A method for mussel adhesive protein synthesis

Abstract

The application provides a mussel mucin synthesis method and relates to the technical field of genetic engineering.The application improves the yield of mussel mucin by carrying out deletion mutation on the N-terminal sequence of the secondary structure of the mussel mucin amino acid sequence such as SEQ ID NO.1.The yield of mussel mucin in the prior art is about 2.57g / L, while the yield of mussel mucin in the application is about 5g / L, which is greatly improved compared with the yield of mussel mucin in the prior art.On this basis, the mussel mucin is modified by dopa through co-expression of tyrosinase, and the expression amount of the tyrosinase is controlled through different intensities of RBS, so as to regulate the modification degree of dopa, improve the yield of mussel mucin, and realize controllable modification of dopa in the mussel mucin, finally obtaining mussel mucin with high yield and different modification degrees of dopa.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for synthesizing mussel adhesive protein. Background Technology

[0002] Mussel adhesive proteins (MAPs) are specialized proteins evolved by marine mussels to adapt to the turbulent coastal environment. These proteins, primarily found in the byssal threads of mussels, firmly anchor the mussel to solid surfaces such as rocks and wood, maintaining stability even under the pressure of seawater. The highly efficient adhesive properties of MAPs have attracted significant interest from scientists, leading to applications and developments in various fields. These proteins contain a large amount of 3,4-dihydroxyphenylalanine (DOPA), endowing them with unique strong adhesiveness, biocompatibility, and biodegradability. Different degrees of DOPA modification in mussel adhesive proteins impart different functions. High-DOPA modified mussel adhesive proteins can be used in medical bioadhesives, wound healing materials, and medical coatings, while low-DOPA modified mussel adhesive proteins are more suitable for skincare products. Based on these characteristics, mussel adhesive proteins and their synthetic analogues show broad application prospects in biomedical engineering, coating corrosion protection, materials science, and other fields. For example, in biomedicine, it is used to develop new medical adhesives and tissue repair materials; in environmental materials, it serves as the basis for marine antifouling coatings, reducing pollution and lowering ship maintenance costs; and in materials science, it has inspired the design of new composite materials, enhancing their moisture resistance, abrasion resistance, and self-healing capabilities.

[0003] Currently, mussel adhesive protein is mainly obtained through tissue extraction. This process includes raw material preparation, pretreatment, crushing and dissolving, extraction and purification, concentration and drying, and quality control. Throughout the process, various indicators must be strictly monitored to ensure the purity, activity, and safety of the final product. However, tissue extraction faces challenges such as high cost, low yield, uncontrollable dopa modification, and stability issues, making it difficult to meet market demand for mussel adhesive protein. Therefore, there is an urgent need for a method to synthesize mussel adhesive protein with high yield and controllable dopa modification. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problems of low yield of mussel adhesive protein synthesis and uncontrollable dopa modification in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a method for synthesizing mussel adhesive protein. This invention increases the expression level of mussel adhesive protein by deleting and mutating the N-terminal sequence of the secondary structure of mussel adhesive protein, as shown in SEQ ID NO.1. Simultaneously, tyrosinase can catalyze the conversion of tyrosine residues in mussel adhesive protein to DOPA with ortho-hydroxyl groups. Therefore, by co-expressing tyrosinase to modify mussel adhesive protein with DOPA, and controlling the expression level of tyrosinase using different intensities of RBS, the degree of DOPA modification is regulated, ultimately yielding high-yield mussel adhesive protein with varying degrees of DOPA modification.

[0006] The first objective of this invention is to provide a method for synthesizing mussel adhesive protein, wherein a gene sequence encoding an amino acid sequence as shown in any one of SEQ ID NO. 2-6 is introduced into a plasmid to form a recombinant plasmid, the recombinant plasmid is introduced into a host bacterium to form a recombinant bacterium, and the recombinant bacterium is inoculated into a fermentation medium for fermentation culture to obtain mussel adhesive protein, wherein the amino acid sequence of the mussel adhesive protein is shown in SEQ ID NO. 12.

[0007] Furthermore, the amino acid sequence of SEQ ID NO.2 is obtained by deleting and mutating the 2nd-3rd amino acids at the N-terminus of the secondary structure of mussel adhesive protein as shown in SEQ ID NO.1. The sequence of SEQ ID NO.2 is as follows:

[0008] MAVCIYFFLVGQIQAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0009] Furthermore, the amino acid sequence of SEQ ID NO.3 is obtained by deleting amino acids 2-12 from the N-terminus of the secondary structure of mussel adhesive protein as shown in SEQ ID NO.1. The sequence of SEQ ID NO.3 is as follows:

[0010] MGQIQAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0011] Furthermore, the amino acid sequence of SEQ ID NO.4 is obtained by deleting amino acids 2-14 from the N-terminus of the secondary structure of mussel adhesive protein as shown in SEQ ID NO.1. The sequence of SEQ ID NO.4 is as follows:

[0012] MIQAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0013] Furthermore, the amino acid sequence of SEQ ID NO.5 is obtained by deleting amino acids 2-16 from the N-terminus of the secondary structure of mussel adhesive protein as shown in SEQ ID NO.1. The sequence of SEQ ID NO.5 is as follows:

[0014] MAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0015] Furthermore, the amino acid sequence of SEQ ID NO.6 is obtained by deleting and mutating amino acids 2-19 from the N-terminus of the secondary structure of mussel adhesive protein as shown in SEQ ID NO.1. The sequence of SEQ ID NO.6 is as follows:

[0016] MIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0017] Furthermore, the sequence of SEQ ID NO.1 is shown below:

[0018] MISAVCIYFFLVGQIQAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0019] Furthermore, the sequence of SEQ ID NO.12 is shown below:

[0020] IPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0021] Furthermore, the recombinant plasmid also includes the gene sequence of tyrosinase, the amino acid sequence of which is shown in SEQ ID NO.11.

[0022] Furthermore, the sequence of SEQ ID NO.11 is as follows:

[0023] MAKYHRLNLQNPAAAPFLESYKKAITVMLQLPPSDARNWYRNAFIH

[0024] TLDCPHGNWWFVVWHRGYTGWFERTVRELSGDPNFAFPYWDWTALPQ

[0025] VPDSFFNGVLDPNNPAFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNR

[0026] GFQSVNDVWQAVRDSPMFFPRGRARTLTRQNPGFDATTRRAVSIGTIRNA

[0027] LAPTDFITFGSGKTANHSESATQGILESQPHNNVHNNIGGFMQDLLSPTDP

[0028] VFFAHHSNIDRLWDVWTRKQQRLGLPTLPTGANLPLWANEPFLFFIGPDG

[0029] KPVAKNKAGDYATIGDFDYNYEPGSGEAVIPAASRPGEMNNK

[0030] Furthermore, the expression of the tyrosinase is regulated by the RBS sequence shown in SEQ ID NO.7-10.

[0031] SEQ ID NO.7: AAGGGCCGAATT

[0032] SEQ ID NO.8: AAGGGGAGAGCC

[0033] SEQ ID NO.9: AAGGTGGTTCAT

[0034] SEQ ID NO.10: AAGAAGGGTTCATAG

[0035] Furthermore, the plasmids include pUC18, pUC19, pGEM-T, pBR322, pET-28a, pET-21b, pBAD, pBBR1MCS-2, pBBR1MCS-5, pSC101, pMAL-c2, and pMAL-p2.

[0036] Furthermore, the host bacteria include Escherichia coli DH5α, Escherichia coli BL21(DE3), Escherichia coli MG1655, Escherichia coli W3110, Escherichia coli TOP10, and Escherichia coli Nissle 1917.

[0037] Furthermore, the fermentation culture temperature is 20-45℃.

[0038] Furthermore, the pH of the fermentation culture is 4-7.

[0039] Furthermore, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and metal ions.

[0040] Furthermore, the inoculation amount of the recombinant bacteria is 1%-5% by volume.

[0041] The beneficial effects of this invention are:

[0042] This invention increases the yield of mussel adhesive protein by deleting and mutating the N-terminal sequence of the secondary structure of mussel adhesive protein, as shown in SEQ ID NO.1. Existing technologies yield mussel adhesive protein at approximately 2.57 g / L, while the highest yield of this invention is approximately 5 g / L, representing a significant improvement over existing technologies. Furthermore, dopa modification of mussel adhesive protein is achieved through co-expression of tyrosinase, and the expression level of tyrosinase is controlled by varying intensities of RBS, thereby regulating the degree of dopa modification. This process not only increases the yield of mussel adhesive protein but also achieves controllable modification of dopa within the protein, ultimately yielding high-yield mussel adhesive protein with different degrees of dopa modification. Attached Figure Description

[0043] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0044] Figure 1 This is the secondary protein structure of mussel adhesive protein;

[0045] Figure 2This study investigated the effect of N-terminal deletion of mussel adhesive protein on its expression level. Specifically, pvfp1 represents a recombinant strain containing pET32a-pvfp; pvfp2 represents a recombinant strain containing pET32a-pvfp-del3; pvfp3 represents a recombinant strain containing pET32a-pvfp-del12; pvfp4 represents a recombinant strain containing pET32a-pvfp-del14; pvfp5 represents a recombinant strain containing pET32a-pvfp-del16; and pvfp6 represents a recombinant strain containing pET32a-pvfp-del19.

[0046] Figure 3 The effect of different intensities of RBS regulation of tyrosinase expression on the degree of dopa modification of mussel adhesive proteins;

[0047] Figure 4 This refers to the yield of mussel adhesive protein produced by fermenting recombinant Escherichia coli in a 5L fermenter. Detailed Implementation

[0048] 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 and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0049] Strain: Escherichia coli Nissle 1917;

[0050] Plasmid: pET32a;

[0051] LB medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L;

[0052] TB medium: tryptone: 24 g / L, yeast extract: 24 g / L, glycerol: 4 mL / L, potassium phosphate (K2HPO4): 9.4 g / L, potassium dihydrogen phosphate (KH2PO4): 2.2 g / L;

[0053] Preparation of competent Escherichia coli cells: First, pick a single colony from a fresh plate and inoculate it into 5 mL of LB medium. Incubate overnight at 37°C with shaking. The next day, take 1 mL of the overnight culture and add it to 100 mL of fresh LB medium. Incubate at 37°C with shaking until OD reaches 100%. 600The concentration is approximately 0.4-0.6. Transfer the culture medium to centrifuge tubes and centrifuge at 4000 rpm for 10 minutes at 4°C, discarding the supernatant. Resuspend the bacterial cells in ice-cold 0.1M CaCl2 solution to a final volume approximately 1 / 10 of the original culture medium volume, then incubate on ice for 30 minutes. Centrifuge again at 4°C for 10 minutes, discarding the supernatant, and resuspend the bacterial cells in ice-cold 0.1M CaCl2 solution to a final volume approximately 1 / 100 of the original culture medium volume. Finally, aliquot the resuspended bacterial solution into sterile 1.5mL centrifuge tubes, approximately 100μL per tube, and immediately store at -80°C for subsequent transformation experiments.

[0054] Purification of mussel foot protein: First, collect bacterial cells and centrifuge at 10,000 rpm for 5 minutes. Collect the bacterial cell precipitate and wash with physiological saline to remove impurities. Next, mechanically disrupt or sonicate the washed cells to release the mucin. Add the disrupted material to an acidic solution to promote mucin dissolution. Subsequently, separate and purify the mussel mucin using affinity chromatography, and further improve purity using precipitation and filtration techniques. The purified mucin solution needs to be concentrated and dried to obtain a powdered product.

[0055] Determination of mussel foot protein yield: First, prepare standard protein solutions and sample solutions of different concentrations; then, add Coomassie Brilliant Blue G-250 dye to each sample and standard, mix well, and incubate at room temperature for 5-15 minutes; next, measure the absorbance at 595 nm using a spectrophotometer; finally, calculate the protein content in the sample based on the standard curve.

[0056] Determination of DOPA Modification: A series of DOPA standard solutions of known concentrations were prepared. Absorbance was measured using a UV-Vis spectrometer at 280 nm. Data Analysis: A standard curve was plotted based on the absorbance of the standards and their known concentrations. The DOPA content in the sample was calculated by comparing the absorbance of the sample.

[0057] Example 1: Construction of recombinant Escherichia coli producing mussel adhesive protein

[0058] (1) Optimization of N-terminal sequence deletion of mussel adhesive protein

[0059] Based on the genomic sequence of *P. perna* mussel adhesive protein, the gene *pvfp* was synthesized by GENEWIZ (Suzhou). The amino acid sequence of the secondary structure of mussel adhesive protein *pvfp* is shown in SEQ ID NO.1 (GenBank: UNW37542.1). The N-terminal sequence in the secondary structure not only participates in the protein translation initiation process but also affects its stability. During the translation initiation stage, the sequences near the ribosome binding site (RBS) and start codon (AUG) in the N-terminal sequence determine the translation efficiency; optimizing these sequences can significantly improve the protein expression level. Therefore, based on the secondary structure of mussel adhesive protein (e.g., *pvfp*), the amino acid sequence of the secondary structure of *pvfp* is shown in SEQ ID NO.1 (GenBank: UNW37542.1). Figure 1 (As shown), primers were designed to delete the N-terminal amino acid sequence of the secondary structure of mussel adhesive protein. Using the synthesized pvfp gene sequence as a template, primers pvfp-del3-F and pvfp-del3-R, pvfp-del12-F and pvfp-del12-R, pvfp-del14-F and pvfp-del14-R, pvfp-del16-F and pvfp-del16-R, and pvfp-del19-F and pvfp-del19-R were designed for PCR amplification, yielding the gene sequences pvfp-del3, pvfp-del12, pvfp-del14, pvfp-del16, and pvfp-del19. These sequences were then analyzed using T5exonuclease-dependent... The assembly (TEDA) method was used to ligate the gene sequences of pvfp, pvfp-del3, pvfp-del12, pvfp-del14, pvfp-del16, and pvfp-del19 into plasmid pET32a, respectively, to construct recombinant expression plasmids pET32a-pvfp, pET32a-pvfp-del3, pET32a-pvfp-del12, pET32a-pvfp-del14, pET32a-pvfp-del16, and pET32a-pvfp-del19.

[0060] (2) Construction of the biosynthetic pathway of Escherichia coli mussel adhesive protein

[0061] The recombinant plasmids pET32a-pvfp, pET32a-pvfp-del3, pET32a-pvfp-del12, pET32a-pvfp-del14, pET32a-pvfp-del16, and pET32a-pvfp-del19 were chemically transformed into *Escherichia coli* Nissle1917 to obtain recombinant strains pvfp1-6 (where pvfp1 indicates the presence of pET32a-pvfp-del3). Recombinant strains of vfp; pvfp2 represents a recombinant strain containing pET32a-pvfp-del3; pvfp3 represents a recombinant strain containing pET32a-pvfp-del12; pvfp4 represents a recombinant strain containing pET32a-pvfp-del14; pvfp5 represents a recombinant strain containing pET32a-pvfp-del16; pvfp6 represents a recombinant strain containing pET32a-pvfp-del19). Select single colonies and inoculate them into 5 mL of LB medium, incubating overnight at 37°C. Inoculate 1% of the culture into 25 mL of TB medium and wait for growth to reach OD. 600 =1. Add 0.2 mM IPTG to induce fermentation, culture for 48 h, centrifuge to collect cells, purify mussel adhesive protein, and determine the content of mussel adhesive protein (e.g., Figure 2 (As shown).

[0062] Table 1 Primers used for N-terminal deletion of the mussel adhesive protein gene pvfp-6.

[0063] Primer Name Sequence (5’-3’) Sequence ID pvfp-del3-F AGGAGATATACATATGGCAGTTTGTATATATTTCT SEQ ID NO.13 pvfp-del3-R ATATATACAAACTGCCATATGTATATCTCCT SEQ ID NO.14 pvfp-del12-F AGGAGATATACATATGGGCCAGATACAGGCTGG SEQ ID NO.15 pvfp-del12-R CCAGCCTGTATCTGGCCCATATGTATATCTCCT SEQ ID NO.16 pvfp-del14-F AGGAGATATACATATGCATACCATTTGAAAAACCCG SEQ ID NO.17 pvfp-del14-R CGGGTTTTTCAAATGGTATGCATATGTATATCTCCT SEQ ID NO.18 pvfp-del16-F AGGAGATATACATATGATTTGAAAAACCCGGACAGTG SEQ ID NO.19 pvfp-del16-R CACTGTCCGGGTTTTTCAAATCATATGTATATCTCCT SEQ ID NO.20 pvfp-del19-F AGGAGATATACATATGACCCGGACAGTGTCCAGTC SEQ ID NO.21 pvfp-del19-R GACTGGACACTGTCCGGGTCATATGTATATCTCCT SEQ ID NO.22

[0064] Example 2: Control of Dopa Modification of Mussel Adhesive Protein

[0065] (1) Construction of a controllable dopa-modified system for Escherichia coli

[0066] Based on the genomic sequence of V. spinosum tyrosinase, the gene Vs-tyr was synthesized by GENEWIZ (Suzhou). The amino acid sequence of Vs-tyr is shown in SEQ ID NO.11. Using the synthesized Vs-tyr gene sequence as a template, primers Vs-tyr-RBS1-F, Vs-tyr-RBS2-F, Vs-tyr-RBS3-F, Vs-tyr-RBS4-F, and Vs-tyr-RBS-R were designed for PCR amplification. The plasmid pET32a-pvfp-del16 of the recombinant strain pvfp5, which had the highest yield, was selected. Then, the Vs-tyr gene with different RBS strengths was ligated using the TEDA method. The plasmid pET32a-pvfp-del16 was inserted to construct recombinant expression plasmids of different intensities of RBS: pET32a-pvfp-del16-Vs-tyr-RBS1, pET32a-pvfp-del16-Vs-tyr-RBS2, pET32a-pvfp-del16-Vs-tyr-RBS3, and pET32a-pvfp-del16-Vs-tyr-RBS4. (The sequences of RBS1-4 are shown in SEQ ID NO.7-10. According to the RBS Calculator website, the intensities are predicted to increase by 2-fold in each order.)

[0067] Table 2 Primers used for optimizing tyrosinase Vs-tyr RBS expression.

[0068]

[0069]

[0070] (2) Detection of DOPA modification degree of mussel adhesive protein produced by recombinant Escherichia coli

[0071] The constructed recombinant plasmids pET32a-pvfp-del16-Vs-tyr-RBS1, pET32a-pvfp-del16-Vs-tyr-RBS2, pET32a-pvfp-del16-Vs-tyr-RBS3, and pET32a-pvfp-del16-Vs-tyr-RBS4 were chemically transformed into *E. coli* Nissle1917 to obtain recombinant strains pvfp7-10. pvfp7 represents the recombinant strain containing pET32a-pvfp-del16-Vs-tyr-RBS1; pvfp8 represents the recombinant strain containing pET32a-pvfp-del16-Vs-tyr-RBS2; pvfp9 represents the recombinant strain containing pET32a-pvfp-del16-Vs-tyr-RBS3; and pvfp10 represents the recombinant strain containing pET32a-pvfp-del16-Vs-tyr-RBS4. Single colonies were selected and inoculated into 5 mL of LB medium and cultured overnight at 37°C. A 1% inoculum was then inoculated into 25 mL of TB medium, and cultured until the OD reached... 600 =1. Add 0.2 mM IPTG to induce fermentation, culture for 48 h, centrifuge to collect cells, purify mussel adhesive protein, and determine the dopa modification degree of mussel adhesive protein (e.g., Figure 3 (As shown).

[0072] Example 3: Feed-in fermentation of recombinant Escherichia coli mussel adhesive protein in a 5L fermenter

[0073] Fermentation production in a 5L fermenter: The recombinant strain pvfp10 constructed in Example 2 was inoculated into 5 mL of LB medium and cultured overnight at 37°C and 220 rpm. The seed culture was prepared with initial OD... 600 A 0.1 mM inoculum was transferred to a 25 mL Erlenmeyer flask containing fermentation medium and incubated at 220 rpm and 30°C for 10 h. Then, a 10% inoculum was transferred to a 5 L fermenter. The initial temperature was set at 37°C and the fermentation speed at 3000 rpm. After 4 h of fermentation, IPTG at a final concentration of 0.2 mM was added to induce gene expression. During fermentation, the pH of the fermentation broth was controlled at approximately 7 using 14% ammonia, and glycerol was fed in to maintain the glycerol content in the fermenter at approximately 1%. Figure 4 It can be seen that the yield of mussel adhesive protein obtained by the recombinant strain pvfp10 after fermentation in a 5L fermenter is 5 g / L (e.g., Figure 4 (As shown).

[0074] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for synthesizing mussel adhesive protein, characterized in that, The gene sequence encoding the amino acid sequence shown in SEQ ID NO.5 is introduced into a plasmid to form a recombinant plasmid. The recombinant plasmid is then introduced into a host bacterium to form a recombinant bacterium. The recombinant bacterium is inoculated into a fermentation medium for fermentation culture to obtain mussel adhesive protein.

2. The method according to claim 1, characterized in that, The recombinant plasmid also includes the gene sequence of tyrosinase.

3. The method according to claim 2, characterized in that, The expression of the tyrosinase is regulated by the RBS sequence shown in SEQ ID NO.7-10.

4. The method according to claim 2, characterized in that, The amino acid sequence of the tyrosinase is shown in SEQ ID NO.

11.

5. The method according to claim 1, characterized in that, The plasmids include pUC18, pUC19, pGEM-T, pBR322, pET-28a, pET-21b, pBAD, pBBR1MCS-2, pBBR1MCS-5, pSC101, pMAL-c2, and pMAL-p2.

6. The method according to claim 1, characterized in that, The host bacteria include Escherichia coli DH5α, Escherichia coli BL21(DE3), Escherichia coli MG1655, Escherichia coli W3110, Escherichia coli TOP10, and Escherichia coli Nissle 1917.

7. The method according to claim 1, characterized in that, The fermentation culture temperature is 20-45℃.

8. The method according to claim 1, characterized in that, The pH of the fermentation culture is 4-7.

9. The method according to claim 1, characterized in that, The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and metal ions.

10. The method according to claim 1, characterized in that, The inoculation amount of the recombinant bacteria is 1%-5% by volume.

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