CN106754466A - It is a kind of for efficient exogenous protein expression and the bacillus subtilis of High Density Cultivation - Google Patents

Abstract

The invention discloses a bacillus subtilis used for high-efficiency exogenous protein expression and high-density cultivation, and belongs to the technical field of bioengineering. The present invention adopts the method of screening and separation to obtain a strain of Bacillus subtilis with high protein expression from the soil, and knocks out its six genes srfC, spoIIAC, amyE, nprE, aprE and bamA to obtain B. subtilis WS5, and the preservation number is CCTCC M 2016536. Bacillus subtilis WS5 was used as the expression host to construct β‑CGTase genetically engineered bacteria. The β-CGTase genetically engineered bacteria were cultured in a 3L fermenter, the foam produced during the fermentation process was significantly reduced, no spores were detected in the microscope, and there were almost no extracellular amylases and proteases during the culture process. CGTase enzyme activity reached 270U/ml, DCW reached 70g/L.

Description

A Bacillus subtilis for high-efficiency foreign protein expression and high-density culture

technical field

The invention relates to a bacillus subtilis used for high-efficiency exogenous protein expression and high-density culture, and belongs to the technical field of bioengineering.

Background technique

Bacillus subtilis (Bacillus subtilis) is a Gram-positive bacterium, because it is easy to isolate and culture, has a relatively clear genetic background, good secretion, and no pathogenicity, it has become an important industrial strain and is more and more popular. It is increasingly used in the production of antibiotics, pharmaceutical proteins and industrial enzyme preparations. B. subtilis 168 is a laboratory type strain containing many mutations. Industrially used strains such as B. subtilis WB600 and B. subtilis WB800 are all derived from B. subtilis 168, and B. subtilis 168 is obtained through a series of mutagenesis screening, and there are mutations in some genes compared to wild strains . The fermentation culture of the above-mentioned model strains reported so far cannot reach a higher cell density, and the expression of foreign proteins of the model strains is lower than that of some wild strains. Bacillus subtilis produces surfactin during the fermentation process. Surfactin is an amphiphilic molecule whose aggregation on the surface of the liquid causes foaming.

Bacteria live in a natural environment full of phages or other viruses for a long time, and have evolved a variety of defense systems, CRISPR/CAS system is one of them, the more common type II CRISPR-CAS acquired immune system (Streptococcuspyogenes and S.thermophilus ), when the phage invades, the original spacer sequence (postospacer) on its genome is inserted as a new spacer sequence after the initiation sequence of the host cell CRISPR sequence, as the first spacer sequence; when the phage invades again, the bacterial The CRISPR sequence is transcribed to produce a long chain of RNA, that is, the crRNA precursor (pre-crRNA), and then the Cas protein complex is cleaved with the assistance of tracrRNA (trans-activating crRNA) to produce mature crRNAs, and finally the tracrRNA-crRNA-Cas9 complex Recognizes and cleaves the site complementary to crRNA. Now usually design 1 sgRNA (small guide RNA) to replace trancrRNA-crRNA to guide Cas9 protein.

When the sgRNA and the Cas9 gene expression plasmid are transformed into Bacillus subtilis, the sgRNA is paired with the complementary sequence corresponding to the gene through the target sequence, and other sequences in the sgRNA form a stem-loop structure, which can be recognized by Cas9, and then the PAM (Protospacer -Adjacent Motif) cuts the DNA double strand of the target gene between 2-3 bases upstream of the sequence. The broken double-strand DNA can undergo accurate genome recombination through the HDR (homology-directed repair) pathway when there is a homologous repair template. If the break occurs in the coding region, and the repair template inserts a few bases (not a multiple of 3) at the break, it will cause the frame shift of the repaired gene reading frame, resulting in the early appearance of a stop codon in the coding region .

Contents of the invention

The purpose of the present invention is firstly to provide a strain of Bacillus subtilis (Bacillus subtilis) WS5 which can be used for high-efficiency expression of foreign protein and high-density culture.

The Bacillus subtilis WS5 was deposited in the China Center for Type Culture Collection on September 29, 2016, with the preservation number CCTCC NO:M 2016536, and the preservation address is Wuhan University, Wuhan, China.

The Bacillus subtilis WS5 uses the CRISPR/Cas9 gene editing system to knock out the six genes of srfC, spoIIAC, amyE, nprE, aprE and bamA of the Bacillus subtilis WS. The production of foam, spores, extracellular amylase and protease during fermentation is reduced.

The Bacillus subtilis WS5 strain has a protein expression level as high as 0.8 mg/ml, which is higher than the 0.2-0.5 mg/ml expression level of the existing model strains. It has high utilization rate of the medium, has good protein secretion ability, and can carry out high-density fermentation culture.

The present invention also provides a recombinant Bacillus subtilis for recombinantly expressing β-CGTase, in which the β-CGTase gene derived from B.circulans is cloned into the PHY300PLK vector, and B.subtilis WS5 is used as the expression host to realize high-efficiency expression of β-CGTase .

The present invention also provides a method for fermenting and producing β-CGTase by using the recombinant Bacillus subtilis, which is to connect the seed culture solution into a 3L fermenter with a liquid volume of 0.9L, control the pH to 7 with 25% ammonia water, and cultivate at a temperature of 30°C , maintain the dissolved oxygen at about 30% by coupling with the stirring speed and adjusting the ventilation rate. When the dissolved oxygen rises rapidly, start to feed the glucose feed solution with a concentration of 50%, and end the culture when the β-CGTase enzyme activity decreases.

Described fermentation medium: calcium sulfate 0.939g/L, magnesium sulfate 7.27g/L, potassium hydroxide 4.13g/L, potassium sulfate 18.2g/L, corn steep liquor 30g/L, phosphoric acid 26.7ml/L, glucose 5g/L L, metal ion PTM solution 5ml/L; composition of metal ion PTM solution: CuSO ₄ ·5H ₂ O 6g/L, KI 0.08g/L, MnSO ₄ ·H ₂ O 0.5g/L, Na ₂ MoO ₃ ·2H ₂ O 0.2g/L, H ₃ BO ₃ 0.02g/L, CoCl ₂ 0.5g/L, ZnCl ₂ 20g/L, FeSO ₄ 7H ₂ O 65g/L, biotin 0.2g/L, H ₂ SO ₄ 5.0g/L.

Apply the constructed recombinant Bacillus subtilis to carry out 3L fermenter culture, during the fermentation process, the foam produced by the β-CGTase genetically engineered bacteria with B.subtilis WS5 as the host is significantly reduced, and the bacterial cell concentration is very high in the late stage of fermentation And in the case of nutrient deficiency, no spores were detected in the microscope. Use amylase screening plate and protease screening plate to detect extracellular amylase and protease in the supernatant of the fermentation broth, and there is almost no transparent circle on the plate obtained after culturing for 24 hours, which proves that there is almost no extracellular amylase and protease in the culture process of B. subtilis WS5 . β-CGTase activity reached 270U/ml (1.2mg/ml) and DCW reached 70g/L when fermented for 70 hours, indicating that B. subtilis WS5 can be cultured at high density and express foreign proteins efficiently, which has high industrial application value.

biological material deposit

Bacillus subtilis WS5 was deposited in the China Center for Type Culture Collection on September 29, 2016, with the preservation number CCTCC NO:M 2016536, and the preservation address is Wuhan University, Wuhan, China.

Description of drawings

Figure 1: Microscopic examination of spores, (a) without knockout of spoIIAC, (b) knockout of spoIIAC.

Figure 2: Transparent circles of amylase and protease, (a) no gene knockout, nprE knockout, nprE and aprE knockout, (b) amyE knockout.

Figure 3: SDS-PAGE diagram of β-CGTase genetically engineered bacteria cultured for 70 hours

detailed description

SEQ ID NO:1: β-CGTase gene sequence

Example 1: Screening strains with high protein expression from collected soil samples

Take soil collected from different locations in different regions (garbage dumps, starch factories, vegetable markets, food factories, grain processing factories, hotels, etc.), mix 2g of soil samples with 9ml of sterile water, add glass beads and shake well Stand still for a while, take 5ml of supernatant and add it to LB medium, 25°C-37°C, 200r/min shaking culture for 2d-3d; 2. Take 10ml of culture solution and add it to another fresh LB medium to continue shaking culture, After repeating this operation twice, it was coated on LB solid flat plate. Inverted culture at 25°C-37°C for 2-3 days, a single colony can be observed and strains with good growth conditions can be screened; 3. Select and inoculate a single colony into a shallow well plate containing LB seed medium through high-throughput, 37°C , 200r/min shaking culture overnight. Then, according to the inoculum amount of 5%, the liquid strains were inserted into the deep-well plate equipped with TB fermentation medium, placed at 26°C-33°C, 200r/min shaking culture for 2-3d, and then used on the enzyme label plate The protein content in the supernatant of the fermentation broth was measured by Coomassie Brilliant Blue detection method, and the strains with high protein expression were initially screened; 4. Inoculate the strains with high protein expression obtained by the above preliminary screening into triangles equipped with 10ml LB medium. Shake culture overnight at 37°C and 200r/min in the bottle, then transfer the liquid bacteria into a conical flask containing 50ml of TB enzyme production medium according to the inoculum amount of 5%, and place it at 26°C-33°C at 200r/min Shaking culture was cultivated for 2-3 days, and then the protein content in the supernatant of the fermentation broth was measured by the Coomassie brilliant blue detection method in a 15ml ground-mouth test tube, and a strain with a protein expression level of up to 0.8mg/ml was obtained by re-screening, which was higher than the existing strain. The expression level of the type strain is high (0.2-0.5mg/ml), identified as Bacillus subtilis by morphology and 16sRNA, and named as B.subtilis WS.

Medium formula:

(1) LB seed medium: 5g/L yeast powder, 10g/L peptone 1%, 10g/L sodium chloride.

(2) TB fermentation medium: 24g/L yeast powder, 12g/L peptone, 5g/L glycerol, 12.54g/L K ₂ HPO ₄ , 2.31g/L KH ₂ PO ₄ .

Embodiment 2: Construction of knockout plasmid

According to the gene sequence of Bacillus subtilis, the sgRNA used to specifically target the srfA, spoIIAC, amyE, nprE, aprE and bamA genes was designed, and the gene sequence of the temperature-sensitive replication origin of PE194 was found from NCBI (NCBIGenBank: M17811.1), Then chemically synthesized sgRNA and PE194 sequence (Genbank accession number JN798465.1: 7790-9126), using primers P1 and P2, PCR to obtain the vector backbone Frag1; using the shuttle plasmid pHY300PLK (Takara, Dalian, China) as a template, using primers P3 and P4, ampicillin screening gene and Escherichia coli replication origin p15A (Genbank accession number D90381.1: 5993-6537) were obtained by PCR, as the vector backbone Frag2; the shuttle plasmid pHY300PLK was used as a template, primers P5 and P6 were used, and tetracycline screening gene was obtained by PCR (Genbank accession number FN806789.3: 27499-28875), as the vector backbone Frag3; using the plasmid pwtcas9-bacterial (Addgene plasmid #44250) as a template, using primers P7 and P8, PCR obtained the cas9 gene from Streptococcuspyogenes (Genbank accession number AE004092 .2:854751-858855), as the vector backbone Frag4. The four fragments Frag1, Frag2, Frag3 and Frag4 were ligated using In-Fusion HD Cloning Plus kits to obtain plasmid PHY300d. The homologous repair arm fragments with XbaI restriction sites were ligated into corresponding restriction sites to obtain knockout plasmids PHY300dsrf, PHY300dspo, PHY300damy, PHY300dnpr, PHY300dapr and PHY300dbam.

Table 1 The connection system of In-Fusion HD Cloning Plus kits is as follows:

The molar ratio of the linearized vector and the purified PCR product fragment is 1:2; make up 10 μl of the system with water. Ligate at 50°C for 20 minutes, ice-bath for 5 minutes, then transform into E.coli JM109 competent cells, apply to LB solid medium (containing 100 μg/mL ampicillin), and culture overnight at 37°C. Pick positive clones, extract plasmids, and perform sequencing verification.

Table 2 Primer sequences

Embodiment 3: Bacillus subtilis transformation method

Dip frozen Bacillus subtilis WS with an inoculation loop, then streak on LB plates, and culture overnight at 37°C for activation. Pick a single colony and inoculate it in 5ml LB liquid medium, culture overnight at 37°C for 18h. Take a certain amount of overnight culture into 4.5ml of GMI, so that the OD600 value reaches 0.1-0.2, leaving 4.5ml of mixed bacterial solution. Shake culture at 200 rpm at 37°C, measure OD600 every 20 minutes, when OD600 reaches 0.4-0.6 (about 60-90 minutes); continue shaking culture for 90 minutes, draw 0.05ml of bacterial liquid into a sterile test tube containing 0.45ml of preheated GMⅡ medium; shaking at 37°C for 90 minutes, at this time, many competent cells formed in the culture; add 1 μg of plasmid (15-20 μl), shake and culture at 37°C for 30 minutes; centrifuge to remove most of the supernatant, resuspend the cells, and spread on Incubate overnight at 37°C on a screening plate containing the corresponding antibiotic.

Medium formula:

(1) 10× minimum salt solution: K ₂ HPO ₄ 14g (K ₂ HPO ₄ ·3H ₂ O 18.34g), KH ₂ PO ₄ 6g, (NH ₄ ) ₂ SO ₄ 2g, sodium citrate (Na ₃ C ₆ H ₅ O ₇ ·2H ₂ O) 1g, MgSO ₄ ·7H ₂ O 0.2g, dissolve in distilled water successively, add water to 100ml.

(2) L-trp solution, 2mg/ml, stored in a brown bottle, sterilized at 113°C for 30min, and wrapped in black paper.

(3) GMI solution: 95ml of 1× minimum salt solution, 1ml of 50% glucose, 0.4ml of 5% hydrolyzed casein, 1ml of 10% yeast juice, 2.5ml of 2mg/ml L-trp.

(4) GMⅡ solution: 97.5ml of 1× minimum salt solution, 1ml of 50% glucose, 0.08ml of 5% hydrolyzed casein, 0.04ml of 10% yeast juice, 0.5ml of 0.5M MgCl ₂ (2.5mM), 0.5ml of 0.1M CaCl ₂ ml (0.5mM), 2mg/ml L-trp0.5ml (5ug/ml).

Example 4: B. subtilis WS gene knockout

The srfC, spoIIAC, amyE, nprE, aprE and bamA genes in the B. subtilis WS genome were knocked out with plasmids PHY300dsrf, PHY300dspo, PHY300damy, PHY300dnpr, PHY300dapr and PHY300dbam, respectively. Using the method in Example 3, the knockout plasmid was transformed into Bacillus subtilis competent cells, spread on LB solid medium (containing 20 μg/mL tetracycline), and cultured overnight at 37°C. Pick positive clones, extract the genome, use the genome as a template colony PCR to amplify the homologous repair fragment, and then carry out XbaI enzyme digestion verification at 37°C. Since the gene knockout strain inserts the XbaI enzyme cutting site at the gene break when performing genome homology repair, it can be cut by XbaI endonuclease, while the wild type will not be cut, and the enzyme digestion is verified to be correct Knockout bacteria were carried out at 51°C to eliminate the knockout plasmid. After knocking out six genes srfC, spoIIAC, amyE, nprE, aprE and bamA in sequence, the gene knockout strain B. subtilis WS5 was obtained.

Table 3 The XhoI digestion system is:

Enzyme digestion reaction at 37°C for 2h.

Embodiment 5: Construction of β-CGTase genetically engineered bacteria

Under the premise of ensuring that the amino acid sequence remains unchanged, the original gene of natural CGTase derived from B.circulans 251 was analyzed by using codon usage frequency analysis software combined with factors such as E. coli codon bias, mRNA secondary structure and GC content. The sequence (Genbank accession number X78145.1) was codon-optimized, and the rare codons in the rest of the sequence except the signal peptide were replaced with the preferred codons in Bacillus subtilis, while taking into account the stability of the mRNA secondary structure. The CGTase gene was chemically synthesized, and connected to the pMD18-T (Takara, Dalian, China) vector through NcoI and HindIII restriction sites to obtain the plasmid βcgt/pMD18-T.

The plasmid used to construct the Bacillus subtilis expression vector is pHY300PLK with the amyQ promoter from B. amyloliquefaciens. Using plasmid pHY300PLK and plasmid βcgt/pMD18-T as templates respectively, primers P1/P2 and P3/P4 were used to PCR amplify the vector fragment and gene fragment with 15bp homologous sequence, and then ligated with In-Fusion HD Cloning Pluskit ligase , the ligation product was transformed into E.coli JM109 competent cells, cultured at 37°C for 8 hours, the picked transformants were shaken in LB containing 100 mg/L ampicillin liquid, the plasmid was extracted, and the expression plasmid βcgt/pHY300PLK was obtained by sequencing verification.

The PCR reaction system of table 4 is:

The reaction procedure was as follows: pre-denaturation at 94°C for 4 min; 30 cycles of 98°C for 10 s, 55°C for 10 s, and 72°C for 1.5 min; extension at 72°C for 10 min, and cooling to 4°C.

Table 5 Primer sequences

The plasmid βcgt/PHY300PLK was transformed into B. subtilis WS5 host bacteria, spread on LB plates containing tetracycline (20mg/L), and incubated at 37°C for 8h. Pick a single colony into liquid LB, culture overnight at 37°C, and save the glycerol tube.

Embodiment 6: β-CGTase genetically engineered bacteria 3L fermentation tank culture

The β-CGTase genetically engineered bacterium constructed in the above-mentioned embodiment 5 was carried out in a 3L fermenter, and the expression of B.subtilis WS5 foreign protein was checked, and the activity of foam, spores, extracellular amylase and protease in the fermentation process was checked. A situation arises. The cultivation process is as follows: inoculate 200 μl of glycerol tube bacterial liquid into a 500 mL Erlenmeyer flask containing 100 mL of seed medium, cultivate at 30°C and 200 r/min for 24 hours, and transfer the above culture liquid into a 3L fermenter with a liquid volume of 0.9L , use 25% ammonia water to control pH 7, culture temperature 30°C, maintain dissolved oxygen at about 30% by coupling with stirring speed and adjusting ventilation, when dissolved oxygen rises rapidly, it indicates that the glucose in the medium has been exhausted, The volume ratio of 50% glucose feed solution was added at the beginning, and the cultivation was terminated when the enzyme activity of β-CGTase decreased.

During the fermentation process, the foam produced by the β-CGTase genetically engineered bacteria with B.subtilisWS5 as the host was significantly reduced, and some foam would be produced in the logarithmic phase of growth, but the foam could be effectively controlled by dropping a very small amount of antifoaming agent The production of spores; the production of spores was not detected in the microscope under the condition of high bacterial concentration and nutrient deficiency in the late stage of fermentation (Fig. 1). The amylase screening plate and protease screening plate were used to detect extracellular amylase and protease in the supernatant of the fermentation broth, and the plate obtained after culturing for 24 hours had almost no transparent circle (Figure 2), indicating that the extracellular amylase in the culture process of B. subtilisWS5 And almost no protease. The β-CGTase activity reached 270U/ml (1.2mg/ml) at 70 hours of fermentation culture, which is the highest level of CGTase from B.circulans 251 in Bacillus subtilis reported so far; DCW reached 70g/L (Figure 3), which is The high density of Bacillus subtilis fermentation cells reported so far indicates that B. subtilis WS5 can be cultured at high density and express foreign proteins efficiently, which has high industrial application value.

Medium formula:

(1) Seed medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L.

(2) Fermentation medium: calcium sulfate 0.939g/L, magnesium sulfate 7.27g/L, potassium hydroxide 4.13g/L, potassium sulfate 18.2g/L, corn steep liquor 30g/L, phosphoric acid 26.7ml/L, glucose 5g /L, metal ion PTM solution 5ml/L; composition of metal ion PTM solution: CuSO ₄ ·5H ₂ O 6g/L, KI 0.08g/L, MnSO ₄ ·H ₂ O 0.5g/L, Na ₂ MoO ₃ · 2H ₂ O 0.2g/L, H ₃ BO ₃ 0.02g/L, CoCl ₂ 0.5g/L, ZnCl ₂ 20g/L, FeSO ₄ 7H ₂ O 65g/L, biotin 0.2g/L, H ₂ SO ₄ 5.0g/L).

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

SEQUENCE LISTING

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Claims (6)

1. A strain of Bacillus subtilis WS5 was deposited in the China Center for Type Culture Collection on September 29, 2016, with the preservation number CCTCC NO:M 2016536, and the preservation address is Wuhan University, Wuhan, China. 2. A strain of recombinant Bacillus subtilis, characterized in that the β-CGTase gene derived from B.circulans is cloned into the PHY300PLK vector, and then transformed into Bacillus subtilis WS5 according to claim 1. 3. a method for producing β-CGTase by recombinant bacillus subtilis fermentation according to claim 2, characterized in that, the seed culture solution is inserted into a 3L fermenter with a liquid filling capacity of 0.9L, and the pH is controlled with 25% ammoniacal liquor 7. The culture temperature is 30°C, and the dissolved oxygen is maintained at about 30% by coupling with the stirring speed and adjusting the ventilation rate. When the dissolved oxygen rises rapidly, start to feed the glucose feed solution with a concentration of 50%. The cultivation was terminated when the viability decreased. 4. The method according to claim 3, characterized in that, fermentation medium: calcium sulfate 0.939g/L, magnesium sulfate 7.27g/L, potassium hydroxide 4.13g/L, potassium sulfate 18.2g/L, corn steep liquor 30g/L, phosphoric acid 26.7ml/L, glucose 5g/L, metal ion PTM solution 5ml/L; composition of metal ion PTM solution: CuSO ₄ ·5H ₂ O 6g/L, KI 0.08g/L, MnSO ₄ ·H ₂ O 0.5g/L, Na ₂ MoO ₃ 2H ₂ O 0.2g/L, H ₃ BO ₃ 0.02g/L, CoCl ₂ 0.5g/L, ZnCl ₂ 20g/L, FeSO ₄ 7H ₂ O 65g/L L, biotin 0.2g/L, H ₂ SO ₄ 5.0g/L. 5. the application of the bacillus subtilis (Bacillus subtilis) WS5 described in claim 1 in fermenting and producing enzyme. 6. The application of the Bacillus subtilis (Bacillus subtilis) WS5 described in claim 1 in producing β-CGTase by fermentation.