Method for improving secretory expression of expansin by bacillus subtilis and application thereof

Abstract

The invention discloses a method for improving secretory expression of expansin by bacillus subtilis and application of the method, and belongs to the technical field of biology. According to the invention, bacillus subtilis G600 capable of successfully secreting and expressing the expansin BsEXLX1 from bacillus subtilis is firstly constructed, the expression level of expansin protein is enhanced through a strong promoter P21 in the strain, and then the most suitable RBS and signal peptide combination is screened, so that the efficient secretory expression of the expansin is optimized, and finally the secretory expression level of the BsEXLX1 is improved by 3.5 times. The transformation method provided by the invention can promote the secretory expression of the expansin, lays a foundation for promoting the biosynthesis of the expansin by transforming bacillus subtilis in metabolic engineering, and has a very good application prospect. The expansin produced by the recombinant strain constructed by the invention is stable in protein expression and high in extracellular secretion amount, the construction method is simple and convenient to use, and a foundation is laid for further large-scale industrial production of expansin.

Description

Technical Field

[0001] The invention relates to a method for improving the secretion and expression of expansin by Bacillus subtilis and application thereof, belonging to the field of biotechnology. Background Art

[0002] Expansins are a family of proteins that can induce plant cell wall relaxation and play a key role in cell wall expansion. In recent years, they have shown broad application prospects in agricultural and industrial production. In agricultural production, understanding the mechanism of action of expansins is expected to produce higher-yielding and more stress-resistant crops, increasing food production to meet growing global demand. In industry, the unique effect of expansins on cellulose can provide new ideas for biomass energy conversion and papermaking process improvements, reducing production costs and improving resource utilization. In summary, expansins have become a hot topic in multidisciplinary research in biotechnology and agronomy, and in-depth research on them has broad application prospects.

[0003] Currently, expansin, a difficult protein to secrete and express, is primarily obtained through bioextraction and microbial fermentation. Bioextraction methods suffer from low yields and difficult purification methods, limiting large-scale production and application. Microbial fermentation, on the other hand, offers milder production conditions and higher production efficiency, offering broad application prospects. Studies have demonstrated heterologous expression of expansin using Pichia pastoris as a host, achieving certain yields. However, Pichia pastoris fermentation uses methanol as a carbon source, and methanol metabolism produces numerous byproducts that may negatively impact Pichia growth and metabolism. Furthermore, methanol, a toxic chemical, poses safety risks in industrial applications. Furthermore, Pichia pastoris cultivation requires a long time, resulting in high fermentation costs. Bacillus subtilis, with its clear genetic background, mature genetic manipulation techniques, rapid growth rate, and absence of endotoxins, is also highly environmentally tolerant compared to fungi and can be grown in inexpensive culture media, resulting in low costs. This makes it an ideal strain for efficient expansin production. There are currently a few reports on the production of expansin by Bacillus subtilis, but none of them have explored the path to increase the secretion of expansin in Bacillus subtilis. Summary of the Invention

[0004] To solve the above problems, the present invention is not limited to single-factor optimization, but takes into account the interaction between expression elements, especially the compatibility between RBS and signal peptide, which is one of the key factors affecting protein expression efficiency and secretion success. The use of a suitable combination of RBS and signal peptide may be able to coordinate the translation rate and secretion efficiency of the expansin protein during expression, thereby solving the multidimensional problems of expansin protein translation and secretion, and thus achieving high efficiency of expansin during secretory expression.

[0005] The first object of the present invention is to provide a method for increasing the secretion and expression of expansin by Bacillus subtilis, comprising the steps of overexpressing an expansin gene expression cassette in a Bacillus subtilis host strain,

[0006] The expansin gene expression frame includes an expansin coding gene sequence and an RBS coding sequence and a signal peptide coding sequence located upstream (before) of the expansin coding gene sequence. The signal peptide coding sequence is located between the RBS coding sequence and the expansin coding gene sequence. The RBS coding sequence is shown in SEQ ID NO.6, and the signal peptide coding sequence is shown in SEQ ID NO.11.

[0007] Furthermore, it contains at least one of the following characteristics:

[0008] (1) The amino acid sequence of expansin is shown in SEQ ID NO. 1;

[0009] (2) The expansin encoding gene sequence is shown in SEQ ID NO.2.

[0010] Furthermore, it contains at least one of the following characteristics:

[0011] (1) The expansin gene expression frame further contains a promoter for initiating gene expression, and the promoter is a constitutive promoter;

[0012] The constitutive promoter includes promoter P 21 ;

[0013] The promoter P 21 The nucleotide sequence is shown in SEQ ID NO.3;

[0014] (2) The Bacillus subtilis host bacteria includes Bacillus subtilis G600.

[0015] Furthermore, it contains at least one of the following characteristics:

[0016] (1) The overexpression is to increase the number of copies of at least one gene expression cassette;

[0017] (2) The overexpression method is free expression or integrated expression;

[0018] When the overexpression method is episomal expression, the expression vector of the expansin gene expression cassette includes pHT-01 plasmid.

[0019] Furthermore, in an embodiment of the present invention, the RBS coding sequence and the signal peptide coding sequence are connected by enzyme cleavage; and the signal peptide coding sequence and the expansin coding gene sequence are connected by enzyme cleavage.

[0020] Furthermore, the expansin encoding gene sequence and the green fluorescent protein encoding gene are connected via a flexible linker. The flexible linker conforms to the general formula (GGGGS)n, where n is an integer between 1 and 10. The linker used in the embodiment of the present invention is a linker with n=1.

[0021] A second object of the present invention is to provide a recombinant Bacillus subtilis for (efficiently) secreting and expressing expansin, wherein the recombinant Bacillus subtilis is modified using a Bacillus subtilis host strain as a starting strain, and the modification comprises:

[0022] An overexpression swollenin gene expression cassette comprises an swollenin encoding gene sequence and an RBS encoding sequence and a signal peptide encoding sequence located upstream of the swollenin encoding gene sequence, wherein the signal peptide encoding sequence is located between the RBS encoding sequence and the swollenin encoding gene sequence, the RBS encoding sequence is shown in SEQ ID NO.6, and the signal peptide encoding sequence is shown in SEQ ID NO.11.

[0023] The third object of the present invention is to provide a method for constructing the recombinant Bacillus subtilis, comprising the following steps:

[0024] A gene expression cassette comprising a promoter, an RBS shown in SEQ ID NO.6, a signal peptide shown in SEQ ID NO.11 and an expansin encoding gene shown in SEQ ID NO.2 was constructed and transformed into a Bacillus subtilis G600 competent cell to obtain the recombinant Bacillus subtilis.

[0025] The fourth object of the present invention is to provide the use of the recombinant Bacillus subtilis in the preparation of expansin.

[0026] A fifth object of the present invention is to provide a method for producing expansin, comprising the step of fermentation production using the recombinant Bacillus subtilis.

[0027] Furthermore, the fermentation temperature is 35-38°C.

[0028] Furthermore, the fermentation includes shake flask fermentation or fed-batch fermentation.

[0029] Furthermore, the fermentation comprises the following steps: inoculating the recombinant Bacillus subtilis into a seed culture medium to obtain a seed liquid, and inoculating the seed liquid into a fermentation culture medium for fermentation.

[0030] Furthermore, the culture was performed at 180-280 rpm.

[0031] Furthermore, the seed liquid inoculation amount is 1-10% (v / v).

[0032] Furthermore, the seed culture medium contains the following components: 3-10 g / L peptone, 5-15 g / L yeast powder, and 5-15 g / L NaCl.

[0033] Furthermore, the fermentation medium contains the following components: peptone 10-15 g / L, yeast powder 20-30 g / L, glycerol 2-5 mL / L, KH2PO4 2.0-2.5 g / L, and K2HPO4 12-13 g / L.

[0034] Beneficial effects of the present invention:

[0035] The present invention has developed a method for promoting the secretion of expansin by Bacillus subtilis. First, an expansin-expressing strain was constructed. Then, through screening of a ribosome binding site (RBS) and a signal peptide combination, the secretion expression of expansin was successfully increased by 3.5 times. The present invention not only effectively increases the extracellular secretion level of the protein but also simplifies the subsequent separation and purification process, thereby reducing the cost of the production process and is expected to be used for subsequent large-scale fermentation production. The recombinant strain constructed by the present invention is simple to prepare, easy to use, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the construction of recombinant expression plasmid and RBS-signal peptide screening plasmid.

[0037] Figure 2 The figure is SDS-PAGE diagram of recombinant protein expression.

[0038] Figure 3 Schematic diagram of the RBS-signal peptide screening principle.

[0039] Figure 4 Figure 2 is the fluorescence intensity diagram of droplet in droplet microfluidics.

[0040] Figure 5 This is a flow cytometry screening chart.

[0041] Figure 6 This is the fluorescence intensity distribution diagram of the RBS-signal peptide combination multiple screening.

[0042] Figure 7 The fluorescence diagram of the recombinant strains containing different RBS-signal peptide combinations.

[0043] Figure 8 The graph shows the yield of recombinant strains containing different RBS-signal peptide combinations. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0045] The materials involved in the present invention are as follows:

[0046] Seed culture medium: peptone 5g / L, yeast powder 10g / L, NaCl 10g / L.

[0047] Fermentation medium: peptone 10 g / L, yeast powder 20 g / L, glycerol 4 mL / L, KH2PO4 2.2 g / L, K2HPO4 12 g / L.

[0048] The sequence involved in the present invention is as follows:

[0049] SEQ ID NO.1: BsEXLX1 amino acid sequence

[0050] MKKIMSAFVGMVLLTIFCFSPQASAAYDDLHEGYATYTGSGYSGGAFLLDPIPSDMEITAINPADLNYGGVKAALAGSYLEVEGPKGKTTVYVTDLYPEGARGALDLSPNAFRKIG NMKDGKINIKWRVVKAPITGNFTYRIKEGSSRWWAAIQVRNHKYPVMKMEYEKDGKWINMEKMDYNHFVSTNLGTGSLKVRMTDIRGKVVKDTIPKLPESGTSKAYTVPGHVQFPE

[0051] SEQ ID NO.2: BsEXLX1 nucleotide sequence

[0052] ATGAAAAAGATCATGAGTGCATTTGTTGGTATGGTTTTGTTGACGATCTTCTGTTTTTCCCCGCAAGCTTCGGCAGCATATGACGACCTGCATGAAGGTTATGCAACGTATACAGGGTCAGGCTATTCAGGAGGAGCTTTCCTGCTGGATCCCATTCCTTCCGATATGGAGATTACTGCAATAAATCCGGCGGATCTCAATTACGGAGGAGTAAAAGCGGCACTTGCCGGCTCTTATTTGGAAGTTGAAGGGCCAAAAGGGAAAACAACCGTATATGTTACTGATCTTTATCCCGAAGGCGCTCGGGGAGCTCTTGATCTGTCACCTAATGCCTTCCGTAAAATCGGCAATATGAAAGACGGAAAAATCAATATTAAATGGCGTGTTGTCAAAGCCCCAATCACCGGCAATTTCACGTACCGGATCAAAGAAGGCAGCAGCAGGTGGTGGGCAGCAATCCAAGTCAGAAATCACAAGTATCCTGTTATGAAAATGGAATATGAAAAGGATGGTAAGTGGATCAACATGGAGAAAATGGACTATAACCATTTTGTGAGTACGAATTTAGGTACTGGCTCTCTCAAAGTCAGAATGACTGACATCCGCGGAAAAGTTGTGAAAGACACCATTCCAAAGCTGCCTGAAAGCGGAACGTCCAAAGCCTATACAGTACCGGGCCATGTTCAGTTTCCTGAATAA

[0053] SEQ ID NO.3: Promoter P 21

[0054] TGCGGAAGTAAACGAAGTGTACGGACAATATTTTGACACTCACAAACCGGCGAGATCTTGTGTTGAAGTCGCGAGACTCCCGAAGGATGCGTTAGTCGAGATCGAAGTTATTGCACTGGTGAAATAATAAGAAAAGTGATTCTGGGAGAGCCGGGATCACTTTTTTATTTACCTTATGCCCGAAATGAAAGCTTTATGACCTAATTGTGTAACTATATCCTATTTTTTCAAAAAATATTTTAAAAACGAGCAGGATTTCAGAAAAAATCGTGGAATTGATACACTAATGCTTTTATATAG

[0055] SEQ ID NO.4: RBS1

[0056] AAGGAGGAATCAC

[0057] SEQ ID NO.5: RBS2

[0058] AAGGAGGTATCAC

[0059] SEQ ID NO.6: RBS3

[0060] CAGGAGGAATCAC

[0061] SEQ ID NO.7: RBS4

[0062] CAGGAGGTATCAC

[0063] SEQ ID NO.8: Signal peptide CiTH

[0064] MGNTRKKVSVIGAGFTGATTAFLIAQKELA

[0065] SEQ ID NO.9: Signal peptide YojL

[0066] MKKKIVAGLAVSAVVGSSMAAAPAEA

[0067] SEQ ID NO.10: Signal peptide YpcP

[0068] MNNNKLLLVDGMALLFRAFFATAVH

[0069] SEQ ID NO.11: Signal peptide AspB

[0070] MKLAKRVSALTPSTTLAITAKA

[0071] SEQ ID NO.12: Signal peptide yobV

[0072] MKLERLLAMVVLLISKKQVQAAELA

[0073] Example 1: Expansin recombinant expression plasmid pHT-P 21 -Build of BsEXLX1

[0074] like Figure 1 As shown, the constructed expansin expression cassette consists of the BsEXLX1 gene and 6×His tag 2. The amino acid sequence of BsEXLX1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. 21 The nucleotide sequence is shown in SEQ ID NO. 3. The recombinant protein can be purified using a nickel column via the 6×His tag.

[0075] In order to enhance the expression of expansin in B. subtilis, the commercial plasmid pHT-01 vector was used, and the σ Η promoter P 21 To express expansin, the recombinant expression plasmid can be constructed smoothly and expressed stably and at high intensity in B. subtills. 21 After being connected to the plasmid vector pHT-01 together with the gene elements, the vector pHT-P was obtained. 21 -BsEXLX1. After the above plasmid was transformed into the B. subtilis G600 strain, the engineered strain BE-0 that can express expansin was obtained. The SDS-PAGE diagram of its secretion expression is shown in the figure below. Figure 2 shown.

[0076] Example 2: Construction of Expansin RBS-Signal Peptide Screening Plasmid

[0077] The SCORE method was used to screen the RBS-signal peptide combination suitable for secretory expression of expansin, where the plasmid construction schematic is shown in the figure. Figure 1 As shown, the principle of this method (such as Figure 3As shown in the figure, the "insert site" includes two parts, namely "RBSLibiary" and "173Sec Signal peptides libiary"), which is a superfolder green fluorescent protein (sfGFP) fused to the C-terminus of the expansin protein sequence. The secretion of expansin is characterized by the fluorescence intensity in the fermentation supernatant. The BsEXLX1 protein sequence was ligated into the screening plasmid to obtain the template plasmid pHT-P21-RBS-SP-BsEXLX1-sfGFP, which mainly consists of the promoter P21, two Eco31Ⅰ restriction sites (GGTCTC), the BsEXLX1-sfGFP fusion protein gene, the replicon, and the resistance genes (AmpR, CmR). An RBS library was constructed, using RBS (AAGGAGATATACC) as the initial RBS. Degenerate primers RBS-F and RBS-R were used, and Eco31Ⅰ restriction sites were added at both ends to obtain an RBS mixed library (MAGGMRGWATCAC), which contains 16 RBSs with different transcriptional strengths. A signal peptide combinatorial library was constructed. 173 signal peptides of the Sec pathway of B. subtilis were downloaded from the website. Primers were designed to add Eco31Ⅰ restriction sites at both ends of the signal peptide fragments. The fragments were amplified from the genome and mixed to obtain a signal peptide library. The template plasmid pHT-P21-RBS-SP-BsEXLX1-sfGFP, RBS library and signal peptide library were connected using the Golden Gate method (Table 3). The Golden Gate mixing system and PCR system methods are shown in Tables 1 and 2. E. coli DH5α was transformed to amplify the plasmid library, and then the plasmid library was extracted from E. coli DH5α.

[0078] The screened signal peptide library consisted of 173 signal peptides of the endogenous Sec secretion pathway of Bacillus subtilis and a RBS library containing 16 different translation strengths, and the library size contained 2768 combinations.

[0079] The operation process of this method is as follows:

[0080] 1. Connect the expansin protein sequence to the screening plasmid to obtain the template plasmid pHT-P 21 -RBS-SP-BsEXLX1-sfGFP, the plasmid consists of promoter P21, two Eco31Ⅰ restriction sites and BsEXLX1-sfGFP fusion protein;

[0081] 2. Construct an RBS library, using RBS (AAGGAGATATACC) as the initial RBS, using degenerate primers RBS-F and RBS-R, with Eco31Ⅰ restriction sites at both ends to obtain an RBS mixed library

[0082] (MAGGMRGWATCAC), the library contains 16 RBSs with different transcriptional intensities;

[0083] 3. Construct a signal peptide library. Download 173 signal peptides of the sec pathway of B. subtilis from the Signal Sequence Database. Design primers to add Eco31Ⅰ restriction sites at both ends of the signal peptide fragments. Amplify the fragments from the genome and mix them to obtain a signal peptide library.

[0084] 4. Use the Golden gate method to transfer the template plasmid pHT-P 21 -RBS-SP-BsEXLX1-sfGFP, RBS library and signal peptide library were connected. The mixed system and PCR program are shown in the following table.

[0085] Table 1 Golden gate connection system

[0086] Eco31Ⅰ endonuclease 1 μL T4 ligase 2μL 10×T4 buffer 1 μL Template plasmid 75ng RBS Library 25ng Signal peptide library 25ng <![CDATA[ddH2O]]> Add to 10 μL

[0087] Table 2 Golden gate reaction conditions

[0088]

[0089] 5. The ligation product was transformed into Escherichia coli for cloning to obtain a mixed plasmid, which was then transformed into B. subtilis G600. After culturing in a culture dish to obtain a single colony, all plaques in the culture dish were scraped and cultured for an appropriate period of time. A droplet microfluidic sorter was used for pretreatment to encapsulate the cells in droplets. After culturing for an appropriate period of time, a flow cytometry sorter was used for rescreening to achieve preliminary screening. The culture fluid obtained after the initial screening was cultured in a culture dish, and the cultured single colony was picked into a 96-well plate for fermentation verification. After 48 hours, the bacterial concentration and total fluorescence intensity of the bacteria were determined using a microplate reader. The fluorescence intensity in the fermentation supernatant was determined after centrifugation at 4000 rpm for 5 minutes using a well plate centrifuge. The droplet microfluidic embedding process is shown in the figure below. Figure 4 As shown in the figure, the flow cytometry instrument screening is as follows Figure 5 As shown in the figure, the total fluorescence intensity distribution of bacteria during rescreening is as follows Figure 6 shown.

[0090] 6. The total fluorescence intensity of the bacteria ranged from 800 to 32,000, and the relative fluorescence intensity of the fermentation supernatant ranged from 1,000 to 30,350. The Bacillus subtilis with the highest fluorescence expression intensity was selected for sequencing, and 6 combinations of RBS-signal peptides were obtained. The nucleotide sequences of the RBS sequences and signal peptides are shown in Table 3.

[0091] Table 3 RBS-signal peptide sequence list

[0092] Serial number Fluorescence value RBS (SEQ ID NO. 4-7) Signal peptide (SEQ ID NO.8-12) 1 16658 AAGGAGGAATCAC CiTH 2 14152 AAGGAGGTATCAC YojL 3 22371 AAGGAGGAATCAC YpC 4 30350 CAGGAGGAATCAC AspB 5 10592 AAGGAGGTATCAC yobV 6 9876 CAGGAGGTATCAC yobV

[0093] Example 3: High-efficiency expression of expansin in recombinant strains

[0094] The engineered strains obtained in Examples 1 and 2 (the control group was the recombinant strain BE-0 prepared in Example 1, and the experimental groups were the recombinant strains prepared in Example 2 containing different RBS-signal peptide combinations listed in Table 3) were inoculated into a seed culture medium containing chloramphenicol (5 mg / L) and cultured with shaking at 37°C and 220 rpm overnight. Subsequently, a 5% inoculum (v / v) was transferred to a fermentation medium containing chloramphenicol (5 mg / L) and cultured with shaking at 37°C and 220 rpm for 12-48 h.

[0095] The method for detecting expansin is as follows: the production of expansin was detected by high performance liquid chromatography (HPLC), Agilent 1260, RID detector, C4 column (Waters), mobile phase A: 1% TFA aqueous solution, mobile phase A: 1% TFA acetonitrile solution, flow rate 0.8 mL / min, column temperature 40°C, injection volume 10 μL, and gradient elution for 35 minutes for each sample.

[0096] The experimental results are as follows Figure 7-8 As shown, the recombinant strain containing the RBS3-signal peptide aspB combination had the strongest fluorescence signal and the highest yield, reaching 196 mg / L.

[0097] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for increasing the secretion and expression of expansin by Bacillus subtilis, characterized in that: The method comprises the steps of overexpressing an expansin gene expression cassette in a Bacillus subtilis host strain, The expansin gene expression frame includes an expansin encoding gene sequence and an RBS encoding sequence and a signal peptide encoding sequence located upstream of the expansin encoding gene sequence. The signal peptide encoding sequence is located between the RBS encoding sequence and the expansin encoding gene sequence. The RBS encoding sequence is shown in SEQ ID NO.6, and the signal peptide encoding sequence is shown in SEQ ID NO.

11.

2. The method according to claim 1, characterized in that Contains at least one of the following characteristics: (1) The amino acid sequence of expansin is shown in SEQ ID NO. 1; (2) The expansin encoding gene sequence is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that Contains at least one of the following characteristics: (1) The expansin gene expression frame further contains a promoter for initiating gene expression, and the promoter is a constitutive promoter; The constitutive promoter includes promoter P 21 ; The promoter P 21 The nucleotide sequence is shown in SEQ ID NO.3; (2) The Bacillus subtilis host bacteria includes Bacillus subtilis G600.

4. The method according to claim 1, wherein Contains at least one of the following characteristics: (1) The overexpression is to increase the number of copies of at least one gene expression cassette; (2) The overexpression method is free expression or integrated expression; When the overexpression method is episomal expression, the expression vector of the expansin gene expression cassette includes pHT-01 plasmid.

5. A recombinant Bacillus subtilis, characterized in that Bacillus subtilis G600 was used as the starting strain for transformation, and the transformation included: An overexpression swollenin gene expression cassette comprises an swollenin encoding gene sequence and an RBS encoding sequence and a signal peptide encoding sequence located upstream of the swollenin encoding gene sequence, wherein the signal peptide encoding sequence is located between the RBS encoding sequence and the swollenin encoding gene sequence, the RBS encoding sequence is shown in SEQ ID NO.6, and the signal peptide encoding sequence is shown in SEQ ID NO.

11.

6. The recombinant Bacillus subtilis according to claim 5, characterized in that Contains at least one of the following characteristics: (1) The amino acid sequence of expansin is shown in SEQ ID NO. 1; (2) The expansin encoding gene sequence is shown in SEQ ID NO. 2; (3) The swollenin gene expression frame also contains a promoter located upstream of the RBS coding sequence; the promoter includes promoter P 21 ; (4) The overexpression method is free expression; during free expression, the expression vector of the expansin gene expression cassette includes pHT-01 plasmid.

7. A method for constructing a recombinant Bacillus subtilis, characterized in that: The following steps are involved: A gene expression cassette comprising a promoter, an RBS shown in SEQ ID NO.6, a signal peptide shown in SEQ ID NO.11 and an expansin encoding gene shown in SEQ ID NO.2 was constructed and transformed into a Bacillus subtilis G600 competent cell to obtain the recombinant Bacillus subtilis.

8. Use of the recombinant Bacillus subtilis according to claim 5 or 6 or the recombinant Bacillus subtilis obtained by the construction method according to claim 7 in the preparation of expansin.

9. A method for producing expansin, characterized in that The method comprises the steps of fermenting the recombinant Bacillus subtilis according to claim 5 or 6 or the recombinant Bacillus subtilis obtained by the construction method according to claim 7.

10. The method according to claim 9, characterized in that Contains at least one of the following characteristics: (1) The fermentation temperature is 35-38°C; (2) Culture at 180-280 rpm; (3) The fermentation includes shake flask fermentation or fed-batch fermentation; (4) The fermentation comprises the following steps: inoculating the recombinant strain into a seed culture medium to obtain a seed liquid, and inoculating the seed liquid into a fermentation culture medium for fermentation.

11. The method according to claim 9 or 10, characterized in that Contains at least one of the following characteristics: (1) The seed culture medium used in the fermentation process contains the following components: peptone 3-10 g / L, yeast powder 5-15 g / L, sodium chloride 5-15 g / L; (2) The fermentation medium used in the fermentation process contains the following components: peptone 10-15 g / L, yeast powder 20-30 g / L, glycerol 2-5 mL / L, KH2PO4 2.0-2.5 g / L, K2HPO4 12-13 g / L.

12. An expansin gene expression cassette, characterized in that The expansin gene expression frame includes an expansin encoding gene sequence and an RBS encoding sequence and a signal peptide encoding sequence located upstream of the expansin encoding gene sequence. The signal peptide encoding sequence is located between the RBS encoding sequence and the expansin encoding gene sequence. The RBS encoding sequence is shown as SEQ ID NO.6, and the signal peptide encoding sequence is shown as SEQ ID NO.

11.

13. A recombinant plasmid containing the expansin gene expression cassette according to claim 12.

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