Construction method of bacillus subtilis chassis strain with improved sucrose isomerase expression capability

Abstract

The invention discloses a construction method of a bacillus subtilis chassis strain with improved sucrose isomerase expression capability, and belongs to the technical field of enzyme engineering and microbial modification. Based on a CRISPRi (clustered regularly interspaced short palindromic repeats i) system and an sgRNA (single guide ribonucleic acid) library of a targeted whole genome, key genes influencing PdSI expression are screened from bacillus subtilis and are combined, regulated and controlled, and a bacillus subtilis chassis strain with improved expression capability is obtained. And then, high-density fermentation is carried out in a 3-L tank, so that efficient recombinant expression of PdSI is realized, and the enzyme activity in the fermentation supernatant can reach 408.23 U.mL <-1 >.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing a Bacillus subtilis chassis strain with improved expression of sucrose isomerase, belonging to the field of enzyme engineering and microbial modification. BACKGROUND

[0002] Bacillus subtilis is a gram-positive, aerobic bacteria, which can form endospores with strong resistance. It can survive in extreme environments such as high temperature, dryness, ultraviolet radiation or nutrient deficiency, and rapidly restore metabolic activity under suitable conditions. Bacillus subtilis is an important model industrial microorganism and GRAS microorganism, which is widely used in the production of industrial enzyme preparations. It has the characteristics of strong protein secretion ability, fast growth, short fermentation period, no obvious codon bias, clear genetic background and mature molecular biology editing means, which is conducive to its genetic modification. Sucrose isomerase (Sucrose isomerase, EC 5.4.99.11) is also known as isomaltulose synthase, which can catalyze the isomerization of sucrose to produce isomaltulose. Isomaltulose has the characteristics of low glycemic index and anti-caries, and is widely used in functional foods and medical fields.

[0003] Mining functional genes that affect the expression level of sucrose isomerase and regulating them is the key to further improve its expression. Constructing a library to screen key genes is an effective strategy. Compared with random mutation and transposon-based methods, CRISPRi-based screening can flexibly select the range of genes covered by the sgRNA library designed by itself. Through high-throughput screening, strains with improved expression of sucrose isomerase are obtained, and their genotypes are identified to obtain genes highly related to expression levels and to modify and regulate them. Studies have shown that overexpression of Sec pathway-related factors, knockout of intracellular or extracellular proteases, and increase of chaperone content can promote the expression of recombinant enzymes. When the recombinant enzyme precursor is in the periplasmic space, extracellular molecular chaperone PrsA can promote its effective folding and avoid degradation by quality control proteins. When PrsA is overexpressed in Bacillus subtilis, the activity of alpha-amylase is significantly improved. When the mature protein is released into the extracellular, signal peptide peptidase degrades the signal peptide, and overexpression of signal peptide peptidase SppA that degrades the signal peptide can improve the enzyme activity of superthermostable amylase Pfa.

[0004] Industrial recombinant bacteria producing sucrose isomerase usually need to add antibiotics during fermentation to maintain the stability of the plasmid. However, the quality specification of food enzyme preparations requires that no antibacterial activity be detected in microbial-derived enzyme preparations. With the increasing awareness of health, people are paying more and more attention to food safety issues. Therefore, it is of great significance to construct sucrose isomerase antibiotic-free marker gene integrated recombinant bacteria. SUMMARY

[0005] The present application provides a method for increasing the expression level of sucrose isomerase in Bacillus subtilis, the method comprising (a) and / or (b):

[0006] (a) inhibiting one or more of the following genes: dltA, yphF, lplD, yqjM, trmR, mtlD, liaH, yjcS, pycA, sinR, ilvA and dltB;

[0007] (b) overexpressing one or more of the following proteins: penicillin-binding protein class A ponA, transcriptional factor sigA, transcriptional factor sigL, ribosome assembly protein cpgA, endoribonuclease rnZ, cell division initiation protein ftsZ, polyglycerolphosphate lipoteichoic acid synthase ltaS, stress protein liaH, chaperone prsA, motor protein secA, ABC transporter oppA and pleiotropic regulator degQ.

[0008] In one embodiment, the method is to integrate sgRNA targeting inhibition of one or more of the following proteins in Bacillus subtilis: phosphomucoid synthesis gene dltA, glycoside bond hydrolysis gene LplD, antioxidant stress gene yqjM, tRNA uridine modification gene trmR, phosphomannitol dehydrogenase gene mtlD, antioxidant stress gene liaH, pyruvate carboxylase gene pycA, Xre family transcriptional regulator sinR, threonine dehydration ilvA, alanine esterification dltB, unknown function gene yjcS, yphF.

[0009] In one embodiment, the recombinant Bacillus subtilis knocks out the dltA gene.

[0010] In one embodiment, the recombinant Bacillus subtilis inhibits the mtlD gene.

[0011] In one embodiment, the recombinant Bacillus subtilis inhibits the mtlD and yqjM genes.

[0012] In one embodiment, the recombinant Bacillus subtilis inhibits the mtlD and sinR genes.

[0013] In an embodiment, the host bacteria include, but are not limited to, B. subtilis WS9D, B. subtilis WS9C; the B. subtilis WS9D is integrated with dCas9 gene in B. subtilis WS9C; the B. subtilis WS9C strain is integrated with comk gene at amyE site of B. subtilis WS9 using CRISPR / Cas9 gene editing system, and the B. subtilis WS strain is knocked out of srfC, spoIIAC, amyE, nprB, nprE, aprE, bpr, mpr and epr using CRISPR / Cas9 gene editing system, which reduces the production of foam, spores, extracellular amylase and protease in the fermentation process (the above-mentioned strains are disclosed in the paper "Modification of B. subtilis Strains, Optimization of Promoters and Efficient Production of Pullulanase").

[0014] In an embodiment, the sucrose isomerase has an amino acid sequence as shown in SEQ ID NO. 1.

[0015] In an embodiment, the sucrose isomerase is expressed by using pUB110 as an expression vector and using the constitutive promoter P amyQ’ The expression of the sucrose isomerase is regulated.

[0016] In an embodiment, the inhibition of gene expression is achieved by integrating sgRNA expression cassette in the genome of B. subtilis WS9D.

[0017] In an embodiment, the sgRNA is expressed by using the constitutive promoter P 43 (nucleotide sequence as shown in SEQ ID NO. 5).

[0018] In an embodiment, the sgRNA spacer sequences for targeting dltA, yphF, lplD, yqjM, trmR, mtlD, liaH, yjcS, pycA, sinR, ilvA and dltB are as shown in SEQ ID NO. 8 to SEQ ID NO. 19.

[0019] In an embodiment, the enhancer is any one of the following: class A penicillin-binding protein ponA, transcription factor sigA, transcription factor sigL, ribosome assembly protein cpgA, endoribonuclease rnZ, cell division initiation protein ftsZ, polyglycerolphosphate lipoteichoic acid synthase ltaS, stress protein liaH, chaperone prsA, motor protein secA, ABC transporter oppA and pleiotropic regulator degQ.

[0020] In one embodiment, the enhancer is pAD123 as an expression vector, and the promoter P glv modulates expression.

[0021] In one embodiment, the nucleotide sequence of the motor protein secA is as shown in SEQ ID NO. 20.

[0022] In one embodiment, the coding gene sequence of the ABC transporter oppA is as shown in SEQ ID NO. 21.

[0023] In one embodiment, the coding gene sequence of the cell division initiation protein ftsZ is as shown in SEQ ID NO. 22.

[0024] In one embodiment, the coding gene sequence of the molecular chaperone prsA is as shown in SEQ ID NO. 23.

[0025] In one embodiment, the coding gene sequence of the class A penicillin-binding protein ponA is as shown in SEQ ID NO. 24.

[0026] The present application also provides a recombinant Bacillus subtilis, in which one or more of the genes dltA, yphF, lplD, yqjM, trmR, mtlD, liaH, yjcS, pycA, sinR, ilvA and dltB are inhibited, and the motor protein secA, the ABC transporter oppA, the cell division initiation protein ftsZ, the molecular chaperone prsA or the class A penicillin-binding protein ponA are expressed.

[0027] In one embodiment, the recombinant Bacillus subtilis further expresses the sucrose isomerase as shown in SEQ ID NO. 1 or a mutant thereof.

[0028] In one embodiment, the mutant is a mutation of valine at position 447 to glutamic acid based on the sequence as shown in SEQ ID NO. 1.

[0029] In one embodiment, one or more copies of the sucrose isomerase or the mutant thereof are integrated into the genome of the recombinant Bacillus subtilis.

[0030] In one embodiment, the coding gene of the sucrose isomerase is integrated into at least one of the lacA, bpr, nprB sites of the genome.

[0031] In one embodiment, the Bacillus subtilis uses the Cre / lox gene editing system to integrate the sucrose isomerase gene.

[0032] In one embodiment, the recombinant Bacillus subtilis is a recombinant strain WS9ED1 obtained by integrating a sucrose isomerase gene at the lacA site.

[0033] In one embodiment, the recombinant Bacillus subtilis is a recombinant strain WS9ED2 obtained by integrating a sucrose isomerase gene at the lacA and bpr sites.

[0034] In one embodiment, the recombinant Bacillus subtilis is a recombinant strain WS9ED3 obtained by integrating a sucrose isomerase gene at the lacA, bpr and nprB sites.

[0035] In one embodiment, the sucrose isomerase is expressed by the promoter P HpaII -P amyQ’ initiating transcription.

[0036] In one embodiment, the promoter P HpaII -P amyQ has a nucleotide sequence as shown in SEQ ID NO. 7.

[0037] The present application also provides a method for preparing a sucrose isomerase by fermentation using the recombinant Bacillus subtilis.

[0038] In one embodiment, the method is to induce the enzyme production of the recombinant Bacillus subtilis in TB medium using maltose.

[0039] In one embodiment, the method is to culture the recombinant Bacillus subtilis at 37-40°C for a period of time, and then to ferment at 32-34°C after cooling.

[0040] In one embodiment, the method is to culture the recombinant Bacillus subtilis in a basic medium for a period of time, and then to start feeding when the dissolved oxygen rebounds.

[0041] In one embodiment, the feeding is to add a feeding medium.

[0042] In one embodiment, the basic medium contains yeast extract powder, corn steep liquor, glycerol, ammonium citrate, MgSO4·7H2O, K2HPO4·3H2O, NaH2PO4·2H2O, ammonium sulfate and trace elements.

[0043] In one embodiment, the feeding medium contains glycerol, yeast extract powder, corn steep liquor, MgSO4·7H2O, maltose and trace elements.

[0044] In one embodiment, the trace elements include Cu 2+ , Ca 2+ , Mn 2+ , Co2+ , Zn 2+ , Na + , Fe 2+ .

[0045] The application also provides the application of the recombinant Bacillus subtilis or the method in producing sucrose isomerase.

[0046] Beneficial effects:

[0047] (1) The application carries out high-throughput screening in the strain B. subtilis WS9D based on the CRISPRi system and the sgRNA library targeting the whole genome, and 14 key genes capable of improving the expression level of PdSI are obtained, and the recombinant expression level is 1.17-2.56 times that of the control strain.

[0048] (2) The application obtains the strain WS9E by knocking out the dltA gene in WS9D, and the PdSI enzyme activity is 1.78 times that of the control strain. Further, 12 sgRNAs of the key genes are integrated into the genome of WS9E, and the enzyme activity of the optimal single-integrated strain WS9EA6S (containing a PdSI expression plasmid) is 1.89 times that of the control strain. In the recombinant strain WS9EA6, other sgRNAs of genes helpful to the enzyme activity are continuously integrated, and the enzyme activity of the double-sgRNA integrated strain WS9EB2S (containing a PdSI expression plasmid) is 1.46 times that of the control strain. Based on rational mining, 12 genes are selected, and the results show that when secA, oppA and ftsZ are overexpressed, the PdSI enzyme activity is 1.58, 1.38 and 1.25 times that of the control strain, respectively.

[0049] (3) The application expresses the sucrose isomerase mutant V447E in the recombinant strain WS9EB2, and constructs the strain B. subtilis WS9F. The high-density fermentation verification is carried out in a 3-L tank, and the supernatant enzyme activity of the strain WS9F can reach 408.23 U·mL -1 -1 after fermentation for 64.50 h, which is 12.35 times that of the shake flask fermentation enzyme activity. On this basis, the V447E expression frame is sequentially integrated into the lacA, bpr and nprB sites of the WS9EB2 genome based on site-specific recombination, and the enzyme activities of the corresponding recombinant strains are 1.51, 4.52 and 4.65 U·mL -1 , respectively. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 XhoI enzyme digestion verification of PCR verification product of dltA gene knockout; wherein, M: DL 10,000 DNA Marker; 1-2: WS9D; 3-4: WS9E.

[0051] Figure 2 The effect of knocking out dltA gene on PdSI expression.

[0052] Figure 3 Nucleic acid electrophoresis verification of single sgRNA integration.

[0053] Figure 4 Expression of PdSI in single sgRNA integration bacteria.

[0054] Figure 5 sgRNA mtlD -sgRNA sinR , sgRNA mtlD -sgRNA yqjM , sgRNA mtlD -sgRNA yjcS and sgRNA mtlD -sgRNA trmR Integration verification and effect on PdSI expression and biomass; wherein (a) is the nucleic acid electrophoresis verification result of PCR fragment; M: DL 10,000 DNA Marker; 1: WS9EA6; 2-5: WS9EB1-WS9EB4; (b) is the expression of PdSI.

[0055] Figure 6 sgRNA mtlD -sgRNA yqjM -sgRNA sinR and sgRNA mtlD -sgRNA yqjM -sgRNA yjcS Integration nucleic acid electrophoresis verification and effect on PdSI expression and biomass; wherein (a) is the nucleic acid electrophoresis verification; M: DL 10,000 DNA Marker; 1: WS9EB2; 2: WS9EC1; 3: WS9EC2; (b) is the expression of PdSI.

[0056] Figure 7 Effect of overexpression of different genes on PdSI expression and biomass.

[0057] Figure 8 B. subtilis WS9F 3-L tank fermentation.

[0058] Figure 9 V447E integration verification and expression level determination; (a) nucleic acid electrophoresis verification of different sites; wherein, M: DL10000 DNA Marker; 1, 3, 5: negative control, 2, 4, 6: experimental group; (b) expression of recombinant bacteria. DETAILED DESCRIPTION

[0059] (1) Medium:

[0060] LB liquid medium (g·L -1 ): Peptone 10, yeast extract 5, NaCl 10; LB solid medium needs to add 15 g·L -1 of agar powder in LB liquid medium.

[0061] TB fermentation medium (g·L -1 ): Peptone 12, yeast extract 24, glycerol 5, K2HPO4·3H2O 16.43 (anhydrous K2HPO412.54), KH2PO4 2.31.

[0062] YN liquid medium (g·L -1 ): Yeast extract 7, nutrient broth 18.

[0063] 3-L tank basic medium (g·L -1 ): Angelus yeast powder 36.00, corn syrup 20.00, glycerol 10.00, ammonium citrate 1.00, MgSO4·7H2O 1.00, K2HPO4·3H2O 14.6, NaH2PO4·2H2O 4.52, ammonium sulfate 2.68, trace element solution 3 mL.

[0064] 3-L tank feeding medium (g·L -1 ): Glycerol 250.00, Angelus yeast powder 41.00, corn syrup 23.00, MgSO4·7H2O 7.89, trace element solution 40.00 mL, maltose 250.00 (sterilized separately).

[0065] Trace element solution (g·L -1 ): CuSO4·5H2O 0.16, CaCl2 0.50, MnSO4·H2O 0.10, CoCl2 0.18, ZnSO4·7H2O 0.18, EDTA-2Na 10.50, FeCl2 8.35.

[0066] The above media are sterilized at 121℃ for 20 min before use.

[0067] (2) B. subtilis transformation method:

[0068] (1) Streak activation: use a loop to pick B. subtilis liquid on LB solid plate, 37℃ inverted culture for 10 h.

[0069] (2) Pick bacteria culture: pick single colony on plate into 10 mL of YN liquid medium, culture at 37℃, 200 r·min -1 for 10 h.

[0070] (3) Subculture induction: The bacteria solution cultured in (2) was subcultured into a new 10 mL YN liquid medium with 5% (v·v -1 ) inoculation amount, and 0.25 mL of 40% (w·v -1 ) xylose solution was added to make the final concentration of xylose 1%, and then the solution was cultured at 37℃, 200 r·min -1 for 4 h.

[0071] (4) Aliquot competent: 170 μL of the already cultured Bacillus subtilis competent was taken with a pre-cooled sterile gun head and was aliquoted into a sterile 1.5 mL EP, and 30 μL of 70% (v·v -1 ) glycerol was added into the 1.5 mL EP, and then the mixture was mixed and placed on ice. After the completion of the aliquot competent, it was immediately stored in a -80℃ ultra-low temperature refrigerator.

[0072] (5) Plasmid transformation: After the Bacillus subtilis competent was thawed on ice, 10 μL of the PCR product or recombinant plasmid was quickly added to the Bacillus subtilis competent cells, and then the mixture was incubated in an ice bath for 20 min, and then was incubated in a constant temperature water bath at 37℃ for 20 min. Then, the mixture was cultured in an air shaker at 37℃, 200 r·min -1 for 2 h, and then was plated on a corresponding resistant LB solid plate, and then was incubated at 37℃ for 10-12 h.

[0073] (III) Sucrose isomerase enzyme activity determination method:

[0074] In 1.80 mL of citrate-phosphate buffer (50 mmol·L -1 , pH 6.0), 200 μL of appropriately diluted enzyme solution was added to make the final concentration of sucrose 200 g·L -1 . The reaction mixture was shaken well, and then was incubated in a 30℃ water bath for 15 min. The reaction was terminated by heating in a boiling water bath for 10 min. Then, the reaction mixture was centrifuged at 12,000 rpm for 10 min at 40℃, and the supernatant was filtered with a 0.22 μm syringe filter. The concentration of isomaltulose in the filtered solution was quantitatively analyzed using an Alliance iS high performance liquid chromatography system (Waters) equipped with a refractive index (RI) detector and a Syncronis Amino chromatographic column (4.6 mm x 25 mm x 5 mm, Thermo Scientific, USA). After the sample (10 μL) was injected, the elution was performed at a flow rate of 0.8 mL·min -1 with 80% (v·v -1 ) acetonitrile aqueous solution, and the column temperature was maintained at 40℃.

[0075] The enzyme activity unit of sucrose isomerase is defined as: the amount of enzyme required to produce 1 μmol of isomaltulose within 1 min at 30°C and pH 6.0 is 1 enzyme activity unit (1 U).

[0076] (IV) The primers and sequences involved in the examples.

[0077] The primers and sequences are shown in Table 1.

[0078] Table 1 Primer Sequences

[0079]

[0080]

[0081]

[0082] Example 1: Screening of key genes affecting the expression of sucrose isomerase

[0083] Based on the B. subtilis whole genome suppression library established by the inventors' team in the early stage, 6,000 sgRNAs were designed for the 4,237 open reading frames currently known in B. subtilis. The obtained sgRNAs can target 4,225 open reading frames, with a coverage rate of 99.70%. In B. subtilis WS9D, CRISPRi technology and sgRNA library targeting the whole genome open reading frame were used to obtain a large number of single genotype expression inhibition strains. The enzyme activity of PdSI was used as the basis for high-throughput screening.

[0084] In B. subtilis WS9D (disclosed in the patent application file with publication number CN118726439A), the pUB110-sim plasmid containing the PdSI gene (nucleotide sequence shown as SEQ ID NO. 2) was transformed, and then coated on a 25 μg·mL -1 kanamycin resistance plate, 200 rpm, 37°C for 12 h. The sgRNA library plasmid was transformed in WS9D-pUB110-sim, and then coated on a 25 μg·mL -1 kanamycin and 5 μg·mL -1 chloramphenicol resistance plate, 200 rpm, 37°C for 12 h.

[0085] 96-well plate culture: 25 μg·mL -1 kanamycin and 5 μg·mL -1 chloramphenicol were added to the LB medium, and 140 μL of the above-mentioned medium was dispensed into each well of a sterile 96-well plate. A sterilized toothpick was used to transfer the bacteria from the 25 μg·mL -1 kanamycin and 5 μg·mL -1LB solid plates with chloramphenicol were used to pick single colonies into 96-well plates, and a diagonal line was set for inoculating control strains to ensure that there were control strains in each row and column. The plates were incubated at 37°C, 750 rpm for 10 h in a 96-well plate incubator shaker. Then, 40 μL of the seed culture was inoculated into 600 μL of TB medium (50 μg·mL -1 kanamycin and 5 μg·mL -1 chloramphenicol) in a 96-well deep plate, and the plates were incubated at 37°C, 750 rpm for 2 h in a 96-well plate incubator shaker, and then at 33°C for 48 h.

[0086] Nearly 3500 single colonies were picked for 96-well plate screening, and then strains with higher enzyme activity than the control strain WS9DA0 (constructed by introducing the pAD123- emptyN20 plasmid into the strain WS9D) were selected for shake flask verification. The sgRNAs of the recombinant strains with improved expression were sequenced and identified. After comparison of the sequencing results, 14 key genes that enhanced the expression of PdSI were screened, and the functions of the corresponding genes and the targeted sgRNA sequences are shown in Table 2.

[0087] Table 2 Function annotation of the key genes obtained by screening and sgRNA sequences

[0088]

[0089]

[0090] Example 2: Effect of knocking out the phospholipid synthesis gene dltA on the expression of sucrose isomerase

[0091] The phospholipid synthesis gene dltA obtained by high-throughput screening in Example 1 was knocked out using the CRISPR / Cas9 editing method. The construction of the knockout strain and the shake flask fermentation process are as follows:

[0092] (1) The plasmid pHYcas9dapr (disclosed in the dissertation “Modification of Bacillus subtilis strains, optimization of promoters, and efficient preparation of pullulanase”) was used as a template, and the primer pair dltA-F / R was used to replace the targeting sgRNA, and then the primer pair pHY-ZT-dltA-F / R was used to amplify the vector backbone.

[0093] (2) The WS9C genome was used as a template, and 19 bp of random insertion sequences were introduced into the primers dltA-upper-R and dltA-lower-F, respectively. The primer sequences are shown in Table 1, and the primer pair dltA-upper-F / R and dltA-lower-F / R was used to amplify the upper and lower homologous arms. The PCR reaction system is shown in Table 3, and the reaction conditions are shown in Table 4.

[0094] (3) The vector backbone and homologous repair sequence were connected by using a one-step cloning kit, transformed into E. coli JM109 competent cells, coated on ampicillin-resistant plates, and cultured for 10-12 h. The plasmid was extracted and sequenced by Ansel Biological Technology Co., Ltd. (Suzhou).

[0095] (4) The pHYcas9dltA was transformed into WS9D competent cells, coated on tetracycline-resistant plates, and subjected to colony PCR with primer pair dltA-verification-F / R. After the PCR product was verified by Xho I digestion, it was subjected to sequencing identification. After the knockout success was verified, the colony was picked on an antibiotic-free LB medium and cultured at 51°C and 200 rpm for 12 h. The obtained colony was blotted onto an antibiotic-free plate and a tetracycline-resistant plate. A single colony that could only grow on the antibiotic-free plate but not on the tetracycline-resistant plate was picked. The obtained plasmid-free strain was B. subtilis WS9E.

[0096] (5) The pUB110-sim plasmid was transformed into the original strain WS9D and the dltA knockout strain WS9E competent cells to obtain strains WS9DS and WS9ES, which were coated on plates containing 25 μg·mL -1 kanamycin, and cultured at 200 rpm and 37°C for 12 h. A single colony was inoculated into 10 mL of LB medium containing 25 μg·mL -1 kanamycin, and cultured at 200 rpm and 37°C for 12 h. Then, 5% of the inoculum was transferred to 50 mL of TB medium containing 25 μg·mL -1 kanamycin, and cultured at 200 rpm and 37°C for 2 h. Then, the temperature was reduced to 33°C, and fermentation was performed for 48 h. The results of the shake flask fermentation are shown in Figure 2 , the PdSI enzyme activity of WS9ES was 1.81 times that of the control strain WS9DS, and knocking out dltA did not substantially affect the growth of the strain.

[0097] Table 3 PCR reaction system

[0098]

[0099] Table 4 PCR reaction program

[0100]

[0101]

[0102] *: The cycle number of the denaturation, annealing, and extension steps was 30.

[0103] Example 3: Effect of integrating a single sgRNA expression frame on sucrose isomerase expression

[0104] The integration of the sgRNA expression frame using the Cre / LoxP system, the construction of the integration strain, and the shake flask fermentation method are as follows:

[0105] (1) The upper and lower homology arms were amplified from the WS9C genome using primers mpr-up-F and mpr-down-R. The linearized vector fragment was amplified from the plasmid pET-24a using primers 24a-F / R by inverse PCR. The two fragments were connected by seamless cloning according to the instructions of the Homologous Recombination Seamless Cloning Kit (CU201-02). The ligation product was transformed into E. coli JM109, and the transformants were plated on LB agar plates containing 25 μg·mL -1 kanamycin.

[0106] (2) The linearized vector fragment was amplified from the plasmid pET-24a-mpr of step (1) using primers mpr-up-R and mpr-down-F by inverse PCR. The sgRNA expression frame and tetracycline resistance cassette fragment were amplified from the recombinant plasmid pET-24a-rodZ containing the rodZ expression frame of SEQ ID NO. 25 using primers p43-F and Tet-66-R. The PCR system and program were referred to Tables 2 and 3, and the two fragments were connected by seamless cloning according to the instructions of the Homologous Recombination Seamless Cloning Kit (CU201-02). The ligation product was transformed into E. coli JM109, and the transformants were plated on LB agar plates containing 25 μg·mL -1 kanamycin.

[0107] (3) The N20 sequence in the plasmid was replaced using two-step PCR with 13 pairs of primers dltA-F / R, etc., and the PCR system and program were referred to Tables 2 and 3, using the plasmid pET-24a-mpr-rodZ of step (2) as the template.

[0108] (4) 10 μL of the sgRNA expression frame integration plasmid constructed in step (3) and sequenced correctly was transformed into the strain WS9E constructed in Example 2, and the transformants were plated on agar plates containing 25 μg·mL -1 tetracycline. The single colony with correct PCR verification was picked into 10 mL LB medium containing 25 μg·mL -1 tetracycline, and competent cells were prepared.

[0109] (5) The Cre recombinase expression plasmid pE194-Cre (disclosed in the patent application document with publication number CN118421548A) was transformed into the competent cells prepared in step (4), and the transformants were plated on agar plates containing 1 mmol·L -1 IPTG and 25 μg·mL -1Incubate overnight at 37°C on kanamycin plates.

[0110] (6) According to the photocopy plate method, the obtained monoclonal bacteria were picked onto the blot and onto the antibiotic-free plate, and the blot contained 25 μg·mL⁻¹. -1 Tet r The resistant plates were selected only from those that could grow on non-resistant plates, and not on plates containing 25 μg·mL⁻¹. -1 Colonies grown on tetracycline plates were streaked onto antibiotic-free plates and incubated at 51°C and 200 rpm for 10 h. The resulting colonies were then blotted onto antibiotic-free plates and plates containing 25 μg / mL of tetracycline. -1 On kanamycin plates, the resulting strains WS9EA0-WS9EA12 successfully integrated a single sgRNA expression cassette.

[0111] (7) Preparation of WS9EA0-WS9EA12 competent cells. Add pUB110-sim plasmid to each cell and spread them onto a plate containing 25 μg / mL of [unclear text - likely a specific plasmid or substrate]. -1 The strain WS9EA0S-WS9EA12 was obtained by incubating on kanamycin-resistant plates at 200 rpm and 37°C for 12 h.

[0112] The construction and fermentation methods for integrating two and three sgRNA strains are the same as above.

[0113] like Figure 3 As shown, nucleic acid gel electrophoresis results indicated that all sgRNAs were successfully integrated, and sequencing confirmed that the sequences were completely correct. Non-target sgRNAs (i.e., sgRNAs) were sequentially integrated into WS9E. CK The 12 sgRNAs obtained from high-throughput screening (N20) and empty N20 are sgRNAs. dltA sgRNA ilvA sgRNA sinR sgRNA dltB sgRNA IplD sgRNA mtlD sgRNA liaH sgRNA yqjM sgRNA yphF sgRNA trmR sgRNA pycA and sgRNA yjcS The recombinant strains WS9EA0 to WS9EA12 were then transformed into the PdSI expression plasmid pUB110-sim, and the resulting recombinant strains were named WS9EA0S to WS9EA12S. Single clones of strains WS9EA0S-WS9EA12S were randomly selected and inoculated into a 25 μg / mL solution. -1Incubate kanamycin in 10 mL LB medium at 200 rpm and 37°C for 12 h. Take 500 μL of the bacterial culture and add it to a solution containing 500 μL of 30% glycerol (v·v). -1 The seed culture was transferred into cryovials and stored at -80°C. A 5% inoculum was then transferred to a solution containing 25 μg / mL of the seed culture. -1 The recombinant bacteria were cultured in 50 mL of TB medium at 200 rpm and 37°C for 2 hours, then the temperature was reduced to 33°C, and fermentation continued for 48 hours. The enzyme activity and biomass of the recombinant bacteria were then measured. Figure 4 As shown, integrated sgRNA sinR sgRNA mtlD sgRNA yqjM sgRNA trmR and sgRNA yjcS The enzyme activities of the corresponding recombinant strains WS9EA3S, WS9EA6S, WS9EA8S, WS9EA10S and WS9EA12S were 1.56, 1.89, 1.73, 1.41 and 1.11 times that of the control strain WS9E0S (4.37U / mL), respectively.

[0114] Example 4: Effect of integrating two sgRNA expression cassettes on sucrase isomerase expression

[0115] CRISPRi can simultaneously inhibit the expression of multiple genes. To further enhance the expression level of PdSI, following the method in Example 3, the expression of the most significantly enhanced sgRNA was increased. mtlD sgRNA was integrated into the integrative strain WS9EA6. sinR sgRNA yqjM sgRNA trmR and sgRNA yjcS Following the same culture conditions as in Example 3, the integration of sgRNA was verified. mtlD -sgRNA sinR sgRNA mtlD -sgRNA yqjM sgRNA mtlD -sgRNA yjcS and sgRNA mtlD -sgRNA trmR The yield of sucrose isomerase in the strain. For example... Figure 5 As shown in (a), nucleic acid gel electrophoresis results indicated that all sgRNAs were successfully integrated, and sequencing confirmed the sequences were completely correct. The successfully integrated strains were named WS9EB1 to WS9EB4. The PdSI expression plasmid pUB110-sim was transformed into the integrated strains, and the resulting strains were named WS9EB1S to WS9EB4S. The recombinant strains were fermented according to the method in Example 3, and the enzyme activity and biomass at the end of fermentation were measured. The results are as follows.Figure 5 (b) shows that the sgRNA mtlD -sgRNA yqjM The enzyme activity of the recombinant strain WS9EB2S is 1.86 times that of the control strain WS9EA6S, and the biomass also increases; the recombinant strain WS9EB1S integrated with sgRNA mtlD -sgRNA sinR The enzyme activity of the recombinant strain WS9EB1S integrated with sgRNA

[0116] Example 5: Effect of integrating three sgRNA expression cassettes on the expression of sucrose isomerase

[0117] Based on the double-sgRNA integrated strain WS9EB2 with the most significant promotion effect constructed in Example 4, the recombinant strains integrated with sgRNA CK (empty N20), sgRNA sinR and sgRNA yjcS were constructed according to the method of Example 3. As shown in Figure 6 (a), the results of nucleic acid gel electrophoresis showed that the above sgRNAs were successfully integrated, and the sequences were completely correct after sequencing verification. The successfully integrated strains were named WS9EC1 and WS9EC2, respectively. The PdSI expression plasmid pUB110-sim was introduced into the integrated strains WS9EC1 and WS9EC2 to obtain the recombinant strains WS9EC1S and WS9EC2S. Fermentation was carried out according to the method of Example 3, and enzyme activity and biomass were determined. The results are shown in Figure 6 (b). The further integration of sgRNA sinR and sgRNA yjcS in WS9EC1S and WS9EC2S did not further improve the expression of PdSI, and the enzyme activity was only 0.86 and 0.83 times that of WS9EB2S.

[0118] Example 6: Construction of recombinant strains for enhancing the expression of key genes of sucrose isomerase and shake flask fermentation

[0119] To find the key genes that enhance the expression of recombinant proteins in B. subtilis, 12 genes were selected based on the changes in the levels of cell membrane proteins before and after the expression of the target protein by the high-yield strain midiBacillus (disclosed in the paper "Membrane modulation of super-secreting "midiBacillus" expressing the major Staphylococcus aureus antigen-a mass-spectrometry-based absolute quantification approach") and the functional annotations of the corresponding genes in the Subtiwiki database. As shown in Table 5, the 12 genes are respectively involved in the pathways of transcription, translation, biosynthetic matrix synthesis, stress response, and secretion. The overexpression of the selected genes can improve the expression level of PdSI in B. subtilis WS9C was verified by a double-plasmid system. The specific steps are as follows: the pUB110-sim plasmid was introduced into the strain WS9C, and then the bacteria were spread on a LB resistance plate containing 25 μg·mL-1kanamycin and incubated at 200 rpm and 37°C for 12 h. Then, the WS9C-pUB110-sim competent cells were prepared, the pAD123-sigA~pAD123-degQ plasmids containing the sigA, sigL, cpgA, rnZ, ftsZ, ltaS, liaH, prsA, secA, oppA, and degQ fragments were introduced into the competent cells, respectively, and then the bacteria were spread on a LB resistance plate containing 25 μg·mL-1kanamycin and 5 μg·mL-1chloramphenicol and incubated at 200 rpm and 37°C for 12 h. The strains were randomly picked and inoculated into 10 mL of LB medium containing 25 μg·mL-1kanamycin and 5 μg·mL-1chloramphenicol, and incubated at 200 rpm and 37°C for 12 h. Then, the seed liquid was transferred into 50 mL of TB medium containing 25 μg·mL-1kanamycin and 5 g / L maltose, and the expression was induced, and the bacteria were incubated at 200 rpm and 37°C for 2 h, and then the temperature was changed to 33°C, and then the fermentation was carried out for 48 h. The results are shown in Table 6. -1 Table 6 Expression levels of PdSI in the recombinant strains -1 Table 7 Expression levels of PdSI in the recombinant strains -1 Table 8 Expression levels of PdSI in the recombinant strains -1 Table 9 Expression levels of PdSI in the recombinant strains -1 Table 10 Expression levels of PdSI in the recombinant strains -1 Table 11 Expression levels of PdSI in the recombinant strains Figure 7 As shown in Table 6, the expression levels of PdSI in the recombinant strains were improved, and the overexpression of the genes secA, oppA, ftsZ, prsA, or ponA can increase the PdSI enzyme activity to 1.58, 1.38, 1.25, 1.15, and 1.10 times that of the control strain WS9C containing the empty plasmid.

[0120] Table 5 Selected positive regulation genes

[0121]

[0122]

[0123] Example 7: High-density fermentation verification of recombinant bacteria B. subtilis WS9F

[0124] The plasmid PHY300PLK-sim containing mutant V447E V447E (publicated in the paper "Enhancing the thermostability of Pantoea dispersa sucrose isomerase using semi-rational strategy"; the enzyme activity of the mutant V447E is significantly improved relative to the wild type) was transformed into the strain B. subtilis WS9EB2 constructed in Example 4, obtaining the recombinant bacteria B. subtilis WS9F. The glycerol tube of the recombinant bacteria was streaked on LB plates containing 25 μg·mL -1 of tetracycline and cultured at 37°C for 12 h. Single colonies of bacteria were picked and inoculated into 10 mL of LB medium containing 25 μg·mL -1 of tetracycline, and cultured at 200 rpm and 37°C for 12 h to preserve the bacteria. The above cultured bacteria were transferred to 50 mL of LB medium containing 25 μg·mL -1 of tetracycline at an inoculation amount of 1.00‰ (v·v -1 ) and cultured at 200 rpm and 37°C for 12 h. A 3-L tank was filled with 900 mL of base medium, and 100 mL of seed liquid was added to it by flame inoculation. Before inoculation, the fermentation temperature of the 3-L tank was measured to be 33°C; after inoculation, the dissolved oxygen and pH changes were timely observed, and the dissolved oxygen value was coupled with the rotation speed and oxygen flow to control the dissolved oxygen value (DO value) in the fermentation process at 20%, and the pH was controlled at 7.00±0.1 by phosphoric acid solution and ammonia solution. After the dissolved oxygen rebounded, the feed medium was opened, and its flow rate was adjusted to 0.01 mL·min -1 , and the subsequent sampling was taken every 4 h according to the growth of the bacterial OD 600 , and the feed flow rate was timely adjusted, each time increased by 0.02 mL·min -1 , until the feed flow rate was 0.12 mL·min -1 .

[0125] As shown in Figure 8 , when the fermentation culture was 79.00 h, the OD 600 was 168.00, at which time the enzyme activity of the fermentation supernatant of the recombinant bacteria WS9F reached the highest value of 679.36 U·mL -1 , which was 16.83 times that of the shake flask level.

[0126] Example 8: Construction and expression of recombinant bacteria with genome-integrating sucrose isomerase

[0127] To maintain plasmid stability, antibiotics need to be added during the fermentation of PdSI recombinant bacteria to provide selection pressure. However, the addition of antibiotics limits the application of sucrose isomerase in the food industry. Therefore, constructing food-grade expression strains that can express PdSI without the addition of antibiotics has broad application prospects. In this experiment, the Cre / LoxP system was used to sequentially integrate the V447E expression cassette (dual promoter P) at the lacA, nprB, and bpr sites of the recombinant bacteria B. subtilis WS9EB2 constructed in Example 4. HpaII -P amyQ’ -V447E- terminator), the resulting 1-copy, 2-copy and 3-copy integrated strains were named WS9ED1, WS9ED2 and WS9ED3 respectively; the nucleotide sequence of the terminator is shown in SEQ ID NO.26.

[0128] (1) Using the WS9C genome as a template, upper and lower homologous arms were amplified using primers lacA-up-F and lacA-down-R. Using plasmid pET-24a as a template, a linearized vector fragment was generated by reverse PCR using primer pair 24a-F / R. (2) pHY300PLK-sim V447E Using pET-24a-rodZ as a template, the V447E expression cassette was amplified with primers SI-F and SI-R. The tetracycline resistance cassette fragment was amplified using primers Tet-71-F and Tet-66-R as a template. The PCR system and procedure are shown in Tables 2 and 3. The three fragments were ligated using seamless cloning, following the instructions of the TransGen Basic Homologous Recombinant Seamless Cloning Kit (CU201-02). The ligation products were transformed into E. coli JM109 and purified using a 25 μg / mL solution. -1 Spread on kanamycin-resistant plates.

[0129] (2) Take 10 μL of the correctly sequenced V447E expression cassette integration plasmid and transform it into WS9EB2 competent cells. In a solution containing 25 μg / mL... -1 Spread the tetracycline-resistant plates and pick single clones that have been verified by PCR to contain 25 μg / mL. -1 competent cells were prepared by adding tetracycline to 10 mL of LB medium.

[0130] (3) Transform the above competent cells to express Cre recombinase plasmid pBE-ST, and plate them with a solution containing 1 mmol·L⁻¹ - 1 IPTG and 25 μg·mL -1 Incubate overnight at 37°C on kanamycin plates.

[0131] (4) According to the photocopy plate method, the obtained monoclonal bacteria were picked onto the blot and onto the antibiotic-free plate, and the blot contained 25 μg·mL⁻¹. -1 Tet r The resistant plates were selected only from those that could grow on non-resistant plates, and not on plates containing 25 μg·mL⁻¹. -1 Colonies grown on tetracycline plates were streaked onto antibiotic-free plates and incubated at 51°C and 200 rpm for 10 h. The resulting colonies were then blotted onto antibiotic-free plates and plates containing 25 μg / mL of tetracycline. -1 On kanamycin plates, the resulting strain WS9EC1 successfully integrated sim at the lacA site. V447E Expression cassettes. Subsequently, the V447E expression cassette was sequentially superimposed and integrated at the nprB and bpr sites of strain WS9ED1.

[0132] The results of PCR verification of the colony of the integrated recombinant strain are as follows: Figure 9 As shown in (a), the upstream and downstream homologous arm gene fragments of the control strain were approximately 2000 bp in size, and the gene fragment after linking to the V447E expression cassette was approximately 4728 bp in size. The size of the target fragment after homologous recombination was consistent with the theoretical value. Therefore, the V447E expression cassette was successfully integrated at different sites. The shake-flask fermentation results are as follows: Figure 9 As shown in (b), the enzyme activities of recombinant strains WS9ED1, WS9ED2, and WS9ED3 during shake-flask fermentation were 1.41, 4.50, and 4.64 U·mL, respectively. -1 As the number of V447E integration copies increases, the expression level of the recombinant strain gradually increases, and it does not significantly affect the growth of the strain.

[0133] 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 method for increasing the expression level of sucrose isomerase in Bacillus subtilis, characterized by, comprises (a) and / or (b): (a) inhibiting one or more genes of dltA, yphF, lplD, yqjM, trmR, mtlD, liaH, yjcS, pycA, sinR, ilvA and dltB; (b) overexpressing one or more of the following proteins: penicillin-binding protein class A ponA, transcription factor sigA, transcription factor sigL, ribosome assembly protein cpgA, endoribonuclease rnZ, cell division initiation protein ftsZ, polyglycerolphosphate lipoteichoic acid synthase ltaS, stress protein liaH, chaperone prsA, motor protein secA, ABC transporter oppA and pleiotropic regulator degQ.

2. The method of claim 1, wherein, The method is to integrate in Bacillus subtilis sgRNA targeting inhibition of one or more of the following proteins: phosphomucoid synthesis gene dltA, glycoside bond hydrolysis gene LplD, antioxidant stress gene yqjM, tRNA uridine modification gene trmR, phosphomannitol dehydrogenase gene mtlD, antioxidant stress gene liaH, pyruvate carboxylation gene pycA, Xre family transcription regulator sinR, threonine dehydration ilvA, alanine esterification dltB, unknown function gene yjcS, yphF.

3. The method according to claim 1 or 2, characterized in that, The sgRNA spacer sequence for targeting dltA, yphF, lplD, yqjM, trmR, mtlD, liaH, yjcS, pycA, sinR, ilvA and dltB is shown as SEQ ID NO. 8-SEQ ID NO.

19.

4. The method according to any one of claims 1 to 3, characterized in that, The sucrose isomerase has the amino acid sequence of (a) or (b): (a) the amino acid sequence shown as SEQ ID NO. 1; (b) the sequence shown as SEQ ID NO. 1 is mutated at position 447 valine to glutamic acid.

5. A recombinant Bacillus subtilis characterized in that, One or more genes of dltA, yphF, lplD, yqjM, trmR, mtlD, liaH, yjcS, pycA, sinR, ilvA and dltB are inhibited, and the motor protein secA, ABC transporter oppA, cell division initiation protein ftsZ, chaperone prsA or penicillin-binding protein class A ponA are expressed.

6. The recombinant B. subtilis of claim 5, wherein, One or more copies of sucrose isomerase or its mutant are integrated on the genome.

7. The recombinant B. subtilis of claim 5 or 6, wherein, The coding gene of the sucrose isomerase is integrated at at least one of the lacA, bpr, nprB sites on the genome.

8. The recombinant B. subtilis of any one of claims 5-7, wherein, The sucrose isomerase or the mutant thereof is under the control of a promoter P HpaII - P amyQ initiates transcription.

9. A method for the fermentative production of sucrose isomerase, characterized in that, The recombinant Bacillus subtilis of any one of claims 5-8 is fermented in a culture medium, and the sucrose isomerase is collected.

10. Use of the recombinant Bacillus subtilis of any one of claims 5-8 or the method of claim 9 in the preparation of sucrose isomerase.

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