Recombinant bacillus subtilis engineering bacterium capable of simultaneously degrading cellulose and hemicellulose as well as construction method and application of recombinant bacillus subtilis engineering bacterium

Abstract

The invention discloses a recombinant bacillus subtilis engineering bacterium capable of simultaneously degrading cellulose and hemicellulose as well as a construction method and application of the recombinant bacillus subtilis engineering bacterium, and belongs to the technical field of biological engineering. The engineering bacterium is classified and named as bacillus subtilis NJAU-Li2025, the engineering bacterium is preserved in the China Center for Type Culture Collection on May 6, 2025, and the preservation number of the engineering bacterium is CCTCC NO: M 2025957. The engineering bacterium disclosed by the invention has efficient cellulase expression and secretion capabilities, retains the degradation activity of host bacteria on hemicellulose, can synergistically degrade cellulose and hemicellulose in pasture, and realizes efficient biotransformation and utilization of cellulose and hemicellulose resources in pasture.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant Bacillus subtilis engineered bacterium capable of simultaneously degrading cellulose and hemicellulose, its construction method, and its application. Background Technology

[0002] Cellulose resources are abundant in nature, representing a rich source of renewable biomass on Earth. In my country, for example, agricultural production generates hundreds of millions of tons of cellulose-rich agricultural byproducts annually, such as wheat bran and straw. However, due to a lack of efficient and low-cost processing and utilization technologies, only a very small portion of these resources is currently being effectively utilized. The long-term accumulation of large amounts of unused cellulose waste not only occupies significant land space but also poses safety hazards; incineration, on the other hand, would cause severe air pollution and ecological damage.

[0003] Cellulose, as an important component of herbivore feed, has broad application prospects in animal husbandry. Naturally cellulose-rich plant-based feeds (such as pasture and straw) typically contain not only cellulose but also large amounts of hemicellulose, pectin, and other soluble polysaccharides. These components are easily dissolved during animal digestion, forming sticky substances that hinder further degradation of feed particles, reducing feed digestibility, and also affecting the absorption efficiency of released nutrients, thus negatively impacting the colonization and reproduction of beneficial bacteria in the gut. Therefore, improving the degradation efficiency of cellulose and hemicellulose is of great significance for improving feed utilization and enhancing animal production performance.

[0004] Microbial-mediated biodegradation, an environmentally friendly, mild, and efficient method of cellulose utilization, has become a research hotspot in this field. Among them, probiotics have shown great potential in the field of feed additives due to their ability to regulate the host's intestinal microecology, produce beneficial metabolites, enhance immunity, and secrete various digestive enzymes (such as proteases, amylases, and cellulases).

[0005] Bacillus subtilis is a typical probiotic, listed as a safe strain in my country's "List of Feed Additives," and also recognized as a "Generally Recognized As Safe" (GRAS) microorganism by the US FDA. It forms heat- and acid / alkali-resistant spores, facilitating processing, storage, and transportation. When used as a feed additive, it germinates and colonizes in the animal gut, improving gut microbiota structure, enhancing disease resistance, and secreting various exogenous enzymes to promote nutrient digestion and absorption. Furthermore, Bacillus subtilis is a mature genetic engineering host with advantages such as a clear genetic background, well-developed expression system, and ease of large-scale fermentation, making it widely used for the efficient expression of exogenous proteins. Existing research has shown that modifying Bacillus subtilis through genetic engineering to overexpress cellulase can significantly enhance its fiber degradation capacity. However, current technology still lacks a recombinant Bacillus subtilis engineered strain that combines high cellulase secretion capacity, good intestinal adaptability, and safety, especially a highly efficient feed probiotic strain capable of stable survival in the acidic environment of the stomach and continuous enzyme production in the intestine.

[0006] Therefore, constructing a recombinant Bacillus subtilis engineered strain that can efficiently express and secrete cellulase, is resistant to gastric acid, has high safety, and is suitable for feed additives is of great significance for improving feed conversion efficiency and promoting the development of green animal husbandry. Summary of the Invention

[0007] In view of this, the present invention provides a recombinant Bacillus subtilis engineered strain capable of simultaneously degrading cellulose and hemicellulose, its construction method and application. The engineered strain provided by the present invention carries a recombinant expression vector containing an exogenous cellulase gene, which has a high efficiency in cellulase expression and secretion, while retaining the host strain's degradation activity for hemicellulose. It can synergistically degrade cellulose and hemicellulose in forage to generate soluble sugars, thereby realizing the efficient bioconversion and utilization of cellulose and hemicellulose resources in forage.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a recombinant Bacillus subtilis engineered strain capable of simultaneously degrading cellulose and hemicellulose. The engineered strain is obtained by genetic engineering of Bacillus subtilis KC7-2 with accession number CCTCC NO: M 20241570, carrying a recombinant expression vector containing an exogenous cellulase gene. This vector enables efficient expression of cellulase and synergistic degradation of hemicellulose during fermentation. The engineered strain is classified as Bacillus subtilis NJAU-Li2025 and was deposited at the China Center for Type Culture Collection on May 6, 2025, with accession number CCTCC NO: M 2025957.

[0009] Preferably, the wild-type parent strain of Bacillus subtilis is Bacillus subtilis KC7-2, which is isolated from the rumen contents of yak and has strong cellulose degradation ability. It was deposited at the China Center for Type Culture Collection on July 15, 2024, with accession number: CCTCC NO: M 20241570.

[0010] Preferably, the cellulase gene has a nucleotide sequence as shown in SEQ ID NO: 1.

[0011] Preferably, the protein encoded by the cellulase gene has the amino acid sequence shown in SEQ ID NO: 2.

[0012] The present invention also provides a recombinant expression vector comprising the cellulase gene described above and operably linked to a promoter capable of initiating transcription in Bacillus subtilis; wherein the recombinant expression vector is a recombinant plasmid constructed based on the pHT304 plasmid.

[0013] Preferably, the promoter is the P43 promoter inherent in the pHT304 vector; the pHT304 plasmid also contains the erythromycin resistance gene (Erm). r () is used as a filter tag.

[0014] Preferably, the recombinant expression vector has a nucleotide sequence as shown in SEQ ID NO: 3.

[0015] This invention also provides a method for constructing recombinant Bacillus subtilis engineered bacteria, comprising the following steps: (1) Preparation of competent cells of Bacillus subtilis KC7-2: The bacterial culture was cultured to OD 600 ≈ 0.6, washed with buffer containing 0.5 M sorbitol, 0.5 M mannitol and 10% glycerol after ice bath; (2) The recombinant expression vector described above was introduced into the competent cells of Bacillus subtilis in step (1) by electroporation. The electroporation parameters were: voltage 2.5 kV / cm, capacitance 25 μF, and resistance 200 Ω. (3) After transformation, the bacteria were thawed at 30℃ for 1 hour, spread on LB plates containing 10 μg / mL erythromycin, and cultured for 48 hours. Single clones were picked and the recombinant engineered bacteria were obtained by PCR verification.

[0016] The present invention also discloses the application of the above-described recombinant Bacillus subtilis engineered strain or the above-described recombinant expression vector in the degradation of forage, straw or plant fiber materials.

[0017] This invention also discloses the application of the above-described recombinant Bacillus subtilis engineered strain in the preparation of animal feed additives or probiotic preparations.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The recombinant Bacillus subtilis engineered strain constructed in this invention not only efficiently expresses and secretes cellulase, but also exhibits high endogenous hemicellulase activity, providing excellent application potential for biomass degradation and feed improvement. Details are as follows: (1) Enzyme activity assays revealed that the cellulase activity of the engineered bacteria reached a peak of 118.4 U after 36 hours of culture; subsequently, the enzyme activity slowly decreased and stabilized after 72 hours, remaining at a relatively high level of approximately 50 U (e.g., ...). Figure 8 Meanwhile, its hemicellulase activity reached a peak of 315.5 nmol / min / mL at 36 h, and remained at approximately 165 nmol / min / mL after 72 h (e.g., Figure 9 This indicates that the strain exhibits highly efficient dual-enzyme synergistic degradation ability, and the enzyme activity lasts for a long time. (2) The test results show (see Figure 6 This recombinant engineered bacteria exhibits strong survival ability in a simulated gastric acid environment (pH = 3.0), maintaining activity under extremely acidic conditions. This characteristic allows it to remain viable after entering the animal's digestive tract via oral administration, facilitating colonization in the intestines and the continuous secretion of cellulase and hemicellulase, significantly improving the in vivo digestibility of fiber components in feed.

[0019] In summary, the recombinant Bacillus subtilis engineered strain provided by this invention exhibits excellent performance in the secretion of cellulase and hemicellulase, especially achieving peak enzyme activity in the late logarithmic growth phase (36 hours) and maintaining effective enzyme activity levels for a long period. Furthermore, it can survive in the acidic pH environment of the stomach, a characteristic that makes it of significant application value in forage improvement, biomass degradation, and probiotic feed additives. Attached Figure Description

[0020] Figure 1 The plasmid map of the recombinant expression vector pHT304-Egls constructed in this invention; Figure 2 This is an electrophoresis image of the PCR verification of the engineered Bacillus subtilis strain provided by this invention; Figure 3 Colony diagram of the recombinant Bacillus subtilis engineered strain provided by this invention on LB agar medium; Figure 4 Gram-stained microscopic images of recombinant Bacillus subtilis engineered bacteria provided by this invention; Figure 5 The growth curve image of the recombinant Bacillus subtilis engineered bacteria provided by this invention; Figure 6 The acid resistance curve of the recombinant Bacillus subtilis engineered strain provided by this invention; Figure 7 The Congo red staining test for cellulose degradation by recombinant Bacillus subtilis engineered bacteria provided by this invention; Figure 8 Cellulase activity of recombinant Bacillus subtilis engineered strain and Bacillus subtilis KC7-2 at different time points in fermentation degradation test provided by the present invention; Figure 9 The hemicellulase activity of the recombinant Bacillus subtilis engineered strain and Bacillus subtilis KC7-2 at different time points in the fermentation degradation experiment provided by the present invention; Figure 10 Cellulose content in samples of recombinant Bacillus subtilis engineered strain provided by this invention after 72 hours of forage fermentation degradation test; Figure 11 The content of hemicellulose in samples of recombinant Bacillus subtilis engineered strain provided by the present invention after 72 hours of forage fermentation degradation test. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] This invention provides a recombinant Bacillus subtilis engineered strain capable of simultaneously degrading cellulose and hemicellulose. This engineered strain is obtained by genetically modifying Bacillus subtilis KC7-2 with accession number CCTCC NO: M 20241570. It carries a recombinant expression vector containing an exogenous cellulase gene, enabling efficient expression of cellulase and synergistic degradation of hemicellulose during fermentation. The engineered strain is classified as Bacillus subtilis NJAU-Li2025 and was deposited at the China Center for Type Culture Collection on May 6, 2025, with accession number CCTCC NO: M 2025957.

[0025] The wild-type parent strain of the aforementioned Bacillus subtilis is Bacillus subtilis KC7-2, isolated from yak rumen contents, which possesses strong cellulose-degrading capabilities. It was deposited at the China Center for Type Culture Collection (CCTCC) on July 15, 2024, with accession number CCTCC NO: M 20241570. The cellulase gene described above has the nucleotide sequence shown in SEQ ID NO: 1.

[0026] The protein encoded by the cellulase gene described above has the amino acid sequence shown in SEQ ID NO: 2.

[0027] The present invention also provides a recombinant expression vector containing the cellulase gene described above and operably linked to a promoter capable of initiating transcription in Bacillus subtilis; the recombinant expression vector is a recombinant plasmid constructed based on the pHT304 plasmid.

[0028] The promoter is the P43 promoter inherent in the pHT304 vector; the pHT304 plasmid also contains the erythromycin resistance gene (Erm). r () is used as a filter tag.

[0029] This invention also provides a method for constructing recombinant Bacillus subtilis engineered bacteria, comprising the following steps: (1) Preparation of competent cells of Bacillus subtilis KC7-2: The bacterial culture was cultured to OD 600 ≈ 0.6, after ice bath, wash with buffer containing 0.5M sorbitol, 0.5M mannitol and 10% glycerol; (2) The recombinant expression vector described above was introduced into the competent cells of Bacillus subtilis in step (1) by electroporation. The electroporation parameters were: voltage 2.5 kV / cm, capacitance 25 μF, and resistance 200 Ω. (3) After transformation, the bacteria were thawed at 30℃ for 1 hour, spread on LB plates containing 10 μg / mL erythromycin, and cultured for 48 hours. Single clones were picked and the recombinant engineered bacteria were obtained by PCR verification.

[0030] The present invention also discloses the application of the above-described recombinant Bacillus subtilis engineered strain or the above-described recombinant expression vector in the degradation of forage, straw or plant fiber materials.

[0031] This invention also discloses the application of the above-described recombinant Bacillus subtilis engineered strain in the preparation of animal feed additives or probiotic preparations.

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments: Example 1 1. Isolation, screening, identification and preservation of wild-type Bacillus subtilis strains 1.1 Isolation and purification of strains: The applicant collected rumen contents samples from healthy yaks in the Ali region for screening cellulase-producing strains. The specific procedures are as follows: In a sterile operating table, a small amount of rumen contents sample was inoculated into 3 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0) and cultured in a shaker at 37°C for 12 h at a shaking speed of 180 r / min. After the culture was completed, the bacterial culture was inoculated onto LB solid medium (containing 1.5% agar) plates using a sterile inoculation loop using the three-zone streak method and cultured in a 37°C incubator for another 12 h.

[0033] After cultivation, observe the colony morphology. Select colonies that are suspected to be Bacillus subtilis, with a rough, opaque surface, a dirty white or slightly yellow color, and irregular edges, perform Gram staining, and observe their morphology under a microscope. At the same time, inoculate the above typical colonies again onto LB agar plates using the three-zone streak method for purification. Repeat the purification process for 3-4 generations until uniform single colonies of consistent morphology and size are obtained.

[0034] Gram-positive colonies that are blue-purple, short rod-shaped, and have spores were selected from the purified colonies and inoculated into nutrient broth medium. The colonies were cultured at 37°C and 180 r / min for 12 h to obtain an overnight culture solution for later use.

[0035] 1.2 Preliminary screening of cellulose-degrading bacteria: Cellulase activity of the purified strains was screened using Congo red staining. Cells from each purified strain were collected using a sterile inoculation loop and inoculated onto equal volumes on cellulase solid medium plates (10 g / L peptone, 10 g / L yeast extract, 10 g / L CMC-Na, 5 g / L NaCl, 1 g / L KH₂PO₄, and 18 g / L agar powder) and incubated at 37°C for 48 h. After incubation, 1 mg / mL Congo red solution was added for staining for 10-15 min, the staining solution was discarded, and unbound dye was eluted with 1 mol / L NaCl solution.

[0036] The diameter of the hydrolysis zone (D) and the colony diameter (d) were observed and measured, and the D / d ratio was calculated. The larger the ratio, the stronger the cellulase production capacity of the strain. After screening, a strain with the largest D / d ratio and the strongest cellulose degradation capacity was obtained and named KC7-2.

[0037] 1.3 Taxonomic identification of strains: Gram staining was performed on the selected strain KC7-2. Microscopic examination revealed that it was Gram-positive, short rod-shaped, and had a central spore, consistent with the morphological characteristics of Bacillus subtilis. Genomic DNA was extracted from this strain and PCR amplification was performed using universal 16S rDNA primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The amplified product was purified and sequenced, yielding a 16S rDNA sequence of approximately 1500 bp. The sequence was submitted to the NCBI database for BLAST alignment, and the results showed that its 16S rRNA gene sequence similarity exceeded 99% with multiple Bacillus subtilis strains. The top 30 sequences with the highest Identity values ​​from the alignment results were selected for multiple sequence alignment using MAFFT software, and a phylogenetic tree was constructed using the Neighbor-Joining method. The results showed that strain KC7-2 clustered with Bacillus subtilis in the same branch and was the most closely related in evolution. The strain was identified as Bacillus subtilis and named Bacillus subtilis KC7-2. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on July 15, 2024, with accession number CCTCC NO: M 20241570, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China.

[0038] Example 2 This example demonstrates whole-genome sequencing and the acquisition of the cellulase gene.

[0039] 2.1 Whole genome sequencing: The whole genome of the preserved strain Bacillus subtilis KC7-2 was sequenced, and the specific steps are as follows: ① DNA extraction: High-quality genomic DNA was extracted using the Qiagen Genomic DNA Extraction Kit; ② Quality Inspection: Visual inspection revealed no foreign matter or degradation; 0.75% agarose gel electrophoresis showed a clear main band with no tailing; Nanodrop detection showed OD... 260 / 280 = 1.85, OD 260 / 230 = 2.1; Qubit quantification: concentration is 50 ng / μL.

[0040] ③ Library construction: 1D sequencing libraries were constructed using the Oxford Nanopore Technologies NBD104 / NBD114 kit, and barcode tags and sequencing adapters were ligated.

[0041] ④ Sequencing: Load the library into the PromethION Flow Cell and use the PromethION sequencer to perform real-time single-molecule long-read sequencing to obtain raw data.

[0042] ⑤ Data quality control and assembly: Fastp was used for raw data quality control and filtering; SPAdes was used for genome assembly; GapFiller was used to fill GAPs in the assembled contigs; Pilon was used for sequence correction; and finally, the complete genome sequence was obtained.

[0043] 2.2 Genome annotation and cellulase gene screening: Systematic annotation of the assembled genome: Using Prokka for gene structure prediction (CDS, tRNA, rRNA, etc.). Functional annotations were performed by comparing data with databases such as CDD, COG, NR, and SwissProt using NCBI Blast+. KEGG pathway analysis using KAAS; The predicted protein sequences were compared with the CAZy database using HMMER3, and the E-value was <1e. -5 This is a significant match.

[0044] CAZy annotation results showed that the genome of this strain contained multiple genes related to cellulose degradation, including: endo-β-1,4-glucanase (EC 3.2.1.4); exo-β-1,4-glucanase (EC 3.2.1.91); and β-glucosidase (EC 3.2.1.21).

[0045] Among them, a gene encoding endonuclease-1,4-β-D-glucanase is located on the negative strand of the genome. After removing three stop codons, its length is 1497 bp. Its nucleotide sequence is shown in SEQ ID NO: 1, and the encoded protein sequence is shown in SEQ ID NO: 2. It has a typical cellulase active domain (GH5) and a signal peptide sequence (SignalP prediction), and has the potential for high-efficiency secretion. This gene was selected as the target cellulase gene for recombinant expression in this invention.

[0046] Example 3 This example demonstrates the construction of an engineered Bacillus subtilis strain with a transcellulase gene.

[0047] 3.1 Construction of recombinant expression vector: Based on the cellulase gene sequence (SEQ ID NO: 1) obtained in Example 2, primers were designed to introduce HindIII (5′ end) and EcoRI (3′ end) restriction sites and protective bases, and the target gene fragment was chemically synthesized; simultaneously, the pHT304 plasmid (containing P) was extracted. 43 The promoter and erythromycin resistance gene were used as the expression vector. The target gene and pHT304 plasmid were double-digested with HindIII and EcoRI, respectively. The target fragment and linearized vector were recovered. The recovered target gene fragment and linearized vector were mixed and ligated overnight using T4 DNA ligase to obtain the recombinant plasmid pHT304-Egls carrying the cellulase gene, i.e., the recombinant expression vector, whose nucleotide sequence is shown in SEQ ID NO: 3. Its structure is as follows: Figure 1 As shown, it includes the following functional elements: Promoter: P 43 The promoter (gray area) is located in the upstream region of the plasmid and is used to drive the efficient transcription of the cellulase gene.

[0048] Cellulase gene (Egls) (purple region): inserted into P 43 Downstream of the promoter, its nucleotide sequence is shown in SEQ IDNO: 1.

[0049] Erythromycin resistance gene (Erm) r(Purple area): As a selection marker, it allows screening of host strains transformed with recombinant plasmids on a medium containing erythromycin.

[0050] ori1030 replicon (blue area): confers autonomous replication ability of plasmid in Bacillus subtilis.

[0051] pMB1 replicon (yellow area): confers the ability of the plasmid to replicate in E. coli.

[0052] 3.2 Preparation of Bacillus subtilis competent cells: ① Pick a typical single colony from the freshly cultured Bacillus subtilis KC7-2 obtained and preserved in Example 1 on an LB solid plate, inoculate it into 3 mL of LB liquid medium, and incubate at 37°C and 200 r / min for 24 h; ② Take 1.5 mL of overnight culture and inoculate it into 150 mL of fresh LB medium (500 mL Erlenmeyer flask), and incubate at 37℃ and 200 r / min until OD. 600 ≈ 0.6; ③ Transfer the above bacterial culture to a 50ml centrifuge tube, incubate on ice for 15 minutes, centrifuge at 4℃, 6000rpm for 1 minute, discard the supernatant, and collect the bacterial cells; ④ Resuspend the bacterial cells in 30 mL of pre-cooled competent cell preparation buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol), incubate on ice for 15 min, centrifuge at 4 °C, 6000 rpm for 1 min, and collect the bacterial cells; ⑤ Resuspend the cells in 15 mL of the above buffer solution, incubate on ice for 15 min, centrifuge at 4°C, 6000 rpm for 1 min, and collect the bacterial cells; ⑥ Finally, resuspend the cells in 2 mL of pre-cooled competent cell preparation buffer, resulting in a cell concentration of approximately 1 × 10⁻⁶. 10 cells / ml; ⑦ Aliquot the prepared competent cells into 1.5mL sterile centrifuge tubes, 100μL per tube, freeze in liquid nitrogen at ultra-low temperature, and store at -70℃ for later use.

[0053] 3.3 Electroconversion and screening of engineered bacteria: ① Take 100 μL of competent cells, add 5 μg of the above pHT304-cel recombinant plasmid, mix well, and transfer to a pre-cooled 0.2 cm electroporation cuvette. Incubate on ice for 5 min. ② Perform electrical conversion according to the parameters of the electro-converter. The specific parameters are: voltage 2.5kV / cm, capacitance 25uF, resistance 200Ω, and electric shock time approximately 5ms. ③ After the electroshock is complete, remove the cup and immediately add 500 μL of LB solution to elute the competent cells. Add the solution to a 1.5 mL sterile centrifuge tube and incubate at 30°C and 250 rpm for 1 h. ④ Spread the revived bacterial culture onto LB agar plates containing 10 μg / mL erythromycin and incubate at 37°C for 24–48 h; ⑤ Pick a single colony and inoculate it into LB liquid medium containing 10 μg / mL erythromycin, and incubate at 30°C until OD500. 600 ≈0.6; ⑥ Take 1 mL of bacterial culture and extract genomic DNA using the Vazyme Bacterial Genomic DNA Extraction Kit (#DC103); ⑦PCR verification confirmed the successful construction of the Bacillus subtilis strain carrying the recombinant plasmid pHT304-cel.

[0054] The PCR validation system is shown in Table 1 below: Table 1

[0055] Primer sequences: Amp-YZF: 5′-CGCTGAGATAGGTGCCTCACTG-3′ (SEQ ID NO: 4); pUC57-M13RTR: 5′-TGGTCATAGCTGTTTCCTGTGTG-3′ (SEQ ID NO: 5).

[0056] The PCR reaction procedure is shown in Table 2 below: Table 2

[0057] Electrophoresis results: The PCR amplification products were analyzed by 1.0% agarose gel electrophoresis, such as... Figure 2 The image shows the PCR verification electrophoresis diagram of the recombinant Bacillus subtilis engineered strain. As can be seen from the image: lane M is the Trans2K® Plus II DNA Marker, displaying standard DNA fragments of different sizes. Lanes 1-9 correspond to the PCR products of nine transformed clones (KC7-2 transformants 1-9), respectively. A clear, specific amplified band is visible at approximately 1200 bp, consistent with the expected amplified fragment size, indicating that these transformed clones contain the target gene and that PCR amplification was successful. Lane NC, representing the untransformed wild-type Bacillus subtilis KC7-2 strain, serves as a negative control. Lane NC shows no amplified band, demonstrating that the target fragment will not be amplified without the recombinant plasmid. This proves that the recombinant plasmid has been successfully introduced into the Bacillus subtilis host cell, and the engineered strain has been successfully constructed.

[0058] In summary, through specific PCR verification, a recombinant engineered bacterium stably expressing cellulase was screened and named Bacillus subtilis NJAU-Li2025, which will be used for subsequent experiments. This strain was deposited at the China Center for Type Culture Collection on May 6, 2025, with accession number CCTCC NO: M 2025957, and the address of the depository is No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0059] Example 4 This embodiment verifies the characteristics and cellulose degradation activity of the recombinant Bacillus subtilis engineered strain of the present invention.

[0060] 4.1 Colony morphology and physiological characteristics: (1) Colony morphology and microscopic characteristics After incubating the engineered strain NJAU-Li2025 on LB agar plates containing 10 μg / mL erythromycin at 37°C for 20 h, it formed irregular colonies with a rough, opaque, dirty white or slightly yellow surface (e.g., Figure 3 As shown in the figure, this is a typical Bacillus subtilis morphology.

[0061] Gram staining and microscopic examination of the strain revealed that the cells were blue-purple, indicating Gram-positive bacteria. The cells were short rod-shaped, with some cells showing oval-shaped spores in the center, consistent with the typical cytological characteristics of Bacillus subtilis (e.g., ...). Figure 4 (As shown).

[0062] To verify its genetic stability, the engineered bacteria were inoculated into LB solid medium containing 10 μg / mL erythromycin using a sterile inoculation loop with a three-zone streak method and cultured at 37℃ for 20 h. The strain was still able to grow normally and form typical colonies, indicating that it has good genetic stability under selective pressure.

[0063] (2) Growth characteristics analysis To evaluate the growth kinetics of the engineered bacteria, its growth curve was measured. The specific method is as follows: Twelve hours prior to inoculation, a single colony of the engineered bacteria was inoculated into LB liquid medium containing 10 μg / mL erythromycin and cultured at 37°C with shaking at 200 rpm for 12 hours to obtain the seed culture. The seed culture was then inoculated into fresh LB liquid medium, and the initial OD was measured. 600 Adjust the concentration to approximately 0.005, and incubate continuously at 37℃ and 200 r / min. Take samples every 1-2 hours to measure the OD value. 600 Values, with incubation time as the x-axis and OD... 600 Plot a growth curve on the ordinate (e.g.) Figure 5 (As shown).

[0064] from Figure 5 As can be seen, the engineered bacteria grew well, entered the logarithmic growth phase after a lag phase of about 4 hours, and reached the stationary phase in 12-18 hours. The overall growth trend was similar to that of wild-type Bacillus subtilis, indicating that the insertion of the exogenous gene did not significantly affect its basic growth characteristics.

[0065] (3) Acid resistance test To assess the survival ability of this engineered bacterium in a low pH environment (simulating gastric acid), an acid resistance test was conducted. The specific method is as follows: Twelve hours prior to inoculation, single colonies of the engineered bacteria were inoculated into LB liquid medium containing 10 μg / mL erythromycin and cultured at 37°C with shaking at 200 rpm for 12 hours to obtain seed culture. The engineered bacteria seed culture was then inoculated at a 1% (v / v) inoculation rate into LB liquid medium at pH 3.0, 4.0, 5.0, and 6.0 (pH adjusted with HCl), and cultured at 37°C with shaking at 200 rpm. After incubation, samples were taken for dilution and plate counting (e.g., ...). Figure 6 (As shown).

[0066] from Figure 6 As can be seen, the recombinant Bacillus subtilis engineered bacteria of this invention can still maintain a certain survival rate under pH 4.0 conditions, and a certain number of live bacteria still exist; indicating that the engineered bacteria have good acid resistance and have the potential to be used as probiotics or feed additives in the animal digestive tract.

[0067] 4.2 Verification of cellulose degradation activity: To verify whether the engineered bacterium NJAU-Li2025 has the ability to degrade cellulose, its cellulase production activity was preliminarily identified using the Congo Red Staining Method.

[0068] The specific procedures are as follows: 24 hours in advance, inoculate a single colony of the engineered bacteria onto an LB agar plate containing 10 μg / mL erythromycin and incubate at 37°C for activation. Using a sterile inoculation loop, pick an equal amount of bacterial cells and inoculate them in the center of a cellulose Congo red agar plate (CMC-Na medium) using the spot inoculation method. Invert the inoculated plate in a 37°C incubator and incubate for 48 hours to allow the strain to grow fully and secrete cellulase. After incubation, add 1 mg / mL Congo red solution to the plate and stain at room temperature for 10-15 minutes. Carefully discard the stain and wash the plate 2-3 times with 1 mol / L NaCl solution for 5 minutes each time to remove any Congo red dye that has not bound to cellulose. Observe the plate against a white background. If the strain produces cellulase, the secreted enzyme will hydrolyze the CMC-Na in the medium, forming a transparent hydrolysis zone. Measure the colony diameter (D) and the diameter of the hydrolysis zone (H). The larger the H / D ratio, the stronger the strain's cellulase production ability and the higher its cellulose degradation activity.

[0069] Figure 7 This image shows the growth and hydrolysis zone formation of the engineered Bacillus subtilis strain on CMC-Na solid medium. As can be seen from the image, the engineered bacterial colonies are round with neat edges, smooth surfaces, and are white or pale yellow in color. A clear, transparent hydrolysis zone appears around the colonies, and the diameter of the hydrolysis zone is significantly larger than the colony diameter. This indicates that the cellulase secreted by the strain can efficiently degrade the surrounding CMC-Na, demonstrating the strain's strong cellulose degradation ability. This provides strong support for further research on the application of the engineered bacteria in cellulose degradation and other related fields.

[0070] Example 5 This embodiment systematically evaluates the cellulase secretion and activity changes of the recombinant Bacillus subtilis engineered strain and Bacillus subtilis KC7-2 at different fermentation time points by preparing fermentation broth samples, preparing cellulase extracts, and detecting cellulase activity.

[0071] Fermentation broth sample preparation: Engineered bacteria NJAU-Li2025 and Bacillus subtilis KC7-2 were inoculated into MRS liquid fermentation medium containing forage samples, with each parallel sample having a volume of 1L and 3 parallel samples were set up; the fermentation system was placed in a constant temperature shaker at 37℃ and 120r / min for 72h; during the fermentation process, samples were taken at 0h, 12h, 24h, 36h, 48h, 60h, and 72h, respectively. After each sampling, the fermentation broth was thoroughly stirred and filtered through several layers of coarse gauze to remove forage residue, and the filtered fermentation broth sample was obtained.

[0072] Preparation of cellulase extract: The fermentation broth sample was filtered through four layers of gauze to remove residue. The filtrate was processed according to the instructions of the cellulase detection kit (Shanghai Yuanye Biotechnology Co., Ltd.). The specific steps are as follows: The filtrate was centrifuged at 3000 r / min for 10 min, and 10 mL of the supernatant was added to a 50 mL volumetric flask. Water was added to bring the volume to the mark to obtain the cellulase extract, which was used for the detection of cellulase activity in the sample.

[0073] Cellulase activity assay: The cellulase activity of the above samples was detected using the Yuanye Cellulase Assay Kit (DNS microplate method). Separate test tubes and control tubes were prepared. 0.1 mL of cellulase extraction solution was added to the test tubes, and 0.1 mL of CES Assay buffer and 0.3 mL of CMC solution were added to the control tubes as substrates. After mixing, the test and control tubes were incubated in a 60°C water bath for 20 min to initiate the enzymatic reaction. After the reaction was complete, 0.3 mL of DNS reagent was quickly added to each tube to terminate the reaction. 0.1 mL of enzyme extraction solution was added to the control tube as a negative control. After mixing, the control and test tubes were heated in a boiling water bath for 5 min to allow the reducing sugar to react with the DNS reagent for colorimetric reaction. After cooling, 280 μL of each tube was sequentially transferred to a 96-well plate. The plate was zeroed using tube "0", and the absorbance of each tube was measured at 540 nm using a microplate reader.

[0074] Preparation of standard curve: Prepare glucose standard solutions (0 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL). According to the kit instructions, add an appropriate amount of DNS reagent to each tube of standard solution, mix well, and heat in a boiling water bath for 6 min. After cooling, measure the absorbance value at a wavelength of 540 nm. Plot the standard curve with glucose concentration (μg / mL) as the x-axis and absorbance value as the y-axis.

[0075] Based on the standard curve, calculate the corresponding glucose concentrations C1 and C0 (unit: μg / mL) from the absorbance values ​​of the test tube and control tube, respectively. Then, according to the kit definition, one unit of cellulase activity is defined as: 1 μg of glucose produced by the hydrolysis of sodium carboxymethyl cellulose by 1 mL of enzyme extract within 1 min at 60°C. Calculation formula: U=k×(C1-C0) / t Where k is a constant and t is the reaction time (min).

[0076] like Figure 8The figure shows the cellulase activity curves of the engineered strain NJAU-Li2025 and Bacillus subtilis KC7-2 at different time points in the fermentation degradation experiment. As can be seen from the figure: the cellulase activity of the engineered strain NJAU-Li2025 reached its peak of 118.4 U at 36 h of growth, then slowly decreased, stabilizing at around 50 U at 72 h; the cellulase activity of Bacillus subtilis KC7-2 reached its peak of 75 U at 24 h of growth, then slowly decreased, stabilizing at around 40 U at 72 h. Therefore, the cellulase activity of NJAU-Li2025 was significantly higher than that of KC7-2 throughout the fermentation process, indicating that NJAU-Li2025 exhibits superior cellulase activity, especially in the mid-fermentation stage, demonstrating better performance than Bacillus subtilis KC7-2 and making it suitable for more efficient degradation of cellulose materials.

[0077] Example 6 This embodiment systematically evaluates the hemicellulase secretion and activity changes of the recombinant Bacillus subtilis engineered strain and Bacillus subtilis KC7-2 at different fermentation time points by preparing fermentation broth samples, preparing hemicellulase extracts, and detecting hemicellulase activity.

[0078] Fermentation broth sample preparation: The engineered bacteria NJAU-Li2025 and Bacillus subtilis KC7-2 of the present invention were inoculated into MRS liquid fermentation medium containing forage samples. Each parallel sample had a volume of 1L, and 3 parallel samples were set up. The fermentation system was placed in a constant temperature shaker at 37℃ and 120r / min for 72h. Samples were taken at 0h, 12h, 24h, 36h, 48h, 60h, and 72h. After each sampling, the samples were thoroughly stirred and filtered through several layers of coarse gauze to remove forage residue, and the filtered fermentation broth sample was obtained.

[0079] Preparation of hemicellulase extract: The fermentation broth sample was centrifuged at 8000g and 4℃ for 15min. After centrifugation, the supernatant was collected to obtain the hemicellulase extract, which was used as the sample to be tested.

[0080] Hemicellulase activity assay: The hemicellulase activity of the above-mentioned samples was detected using a hemicellulase activity assay kit (Shanghai Baishengyue Biotechnology Co., Ltd.). The specific steps are as follows: Set up test tubes and control tubes respectively. Add 0.2 mL of the sample to the test tube and control tube respectively, and then add 0.3 mL of buffer solution as substrate. Add 0.1 mL of reagent one (containing xylan) from the kit to the test tube, mix well, and incubate the reaction tube in a 50℃ water bath for 15 min. Add 0.1 mL of reagent one as a negative control to the control tube. Finally, add 0.3 mL of DNS reagent to the reaction tube to terminate the reaction, and heat the reaction tube in a boiling water bath for 5 min to allow the reducing sugar to react with the DNS reagent to produce a colorimetric reaction. After cooling to room temperature, use a spectrophotometer to measure the absorbance (A) at a wavelength of 540 nm. Calculate ΔA = A_test - A_control. Set up one control tube for each test tube.

[0081] The standard curve equation given in the instruction manual is: y = 2.5554x - 0.002, where R² = 0.9983.

[0082] Formula for calculating hemicellulase activity: According to the kit definition, the unit of hemicellulase activity is: the amount of enzyme required to break down hemicellulose to produce 1 nmol of reducing sugar per minute per milliliter of liquid sample at 50°C and pH 4.8 is one unit of hemicellulase activity.

[0083] The formula for calculating enzyme activity is: Hemicellulase activity (nmol / min / mL) = 435 × (ΔA + 0.002), Where ΔA is the absorbance of the sample minus the absorbance of the blank control.

[0084] like Figure 9 The figure shows the hemicellulase activity curves of the engineered strain NJAU-Li2025 and Bacillus subtilis KC7-2 at different time points during the fermentation degradation experiment. As can be seen from the figure, the hemicellulase activity of the engineered strain NJAU-Li2025 reached a peak of 315.5 nmol / min / mL at 36 h, then stabilized at around 165 nmol / min / mL at 72 h. Similarly, the hemicellulase activity of Bacillus subtilis KC7-2 reached a peak of 300 nmol / min / mL at 24 h, then slowly decreased, stabilizing at around 120 nmol / min / mL at 72 h. Therefore, it is evident that the hemicellulase activity of NJAU-Li2025 was consistently higher than that of KC7-2 throughout the fermentation process, especially showing a significant advantage in the middle stage of fermentation (36-72 h). This indicates that NJAU-Li2025 can maintain higher enzyme activity over a longer fermentation period, exhibiting better stability and persistence.

[0085] Example 7 This embodiment describes the ability of the recombinant Bacillus subtilis engineered strain of the present invention to degrade cellulose and hemicellulose in forage samples during fermentation using the differential method.

[0086] (1) Preparation of fermentation broth samples The engineered strain NJAU-Li2025 of the present invention was inoculated into MRS liquid fermentation medium containing forage samples, with each parallel sample having a volume of 1L and 3 parallel samples were set up; the fermentation system was placed in a constant temperature shaker at 37℃ and 120 r / min for 72h; samples were taken at the beginning of fermentation (0h) and the end of fermentation (72h), and the samples were thoroughly stirred after each sampling and collected for subsequent detection.

[0087] (2) Detection of cellulose and hemicellulose content Reagent kits: Cellulose content test kit (Beijing Solarbio Science & Technology Co., Ltd.), Hemicellulose content test kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0088] ① Determination of cellulose content: Fermentation broth treatment: Filter the fermentation broth through filter paper or gauze to separate the solid fermentation substrate (residue) and the liquid portion (filtrate). Retain the solid fermentation substrate and discard the filtrate. Place the separated fermentation residue in a drying device and dry it at 40°C to constant weight. Cool it to room temperature and store it properly as a sample for subsequent analysis.

[0089] Preparation of coarse cell walls: Weigh approximately 0.3g (W1) of sample, add 1mL of extraction buffer I, and homogenize rapidly at room temperature; place the sample in a 90°C water bath and heat for 20min (cover with sealing film to prevent cap bursting), cool to room temperature, centrifuge at 6000g, 25°C for 10min, and discard the supernatant; wash the precipitate twice each with 1.5mL of extraction buffer I and acetone, in the order of extraction buffer I-acetone-extraction buffer I-acetone, then shake for 2min, centrifuge at 6000g, 25°C for 10min, and discard the supernatant. The precipitate is the coarse cell wall.

[0090] Preparation of cell wall material (CWM): Add 1 mL of extraction solution II to the above-mentioned crude cell wall, soak for 15 h, centrifuge at 6000 g, 25 °C for 10 min, and discard the supernatant; wash the obtained precipitate twice with distilled water (vortex for about 2 min each time, then centrifuge at 6000 g, 25 °C for 10 min, and discard the supernatant); dry the precipitate at 60 °C to obtain cell wall material (CWM), weigh it and record the mass as W2.

[0091] Extraction and determination of cellulose: Weigh 5 mg (W3) of dried CWM, add 0.5 mL of distilled water, and homogenize thoroughly; transfer the homogenate to an EP tube and bring the volume to 0.5 mL with distilled water; place the homogenate in an ice-water mixture, slowly add 0.75 mL of concentrated sulfuric acid, and slowly mix. Let it stand in an ice-water bath for 30 min, centrifuge at 8000 g and 4℃ for 10 min, take the supernatant, and dilute the supernatant 20 times with distilled water to obtain the test solution.

[0092] Prepare glucose standard solutions (0.09 mg / mL, 0.08 mg / mL, 0.07 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, 0.00625 mg / mL) according to the kit instructions. Set up test tubes, standard tubes, and blank tubes. Add 0.3 mL of the sample to be tested to the test tube, 0.3 mL of the standard solution to the standard tube, and 0.3 mL of distilled water to the blank tube. Add 0.07 mL of working solution and 0.63 mL of concentrated sulfuric acid to all reaction tubes, mix well, and place in a 95°C water bath for color development. After cooling to room temperature, measure the absorbance (A) at 620 nm using a spectrophotometer. Calculate ΔA = Atest - Ablank, ΔAstandard = Astandard - Ablank. Set up a control tube for each test tube.

[0093] Cellulose content calculation: Plot a standard curve with glucose concentration (μg / mL) on the x-axis and absorbance value on the y-axis. Measure the absorbance value of the diluted test solution and substitute it into the standard curve to calculate the corresponding glucose concentration x (mg / mL).

[0094] The cellulose content of the forage sample with added Bacillus subtilis was calculated at 0h and 72h according to the following formula.

[0095] Cellulose (mg / g mass) = x × V 提取 ×20× ÷1.11=22.52×x× ÷W1 like Figure 10The figure shows the changes in cellulose content in forage samples at 0 h and 72 h during the fermentation degradation experiment using the engineered bacteria. As can be seen from the figure, at 0 h (the start of fermentation), the cellulose content in the forage sample was 175.20 mg / g dry weight; after 72 h of fermentation, the cellulose content in the forage sample decreased to 63.43 mg / g dry weight. Bacillus subtilis significantly reduced the cellulose content in the forage at 72 h, with a degradation rate of 63.79%. This demonstrates that the engineered bacteria NJAU-Li2025 of this invention has a highly efficient cellulose degradation capacity, significantly reducing the cellulose content in forage in a short time, thereby improving the nutritional value and digestibility of forage.

[0096] ② Hemicellulose content determination: Sample preparation: Take 0.05g of the sample to be tested, add 1mL of 80% ethanol to the test tube, and mix thoroughly; heat the mixture in a 90℃ water bath for 10min, then centrifuge at 8000g for 10min, and retain the precipitate; add 1mL of distilled water to the precipitate, mix again, centrifuge at 8000g for 10min, discard the supernatant, repeat this step three times, and finally take the precipitate and dry it to constant weight to obtain the sample to be tested in the test tube.

[0097] Sample pretreatment: Grind the dried sample to constant weight thoroughly and pass it through a 30-50 mesh sieve to ensure that the sample is uniform.

[0098] Preparation of standard solutions and setup of test tubes: According to the kit instructions, prepare a series of D-xylose standard solutions (2.5 mg / mL, 2 mg / mL, 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL) as standard tubes; set up test tubes, standard tubes and blank tubes respectively.

[0099] Specific operating steps: Add 0.5 mL of 80% ethanol to each of the test tube and blank tube, heat in a 90℃ water bath for 60 min, and allow to cool naturally to room temperature; add 0.5 mL of extraction solution II, mix thoroughly, centrifuge at 8000 g for 10 min, and collect the supernatant for testing; add 0.125 mL of the corresponding supernatant to each of the test tube and blank tube, and add 0.125 mL of standard solution to each standard tube. Add 0.125 mL of reagent II and 0.75 mL of distilled water to all reaction tubes, vortex to mix, and heat in a 90℃ water bath for 5 min, then allow to cool naturally. Measure the absorbance (A) at a wavelength of 540 nm using a spectrophotometer, and calculate ΔA = Atest - Ablank and ΔAstandard = Astandard - Ablank. Set up a control tube for each test tube.

[0100] Hemicellulose content calculation: A standard curve was plotted with D-xylose concentration (y, mg / mL) on the x-axis and absorbance value on the y-axis. The absorbance value of the diluted test solution was substituted into the standard curve to calculate the corresponding D-xylose concentration x (mg / mL). The hemicellulose content of the forage sample supplemented with Bacillus subtilis of this invention at 0h and 72h was calculated using the following formula: Hemicellulose (mg / g dry weight) = y × V_total sample ÷ W × F = y ÷ W × F Vtotal: Volume of extraction solution added (1 mL); W: Sample mass (g); F: Dilution factor.

[0101] like Figure 11 The figure shows the changes in hemicellulose content in forage samples at 0 h and 72 h during the fermentation degradation experiment using the engineered bacterium NJAU-Li2025. As can be seen from the figure, at 0 h (the start of fermentation), the hemicellulose content in the forage sample was 381.65 mg / g dry weight; after 72 h of fermentation, the hemicellulose content decreased to 184.10 mg / g dry weight. This indicates that the engineered bacterium significantly reduced the hemicellulose content in forage within 72 h, with a degradation rate of 51.76%. This demonstrates that the engineered bacterium NJAU-Li2025 of this invention has a highly efficient hemicellulose degradation capacity, significantly reducing the hemicellulose content in forage within a short period, which helps improve resource utilization efficiency in forage and further enhances its nutritional value and digestibility.

[0102] The foregoing has provided a detailed description of the recombinant Bacillus subtilis engineered strain capable of simultaneously degrading cellulose and hemicellulose, its construction method, and its applications. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of these embodiments are merely illustrative of the methods and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of this invention.

Claims

1. A recombinant Bacillus subtilis engineered bacterium capable of simultaneously degrading cellulose and hemicellulose, characterized in that, The engineered strain was obtained by genetic engineering of Bacillus subtilis KC7-2 with accession number CCTCC NO: M 20241570. It carries a recombinant expression vector containing a cellulase gene derived from strain KC7-2, which can efficiently express cellulase and synergistically degrade hemicellulose during fermentation. The engineered strain is classified as Bacillus subtilis NJAU-Li2025 and was deposited at the China Center for Type Culture Collection on May 6, 2025, with accession number CCTCC NO: M 2025957.

2. The recombinant Bacillus subtilis engineered strain according to claim 1, characterized in that, The wild-type parent strain of Bacillus subtilis is Bacillus subtilis KC7-2, which is isolated from the rumen contents of yak and has a strong cellulose degradation ability. Its preservation number is CCTCC NO: M 20241570.

3. The recombinant Bacillus subtilis engineered strain according to claim 1, characterized in that, The cellulase gene has the nucleotide sequence shown in SEQ ID NO:

1.

4. The recombinant Bacillus subtilis engineered strain according to claim 3, characterized in that, The protein encoded by the cellulase gene has the amino acid sequence shown in SEQ ID NO:

2.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the cellulase gene as described in claim 3 or 4 and is operatively linked to a promoter capable of initiating transcription in Bacillus subtilis; the recombinant expression vector is a recombinant plasmid constructed based on the pHT304 plasmid.

6. The recombinant expression vector according to claim 5, characterized in that, The promoter is P, which is inherent in the pHT304 plasmid. 43 Promoter; the pHT304 plasmid also contains the erythromycin resistance gene (Erm). r () is used as a filter tag.

7. The recombinant expression vector according to claim 6, characterized in that, The recombinant expression vector has the nucleotide sequence shown in SEQ ID NO:

3.

8. A method for constructing recombinant Bacillus subtilis engineered bacteria, characterized in that, Includes the following steps: (1) Preparation of competent cells of Bacillus subtilis KC7-2: The bacterial culture was cultured to OD 600 ≈ 0.6, after ice bath, wash with buffer containing 0.5 M sorbitol, 0.5 M mannitol and 10% glycerol; (2) The recombinant expression vector described in claim 5, 6 or 7 is introduced into the competent cells of Bacillus subtilis in step (1) by electroporation. The electroporation parameters are: voltage 2.5 kV / cm, capacitance 25 μF, and resistance 200 Ω. (3) After transformation, the bacteria were thawed at 30℃ for 1 hour, spread on LB plates containing 10 μg / mL erythromycin, and cultured for 48 hours. Single clones were picked and the recombinant engineered bacteria were obtained by PCR verification.

9. The application of the recombinant Bacillus subtilis engineered strain according to any one of claims 1-4 or the recombinant expression vector according to any one of claims 5-7 in the degradation of forage, straw or plant fiber materials.

10. The use of the recombinant Bacillus subtilis engineered strain according to any one of claims 1-4 in the preparation of animal feed additives or probiotic preparations.

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