A bacillus subtilis chassis cell suitable for high-density fermentation enzyme production

Abstract

The application discloses a Bacillus subtilis chassis cell suitable for high-density fermentation enzyme production and belongs to the field of genetic engineering and enzyme engineering. On the basis of B.subtilis 168, four chassis cells BS1 to BS4 with high robustness and resistance to autolysis in the later fermentation stage and one chassis cell BS5 with combined knockout are obtained by knocking out genes lytC, sigD, mraZ and sigE. An expression vector pMA5-glsA is constructed to express L-glutamine enzyme in the constructed chassis cells, wherein the 5-L fermenter enzyme activity of BS5 is 1.98 times that of B.subtilis 168, and on the basis of the increase of cell biomass, the unit cell enzyme activity is also increased. The chassis strain in the application is more suitable for industrial production than the industrial model strain and has very great application prospect and industrial value.

Description

Technical Field

[0001] This invention relates to a Bacillus subtilis chassis cell suitable for high-density fermentation and enzyme production, belonging to the field of microbiology. Background Technology

[0002] The core of catalytic production in microbial cell factories is the chassis cell, which is an important functional element in synthetic biology. Bacillus subtilis is a commonly used model strain in industrial fermentation. It is a Generally Recognized as Safe (GRAS) strain approved by the U.S. Food and Drug Administration, possessing a clear genetic background and exhibiting good operability and safety.

[0003] Traditional methods for producing L-glutaminase face a number of challenges. Among these, the selection and construction of chassis cells, which are responsible for synthesizing L-glutaminase, is of paramount importance. In many cases, choosing appropriate chassis cells is crucial for efficient L-glutaminase production. However, traditional chassis cell methods typically focus on modifying the product's metabolic pathways, and the cells themselves suffer from drawbacks such as slow growth, autolysis, and low yield, which limit the large-scale production of L-glutaminase.

[0004] Secondly, genes in chassis cells that are not related to L-glutaminase production may compete for limited cellular resources, reducing the synthesis rate of L-glutaminase. Therefore, non-essential growth metabolism in chassis cells needs to be optimized to promote L-glutaminase synthesis and thus improve production efficiency.

[0005] To address these issues, this invention provides an innovative method for constructing chassis cells, which includes lifespan engineering to increase cell biomass, mitigating late-stage cell autolysis to ensure extended high-yield periods, cre-lox genome editing to streamline unnecessary metabolic pathways, and genetic engineering to enhance L-glutaminase production. This comprehensive approach significantly improves L-glutaminase production efficiency, providing high-quality enzyme preparation for a wide range of applications.

[0006] Existing research has focused on increasing L-glutaminase production through modifications to single metabolic pathways or expression elements, but these studies have overlooked the importance and universality of the physiological properties of the chassis cells themselves in industrial production. Therefore, the modification of Bacillus subtilis chassis cells will be the focus of this invention. Summary of the Invention

[0007] To address the issues of autolysis and excessively high minimum maintenance energy coefficient in the later stages of Bacillus subtilis chassis cell fermentation, this invention provides a highly robust production-oriented Bacillus subtilis chassis cell by knocking out one or more genes, such as lytC, sigD, mraZ, and sigE, from the Bacillus subtilis genome.

[0008] The present invention provides a recombinant Bacillus subtilis strain, wherein one or more of the following genes are knocked out: peptidoglycan hydrolase-related genes, cell division-related genes, or spore synthase-related genes ... spore synthase-related genes,

[0009] In one embodiment, the peptidoglycan hydrolase-related gene includes lytC or sigD.

[0010] In one embodiment, the cell division-related gene is mraZ.

[0011] In one embodiment, the spore synthase-related gene is the spore stage 3 synthase sigE.

[0012] In one embodiment, the recombinant bacterium is obtained by knocking out the lytC gene from the genome sequence of Bacillus subtilis B. subtilis.168, and is named BS1; the nucleotide sequence of the lytC gene is shown in SEQ ID NO.1.

[0013] In one embodiment, the recombinant bacteria is obtained by knocking out the sigD gene on the genome sequence of Bacillus subtilis B. subtilis.168, and is named BS2; the nucleotide sequence of the sigD gene is shown in SEQ ID NO.2.

[0014] In one embodiment, the recombinant bacteria is obtained by knocking out the mraZ gene on the genome sequence of Bacillus subtilis B. subtilis.168, and is named BS3; the nucleotide sequence of the mraZ gene is shown in SEQ ID NO.3.

[0015] In one embodiment, the recombinant bacterium is obtained by knocking out the sigE gene on the genome sequence of Bacillus subtilis 168, and is named BS1; the nucleotide sequence of the sigE gene is shown in SEQ ID NO.4.

[0016] In one embodiment, the recombinant bacteria is obtained by knocking out the genes lytC, sigD, mraZ, and sigE in the B. subtilis 168 sequence, and the recombinant bacteria is named BS5.

[0017] The present invention also provides a method for preparing any of the above-described recombinant strains, comprising the following steps:

[0018] (1) Taking the sequence shown in SEQ ID NO.1 as an example, knockout primers were designed, and PCR amplification was performed to obtain 800bp fragments upstream and downstream of the target gene and the lox66-zeo-lox71 gene fragment. The gene was ligated by fusion PCR and amplified to obtain the gene fragment sequence SEQ ID NO.5.

[0019] (2) Transform the recombinant gene fragment constructed in step (1) into the host cell.

[0020] (3) After verifying the recombinant strain from step (2) by colony PCR, the strain was introduced into the pDG148 plasmid to eliminate zeo resistance.

[0021] (4) The correct transformant from step (3) is cultured at 52°C to eliminate the pDG148 plasmid.

[0022] The present invention also provides recombinant Bacillus subtilis expressing L-glutaminase.

[0023] In one embodiment, the recombinant Bacillus subtilis expresses the L-glutaminase using pMA5 as a vector.

[0024] In one embodiment, the L-glutaminase is derived from Lactobacillus reuteri DSM20016 and has the amino acid sequence shown in Genbank accession number ABQ83511.

[0025] The present invention also provides the application of the recombinant Bacillus subtilis in the fermentation production of L-glutamine.

[0026] The present invention also provides the application of the recombinant Bacillus subtilis strain in improving protein expression levels.

[0027] Beneficial effects:

[0028] (1) In this invention, genes that have a decisive influence on the biomass, growth rate, minimum maintenance energy coefficient, and enzyme production capacity of Bacillus subtilis were screened from lytC, sigD, mraZ, sigE, spolE, sigD, sigA, ponA, sdpC, skfA, spooA, xpF, flgD, ppsE, and lytB. By knocking out the relevant genes, a recombinant Bacillus subtilis with improved enzyme production capacity was constructed.

[0029] (2) The biomass of the recombinant Bacillus subtilis BS5 constructed in this invention increased by 40% compared to the original B. subtilis 168; OD during the later stage of the culture process600 The decrease was slower than that of the original strain; the maintenance energy coefficient was reduced by 25% compared to the original strain B. subtilis168.

[0030] (3) The recombinant Bacillus subtilis strain provided by this invention increases the yield of L-glutaminase. In batch fermentation, the yield of L-glutaminase can be nearly doubled, demonstrating excellent production capacity. After measuring enzyme activity and cell growth and production during fermentation in shake flasks and 5-L tanks, the expression of L-glutaminase was better when the recombinant strain BS5 of this invention was used as the host, increasing by 97.5% compared with the original strain. Moreover, the yield did not fluctuate or decrease significantly in the later stage of fermentation. The fermentation process is easy to control and is conducive to industrial production.

[0031] (4) The Bacillus subtilis BS5 obtained in this invention is more suitable for enzyme production than the wild type, and is more conducive to the flexibility of the production process. Attached Figure Description

[0032] Figure 1 Growth curve of recombinant chassis strain cultured in LB medium for 24 h.

[0033] Figure 2 The number of viable cells of the recombinant chassis strain cultured in LB medium for 24 h.

[0034] Figure 3 The enzyme activity of L-glutaminase produced by shake-flask fermentation of chassis strains was measured.

[0035] Figure 4 The growth, enzyme activity, and unit cell enzyme activity of the 5-L fermenter strain of the chassis strain during fermentation to produce L-glutaminease were studied. Detailed Implementation

[0036] The culture media involved in the following examples are as follows:

[0037] (1) LB liquid medium: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L.

[0038] (2) LB solid medium: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L.

[0039] (3) TB medium: yeast extract 24 g / L, peptone 12 g / L, glycerol 5 g / L, K2HPO4 12.54 g / L, KH2PO4 2.31 g / L.

[0040] The detection methods involved in the following embodiments are as follows:

[0041] L-glutamate fermentation process and enzyme activity assay method for recombinant strains:

[0042] For the shake-flask stage, the recombinant strain was inoculated into LB medium containing appropriate antibiotics and incubated at 30°C for 30 hours. Interim experiments were conducted in a 5-L bioreactor using a DO-stat feeding strategy. L-glutaminase activity was determined by terminating the reaction with 80 μL of 20% trichloroacetic acid after incubation at 55°C for 5 minutes. The reaction mixture (1 mL) contained 900 μL of 200 mmol / L L-glutamine and 20 μL of L-glutaminase. The L-glutamate concentration in the supernatant obtained after centrifugation was determined using a biosensor analyzer (Jinan Yanke Instrument Co., Ltd.). One unit (U) of L-glutaminase activity is defined as the production of 1 μmol / L per minute. -1 The amount of enzyme required to produce glutamate. One unit of L-glutaminase enzyme activity per unit cell (U / OD) 600 Defined as producing 1 μmol / L per minute -1 Enzyme requirement for glutamate / cell OD 600 The value.

[0043] Example 1: Scarless Gene Knockout Method

[0044] The target gene's base sequence was retrieved from NCBI, and 800 bp sequences before and after the target gene were obtained. Primers were then designed. Taking the gene mraZ as an example, the upstream and downstream homologous arms of mraZ on the genome were obtained by PCR amplification using the B. subtilis 168 genome as a template. The resistance selection marker expression cassette lox71-zeo-lox66 fragment was amplified from plasmid p7Z6.

[0045] The overlap extension PCR technique was used to fuse three DNA fragments into a single long DNA fragment. This method consists of three steps: First, using the B. subtilis 168 genome as a template, PCR amplification was performed to obtain the upstream and downstream homologous arms of the target gene. Using plasmid p7Z6 as a template, PCR amplification was performed to obtain the resistance selection marker fragment 1ox71-zeo-lox66.

[0046] Table 1. PCR reaction system for the first step

[0047]

[0048] PCR instrument settings: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, annealing for 45 s (annealing temperature determined by primers), 72℃ extension for 30 s (extension time determined by gene size, 30 s / kb), 30 cycles; 72℃ extension for 10 min, storage at 4℃. Add an appropriate amount of Goldview nucleic acid staining solution to the PCR amplification product, apply to a gel, and run nucleic acid electrophoresis. Remove the gel and place it in a gel imaging system for observation and recording the results. Cut the nucleic acid bands and place them in 1.5 mL EP tubes. Recover the DNA product according to the instructions of the agarose gel DNA recovery kit to obtain the upstream homologous arm fragment, the 1ox71-zeo-lox66 fragment, and the downstream homologous arm fragment.

[0049] Table 2. PCR reaction system for the second step

[0050]

[0051] PCR instrument settings: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, annealing for 45 s (annealing temperature determined by primers), 72℃ extension for 80 s, 15 cycles, 72℃ extension for 10 min, storage at 4℃. The second step is the pre-fusion of the three DNA fragments. The product is run on a validation gel, and after successful validation, it is directly used as the template for the third step of amplification.

[0052] Table 3. PCR reaction system for step 3

[0053]

[0054] The amplification conditions were the same as in the first step. After purification, the PCR product yielded three recombinant fragments: an upstream homologous arm, a 1ox71-zeo-lox66 segment, and a downstream homologous arm.

[0055] The designed primer sequences are shown in Table 4.

[0056] Table 4 Primer sequences

[0057]

[0058]

[0059] Three DNA fragments were overlapped and extended into a single recombinant DNA fragment sequence of approximately 2.2 kb (SEC ID NO. 5); this recombinant DNA fragment was then introduced into B. subtilis 168 competent cells. The specific steps were as follows:

[0060] (1) Streak the preserved B. subtilis 168 on an LB agar plate and incubate overnight at 37°C for 12 h. Pick a single colony of B. subtilis 168 from the plate and transfer it to 10 mL of LB medium. Incubate at 37°C and 200 r / min on a shaker for 12 h.

[0061] (2) Transfer 200 μL of the activated bacterial culture to 10 mL of SPI medium and incubate at 37°C and 200 r / min for 4.5 h. Transfer 1 mL of the bacterial culture to 10 mL of SPII medium and continue to incubate at 37°C and 200 r / min for 1.5-2 h.

[0062] (3) Quickly remove the culture flask, add 100 μL of 100×EGTA solution, and incubate in a shaker at 37℃ and 200 r / min for 10 min. Remove the culture flask again and dispense 500 μL of bacterial culture into a 1.5 mL sterile EP tube.

[0063] (4) Add an appropriate amount of plasmid or DNA fragment (10 μL), mix gently, and incubate in a shaker at 37°C and 200 r / min for 2 h.

[0064] (5) Centrifuge the bacterial cells at 5000 r / min for 5 min, discard the supernatant, and resuspend the bacterial cells in approximately 100 μL of supernatant. Spread the resuspended cells onto a substrate containing 30 mg / L Zeo r Zeo, with the target gene knocked out, was obtained by culturing on LB resistant plates at 37°C for 16 hours. r Transformer.

[0065] The thermosensitive plasmid pDG148 contains Cre recombinase. Recombination occurs at the lox71 and lox66 sites under the action of Cre recombinase, thereby excising the resistance gene zeo. Therefore, transferring pDG148 into Zeo... r The competent cells of the transformants were coated with a solution containing 50 mg / L Kan r On LB resistance plates, Zeo-free samples were obtained. r The transformants were then picked and placed on antibiotic-free LB plates and cultured at 51°C for 48 hours to remove pDG148, thus obtaining the antibiotic-free target gene knockout strain BS3.

[0066] Example 2: Construction of single and multiple knockout gene strains

[0067] Following the method in Example 1, strain BS1 (with the lytC gene knocked out), strain BS2 (with the sigD gene knocked out), strain BS3 (with the mraZ gene knocked out), and strain BS4 (with the sigE gene knocked out) were constructed. Based on strain BS1, the sigD, sigE, and mraZ genes were further knocked out to obtain strain BS5. The gene knockout was verified using colony PCR or bacterial culture PCR; the reaction system is shown in Table 4.

[0068] Table 4. PCR reaction system for bacterial colonies and bacterial suspensions

[0069]

[0070] Amplification conditions: 95℃ pre-denaturation for 15 min, 95℃ denaturation for 30 s, annealing for 30 s (annealing temperature determined by primers), 72℃ extension for 1 min (extension time determined by gene size, 1 min / kb), 28 cycles, 72℃ extension for 10 min, and storage at 4℃. PCR products were verified using a 1% agarose gel electrophoresis.

[0071] Example 3: Construction of L-glutaminase-derived genetically engineered bacteria from Lactobacillus reuteri

[0072] The plasmid used to express the L-glutaminase vector was pMA5, carrying the promoter PipaII (SEQ ID NO. 6). Using the *Lactobacillus reuteri* genome as a template, the glsA gene (SEQ ID NO. 7) was amplified. The vector fragment and gene fragment, each containing a 15 bp homologous sequence, were amplified by PCR using primers P1 / P2 and P3 / P4. These fragments were then ligated using In-Fusion HD Cloning Pluskit ligase. The ligation product was transformed into *E. coli* JM109 competent cells and cultured at 37°C for 8 hours. Transformants were selected and cultured with shaking in LB broth containing 100 mg / L ampicillin. The plasmid was extracted, and sequencing confirmed the expression plasmid pMA5-glsA.

[0073] The PCR reaction system is as follows:

[0074] 5×PrimeSTAR Buffer(Mg2+Plus)10mL;

[0075] dNTP (10 mmol / L) 4 μL;

[0076] Template (50 pmol / L) 0.5 μL;

[0077] PCR primer 1 0.5 μL;

[0078] PCR primer 2, 0.5 μL;

[0079] PrimeSTAR HSDNA Polymerase 0.5μL;

[0080] Add ddH2O to bring the system to 50 μL.

[0081] The reaction procedure is as follows:

[0082] Pre-denaturation at 94℃ for 4 min, followed by 98℃ for 10 s, 55℃ for 10 s, and 72℃ for 1.5 min, for 30 cycles. Then, extend the temperature to 72℃ for 10 min and cool down to 4℃.

[0083] Table 5 Primer sequences

[0084]

[0085] The plasmid pMA5-glsA was transformed into strains BS1-BS5 constructed in Examples 1-2, respectively. The transformants were placed on LB agar plates containing kanamycin (50 mg / L) and incubated at 37°C for 8 h. Single colonies were picked and incubated overnight at 37°C. The glycerol tubes used to verify the correct strains were stored and named BS1-glsA to BS5-glsA, respectively.

[0086] Example 4: Production of L-glutaminase by shake-flask fermentation of genetically engineered bacteria

[0087] The L-glutaminase recombinant bacteria constructed in Example 3 were cultured in shake flasks to examine L-glutaminase expression. The culture process was as follows: 10 μL of glycerol culture was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of LB medium and cultured at 37°C and 200 rpm for 8-10 h. The above culture was then inoculated into a 250 mL Erlenmeyer flask containing 50 mL of TB at a 5% (v / v) inoculation rate and cultured at 30°C and 200 rpm for 36 h. Intracellular L-glutaminase activity was then measured. The enzyme activity was 16.79 U / mL with *B. subtilis* 168 as the host bacterium, 20.43 U / mL with BS1, 24.17 U / mL with BS2, 23.28 U / mL with BS3, 14.17 U / mL with BS4, and 34.17 U / mL with BS5. The unit cell enzyme activity was 2.83 U / mL for *B. subtilis* 168, 2.94 U / mL for BS1, 3.17 U / mL for BS2, 3.24 U / mL for *B. subtilis* 168, 2.08 U / mL for BS4, and 4.56 U / mL for BS5.

[0088] The results showed that knocking out genes lytC, sigD, and mraZ had an increasing effect on enzyme production by B. subtilis 168, while BS4 had a slight decrease. The combined knockout of the four genes had a significant effect on enzyme production by B. subtilis 168.

[0089] Example 5: Fermentation of genetically engineered bacteria using Bacillus subtilis mutant as host in a 5-L tank

[0090] Five recombinant L-glutaminase strains constructed in Example 3 were subjected to 5-L fermentation to examine the expression of L-glutaminase and the growth and autolysis of the recombinant bacteria. The culture process was as follows: 200 μL of glycerol culture was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of LB medium. The culture was incubated at 37°C and 200 rpm for 8-10 h. The culture was then transferred to a 5L fermenter with a volume of 1.5 L. Initial OD after inoculation was measured. 600 The value was 1.41. The pH was controlled at 7.0 with ammonia and 20% phosphoric acid, and the culture temperature was 30℃. The dissolved oxygen was maintained at around 30% by coupling with the stirring speed and adjusting the aeration rate. When the dissolved oxygen content was higher than 40% during the logarithmic growth phase of fermentation, the feed rate was increased at 15 mL / h. When the dissolved oxygen content began to increase again, the feed rate was increased. The culture was ended when the L-glutaminase activity decreased.

[0091] Fermentation medium: yeast powder 15g / L, corn steep liquor 25g / L, glucose 12g / L, diammonium hydrogen citrate 1g / L, Na2SO3 2g / L, (NH4)2SO4 2.68g / L, K2HPO4·3H2O 19.2g / L, NaH2PO4·H2O 4g / L, MgSO4·7H2O 1g / L, metal ion PTM solution 3ml / L.

[0092] Feeding medium: glucose 500 g / L, MgSO4·7H2O 7.89 g / L, (NH4)2HPO4 63.36 g / L, metal ion PTM solution 40 mL / L.

[0093] Metal ion PTM solution: CuSO4·5H2O 6g / L, KI 0.08g / L, MnSO4·H2O 0.5g / L, Na2MoO3·2H2ON 0.2g / L, H3BO3 0.02g / L, CoCl2 0.5g / L, ZnCl2 20g / L, FeSO4·7H2O 65g / L, Biotin 0.2g / L, H2SO4 5.0g / L.

[0094] When B. subtilis 168 was used as the host bacteria, the L-glutaminase activity was 1422.9 U / mL after 72 h of fermentation. When BS1 was used as the host bacteria, the L-glutaminase activity was 1955.5 U / mL after 72 h of fermentation. When BS2 was used as the host bacteria, the L-glutaminase activity was 1684.5 U / mL after 72 h of fermentation. When BS3 was used as the host bacteria, the L-glutaminase activity was 1870.7 U / mL after 72 h of fermentation. When BS4 was used as the host bacteria, the L-glutaminase activity was 1885.6 U / mL after 72 h of fermentation. When BS5 was used as the host bacteria, the L-glutaminase activity was 2817.4 U / mL after 72 h of fermentation.

[0095] During fermentation, the growth and sugar consumption of the strains were recorded. After fermentation, the highest unit cell enzyme activity was recorded. *B. subtilis* 168 unit cell enzyme activity: 14.8 U / mL; BS1: 15.1 U / mL; BS2: 15.7 U / mL; *B. subtilis* 168 unit cell enzyme activity: 16.2 U / mL; BS4: 16.8 U / mL; BS5: 19.6 U / mL.

[0096] Comparative Example 1:

[0097] The specific implementation method is the same as in Examples 1-4, except that lytC is replaced with the peptidoglycan hydrolase-related gene xpF (shown in SEQ ID NO. 8). Results showed that the biomass of the recombinant strain decreased significantly in the later stages of fermentation, and OD... 600 Reduced by 15%.

[0098] Comparative Example 2:

[0099] The specific implementation method is the same as in Examples 1-4, except that mraZ is replaced with the cell division-related gene yluC (shown in SEQ ID NO. 9). Results showed that the biomass of the recombinant strain decreased significantly in the later stages of fermentation, and OD... 600 Reduced by 18%.

[0100] Comparative Example 3:

[0101] The specific implementation method is the same as in Examples 1 to 4, except that sigE is replaced with the spo0A gene (shown in SEQ ID NO. 10), which is related to spore formation. The results show that the recombinant strain grows slowly and the growth rate is reduced by 20%.

[0102] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant Bacillus subtilis strain, characterized in that, The recombinant bacteria had peptidoglycan hydrolase-related genes knocked out. lytC and sigD Cell division-related genes mraZ spore synthase-related genes sigE Using pMA5 as the expression vector, L-glutaminase with the amino acid sequence shown in Genbank accession number ABQ83511 was expressed.

2. The recombinant Bacillus subtilis strain according to claim 1, characterized in that, The gene lytC The nucleotide sequence is shown in SEQ ID NO.1; the gene sigD The nucleotide sequence is shown in SEQ ID NO.2; the gene mraZ The nucleotide sequence is shown in SEQ ID NO.3; the gene sigE The nucleotide sequence is shown in SEQ ID NO.

4.

3. A method for increasing the protein expression level of Bacillus subtilis, characterized in that, Knock out Bacillus subtilis genome lytC , sigD , mraZ and sigE Gene.

4. The method according to claim 3, characterized in that, The Bacillus subtilis strain is Bacillus subtilis 168.

5. The application of the recombinant Bacillus subtilis strain according to claim 1 or 2 or the method according to claim 3 or 4 in the fermentation production of protein products.

6. The use of the recombinant Bacillus subtilis according to claim 1 or 2 in the fermentation production of L-glutamine.

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