Bacillus subtilis engineering bacterium for efficiently expressing endo-1, 3-1, 4-beta-D-glucanase based on homologous recombination of lactose operon promoter

Abstract

The invention discloses a bacillus subtilis engineering bacterium for efficiently expressing endo-1, 3-1, 4-beta-D-glucanase based on homologous recombination of a lactose operon promoter, and belongs to the technical field of genetic engineering. The invention discloses a bacillus subtilis engineering bacterium for efficiently expressing endo-1, 3-1, 4-beta-D-glucanase based on homologous recombination of a lactose operon promoter and a construction method of the bacillus subtilis engineering bacterium. A bacillus subtilis 168 strain is used as a starting strain, a promoter region of an endonucle-1, 3-1, 4-beta-D-glucanase gene is knocked out, an escherichia coli lactose operon promoter sequence is integrally expressed, and the homologous recombinant bacillus subtilis for efficiently expressing the endonucle-1, 3-1, 4-beta-D-glucanase gene protein is obtained by virtue of lactose and IPTG (isopropyl-beta-d-thiogalactoside) induction. The recombinant bacterium can be applied to the fields of feed processing and biological energy.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a Bacillus subtilis engineered strain that efficiently expresses endonuclease-1,3-1,4-β-D-glucanase based on homologous recombination with the lactose operon promoter. Background Technology

[0002] β-glucan is a class of non-starch linear polysaccharides widely distributed in plant cell walls, bacterial capsules, and fungal cell walls. In gramineous crops, β-glucan forms a main chain through β-1,4 bonds and is interspersed with β-1,3 bonds, forming a semi-rigid three-dimensional network structure. This structure imparts mechanical strength to the cell wall, but also causes it to bind with water molecules in the animal digestive tract, forming a highly viscous gel that significantly inhibits the digestion and absorption of nutrients.

[0003] Endo-1,3-1,4-β-D-glucanase is a key industrial enzyme that can specifically cleave the 1,3- and 1,4-glycosidic bonds in β-glucan. It is widely used in feed processing and bioenergy. Its high catalytic efficiency stems from the acid-base catalysis involving conserved amino acids (such as glutamic acid and aspartic acid) in the active center.

[0004] Endoglucanases degrade long-chain β-glucans into oligosaccharide fragments and a small amount of monosaccharides by randomly breaking β-1,4 glycosidic bonds. These products play multiple functions in biological processes: they participate in the dynamic remodeling of cell walls in plants; they are further utilized as carbon sources in microbial metabolism; and in animal nutrition, they improve the digestibility of starch and protein by reducing chyme viscosity (by 60% to 80%).

[0005] Endoglucanases (I-1,3-1,4-β-D-glucanases) are widely found in nature, primarily originating from three main groups of organisms: microorganisms, plants, and fungi. Microbial enzymes are widely used in the feed industry due to their high yield and heat resistance; plant-derived enzymes are mainly used for the degradation of β-glucan in brewing processes; while enzymes secreted by white-rot fungi work synergistically with lignin peroxidase to increase β-glucan conversion rates to up to 85% in biomass pretreatment.

[0006] Pigs and other poultry lack the endogenous ability to synthesize endo-1,3-1,4-β-D-glucanase in their digestive systems. When fed grain feeds primarily composed of barley (3%–8% β-glucan content) or oats (4%–10% β-glucan content), the linear molecular chains of β-1,3-1,4-glucan form a highly branched network structure through alternating β-1,4 and β-1,3 bonds. This polysaccharide forms a three-dimensional gel network in the animal's gastrointestinal tract through hydrogen bonding between its C3 hydroxyl groups and water molecules, resulting in a significant increase in chyme viscosity. This creates a physical barrier that hinders the effective contact between digestive enzymes (such as trypsin and lipase) and substrates. High-viscosity chyme reduces the contact area between protein and digestive enzymes by 35%–40%, leading to a 15%–20% decrease in crude protein digestibility in the terminal ileum, a 12%–18% reduction in fat absorption, and significantly inhibiting the absorption efficiency of fat-soluble vitamins (such as vitamins A and D).

[0007] Studies have shown that adding 500–1000 U / kg of endo-1,3-1,4-β-D-glucanase (such as LicH enzyme derived from Bacillus subtilis, with a specific activity of 320 U / mg) to animal feed can specifically recognize and cleave β-1,4 glycosidic bonds, degrading long-chain β-glucan into oligosaccharide fragments with a molecular weight <5 kDa. This enzymatic hydrolysis can reduce intestinal chyme viscosity by 60%–80%, thereby improving the utilization rate of small intestinal villi surface area and the efficiency of nutrient absorption. Ultimately, feed conversion ratio increases by 7%–10%, and daily weight gain increases by 8%–12%. In addition, enzymatic hydrolysis releases starch and protein encapsulated in cell walls, increasing the metabolizable energy value of feed by 0.5–0.8 MJ / kg and crude fiber digestibility by 9%–11%, thereby reducing feed costs per unit weight gain by 3%–5% and significantly improving the economic benefits of large-scale farming.

[0008] Bacillus subtilis ( Bacillus subtilis It is a Gram-positive rod-shaped bacterium (0.7~0.8μM ×2~3μM), without a capsule, relying on peritrichous flagella for movement, and its colonies are dirty white or slightly yellow, with a rough and opaque surface. It can form highly resistant endospores in nutrient-deficient or extreme environments.

[0009] As a GRAS (Generally Recognized as Safe) beneficial microorganism certified by the US FDA in 1999, it has been applied on a large scale with an annual production capacity of over 100,000 tons in feed additives, aquaculture, and the food industry. Bacillus subtilis naturally does not carry pathogenic toxin genes or endotoxins; its cell wall structure is simplified, consisting only of multiple layers of peptidoglycan and teichoic acid, completely avoiding the risk of lipopolysaccharide contamination from the outer membrane of Gram-negative bacteria.

[0010] The 4.2 Mb circular genome of Bacillus subtilis 168 was fully sequenced in 1997, and sophisticated gene-editing tools were established. Furthermore, its codon bias is extremely low; by optimizing rare codons, the expression level of exogenous genes can be increased by 1.5 times compared to the E. coli system. In addition, it possesses two highly efficient secretion systems, Sec and Tat. The Sec pathway recognizes signal peptides such as PhoD and AprE, directly secreting folded, mature, active proteins extracellularly, while the Tat pathway transports complex proteins containing disulfide bonds, significantly reducing inclusion body formation and subsequent refolding costs. These characteristics make it an excellent chassis cell for expressing exogenous proteins.

[0011] Homologous recombination is a method for precisely editing the genome using the cell's natural DNA repair mechanisms. Its basic principle is to introduce a double-strand break in DNA within the cell, typically through physical means such as electroshock or ultrasound, chemical means such as drug induction, or the use of specific nucleases such as CRISPR / Cas9. This break provides an entry point for subsequent recombination. Subsequently, a foreign DNA fragment is introduced as a repair template, containing homologous sequences at both ends that are identical or similar to the target genomic region. Under the cell's homologous recombination mechanism, the homologous sequences of the foreign DNA fragment pair with the homologous sequences of the broken DNA, enabling DNA strand invasion and exchange. This process allows the foreign DNA fragment to precisely replace the gene at the original site, thereby achieving the insertion, knockout, or repair of a specific gene.

[0012] In recent years, advancements in homologous recombination technology have made vector construction more efficient. Seamless cloning technology achieves efficient vector construction by adding short homologous sequences to both ends of the target gene sequence, further improving the flexibility and accuracy of the recombination process. It avoids the dependence on restriction endonucleases and DNA ligases in traditional cloning techniques, significantly simplifies experimental procedures, and reduces potential operational errors.

[0013] Example 1 of homologous recombination of Bacillus subtilis: Tian et al. constructed a recombinant Bacillus subtilis 168-CLE using homologous recombination technology. The PEDV-AJ1102 COE region of the spike protein was displayed on the spore surface, which could generate specific systemic and mucosal immune responses. This study laid the foundation for the development of oral vaccines against porcine epidemic diarrhea virus.

[0014] Example 2 of homologous recombination in Bacillus subtilis: Nie Libo et al. integrated the cellulase gene CelKg, promoter gene P43, and homologous fragments M1 and M2 into wild-type B. subtilis LN using homologous recombination technology to construct the recombinant strain B. subtilis Kpg. The recombinant strain significantly reduced the cellulose content (P<0.05) in bran fermentation, while increasing the true protein content in the fermented feed.

[0015] Example 3 of Homologous Recombination in Bacillus subtilis: The inventors of this project previously used the pHY300PLK plasmid for homologous recombination technology to replace the coding region of the cellulose-degrading enzyme gene in Bacillus subtilis, thereby achieving the expression of the Met-rich zein (Zein) gene. The recombinant vector pHY300PLK-Z was constructed through seamless cloning and electroporated into Bacillus subtilis, successfully replacing the target gene region through homologous recombination. After multiple isolation and purification processes, recombinant bacteria without the resistance vector were obtained. Under the induction of cellulose and methionine, highly efficient Zein gene expression and a significant increase in Met content were achieved, providing new potential for enhancing the application of Bacillus subtilis as a feed additive in animal production.

[0016] Existing examples of homologous recombination in Bacillus subtilis all suffer from low expression efficiency. Achieving efficient expression of the endo-1,3-1,4-β-D-glucanase gene inherent in Bacillus subtilis remains an unsolved technical challenge.

[0017] The lactose operon is a classic model of gene expression regulation in prokaryotes. The operon consists of four core genes (lacZ, lacY, lacA, and lacI) and regulatory elements (promoter and operator).

[0018] The functional activation of the lactose operon depends on the presence of an inducer. When lactose or its analogues (such as isolaxose IPTG) are present in the environment, these molecules bind to the inducer-binding domain of the LacI repressor protein and induce a conformational change, causing the repressor protein tetramer to dissociate and lose its ability to bind to the operated gene. At this point, RNA polymerase recognizes the conserved sequences of the promoter, namely -10 (TATGTT) and -35 (TTGACA), via the σ factor and forms an open promoter complex to activate gene transcription.

[0019] The biological significance of the lactose operon lies not only in its metabolic function but also in its universality as a molecular regulatory paradigm. Its "inducibility" mechanism reveals the molecular logic of prokaryotes adapting to environmental changes: achieving rapid gene switching responses through allosteric regulation of repressor proteins. At the application level, the lactose operon has become a core tool in genetic engineering, and its modular design of promoters and operators is widely used for the controlled expression of exogenous proteins. However, it remains unclear whether the promoter and operator modules of the lactose operon can function effectively in Bacillus subtilis.

[0020] Lactose operon promoters are widely used in microbial expression systems due to their induction flexibility and high expression efficiency. Bacillus subtilis, with its low culture cost, rapid growth, and strong protein secretion capacity, is an ideal host for exogenous protein expression. Endoglucanases (1,3-1,4-β-D-glucanases) have broad application prospects in food processing, feed additive development, and biomass conversion. Summary of the Invention

[0021] The purpose of this invention is to provide a highly efficient Bacillus subtilis strain expressing endonuclease-1,3-1,4-β-D-glucanase based on homologous recombination with the lactose operon promoter, thereby addressing the problems existing in the prior art. This invention utilizes homologous recombination genetic engineering to significantly enhance the expression of the target enzyme gene by replacing the natural promoter of Bacillus subtilis endonuclease with a lactose operon promoter.

[0022] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a recombinant expression cassette for expressing endonuclease-1,3-1,4-β-D-glucanase, comprising upstream and downstream homologous arms of the promoter region of the endonuclease-1,3-1,4-β-D-glucanase encoding gene and the Lac promoter sequence.

[0023] The second technical solution of the present invention is a recombinant expression vector for expressing endonuclease-1,3-1,4-β-D-glucanase, wherein the recombinant expression vector uses pHY300PLK as a backbone and includes the recombinant expression cassette.

[0024] The third technical solution of the present invention is a recombinant Bacillus subtilis expressing endoglucanase, using Bacillus subtilis 168 as the starting strain, and comprising the recombinant expression cassette or the recombinant expression vector.

[0025] The fourth technical solution of the present invention is the application of the recombinant expression cassette, the recombinant expression vector, or the recombinant Bacillus subtilis in the production of endo-1,3-1,4-β-D-glucanase.

[0026] The fifth technical solution of the present invention is a method for preparing endo-1,3-1,4-β-D-glucanase, which utilizes the recombinant Bacillus subtilis to produce endo-1,3-1,4-β-D-glucanase under the induction of isopropyl-β-D-thiogalactoside or lactose.

[0027] Based on the above technical solution, the present invention has the following technical effects: This invention utilizes homologous recombination to replace the promoter region of the Bacillus subtilis endonuclease gene with the Lac promoter sequence of Escherichia coli. This homologous recombination Lac sequence can be induced to express the endonuclease gene using lactose and IPTG substrates, resulting in highly efficient expression of the endonuclease-1,3-1,4-β-D-glucanase homologous recombinant Bacillus subtilis.

[0028] This homologous recombinant Bacillus subtilis produces a large amount of endo-1,3-1,4-β-D-glucanase, which has great application prospects in feed processing and bioenergy.

[0029] This invention establishes a technical method for the efficient expression of Bacillus subtilis endogenous proteins based on lactose and IPTG substrate induction with Lac promoter as the homologous recombination exogenous gene, which has important theoretical and practical guiding significance for subsequent Bacillus subtilis genetic engineering technology. Attached Figure Description

[0030] Figure 1 For the identification of Bacillus subtilis strains. Wherein, M: DL5000 DNA Marker; 1: blank control, H2O; 2: negative control, Escherichia coli; 3: positive control, Bacillus subtilis; 4-11: colonies isolated from petri dishes.

[0031] Figure 2 This study aimed to amplify the upstream homologous arm of the endo-1,3-1,4-β-D-glucanase promoter region of Bacillus subtilis 168 by PCR. In the diagram, M represents the DL2000 DNA Marker; 1 represents the negative control (E. coli); 2 represents the blank control (H2O); and 3-5 represent the upstream homologous arms.

[0032] Figure 3 This study aimed to amplify the downstream homologous arms of the endo-1,3-1,4-β-D-glucanase promoter region of Bacillus subtilis 168 by PCR. In the diagram, M represents the DL2000 DNA Marker; 1 represents the negative control (E. coli); 2 represents the blank control (H2O); and 3-5 represent the downstream homologous arms.

[0033] Figure 4 The Lac promoter sequence of *E. coli* was amplified by PCR. M: DL2000 DNA Marker; 1: Negative control, *Bacillus subtilis*; 2: Blank control, H2O; 3-5: Lac promoter (LacI+LacZ promoter, 1245bp).

[0034] Figure 5Homologous recombination expression cassettes were constructed for overlap PCR. M: DL5000 DNA Marker; 1: Blank control, H2O; 2-3: Upstream homologous arm - Lac - Downstream homologous arm, 2362 bp.

[0035] Figure 6 The recombinant vector pHY300PLK-Lac was validated by electrophoresis. M: DL5000 DNA Marker; 1: pHY300PLK; 2-5: pHY300PLK-Lac.

[0036] Figure 7 Sequencing alignment of the recombinant vector pHY300PLK-Lac. Wherein, 1: gel-recovered fragment from fusion PCR; 2: target replacement fragment sequence; 3-4: PCR products of the recombinant vector pHY300PLK-Lac.

[0037] Figure 8 The results of plate culture of Bacillus subtilis positive recombinants by electroporation are shown. Among them, (a) pHY300PLK; (b) pHY300PLK-Lac.

[0038] Figure 9 The results show the PCR identification of Bacillus subtilis transformed by recombinant plasmids. M: DL5000 DNA Marker; 1: Blank control, water; 2: Negative control, Escherichia coli; 3: Positive control, Bacillus subtilis; 4-11: Electroporated colonies isolated from the culture plate.

[0039] Figure 10 The sequence identification results are for Bacillus subtilis transformed by recombinant plasmids. Among them, 1: gel recovery fragment of fusion PCR; 2: PCR product of recombinant vector pHY300PLK-Lac; 3: gel recovery fragment of electroporated positive recombinant bacteria.

[0040] Figure 11 This study aims to identify the upstream and downstream homologous arm exchanges of single colonies after five generations of electroporation. In the figures, M: DL5000 DNA Marker; 1: Blank control: H2O; 1-7: Upstream homologous arm exchange identification; 2-7: Bacterial samples; 8: Blank control: H2O; 8-14: Downstream homologous arm exchange identification; 9-14: Bacterial samples.

[0041] Figure 12 The initial screening results are obtained from PCR amplification of single colonies of homologous recombinant bacteria with double crossover. Wherein, M: DL5000 DNA Marker; 1: Blank control: H2O; 2: Negative control: Bacillus subtilis; 3-8: Bacterial samples.

[0042] Figure 13The results show the purification of single colonies from homologous recombinant bacteria after double exchange. Wherein, M: DL5000 DNA Marker; 1: Blank control: H2O; 2: Negative control: Bacillus subtilis; 3-12: Bacterial samples.

[0043] Figure 14 This is the result of further screening for homologous recombinant Bacillus subtilis clones without resistance vectors. 1: LB solid medium; 2: TET solid medium.

[0044] Figure 15 The growth curve of homologous recombinant bacteria was determined.

[0045] Figure 16 This is the result of Lac gene stability assay after passage of homologous recombinant bacteria. 1-3: Recombinant bacterial PCR products; 4: Target replacement fragment sequence.

[0046] Figure 17 This study analyzed the expression of lactose-induced homologous recombinant bacterial endo-1,3-1,4-β-D-glucanase mRNA. Note: Different letters indicate significant differences between groups (p<0.05), n=4; the same letter indicates no significant difference.

[0047] Figure 18 Analysis of IPTG-induced homologous recombinant bacterial endonuclease-1,3-1,4-β-D-glucanase mRNA expression. Note: Different letters indicate significant differences between groups (p<0.05), n=4; the same letter indicates no significant difference.

[0048] Figure 19 This study analyzed the activity of endo-1,3-1,4-β-D-glucanase in recombinant bacteria induced by lactose and IPTG. In the figures, A represents the activity of endo-1,3-1,4-β-D-glucanase in recombinant bacteria induced by lactose; B represents the activity of endo-1,3-1,4-β-D-glucanase in recombinant bacteria induced by IPTG. BS: wild-type Bacillus subtilis; rBS: recombinant Bacillus subtilis; rBS(2%Lac): recombinant Bacillus subtilis induced by 2% lactose for 8 h; rBS(0.4μM IPTG): recombinant Bacillus subtilis induced by 0.4μM IPTG for 3 h. Different letters indicate significant differences between groups (p<0.05), n=4; the same letter indicates no significant difference. Detailed Implementation

[0049] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0050] This invention provides a recombinant expression cassette for expressing endonuclease-1,3-1,4-β-D-glucanase, comprising upstream and downstream homologous arms of the promoter region of the endonuclease-1,3-1,4-β-D-glucanase encoding gene and the Lac promoter sequence.

[0051] In some specific implementations, the nucleotide sequence is shown in SEQ ID NO.4.

[0052] This invention also provides a recombinant expression vector for expressing endonuclease-1,3-1,4-β-D-glucanase, wherein the recombinant expression vector uses pHY300PLK as a backbone and includes the recombinant expression cassette.

[0053] This invention also provides a recombinant Bacillus subtilis expressing endoglucanase, using Bacillus subtilis 168 as the starting strain, and comprising the recombinant expression cassette or the recombinant expression vector.

[0054] This invention also provides the application of the recombinant expression cassette, the recombinant expression vector, or the recombinant Bacillus subtilis in the production of endo-1,3-1,4-β-D-glucanase.

[0055] This invention also provides a method for preparing endo-1,3-1,4-β-D-glucanase, which utilizes the recombinant Bacillus subtilis to generate endo-1,3-1,4-β-D-glucanase under the induction of isopropyl-β-D-thiogalactoside or lactose.

[0056] In some specific embodiments, the concentration of the isopropyl-β-D-thiogalactoside in the culture medium is 0.1~1 μM.

[0057] In some specific implementations, the lactose concentration in the culture medium is 0.5% to 2.5% by mass.

[0058] This invention uses Bacillus subtilis strain 168 as the starting strain. Homologous recombination was used to knock out the promoter region of the endonuclease-1,3-1,4-β-D-glucanase gene and integrate the expression of the Escherichia coli lactose operon promoter sequence. Lactose and IPTG induction were used to obtain homologous recombinant Bacillus subtilis strains that efficiently express the endonuclease-1,3-1,4-β-D-glucanase gene protein. This recombinant strain can be applied in feed processing and bioenergy.

[0059] The recombinant Bacillus subtilis was prepared by knocking in the promoter region of the endo-1,3-1,4-β-D-glucanase gene into the lactose operon promoter sequence of *Escherichia coli*. This recombinant Bacillus subtilis can be induced by lactose and IPTG to express the endo-1,3-1,4-β-D-glucanase gene in large quantities. qRT-PCR showed that the expression of β-1,3-1,4-glucanase mRNA in the recombinant bacteria after lactose and IPTG induction increased by 8.14 and 10.03 times, respectively, compared to the uninduced group. Enzyme activity assays using a β-1,3-1,4-glucanase activity kit showed that the enzyme activity of the recombinant bacteria after lactose and IPTG induction increased by 8.77 and 19.83 times, respectively, compared to the uninduced group.

[0060] The 550bp sequences of the upstream and downstream homologous arms of the promoter sequence of the endonuclease-1,3-1,4-β-D-glucanase gene are shown in SEQ ID NO.1 (NCBI Reference Sequence: NC_000964 Region4012866-4013415) and SEQ ID NO.2 (NCBI Reference Sequence: NC_000964 Region4012011-4012560).

[0061] The Lac promoter sequence of Escherichia coli is 1205 bp (Escherichia coli CGMCC: 1.12883, NCBI Reference Sequence: CP125731 Region 3630860-3631939), as shown in SEQ ID No. 3.

[0062] This invention provides a method for constructing the above-mentioned homologous recombinant Bacillus subtilis, comprising the following steps: The upstream and downstream homologous arms of the promoter of the β-1,3-1,4-glucanase gene were artificially synthesized, and the Lac promoter sequence of Escherichia coli (1205 bp) was artificially synthesized. The homologous recombination expression cassette sequence (upstream homologous arm-Lac-downstream homologous arm) was obtained by overlapping PCR.

[0063] Using the seamless splicing PCR method, the above homologous recombination expression cassette was ligated into the BamHI site of the Escherichia coli-Bacillus subtilis shuttle expression vector pHY300PLK to construct the homologous recombination expression vector, named pHY300PLK-Lac.

[0064] The homologous recombination expression vector pHY300PLK-Lac was electrotransformed into competent Bacillus subtilis 168 using conventional methods.

[0065] The successfully electroporated competent Bacillus subtilis was further passaged using LB liquid medium and agar medium containing tetracycline. At the same time, the pHY300PLK-Lac recombinant sequence was amplified by PCR. After multiple rounds of streak plate culture and PCR amplification, positive electroporated clones were obtained through screening.

[0066] Positive electroporation clones were further passaged in LB liquid medium and agar medium containing tetracycline. At the same time, PCR was used to amplify the genome-Lac recombination sequence, and positive clones containing the pHY300PLK-Lac plasmid and undergoing homologous recombination were screened to obtain positive clones.

[0067] The strain was simultaneously inoculated into both antibiotic-free LB solid medium and Tet-containing LB solid medium. Single colonies that formed on LB plates but did not grow at all on Tet plates were selected to obtain homologous recombination genetically engineered strains without plasmid residues that lost the pHY300PLK-Lac plasmid through homologous recombination. These were the target strains.

[0068] The growth curve of the target strain was not significantly different from that of the wild strain, and gene sequencing showed that the Lac sequence introduced after 20 passages of the target strain had genetic stability.

[0069] The obtained homologous recombinant strains were cultured in LB agar and induced with 0.5%, 1%, 1.5%, 2%, and 2.5% lactose for 1, 2, 4, 8, and 12 h to express endonuclease-1,3-1,4-β-D-glucanase. Bacterial RNA was extracted from each group, reverse transcribed, and qRT-PCR was used to detect endonuclease-1,3-1,4-β-D-glucanase gene expression. Relative quantitative analysis was performed. The results showed that after 8 hours of induction with 2% lactose, the mRNA expression level of the endoglucanase gene was significantly higher than that of the LB control group, increasing by 8.14 times.

[0070] The enzyme activity of the recombinant bacteria was determined by the β-1,3-1,4-glucanase activity assay kit. After 8 hours of induction with 2% lactose, the enzyme activity of the recombinant bacteria was 8.77 times higher than that of the uninduced group.

[0071] The obtained homologous recombinant strains were cultured in LB agar and induced to express endo-1,3-1,4-β-D-glucanase for 1, 3, and 6 hours with the addition of 0.1 μM, 0.4 μM, 0.7 μM, and 1 μM IPTG, respectively. qRT-PCR analysis showed that 0.4 μM IPTG induction for 3 hours significantly increased the expression of the target gene. The mRNA expression level in the recombinant bacteria after induction increased by 10.03 times compared to before induction. Enzyme activity assays using a β-1,3-1,4-glucanase activity kit showed that the enzyme activity of the recombinant bacteria after IPTG induction increased by 19.83 times compared to the uninduced group.

[0072] This invention utilizes overlap PCR technology to construct a homologous recombination expression cassette integrating the lactose operon promoter with the upstream and downstream homologous arms of the endonuclease-1,3-1,4-β-D-glucanase gene. Furthermore, seamless cloning technology is applied to construct a homologous recombination expression vector, which is then introduced into *Bacillus subtilis* via electroporation. Through continuous passage screening, antibiotic-free vector-free homologous recombination strains are obtained, and the genetic stability and protein expression levels of the recombinant strains are assessed. This invention not only provides a new strategy for improving the industrial production efficiency of *Bacillus subtilis* endonuclease but also provides a technical method for the application of the lactose operon promoter in the efficient expression of exogenous genes in *Bacillus subtilis*.

[0073] This invention relates to a Bacillus subtilis engineered strain that efficiently expresses endonuclease-1,3-1,4-β-D-glucanase based on homologous recombination of the lactose operon promoter and its construction method: using homologous recombination, the promoter region of the Bacillus subtilis 168 endonuclease gene is replaced by the Escherichia coli lactose operon promoter sequence, and homologous recombinant Bacillus subtilis expressing endonuclease-1,3-1,4-β-D-glucanase is obtained by induction with lactose and IPTG.

[0074] The starting strain of this invention, Bacillus subtilis 168 (ATCC 23857), was purchased from the Shanghai Center for Microbial Culture Collection (SHBCC), strain number SHBCC D25186.

[0075] Example 1 Construction of the integrated expression vector pHY300PLK-Lac: Identification of Bacillus subtilis 168 strain using PCR amplification of the rpoA gene: 0.1 g of Bacillus subtilis 168 bacterial powder was dissolved in 10 mL of LB liquid medium and serially diluted to 10⁻⁶. -3 ~10 -5After adjusting the concentration, the culture was plated on LB agar plates and incubated at 37°C for 18 h. Typical Bacillus subtilis colonies (2-3 mm in diameter, with irregular snowflake-like edges) were picked and inoculated into LB liquid medium. The culture was then incubated at 37°C with shaking at 180 rpm for 12 h. 1 μL of the bacterial culture was used directly as a template for PCR amplification using universal rpoA primers. The target band was verified by 1.5% agarose gel electrophoresis. After gel purification, the band was sequenced, and strains with >99% sequence identity to the rpoA gene in NC_000964 were screened. The strains were then added to a final concentration of 50% sterile glycerol and stored at -80°C for later use (see [link to relevant documentation]). Figure 1 ).

[0076] The upstream and downstream homologous arms of the promoter region of the Bacillus subtilis 168 endonuclease-1,3-1,4-β-D-glucanase-encoding gene were amplified by PCR to obtain sequence 1 (see [link to sequence 1]). Figure 2 ) and sequence 2 (see Figure 3 ), as shown in SEQ ID NO.1 (NCBIReference Sequence: NC_000964 Region4012866-4013415) and SEQ ID NO.2 (NCBIReference Sequence: NC_000964 Region4012011-4012560).

[0077] SEQ ID NO.1:。

[0078] SEQ ID NO.2:

[0079] The artificially synthesized Lac promoter sequence, and the agarose gel electrophoresis results are shown in [see figure]. Figure 3 As shown.

[0080] Referring to commercially available vectors such as pET28a and pHT43, the effective sequence length of the lactose operon promoter was determined; homologous sequences of the effective promoter sequence were found in *E. coli*, and *E. coli* genomic DNA was extracted as a template for PCR amplification using appropriate primers to obtain sequence 3 (see [link]). Figure 4 Sequence 3, 1205 bp (Escherichia coli CGMCC: 1.12883, NCBI Reference Sequence: CP125731 Region 3630860-3631939), is shown in SEQ ID NO. 3. Sequence 3 was validated by 1.5% agarose gel electrophoresis and then purified by gel recovery.

[0081]

[0082] The upstream homologous arm-Lac-downstream homologous arm tandem fragment (homologous recombination expression cassette sequence as shown in SEQ ID NO.4) was obtained using overlap PCR. The successfully tandemly bound band was excised and recovered for subsequent experiments (see [link to experimental data]). Figure 5 ).

[0083]

[0084] The application of seamless cloning methods utilizes Bam The HI restriction endonuclease cleavage site was ligated to the pHY300PLK and homologous recombination expression cassette sequence to construct the recombinant vector pHY300PLK-Lac for homologous recombination. The empty vector was 4870 bp, the recombinant expression cassette sequence was 2305, and the recombinant vector was 7177 bp (see [link]). Figure 6 ).

[0085] Sequencing validation of the recombinant vector pHY300PLK-Lac: PCR amplification was performed using pHY300PLK-Lac as a template, and the sample was then analyzed. The sequencing results were aligned with the expected full-length target replacement fragment (upstream homologous arm - Lac - downstream homologous arm) using DNAMAN software. Results are as follows: Figure 7 As shown, the target replacement fragment carried by the recombinant vector pHY300PLK-Lac has no mismatch.

[0086] Example 2 pHY300PLK-Lac electroporation of Bacillus subtilis and screening of positive clones: Preparation of competent cells of Bacillus subtilis: Remove the frozen Bacillus subtilis culture from the -80℃ freezer, thaw it on ice, and then streak the culture onto LB solid medium using an inoculation loop. Incubate overnight at 37℃. Pick a single colony and inoculate it into 20 mL of LB liquid medium, incubating at 37℃ and 180 rpm for 12-16 h. Transfer 2.5 mL of the culture to 40 mL of growth medium (the volume of the culture is 1 / 16 of the growth medium volume), and incubate at 37℃ and 180 rpm for 4-5 h. After incubating the culture on ice for 10 min, aliquot 20 mL into two 50 mL centrifuge tubes. Centrifuge at 4℃ and 5000 rpm for 7 min. Discard the supernatant, resuspend the bacterial pellet in 40-50 mL of pre-chilled electroporation wash buffer, mix well by pipetting, and centrifuge again at 4℃ and 5000 rpm for 12-15 min. Repeat 4 times. Discard the supernatant, resuspend the precipitate in 1 mL of electroporation wash buffer in each centrifuge tube, and aliquot 70 μL into 1.5 mL EP tubes. It can be used immediately or frozen at -80°C; the conversion efficiency is not significantly affected within one month.

[0087] Electroporation of Bacillus subtilis using pHY300PLK-Lac: Take out the prepared competent Bacillus subtilis cells. If taken from a -80℃ freezer, place them on ice for slow freezing. In a clean bench, add an appropriate amount of the extracted recombinant vector pHY300PLK-Z, adding 0.3-1.5 μL depending on its concentration (optimal vector addition is 1 μg). Gently pipette the vector and competent cells together, then transfer to a sterile electroporation cuvette (1 mm electrode spacing) pre-cooled at -20℃ for at least 30 min. Keep the movements as small as possible throughout the process. After the cuvette is in an ice bath for 5 min, wipe the electrode surface dry with paper and place the cuvette into the electroporator for electroporation. Electroporation conditions: 2.2 kV, 25 μF, 200 Ω, 4 ms. Immediately after electroporation, remove the cuvette and pipette 1 mL of reconstitution solution into it. Mix well and transfer to a 1.5 mL EP tube. Incubate at 37℃ and 90 rpm for 3-6 h. Centrifuge at 7000 rpm for 2 min, resuspend the precipitate in 200 μL of supernatant, mix thoroughly by pipetting, and spread onto LB solid medium containing 10-15 μg / mL tetracycline. Incubate overnight at 37℃ (inverted) for 12-16 h. The next day, pick several single colonies from the plate and inoculate them into LB liquid medium for subsequent detection.

[0088] Screening and identification of positive clones of Bacillus subtilis by electroporation: The recombinant vector pHY300PLK-Lac was transformed into competent Bacillus subtilis cells using electroporation. The bacterial culture was then plated onto LB agar plates containing 15 μg / mL tetracycline. The transformation results are shown below. Figure 8 As shown.

[0089] Sequencing identification of electroporated positive clones: After resuscitation and culture, single colonies of the recombinant bacteria were randomly selected and inoculated into LB liquid medium containing 100 μg / mL ampicillin. The culture was incubated at 37℃ and 220 rpm with shaking until the logarithmic growth phase (OD600≈0.6). One μL of the bacterial culture was used as a template for PCR amplification and identification using primers (see...). Figure 9 The sequencing results were then sent for sequencing. The sequencing data was compared with the full-length sequence (upstream homologous arm - Lac - downstream homologous arm) using DNAMAN software. The results are as follows: Figure 10 As shown, the recombinant bacteria obtained by electroporation showed no mismatches.

[0090] Example 3 Screening for positive clones of homologous recombinant Bacillus subtilis containing transformation vectors: After transforming the pHY300PLK-Lac homologous recombination vector into Bacillus subtilis, the rate of natural homologous recombination is extremely low, necessitating effective screening to obtain homologous recombination strains from pHY300PLK-CLP-transformed positive strains that have never undergone homologous recombination. This invention obtains homologous recombination-positive clones of Bacillus subtilis through continuous culture and PCR identification of pHY300PLK-CLP-transformed positive strains.

[0091] Methods for screening and identifying electroporation bacteria through passage and homologous recombination: Electroporation subculturing: Bacterial suspensions that tested positive for both bacterial strain identification and recombinant fragment verification were streaked into LB solid medium (1.5% agar) containing 10 μg / mL tetracycline and incubated at 37°C for 12-16 h to obtain single colonies. Uniformly morphologically selected single colonies were inoculated into LB liquid medium (10 μg / mL tetracycline) containing 3 mL in sterile EP tubes and cultured at 37°C with shaking at 220 rpm for 12 h until the logarithmic growth phase, serving as the primary generation (P0) seed culture. Subcultured continuously at a 1% inoculum (30 μL of bacterial suspension) up to the 5th generation (P5), using LB liquid medium containing 10 μg / mL tetracycline throughout, subculturing every 12 h.

[0092] Homologous recombination screening and identification method during the passage of electroporation bacteria: When the strain reaches the 5th generation, a portion of the bacterial culture is taken every other generation for the following three PCR identifications: upstream homologous arm exchange, downstream homologous arm exchange, and plasmid identification. The PCR products are analyzed by agarose gel electrophoresis. If both homologous arm exchange PCR results are negative, the strain is passaged further, ensuring a positive plasmid identification result to prevent plasmid loss during passage. Identification is performed every other generation. If either upstream or downstream homologous arm exchange PCR result is positive, the result is recorded, and the strain is passaged intensively. Identification is performed every other generation until both homologous arm exchange and plasmid PCR results are positive. The results of upstream and downstream homologous arm exchange of single colonies after passage 5 of electroporation bacteria are shown in the figure. Figure 11 .

[0093] Initial screening for PCR amplification of single colonies of homologous recombinant bacteria with double homologous arm substitution: Bacterial cultures that were screened and confirmed to be positive for both upstream and downstream homologous arm substitution in the above experiments were inoculated into LB agar containing tetracycline and streaked at 37°C for 12-16 h. The colonies were then identified using the three primer pairs described above as templates. Single colonies with positive identification results were selected and continuously passaged and streaked on LB agar containing tetracycline. A large number of single colonies were randomly selected and subjected to full-length homologous exchange PCR identification. If only a 2812 bp band (the promoter region sequence of the wild-type Bacillus subtilis endonuclease gene, a negative band) appeared on electrophoresis, further passage was performed. Full-length exchange identification PCR was performed every other generation until the electrophoresis results showed two bands: a 1902 bp band (the expected Lac promoter region sequence of Bac subtilis undergoing homologous recombination, a positive band) and a 2812 bp band. This indicated that Bac subtilis undergoing homologous recombination had appeared on the culture plate, but it was mixed with wild-type Bac subtilis and required further purification (see [link]). Figure 12 ).

[0094] Purification results of single colonies of homologous recombinant bacteria undergoing double crossover: Colonies showing double bands (2812 bp and 1902 bp) on electrophoresis were screened, streaked on LB solid medium containing tetracycline, and incubated overnight at 37°C for 12-16 h. The next day, several single colonies were selected and subjected to double crossover identification PCR, and simultaneously inoculated into LB liquid medium containing tetracycline and incubated at 37°C. The PCR products were analyzed by agarose gel electrophoresis, and colonies with the most prominent positive bands were observed and selected for streaking again. The above steps were repeated until the electrophoresis results showed only a positive band of 1902 bp, indicating that the recombinant strain had successfully undergone homologous recombination (see...). Figure 13 ).

[0095] Example 4 Screening for positive clones of homologous recombinant Bacillus subtilis that have lost their transformation vector: By simultaneously inoculating recombinant strains purified through multiple generations onto antibiotic-free LB solid medium and LB-Tet solid medium containing tetracycline (Tet), single colonies that grew well on LB plates but showed no growth on Tet plates were screened, indicating that homologous recombinant strains that had lost the pHY300PLK-Lac plasmid through passage were obtained (see...). Figure 14 ).

[0096] Example 5 Growth curves and exogenous gene stability analysis of homologous recombinant strains: The results of the growth curve measurement are as follows: Figure 15As shown, the growth patterns of the recombinant bacteria during the logarithmic growth phase, stationary phase, and decline phase are basically consistent with those of the wild-type strain.

[0097] Using the 20th generation recombinant bacterial culture as a template, PCR amplification was performed using full-length validation primers. The product was validated by agarose gel electrophoresis, purified by gel excision, and sequenced by Qingke Biotechnology. Figure 16 As shown, sequence alignment using DNAMAN software showed that the recombinant strain sequence was identical to the original design sequence in nucleotides, and no base deletions, insertions, or mismatch mutations were detected, confirming that the target gene can still be stably inherited under the absence of resistance selection pressure.

[0098] Example 6 Establishment of lactose and IPTG-induced expression conditions for homologous recombinant Bacillus subtilis endo-1,3-1,4-β-D-glucanase: Lactose at concentrations of 0.5%, 1%, 1.5%, 2%, and 2.5% (w / w) induced the expression of endonuclease-1,3-1,4-β-D-glucanase for 1, 2, 4, 8, and 12 h, respectively. Bacterial RNA was extracted from each group, reverse transcribed, and the expression of the endonuclease-1,3-1,4-β-D-glucanase gene was detected by qRT-PCR. Relative quantitative analysis was performed. Results showed that after 8 hours of 2% lactose induction, the mRNA expression level of the endoglucanase gene was significantly higher than that of the LB control group, increasing by 8.14 times (see...). Figure 17 ).

[0099] The obtained homologous recombinant strains were cultured in LB agar and induced to express endo-1,3-1,4-β-D-glucanase at 0.1 μM, 0.4 μM, 0.7 μM, and 1 μM IPTG for 1, 3, and 6 h, respectively. qRT-PCR analysis showed that 0.4 μM IPTG induction for 3 h significantly increased the expression of the target gene. The mRNA expression level in the recombinant bacteria after induction increased 10.03-fold compared to before induction (see...). Figure 18 ).

[0100] The enzyme activity of the recombinant bacteria, as determined by a β-1,3-1,4-glucanase activity assay kit, was 8.77 times higher than that of the uninduced group after 8 hours of 2% lactose induction; and 19.83 times higher than that of the uninduced group after IPTG induction (see [link to kit]). Figure 19 ).

[0101] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A recombinant expression cassette for expressing endonuclease-1,3-1,4-β-D-glucanase, characterized in that, This includes the upstream and downstream homologous arms of the promoter region of the gene encoding endonuclease-1,3-1,4-β-D-glucanase and the Lac promoter sequence.

2. The recombinant expression cassette according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A recombinant expression vector for expressing endonuclease-1,3-1,4-β-D-glucanase, characterized in that, The recombinant expression vector uses pHY300PLK as its backbone and includes the recombinant expression cassette as described in claim 1 or 2.

4. A recombinant Bacillus subtilis strain expressing endoglucanase, characterized in that, The starting strain is Bacillus subtilis 168, and it contains the recombinant expression cassette of claim 1 or 2 or the recombinant expression vector of claim 3.

5. The use of the recombinant expression cassette of claim 1 or 2, the recombinant expression vector of claim 3, or the recombinant Bacillus subtilis of claim 4 in the production of endo-1,3-1,4-β-D-glucanase.

6. A method for preparing endo-1,3-1,4-β-D-glucanase, characterized in that, Using the recombinant Bacillus subtilis according to claim 4, an endoglucanase, endo-1,3-1,4-β-D-glucanase, is generated under the induction of isopropyl-β-D-thiogalactoside or lactose.

7. The preparation method according to claim 6, characterized in that, The concentration of the isopropyl-β-D-thiogalactoside in the culture medium is 0.1~1 μM.

8. The production method according to claim 6, characterized in that, The lactose concentration in the culture medium is 0.5% to 2.5% by mass.

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