the TGA and SECIS sequence of the Sec insertion site, and the method is successfully used for engineering selenoprotein, and the specific implementation method is as follows:

1 substitution of the positions of fdhF and sfGFP in example 1 with lactic acid bacteria GPX (SEQ ID NO. 29) by means of PCR amplification and information cloning in example 1 gave GPX expression vector pTRKH2-p8G23G

2 Introduction of Sec insertion site TGA and SECIS sequences into GPX by primer-mediated PCR, since Sec replaces Cys, primers were designed for multiple sites for introduction of TGA and SECIS, primer design using primers and mutation sites see Table 2 for GPx mutants of Table 2

3 Different mutants were electrotransformed into NZ9000/SBIP competence, positive strains were screened for dual resistance to red and chloramphenicol, expanded culture was performed as described in example 1, and expression of GPX mutants was verified by SDS-PAGE and enzyme activity. As shown in FIG. 13, SDS-PAGE results after Nisin and selenium-rich induction, each of the induced mutants (lanes 5,6,7,8,9 correspond to NZ9000/SBIP-GPX-C36U、NZ9000/SBIP-GPX-C63U、NZ9000/SBIP-GPX-C81U、NZ9000/SBIP-GPX-L156U、NZ9000/SBIP-GPX-L156-Ter crude enzyme solutions, respectively) was seen to have a pale band at the band of interest (about 17 kDa); meanwhile, the enzyme activity detection result is shown in fig. 14, and it can be seen that each GPX mutant has enzyme activity and certain difference.

The combination of the results of enzyme activity detection and SDS-PAGE results shows that the method constructed in example 1 successfully expresses GPX introduced into Sec insertion site and SECIS sequence in lactic acid bacteria, and the expressed GPX has a certain functional activity.

Example 2 illustrates that the method for synthesizing selenoprotein by site-directed insertion of selenocysteine into lactobacillus constructed in example 1 can be applied to engineering of selenoprotein. Meanwhile, through engineering transformation, several recombinant selenium-enriched lactobacillus NZ9000/SBIP-GPX-CNNU capable of expressing the transformed selenoprotein are obtained. (NN represents the site of mutation)

Example 3

Construction of novel selenium-enriched functional lactobacillus for recombinant expression of selenoprotein fdhF

The method established in the embodiment 1 is used for constructing a selenium-enriched lactobacillus capable of recombinantly expressing GshF and selenoprotein fdhhF, and the specific steps are as follows:

1 construction of SBIP pathway plasmid pNZ8148-pC1ABD, fdhF expression vector pTRKH2-p8G23FS containing Gshf according to the procedure of example 1, electric shock transformation of the plasmid into the competent lactic acid bacteria NZ9000 to give strain NZ9000/SBIP-fdhF

2 Optimizing the induction conditions of NZ9000/SBIP-fdhF comprising the steps of

1) The growth curve of recombinant selenium-enriched functional lactic acid bacteria NZ9000/SBIP-fdhF was measured, and the result is shown in FIG. 15 that the growth activity of NZ9000/SBIP-fdhF is superior to that of the strain containing double empty plasmid.

2) The selenium-rich effect of NZ9000/SBIP-fdhF is optimized by optimizing the addition type of amino acid in the culture medium, and the selenium-rich color difference result is shown in FIG. 16: NZ9000/SBIP-fdhF was milky as normal to lactic acid bacteria in the absence of induction (panels a- ①) and 30ng/mL of Nisin induction (panels a- ②); the color was a darker orange under selenium-rich induction of 30ng/mL Nisin and 8 μg/mL Na ₂SeO₃ (FIGS. a- ③); FIGS. a- ④ maintain the same Nisin and selenium-rich induction as the previous three groups, and were incubated in GGC-GM17 medium with final concentrations of Glu, gly, cys amino acids of 10mM each, with darkening of the selenium-rich color. The selenium-rich effect of the cells after Glu, gly, cys are added into the GM17 culture medium is different: the Gly-GM17 culture medium and the GM17 culture medium have no obvious difference on the selenium-rich color of the thalli (see tubes ③ and ⑥); glu-GM17 and GGC-GM17 have similar selenium-rich effects on thalli, and are the redest (see tubes ④ and ⑦); selenium enrichment of Cys-GM17 is reddish (see tube ⑤) and is shallower than GM17 and other amino acids added, possibly due to the reducing nature of Cys.

3 SDS-PAGE and enzyme activity verification of recombinant lactic acid bacteria NZ9000/SBIP-fdhF under different conditions:

SDS-PAGE results of NZ9000/SBIP-fdhF under different induction conditions are shown in FIG. 17: lane 1 is crude enzyme solution of double empty plasmid strain NZ 9000/p-p; lane 2 is crude enzyme solution collected after induction of NZ9000/SBIP-fdhF in GGC-GM17 medium and selenium enrichment; lane 3 is crude enzyme solution collected under the induction of selenium enrichment by NZ 9000/SBIP-fdhF; lane 4 is crude enzyme solution collected after Nisin induction by NZ 9000/SBIP-fdhF; lane 5 is crude enzyme solution collected without induction by NZ 9000/SBIP-fdhF. The crude enzyme solution of NZ9000/SBIP-fdhF under four culture conditions (lanes 2-5) can see obvious target bands at the expected positions of theory, which indicates that SelD, selA, selB, FDH and GshF are expressed in NZ 9000/SBIP-fdhF; and lanes 2 and 3 show that the target bands are more obvious than lanes 4 and 5, thus the effect of selenium-enriched induction or NZ9000/SBIP-fdhF expression added into 3G medium is better. The results of the enzyme activity detection are shown in FIG. 18, wherein the enzyme activity of the fdhF crude enzyme solution can reach 28.1146mU/mg after Nisin and selenium-rich induction are added in the GGC-GM17 medium, and the difference is obvious compared with the wild type.

The method established in the embodiment 1 is used for successfully obtaining a novel lactobacillus with a selenium-rich function; after the induction condition is optimized, the selenoprotein fdhF expressed by the recombinant lactobacillus has good biological activity.

The above three examples describe the preferred embodiments of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments, and various modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and these simple modifications all fall within the scope of the present invention.

In addition, the specific features and steps described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in detail.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for synthesizing selenoprotein by inserting selenocysteine into lactobacillus at fixed point is characterized in that: recombinant expression of selenocysteine synthesis and insertion pathway (SBIP) element SelA, selB, selC, selD in lactobacillus and addition of selenium donor bifunctional glutathione synthetase GshF, as shown in sequence table SEQ ID NO.1, 2,3 and 6, realizes efficient site-directed insertion expression of selenoprotein, comprising the following steps:

(2) Constructing a plasmid containing GshF and a target selenoprotein gene;

(3) Introducing the plasmids constructed in the steps (1) and (2) into lactobacillus together through electric shock transformation to construct a gene recombinant strain for expressing the target selenoprotein;

2. The method according to claim 1, characterized in that the gene of the key acting element of the selenocysteine synthesis and insertion pathway (SBIP) of escherichia coli is integrated in the form of a plasmid into lactococcus lactis NZ 9000; meanwhile, another compatible plasmid is used for loading difunctional glutathione peroxidase GshF capable of enhancing the generation of a selenium donor and target selenoprotein containing SECIS, and the difunctional glutathione peroxidase GshF and the target selenoprotein are introduced into lactococcus lactis NZ9000 containing a SBIP pathway to realize the expression of the selenoprotein in lactobacillus, and the specific steps are as follows:

A. Construction of helper plasmid pNZ8148-pC1ABD containing SBIP pathway element genes;

B. construction of a lactic acid bacterium NZ9000/SBIP containing a SBIP pathway;

C. construction of an expression vector containing a desired selenoprotein and a bifunctional glutathione synthetase GshF required for enhancing selenium donor production;

D. Construction of lactococcus lactis NZ9000/SBIP-NN with SBIP pathway genes and a target selenoprotein gene;

E. Verifying the expression of the target selenoprotein;

Wherein:

the construction step of the helper plasmid pNZ8148-pC1ABD containing SBIP pathway element genes in the step A comprises the following steps:

1) E, extracting the whole genome of the color MG 1655;

3) Ligation of SBIP pathway element genes;

4) Verification of the pathway plasmid pNZ8148-pC1 ABD;

The construction steps of the lactobacillus NZ9000/SBIP containing Sec synthesis and insertion pathways in the step B comprise the following steps:

1) Preparing competent cells of lactobacillus NZ 9000;

3) Verification of lactococcus lactis NZ9000/SBIP

4) Preparation of the competent lactococcus lactis NZ 9000/SBIP;

the step C comprises the steps of constructing an expression vector containing the target selenoprotein and the bifunctional glutathione synthetase GshF required for enhancing the generation of a selenium donor, and the method comprises the following steps of:

1) Synthesizing a carrier containing GshF;

2) Obtaining a target selenoprotein gene sequence and preparing fragments;

4) Verifying a target selenoprotein expression vector;

The construction of the lactococcus lactis NZ9000 having the SBIP pathway gene and the selenoprotein gene of interest in the step D comprises the following steps:

2) Validation of lactococcus lactis NZ9000/SBIP-NN (NN represents a selenoprotein of interest);

the expression verification of the selenium protein of the step E comprises the following steps:

1) Preparing a target selenoprotein crude enzyme solution;

3. The method according to claim 1, wherein the vector expressing the SBIP pathway of escherichia coli in lactobacillus in step (1) includes, but is not limited to, e.coli MG1655, the promoter for initiating transcription of SBIP element gene SelA, selB, selD is constitutive and has the sequence shown in SEQ ID No. 4; the promoter used for promoting SelC transcription is inducible, and pNis promoter is used, and the sequence is shown in SEQ ID NO. 5.

4. The method of claim 1, wherein the selenoprotein comprises a selenocysteine insertion sequence (SECIS).

5. The method of claim 1, wherein step (4) enhances selenoprotein synthesis by adding selenium during culturing; inducing SelC transcription by adding Nisin; the constitutive promoter is used to start the transcription of the target selenoprotein and GshF gene, so as to realize the expression of the target selenoprotein, and the used promoter sequences are shown in SEQ ID NO.7 and 8.

6. Use of the method of claim 1 for engineering selenoprotein by introducing TGA codon and SECIS sequence simultaneously into the coding region of the modified selenoprotein gene, wherein the selenoprotein is a protein having cysteine in its active site.

7. The use of claim 6, wherein the simultaneous introduction of TGA codon and SECIS sequence in the coding region of the modified selenoprotein gene is the introduction of Sec codon and SECIS into a protein without the site and sequence, the active center of which contains a cysteine.