Construction method and application of genome-integrated bacillus subtilis

Abstract

The invention discloses a construction method and application of genome-integrated bacillus subtilis, and belongs to the technical field of gene engineering. According to the invention, a maltogenic amylase amyM gene is sequentially integrated to seven different sites of a bacillus subtilis WS9C genome by using a Cre / lox gene editing system, so that the bacillus subtilis WS9C7 is constructed. The bacillus subtilis WS9C7C is constructed by taking bacillus subtilis WS9C7 as an expression host and overexpressing a quorum sensing regulatory factor ComA. Under the condition of no antibiotic addition, when the constructed genetically engineered bacterium is subjected to shake flask fermentation for 60 hours, the enzyme activity of AmyM can reach 652 U / mL-848 U / mL, and when the genetically engineered bacterium is subjected to 3-L tank fermentation, the enzyme activity of AmyM can reach 6988 U / mL-10847 U / mL, so that the genetically engineered bacterium has good industrial application potential.

Description

Technical Field

[0001] The invention relates to a construction method and application of a genome-integrated Bacillus subtilis, belonging to the technical field of genetic engineering. Background Art

[0002] Bacillus subtilis is a food safety microorganism with many industrially advantageous characteristics such as strong protein secretion ability, high-density fermentation, and clear genetic background. Therefore, it has been widely used in the food industry as a valuable host strain for expressing heterologous proteins.

[0003] In Bacillus subtilis, food-grade expression of recombinant proteins is usually achieved through nutritional deficiency selection markers and toxin-antitoxin systems, both of which require plasmids carrying the target gene. There is a risk of plasmid loss during high-density fermentation, and rapid cell proliferation and high density affect the expression level of recombinant proteins. How to achieve efficient food-grade expression of AmyM remains an urgent problem to be solved. Summary of the invention

[0004] The present invention provides a food-safe recombinant Bacillus subtilis, which overexpresses any one of the enzymes (a), (b), and (c);

[0005] (a) AmyM maltogenic amylase from Bacillus stearothermophilus;

[0006] (b) α-amylase AmyS from Bacillus stearothermophilus;

[0007] (c) Sucrose isomerase SI from Pantoea dispersa.

[0008] In one embodiment, the sequence of the gene amyM encoding the maltogenic amylase AmyM is shown as SEQ ID NO.1.

[0009] In one embodiment, the gene sequence encoding the α-amylase AmyS is shown as SEQ ID NO.2.

[0010] In one embodiment, the gene sequence encoding the sucrose isomerase SI is shown as SEQ ID NO.3.

[0011] In one embodiment, the recombinant Bacillus subtilis is prepared by integrating the maltogenic amylase gene amyM from Bacillus stearothermophilus into the nprE, nprB, bpr, mpr, aprE, spoIIAC and srfC sites of the Bacillus subtilis WS9C genome in sequence using the Cre / lox gene editing system to obtain Bacillus subtilis WS9C7 having 7 copies of the amyM gene.

[0012] In one embodiment, the integration is the integration of the maltogenic amylase expression cassette; the maltogenic amylase expression cassette comprises a promoter P HapII-amyQ’ fragment, amyM gene fragment and terminator fragment.

[0013] In one embodiment, the promoter P HapII-amyQ’ The nucleotide sequence is shown in SEQ ID NO.4.

[0014] In one embodiment, the nucleotide sequence of the maltogenic amylase gene amyM is shown as SEQ ID NO.1.

[0015] In one embodiment, the recombinant Bacillus subtilis also integrates and expresses the comK gene in the genome.

[0016] In one embodiment, the comK gene is driven by the promoter P xylA Start transcription.

[0017] In one embodiment, the Bacillus subtilis also integrates and expresses a comK gene in its genome.

[0018] In one embodiment, the comK gene is integrated and expressed at the amyE locus.

[0019] In one embodiment, the Bacillus subtilis WS9C is disclosed in the paper "Construction of a Bacillus subtilis chassis strain with improved recombinant protein expression ability based on rational mining and library screening".

[0020] The present invention also provides a method for integrating one or more genes into the Bacillus subtilis genome, comprising the following steps:

[0021] (1) constructing an integration expression frame containing "integration site upstream homology arm-gene expression frame-lox66-Tet-lox71 fragment-integration site downstream homology arm";

[0022] (2) transforming the integration expression cassette constructed in step (1) into the target strain;

[0023] (3) The Cre recombinase expression plasmid pE194-Cre was transformed into the competent cells of the recombinant strain constructed in step (2), and the transformants were screened and verified by kanamycin resistance (addition of 1 mM IPTG) to obtain a recombinant strain containing the plasmid pE194-Cre and eliminating the lox66-Tet-lox71 fragment;

[0024] (4) The recombinant strain constructed in step (3) is heat-treated to eliminate the plasmid pE194-Cre.

[0025] In one embodiment, the method further comprises repeating steps (1) to (4) to achieve integrated expression of the gene expression cassette at different sites.

[0026] In one embodiment, the method specifically comprises the following steps:

[0027] (1) Using the WS9C genome as a template to amplify the upstream and downstream fragments of the homologous repair arm of the nprE site, the maltogenic amylase gene amyM expression cassette including the promoter P HapII-amyQ’ fragment, amyM gene fragment and terminator fragment; using plasmid pHY300PLK as a template, PCR amplification of the lox66-Tet-lox71 fragment; connecting the upstream fragment of the nprE site homologous repair arm, the amyM expression cassette fragment, the lox66-Tet-lox71 fragment and the downstream fragment of the nprE site homologous repair arm by overlapping PCR to obtain a complete integration fragment; the plasmid pHY300PLK is disclosed in the paper "Study on the transformation of Bacillus subtilis strains, promoter optimization and efficient preparation of pullulanase";

[0028] (2) transforming the integration fragment constructed in step (1) into Bacillus subtilis WS9C competent cells, screening and verifying the transformants by tetracycline resistance, and obtaining a recombinant strain of Bacillus subtilis with the amyM expression cassette and the lox66-Tet-lox71 fragment integrated at the nprE site;

[0029] (3) The Cre recombinase expression plasmid pE194-Cre was transformed into the competent cells of the recombinant strain constructed in step (2), and the transformants were screened and verified by kanamycin resistance (adding 1mM IPTG) to obtain a recombinant strain containing the plasmid pE194-Cre and eliminating the lox66-Tet-lox71 fragment. The Cre recombinase expression plasmid is disclosed in the paper "Food-grade expression and application research of glutamate decarboxylase in Bacillus subtilis". The Cre recombinase gene cre is expressed by the promoter P grac Initiate transcription;

[0030] (4) heat-treating the recombinant strain carrying the Cre recombinase expression plasmid pE194-Cre constructed in step (3) at 51° C. to eliminate the plasmid pE194-Cre, and after verification, obtaining a plasmid-free Bacillus subtilis recombinant strain WS9C1 that has integrated one copy of the maltogenic amylase gene amyM;

[0031] (5) The integration sites are replaced with nprB, bpr, mpr, aprE, spoIIAC and srfC, and the maltogenic amylase gene amyM is sequentially integrated into the Bacillus subtilis WS9C1 genome described in step (4) according to steps (1-4), to obtain recombinant Bacillus subtilis strains WS9C2, WS9C3, WS9C4, WS9C5, WS9C6 and WS9C7 carrying 2, 3, 4, 5, 6, and 7 copies of the maltogenic amylase gene amyM, respectively.

[0032] In one embodiment, the Bacillus subtilis also expresses the quorum sensing regulator ComA.

[0033] In one embodiment, the nucleotide sequence of the gene encoding the quorum sensing regulatory factor ComA is shown as SEQ ID NO.5.

[0034] In one embodiment, the quorum sensing regulator ComA is expressed through the promoter P groE Start transcription.

[0035] In one embodiment, the promoter P groE The nucleotide sequence is shown in SEQ ID NO.6.

[0036] In one embodiment, the expression of the quorum sensing regulator ComA is achieved by integrating the gene comA into the epr site of the Bacillus subtilis WS9C7 genome through steps (1) to (4) to obtain Bacillus subtilis WS9C7C, thereby further improving the expression level of maltogenic amylase.

[0037] The present invention also provides the use of the Bacillus subtilis WS9C7 in expressing proteins for food production.

[0038] In one embodiment, the protein for food production includes but is not limited to amylase, saccharifying enzyme, protease, pectinase, polyphenol oxidase, lipase, lipoxygenase, glucose oxidase, peroxidase, and catalase.

[0039] The invention also provides application of the Bacillus subtilis in food industry or feed industry.

[0040] Beneficial effects: The recombinant Bacillus subtilis strains WS9C7 and WS9C7C constructed by the Cre / lox gene editing system of the present invention do not contain resistance gene screening markers, and are used for the fermentation production of maltogenic amylase, achieving the efficient expression of food-grade maltogenic amylase. The enzyme activities of the recombinant Bacillus subtilis strains WS9C7 and WS9C7CAmyM fermented for 60 hours in a shake flask can reach 652U / mL and 848U / mL, respectively. In a 3-L tank, the highest enzyme activities of AmyM of the recombinant Bacillus subtilis strains WS9C7 and WS9C7C can reach 6988U / mL and 10847U / mL, respectively. It is of great value for the application of maltogenic amylase in starch saccharification, food baking, flour modification and other fields. In addition, the overexpression of ComA also has a significant promoting effect on the expression of other food enzymes such as sucrose isomerase and α-amylase in Bacillus subtilis, and has guiding significance for improving the expression of food enzymes in Bacillus subtilis. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Sequence map of the transformation fragment after integration of amyM at the nprE site.

[0042] Figure 2 This is the sequence map of the comA transformation fragment integrated into the epr site.

[0043] Figure 3 This is the plasmid map of Cre expression plasmid pE194-Cre.

[0044] Figure 4 The AmyM enzyme activity of the recombinant strain in the shake flask, the SDS-PAGE analysis results and the growth rate of AmyM activity.

[0045] Figure 5 The fermentation process curve of the recombinant strain WS9C7 in a 3-L tank and the SDS-PAGE diagram of the fermentation supernatant; wherein, Lanes: M, Marker; 1-13, 3-L tank fermentation at 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100 and 108h, the fermentation supernatant was diluted 5 times.

[0046] Figure 6 The fermentation process curve of the recombinant strain WS9C7C in a 3-L tank and the SDS-PAGE image of the fermentation supernatant; wherein, Lanes: M, Marker; 1-13, 3-L tank fermentation at 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100 and 108h, the fermentation supernatant was diluted 5 times.

[0047] Figure 7 is the relative enzyme activity of the recombinant strains WS9CC-SI and WS9CC-AmyS. DETAILED DESCRIPTION

[0048] Culture medium formula:

[0049] (1) LB liquid medium: 5 g / L yeast powder, 10 g / L sodium chloride, and 10 g / L peptone. LB solid medium: Add 2% (w / v) agar powder to LB liquid medium.

[0050] (2) TB medium: yeast powder 24 g / L, peptone 12 g / L, glycerol 5 g / L, potassium dihydrogen phosphate 2.31 g / L, and potassium hydrogen phosphate 12.54 g / L.

[0051] (3) 3-L tank medium: industrial yeast powder 30 g / L, glycerol 5 g / L, Na2SO3 2 g / L, MgSO4·7H2O 1 g / L, ammonium citrate 1 g / L, (NH4)2SO4 2.68 g / L, NaH2PO4·H2O 4 g / L, K2HPO4 14.6 g / L, metal ion solution 3 mL / L. Feed medium: industrial yeast powder 300 g / L, D-glucose 300 g / L, MgSO4·7H2O 7.89 g / L, metal ion solution 40 mL / L.

[0052] Bacillus subtilis transformation method

[0053] Use an inoculation loop to pick up the bacterial solution of Bacillus subtilis WS9C or the recombinant strain WS9C7 constructed in Example 1 stored in the glycerol tube, streak it on the LB solid plate and culture it overnight at 37°C and 200rpm to activate the strain. Pick a single colony on the LB solid plate and inoculate it into 10mL of LB liquid culture medium and culture it overnight at 37°C and 200rpm. The next day, transfer the fermentation broth to 10mL of new LB liquid culture medium at a 5% inoculation amount, and add 250μL of 400g·L -1 xylose, cultured at 37°C, 200rpm for 4h to grow to the logarithmic phase. Finally, the cells were aliquoted on ice, with 170μL competent cells and 30μL 70% glycerol in each tube, and stored in a -80°C refrigerator for later use.

[0054] After 500 μL of competent cells were thawed on ice, the DNA fusion fragment constructed in Example 1 or 2 was added to the competent cells and mixed, and then incubated on ice for 20 min; then transferred to a 37°C water bath for 20 min; the system was placed on a shaker at 37°C, 200 rpm and cultured for 1.5 h; centrifuged at 5000 rpm, 4 min, part of the supernatant was discarded, the system was reduced to 100 μL and the cells were resuspended, applied to a screening plate containing tetracycline resistance, and cultured overnight at 37°C.

[0055] Maltogenic amylase activity assay

[0056] The 3,5-Dinitrosalicylic acid method (DNS) was used. The hydrolysis product of starch (reducing sugar) can react with 3,5-dinitrosalicylic acid to form a brown-red amino complex under heating conditions, and the depth of its color can be used to characterize the hydrolysis activity. A 1.0% (m / v) soluble starch solution was used as a substrate (dissolved in water). The buffer was a 50 mM phosphate buffer at pH 5.5. The specific steps of the reaction were as follows: 1 mL of soluble starch solution was mixed with 900 μL of buffer and then placed in a 60°C water bath for preheating for 10 min; 100 μL of appropriately diluted enzyme solution sample was added, shaken and mixed, and reacted for 10 min; 3 mL of DNS was added to terminate the reaction, and the reaction system was placed in boiling water for 7 min to develop color; it was cooled on ice, 10 mL of distilled water was added, and the absorbance was measured at 540 nm by a UV spectrophotometer after shaking and mixing. Definition of enzyme activity unit: 1U is defined as the amount of enzyme required to catalyze the production of 1 μmol of glucose per minute.

[0057] Example 1: Construction of multi-copy amyM recombinant strains using genomic integration

[0058] (1) Using plasmid pUB-amyM3 as a template (the plasmid was disclosed in the paper “High level food-grade expression of maltose amylase in Bacillus subtilis through dal gene auxotrophic selection marker”), primers P1 and P2 described in Table 2 were used to amplify the amyM expression cassette fragment Frag1.

[0059] (2) Using plasmid pHY300PLK as a template (the plasmid is disclosed in the paper "Study on the transformation of Bacillus subtilis strains, promoter optimization and efficient preparation of pullulanase"), primers P3 and P4 described in Table 2 were used to amplify the lox71-Tet-lox66 fragment Frag2. The amyM expression cassette fragment Frag1 constructed in step (1) and the lox71-Tet-lox66 fragment Frag2 were connected by overlapping PCR to obtain Frag3.

[0060] (3) Using the Bacillus subtilis WS9C genome as a template, primers P5 and P6 described in Table 2 were used to amplify the upstream fragment Frag4 of the nprE homologous repair arm. Using the Bacillus subtilis WS9 genome as a template, primers P7 and P8 described in Table 2 were used to amplify the downstream fragment Frag5 of the nprE homologous repair arm. The upstream fragments Frag4, Frag3 and the downstream fragment Frag5 were connected by overlapping PCR to obtain Frag6.

[0061] The above reaction system is shown in Table 1.

[0062] Table 1 Reaction system

[0063] 2×Phanta Max Master Mix 25μL Template DNA 1μL Upstream primer (20 μM) 1μL Downstream primer (20 μM) 1μL <![CDATA[ddH2O]]> Make up the system to 50 μL

[0064] The reaction procedure was as follows: pre-denaturation at 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for the corresponding time; extension at 72°C for 5 min, and cooling to 12°C.

[0065] Table 2 Primer sequences

[0066]

[0067] (4) The fragment Frag6 constructed in step (3) was transformed into Bacillus subtilis WS9C competent cells by chemical transformation, spread on LB solid medium (containing 100 μg / mL tetracycline), and cultured at 37°C overnight. The clones were picked for colony PCR verification and sequencing verification to obtain a recombinant strain integrating the amyM expression cassette and the lox71-Tet-lox66 fragment.

[0068] (5) Prepare competent cells of the strain verified to be correct in step (4), transfer the plasmid pE194-Cre (the plasmid is disclosed in the paper "Food-grade expression and application research of glutamate decarboxylase in Bacillus subtilis") into it, spread it on LB solid medium (containing 30 μg / mL kanamycin and 1 mM IPTG), and culture it at 37°C overnight. Pick clones for verification, and obtain a strain with the lox71-Tet-lox66 fragment removed from the genome.

[0069] (6) The strain verified to be correct in step (5) was heat treated at 51°C for 12 h to eliminate the plasmid pE194-Cre. The correct transformant, i.e., the recombinant Bacillus subtilis strain WS9C1 integrating one copy of the amyM expression cassette, was placed in a glycerol tube and stored at -80°C.

[0070] (7) According to steps (1) to (6), the amyM expression cassette is cumulatively integrated into the nprB, bpr, mpr, aprE, spoIIAC and srfC sites of the Bacillus subtilis WS9C genome in sequence. The primers involved are shown in Table 3, and the Bacillus subtilis recombinant strains WS9C2 (two copies of the amyM expression cassette are integrated at the nprE site and the nprB site, respectively) and WS9C3 (three copies of the amyM expression cassette are integrated at the nprE site, the nprB site and the bpr site, respectively) with 2, 3, 4, 5, 6 and 7 copies of the amyM expression cassette integrated are obtained, respectively. WS9C4 (4 copies of amyM expression cassettes were integrated at the nprE site, nprB site, bpr site and mpr site, respectively), WS9C5 (5 copies of amyM expression cassettes were integrated at the nprE site, nprB site, bpr site, mpr site and aprE site, respectively), WS9C6 (6 copies of amyM expression cassettes were integrated at the nprE site, nprB site, bpr site, mpr site, aprE site and spoIIAC, respectively) and WS9C7 with a total of 7 copies of amyM expression cassettes integrated at the above sites were placed in glycerol tubes and stored at -80°C.

[0071] Table 3 Primer sequences

[0072]

[0073] Example 2: Construction of comA overexpression recombinant strain using genomic integration

[0074] (1) Using the genome of Bacillus subtilis WS9C as a template (disclosed in the paper "Construction of a Bacillus subtilis chassis strain with improved recombinant protein expression ability based on rational mining and library screening"), the comA gene fragment was amplified using primers P33 and P34 described in Table 4 and named Frag7. The comA gene encodes the quorum sensing regulator ComA, which regulates a variety of genes with different functions by binding to the gene promoter region, involving fatty acid metabolism, protein degradation and ability development.

[0075] (2) Using the genome of Bacillus subtilis WS9C as a template, the promoter P was amplified using primers P35 and P36 described in Table 4. groE The fragment was named Frag8. Frag7 and Frag8 were connected by overlapping PCR to obtain P groE -comA, named Frag9.

[0076] (3) Using the fragment Frag3 constructed in Example 1 as a template, primer P37 described in Table 4 and primer P4 described in Table 1 of Example 1 were used to amplify the T-lox71-Tet-lox66 fragment (named Frag10). Fragments Frag9 and Frag10 were connected by overlapping PCR to obtain P groE -T-lox71-Tet-lox66, named Frag11.

[0077] (3) Using the Bacillus subtilis WS9C genome as a template (disclosed in the paper "Construction of a Bacillus subtilis chassis strain with improved recombinant protein expression ability based on rational mining and library screening"), primers P38 and P39 described in Table 4 were used to amplify the upstream fragment Frag12 of the epr homology repair arm. Using the Bacillus subtilis WS9 genome as a template, primers P40 and P41 described in Table 4 were used to perform PCR amplification to obtain the downstream fragment Frag13 of the epr homology repair arm. The upstream fragments Frag12 and Frag11 and the downstream fragment Frag13 were connected by overlapping PCR to obtain Frag14.

[0078] (4) According to steps (4) to (6) in Example 1, Frag14 was transformed into Bacillus subtilis WS9C7 competent cells to obtain the recombinant Bacillus subtilis strain WS9C7C integrated with the comA expression cassette, which was placed in a glycerol tube and stored at -80°C.

[0079] Table 4 Primer sequences

[0080]

[0081] Example 3: Recombinant strain expressing maltogenic amylase is used for fermentation production of maltogenic amylase

[0082] The recombinant Bacillus subtilis strains WS9C1, WS9C2, WS9C3, WS9C4, WS9C5, WS9C6, WS9C7 and WS9C7C constructed in Example 1 and Example 2 were respectively subjected to shake flask fermentation culture, and the bacterial liquid was aspirated from the glycerol tube at an inoculum size of 2‰ and inoculated into 10 mL LB medium, and cultured at 37°C, 200 rpm, for 8-10 h to obtain seed liquid; the seed liquid was inoculated into 50 mL TB fermentation liquid medium at an inoculum size of 5%, and cultured at 37°C, 200 rpm, for 2 h, and then transferred to 33°C, 200 rpm and cultured for 60 h.

[0083] The AmyM enzyme activity in the fermentation supernatant of the recombinant strain was determined, SDS-PAGE analysis was performed, and the enzyme activity growth rate of the strain was calculated. For example, the enzyme activity growth rate of strain WS9C2 compared with strain WS9C1 was (309-160) U / mL÷160 U / mL=0.9, and the enzyme activity growth rate of strain WS9C3 compared with strain WS9C2 was (400-309) U / mL÷309 U / mL=0.3. The results showed that after 60 h of fermentation, the AmyM enzyme activities in the fermentation supernatants of the recombinant Bacillus subtilis strains WS9C1, WS9C2, WS9C3, WS9C4, WS9C5, WS9C6 and WS9C7 reached 160 U / mL, 309 U / mL, 400 U / mL, 453 U / mL, 558 U / mL, 567 U / mL and 652 U / mL, respectively, which were 1, 1.9, 2.5, 2.8, 3.5, 3.5 and 4.1 times the AmyM activity of the recombinant Bacillus subtilis strain WS9C1 ( Figure 4 a).

[0084] SDS-PAGE analysis of the crude enzyme solution of the recombinant strain revealed that the theoretical molecular weight of the AmyM band was 69 kDa, and the band width was proportional to the AmyM activity ( Figure 4 c). As the copy number increases, the growth rate of AmyM activity generally decreases, which may be due to the increasing imbalance between exogenous protein synthesis and cellular metabolism ( Figure 4 b) In addition, to avoid inserting too many identical fragments into the WS9C genome, which would lead to strain instability, we did not construct strains with a higher number of amyM copies and used WS9C7 in subsequent studies.

[0085] The AmyM enzyme activity of the strain WS9C7C constructed according to the method of Example 2 reached 847 U / mL, which was 1.3 times that of the strain WS9C7, indicating that overexpression of ComA can further promote the improvement of AmyM enzyme activity.

[0086] Example 4: Fermentation of recombinant strains in 3-L tanks to produce AmyM

[0087] The recombinant Bacillus subtilis strains WS9C7 and WS9C7C constructed in Examples 1 and 2 were respectively subjected to 3-L tank fermentation culture. The bacterial liquid was streaked from the glycerol tube onto an antibiotic-free plate and incubated at 37°C for 12 hours for activation; a single colony was picked into 10 mL LB medium and incubated at 37°C, 200 rpm, for 8-10 hours; the seed liquid was inoculated into 100 mL LB fermentation liquid medium at an inoculation rate of 2‰, and incubated at 37°C, 200 rpm, for 10 hours; 100 mL of fermentation liquid was inoculated into 900 mL 3-L tank medium for fermentation, and the fermentation parameters were specifically 37°C, pH 7.0, and a rotation speed (300-700 rpm) coupled with dissolved oxygen (30%). Samples were taken regularly during the fermentation process to measure OD 600 The AmyM enzyme activity was measured, and the AmyM in the sample supernatant was analyzed by SDS-PAGE. The results showed that when the cell growth rate remained unchanged, the feed solution was added at an initial rate of 0.02 mL / min and gradually increased to a final rate of 0.1 mL / min. After adding the feed, the dissolved oxygen and stirring speed were maintained at the set values, and the strain grew normally and stably. Figure 5 As shown in a, after 91 h of culture in a 3-L fermenter, OD 600 The maximum value reached 160. AmyM activity continued to increase, reaching a maximum of 6,988U / mL after 85h. The supernatant of the fermentation sample was analyzed by SDS-PAGE, and it was found that the production of AmyM continued to increase during the fermentation process ( Figure 5 b).

[0088] Example 5: General analysis of the effect of ComA overexpression on promoting heterologous protein production

[0089] Referring to steps (4) to (6) in Example 1, Frag14 constructed in Example 2 was transformed into Bacillus subtilis WS9C competent cells to obtain the Bacillus subtilis recombinant strain WS9CC integrated with the comA expression cassette.

[0090] The plasmids pUB110-SI (disclosed in the paper "Expression of Pantoea dispersa sucrose isomerase in Bacillus subtilis and preparation of isomaltulose") and pUB110-AmyS (disclosed in the paper "Construction of a Bacillus subtilis chassis strain with improved recombinant protein expression ability based on rational mining and library screening") were transformed into Bacillus subtilis WS9CC, respectively, to obtain recombinant Bacillus subtilis strains WS9CC-SI and WS9CC-AmyS expressing sucrose isomerase SI and α-amylase AmyS, respectively; the plasmids pUB110-SI and pUB110-AmyS were transformed into WS9C competent cells, respectively, to obtain control strains WS9C-SI and WS9C-AmyS, respectively.

[0091] The recombinant strains of Bacillus subtilis WS9CC-SI and WS9CC-AmyS and the control strains WS9C-SI and WS9C-AmyS were fermented in shake flasks according to the method of Example 3. The culture medium and fermentation conditions were the same, except that the fermentation time was adjusted to 48 hours. The method for determining the enzymatic activity of sucrose isomerase SI specifically refers to the paper "Expression of Pantoea dispersa sucrose isomerase in Bacillus subtilis and preparation of isomaltulose". The method for determining the enzymatic activity of α-amylase specifically refers to the paper "Construction of Bacillus subtilis chassis strains with improved recombinant protein expression ability based on rational mining and library screening". Figure 6 As shown, the recombinant protein activities of WS9CC-SI and WS9CC-AmyS were increased by 2.0 and 1.2 times, respectively, compared with the control strains. This indicates that overexpression of ComA enhanced the expression of sucrose isomerase and α-amylase, indicating that ComA has good versatility for the industrial production of recombinant proteins in Bacillus subtilis.

[0092] In the starch industry, maltogenic amylase can be used alone or together with other amylopectin debranching enzymes to produce high maltose syrup; when maltogenic amylase is used in the food baking industry, it can be added to the dough in an appropriate amount, which can significantly improve the anti-aging ability of bread; in addition, adding maltogenic amylase to flour can change the organizational structure of flour products such as steamed bread, increase the volume and reduce the hardness, and has a very good effect in flour modification and processing. Bacillus subtilis has a food-grade status and a mature genetic operating system. The present invention develops the Bacillus subtilis expression system originally containing resistance genes into a food-grade expression system of maltogenic amylase, while eliminating antibiotic resistance, increasing the expression of enzyme activity, so that the constructed food-grade expression system can be taken by the human body, or used as an expression and production platform for food enzymes.

[0093] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone 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 based on the definition of the claims.

Claims

1. A food-safe recombinant Bacillus subtilis, characterized in that: Any one of the enzymes described in (a), (b), or (c) is integrated and expressed; (a) AmyM maltogenic amylase from Bacillus stearothermophilus; (b) α-amylase AmyS from Bacillus stearothermophilus; (c) Sucrose isomerase SI from Pantoea dispersa.

2. The recombinant Bacillus subtilis according to claim 1, characterized in that The gene sequence of amyM encoding the maltogenic amylase AmyM is shown in SEQ ID NO.1; the gene sequence encoding the α-amylase AmyS is shown in SEQ ID NO.2; and the gene sequence encoding the sucrose isomerase SI is shown in SEQ ID NO.

3.

3. The recombinant Bacillus subtilis according to claim 1 or 2, characterized in that The maltogenic amylase gene amyM is integrated into the following one or more sites of the Bacillus subtilis genome using the Cre / lox gene editing system: nprE, nprB, bpr, mpr, aprE, spoIIAC and srfC.

4. The recombinant Bacillus subtilis according to claim 3, characterized in that The maltogenic amylase gene is expressed by the promoter P shown in SEQ ID NO.4 HapII-amyQ’ Regulate expression.

5. The recombinant Bacillus subtilis according to any one of claims 1 to 4, characterized in that The comK gene was also integrated and expressed in the genome.

6. The recombinant Bacillus subtilis according to claim 5, characterized in that The comK gene is driven by the promoter P xylA Start transcription.

7. The recombinant Bacillus subtilis according to any one of claims 1 to 6, characterized in that Bacillus subtilis WS9C was used as the host.

8. A method for integrating one or more genes into the genome of Bacillus subtilis, characterized in that: The steps include: (1) constructing an integration expression frame containing "integration site upstream homology arm-gene expression frame-lox66-Tet-lox71 fragment-integration site downstream homology arm"; (2) transforming the integration expression cassette constructed in step (1) into the target strain; (3) The Cre recombinase expression plasmid pE194-Cre was transformed into the competent cells of the recombinant strain constructed in step (2), and the transformants were screened and verified by kanamycin resistance (addition of 1 mM IPTG) to obtain a recombinant strain containing the plasmid pE194-Cre and eliminating the lox66-Tet-lox71 fragment; (4) The recombinant strain constructed in step (3) is heat-treated to eliminate the plasmid pE194-Cre.

9. Use of the method according to claim 8 in constructing genetically engineered bacteria.

10. Use of the recombinant Bacillus subtilis according to any one of claims 1 to 7 in expressing proteins for food production.

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