Biotin ligase mutant and application thereof

Abstract

The invention discloses a biotin ligase mutant and application thereof, and belongs to the technical field of gene engineering. According to the invention, a series of biotin ligase mutants are prepared by docking biotin ligase and substrate molecules thereof and carrying out multiple mutations on screened 10 amino acids with binding force acting sites. On the basis, a biotin ligase mutant is connected to escherichia coli through a homologous recombination technology, a series of escherichia coli recombinant bacteria with high biotin yield are obtained, and the biotin yield reaches 0.873 mg / L to the maximum after 24-hour fermentation.

Description

Technical Field

[0001] The invention relates to a biotin ligase mutant and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Biotin, also known as vitamin B7 or coenzyme R, is a water-soluble vitamin belonging to the B vitamin family. Its molecular structure contains a fused thiazolidine ring and a furan ring. It is an important coenzyme for various carboxylases and is widely involved in basic metabolic processes such as fatty acid synthesis, amino acid metabolism, and glucose production. Due to its important role in cell proliferation, gene expression regulation, and maintenance of immune function, biotin is widely used in food additives, nutritional supplements, animal feed, and cosmetics, with significant effects on skin health, hair growth, and nail strengthening. Biotin is also used in molecular biology, often for labeling molecules such as proteins and nucleic acids, and plays a key role in affinity purification and bioassay techniques.

[0003] Industrial production of biotin primarily relies on two pathways: microbial fermentation and chemical synthesis. Microbial fermentation typically utilizes bacteria such as Escherichia coli and Pichia pastoris, enhancing their biotin synthesis capacity through genetic engineering to achieve efficient production. BirA is a protein with both catalytic and transcriptional regulatory functions. It acts as a biotin ligase, catalyzing and mediating protein biotinylation reactions, linking biotin to specific proteins. It also acts as a biotin-induced inhibitor, acting as a biotin synthesis regulator and negatively regulating biotin synthesis.

[0004] Escherichia coli has significant advantages in biotin production. Because it has a complete biotin biosynthesis pathway, it can be directly enhanced by metabolic engineering means, and its genetic manipulation system is extremely mature, with a rich synthetic biology toolbox, which can efficiently perform gene editing, pathway optimization and expression regulation. In addition, Escherichia coli grows rapidly, can use cheap carbon sources, and is adaptable to high-density culture, which significantly reduces industrial production costs. In terms of industrial applications, Escherichia coli is a generally recognized as safe (GRAS) strain, and there are a large number of fermentation processes and regulatory information, which facilitates large-scale and compliant production. Patent CN118207171B discloses a biotin ligase mutant. Escherichia coli expressing this mutant has the ability to produce biotin with a yield of 0.156 mg / L. The biotin yield of this method is still relatively low.

[0005] Therefore, developing a BirA mutant to further increase the production of biotin in Escherichia coli has extremely high practical and economic value. Summary of the Invention

[0006] To address this issue, the present invention prepared a series of biotin ligase mutants by docking biotin ligase with its substrate molecules and then multiple mutations of the 10 amino acids with binding sites identified. Furthermore, homologous recombination was used to connect these biotin ligase mutants into Escherichia coli, resulting in a series of recombinant E. coli strains with high biotin production.

[0007] The first object of the present invention is to provide a biotin ligase mutant, which has one or more amino acid mutations at positions 118, 119, 121, 124, 172, 175, 176, 208, 220, and 221 based on the amino acid sequence shown in SEQ ID NO.1.

[0008] In one embodiment, corresponding to the amino acid sequence shown in SEQ ID NO.1, the arginine at position 118 is mutated to alanine; or,

[0009] Arginine at position 119 is mutated to valine; or

[0010] Arginine at position 121 is mutated to glycine; or

[0011] Phenylalanine at position 124 is mutated to alanine; or

[0012] Lysine at position 172 is mutated to serine; or

[0013] Aspartic acid at position 175 is mutated to valine; or

[0014] Leucine at position 176 is mutated to glycine; or

[0015] Asparagine at position 208 is mutated to tryptophan; or

[0016] Asparagine at position 220 is mutated to alanine; or

[0017] The glutamine at position 221 was mutated to glycine.

[0018] In one embodiment, the mutations are present simultaneously:

[0019] Arginine at position 121 is mutated to glycine, and aspartic acid at position 175 is mutated to valine; or

[0020] Arginine at position 121 is mutated to glycine, and asparagine at position 208 is mutated to tryptophan; or

[0021] Arginine at position 118 is mutated to alanine, aspartic acid at position 175 is mutated to valine, and asparagine at position 208 is mutated to tryptophan; or

[0022] Arginine at position 118 is mutated to alanine, and arginine at position 121 is mutated to glycine; or

[0023] The arginine at position 118 was mutated to alanine, the arginine at position 121 was mutated to glycine, the aspartic acid at position 175 was mutated to valine, and the asparagine at position 208 was mutated to tryptophan.

[0024] The second object of the present invention is to provide a polynucleotide encoding any of the above-mentioned biotin ligase mutants.

[0025] The third object of the present invention is to provide a vector carrying the above-mentioned polynucleotide.

[0026] In one embodiment, the vectors include pPIC series vectors, pPICZ series vectors, pPICZα and pET series vectors.

[0027] A fourth object of the present invention is to provide cells expressing any of the above-mentioned biotin ligase mutants, wherein the cells include Bacillus subtilis and Escherichia coli.

[0028] In one embodiment, the E. coli is E. coli MG1655.

[0029] A fifth object of the present invention is to provide a genetically engineered Escherichia coli strain, wherein the biotin ligase of the strain has one or more amino acid mutations at positions 118, 119, 121, 124, 172, 175, 176, 208, 220, and 221 based on SEQ ID NO. 1;

[0030] Preferably, the arginine at position 118 is mutated to alanine; or,

[0031] Arginine at position 119 is mutated to valine; or

[0032] Arginine at position 121 is mutated to glycine; or

[0033] Phenylalanine at position 124 is mutated to alanine; or

[0034] Lysine at position 172 is mutated to serine; or

[0035] Aspartic acid at position 175 is mutated to valine; or

[0036] Leucine at position 176 is mutated to glycine; or

[0037] Asparagine at position 208 is mutated to tryptophan; or

[0038] Asparagine at position 220 is mutated to alanine; or

[0039] Glutamine at position 221 is mutated to glycine; or

[0040] Arginine at position 121 is mutated to glycine, and aspartic acid at position 175 is mutated to valine; or

[0041] Arginine at position 121 is mutated to glycine, and asparagine at position 208 is mutated to tryptophan; or

[0042] Arginine at position 118 is mutated to alanine, aspartic acid at position 175 is mutated to valine, and asparagine at position 208 is mutated to tryptophan; or

[0043] Arginine at position 118 is mutated to alanine, and arginine at position 121 is mutated to glycine; or

[0044] The arginine at position 118 was mutated to alanine, the arginine at position 121 was mutated to glycine, the aspartic acid at position 175 was mutated to valine, and the asparagine at position 208 was mutated to tryptophan.

[0045] A sixth object of the present invention is to provide use of any of the above-mentioned biotin ligase mutants, or the above-mentioned polynucleotides, or the above-mentioned polynucleotide vectors, or the above-mentioned cells, or the above-mentioned genetically engineered Escherichia coli in the preparation of biotin.

[0046] A seventh object of the present invention is to provide a method for preparing biotin using Escherichia coli, wherein biotin is prepared by fermentation using the above-mentioned genetically engineered Escherichia coli bacteria;

[0047] Fermentation medium (L): glucose 10 g, CoCl2·6H2O 2 mg, KH2PO4 7.5 g, ZnSO4·7H2O 2 mg, yeast powder 3 g, CaCl2 4 mg, citric acid 1.8 g, vitamin B1 1.5 mg, MgSO4·7H2O 2 g, CuSO4 0.5 mg, FeSO4·7H2O 70 mg, MnSO4·H2O 10 mg;

[0048] The fermentation conditions were 200 rpm and 37°C for 24 h.

[0049] An eighth object of the present invention is to provide use of any of the above-mentioned biotin ligase mutants in protein labeling and detection or preparation of antibody-drug conjugates or cell surface marker imaging;

[0050] Wherein, protein labeling and detection is to use the biotin ligase mutant to biotinylate proteins, including ELISA, Western blot, and immunofluorescence;

[0051] The preparation of antibody-drug conjugates is to connect the drug molecule to the antibody using the biotin ligase mutant to prepare a targeted drug;

[0052] Cell surface marker imaging is the combination of the biotin ligase mutant and fluorescently labeled streptavidin to achieve the visualization of proteins on the surface of living cells.

[0053] In one embodiment, the biotin ligase mutant can be used to prepare ELISA, Western blot, and immunofluorescence detection kits.

[0054] Beneficial effects of the present invention

[0055] The present invention prepares a series of biotin ligase mutants by docking biotin ligase with its substrate molecules and multiple mutations of 10 amino acids with binding sites. Based on this, homologous recombination technology is used to connect these biotin ligase mutants into Escherichia coli, resulting in a series of recombinant E. coli strains with high biotin production.

[0056] Specifically,

[0057] (1) When the recombinant E. coli prepared in this application has a single-point mutation in the biotin ligase, the biotin content reaches a maximum of 0.662 mg / L after 24 hours of fermentation;

[0058] (2) When the biotin ligase combination mutations were present in the recombinant Escherichia coli prepared in the present application, the biotin content reached a maximum of 0.873 mg / L after 24 hours of fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the simulation results of biotin ligase BirA and ATP molecules; red represents amino acid sites with binding ability. DETAILED DESCRIPTION

[0060] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0061] The present invention uses molecular docking software to analyze the binding sites of biotin ligase BirA (amino acid sequence shown in SEQ ID NO.1) and ATP. By mutating the amino acids in the binding site to hydrophilic amino acids or amino acids that are less likely to form a dimer structure, the binding pocket of biotin ligase BirA is opened, thereby weakening the binding effect of biotin ligase with ATP and bioin-5'-AMP.

[0062] In one embodiment of the present invention, the 10 sites screened are 118th, 119th, 121st, 124th, 172nd, 175th, 176th, 208th, 220th, and 221st.

[0063] In one embodiment of the present invention, the hydrophilic amino acid or the amino acid that is not likely to form a dimer structure is one of glutamic acid, threonine, serine, tryptophan, glycine, tyrosine, alanine, and valine.

[0064] In one embodiment of the present invention, a large number of amino acid mutation attempts were made on the 10 sites screened, namely R118V, R118T, R118W, R118Y, R118A, R119A, R119V, R119E, R119Y, R119T, R121E, R121T, R121S, R121W, R121G, R121A, F124T, F124Y, F124A, F124V, F124S, K172E, K172E, and K172S. 2S, K172G, K172A, K172V, D175E, D175S, D175G, D175A, D175V, L176W, L176A, L176E, L176V, L176G, N208E, N208 T, N208W, N208G, N208A, N208V, N220A, N220V, N220G, N220W, N220T, Q221T, Q221S, Q22G, Q221Y, Q221A, Q221V.

[0065] In one embodiment of the present invention, after the above-mentioned biotin ligase mutation, the strain survival rate is low and the growth is slow, such as recombinant bacteria expressing mutants R118V, R119Y, R119T, N208E, etc.; there are also recombinant bacteria whose yield is not significantly improved, such as recombinant bacteria expressing mutants R118W, K172A, K172E, K172T, Q221Y, Q221A, etc.

[0066] In one embodiment of the present invention, the recombinant strains expressing the mutants R118A, R119V, R121G, F124A, K172S, D175V, L176G, N208W, N220A, and Q221G have the highest yield and better growth performance among the 10 sites.

[0067] In one embodiment of the present invention, the above 10 sites are divided into three regions, namely 118-124, 172-176, and 208-220.

[0068] In one embodiment of the present invention, one mutant with a higher biotin production capacity is selected from the three regions in sequence for combined mutation to prepare a combined mutation recombinant strain.

[0069] In one embodiment of the present invention, the biotin content of the recombinant bacteria expressing single point mutations and the recombinant bacteria expressing combined mutations is significantly increased after 24 hours of fermentation, which is better than that of the recombinant bacteria without mutations.

[0070] In one embodiment of the present invention, the aforementioned biotin ligase mutant has a weaker binding affinity with ATP and bioin-5'-AMP, thereby increasing the amount of bioin-5'-AMP released and no longer undergoes specific modification, enabling it to label adjacent proteins, thereby improving the efficiency and scope of biological labeling.

[0071] In one embodiment of the present invention, the biotin ligase mutant can be prepared by genetic engineering methods, such as plasmid overexpression, protein expression, and purification.

[0072] In one embodiment of the present invention, the biotin ligase mutant can be used for protein labeling and detection or preparation of antibody-drug conjugates or cell surface marker imaging.

[0073] In one embodiment of the present invention, protein labeling and detection is to use the biotin ligase mutant for biotinylation of proteins, including ELISA, Western blot, and immunofluorescence.

[0074] In one embodiment of the present invention, the preparation of antibody-drug conjugates is to use the biotin ligase mutant to connect drug molecules to antibodies to prepare targeted drugs.

[0075] In one embodiment of the present invention, cell surface marker imaging is the combination of the biotin ligase mutant and fluorescently labeled streptavidin to achieve visualization of proteins on the surface of living cells.

[0076] In one embodiment of the present invention, the biotin ligase mutant can be used to prepare ELISA, Western blot, and immunofluorescence kits.

[0077] The raw materials used in the embodiment are:

[0078] The nucleotide sequence was synthesized by GenScript Biotech Co., Ltd.;

[0079] pKD46, product number YH057, was purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0080] Test method:

[0081] Preparation of seed culture medium (L): 10 g tryptone, 5 g yeast extract, 10 g NaCl.

[0082] Preparation of fermentation medium (L): glucose 10 g, CoCl2·6H2O2 mg, KH2PO4 7.5 g, ZnSO4·7H2O2 mg, yeast powder 3 g, CaCl2 4 mg, citric acid 1.8 g, vitamin B1 1.5 mg, MgSO4·7H2O 2 g, CuSO4 0.5 mg, FeSO4·7H2O 70 mg, MnSO4·H2O 10 mg.

[0083] Biotin detection method:

[0084] The recombinant bacteria were inoculated into a seed culture medium and cultured overnight at 200 rpm and 37°C to obtain a seed solution. The seed solution was inoculated into a fermentation medium at 1% v / v and cultured at 200 rpm and 37°C for 24 h to obtain a fermentation solution. The fermentation solution was centrifuged to obtain a supernatant, and the biotin content in the supernatant was detected by liquid chromatography-mass spectrometry.

[0085] Example 1: Construction of biotin ligase mutants

[0086] 1. Active site screening

[0087] The amino acid sequence of biotin ligase BirA is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2. By molecular simulation of the interaction between BirA and ATP (https: / / alphafoldserver.com and pyMOL), 10 amino acid sites were screened and the results are as follows Figure 1 As shown, they are No. 118, 119, 121, 124, 172, 175, 176, 208, 220, and 221 respectively.

[0088] 2. Active site mutation

[0089] Point mutations were performed on the above sites, and the specific mutations are shown in Table 1.

[0090] Table 1 Mutation sites

[0091]

[0092]

[0093] Based on the biotin ligase BirA mutants shown in Table 1, GenScript Biotech Co., Ltd. was commissioned to synthesize the corresponding nucleotide sequence. The nucleotide sequence was then integrated into Escherichia coli MG1655 via homologous recombination technology (using the pKD46 plasmid; the upstream homology arm sequence 5'-3' is shown as SEQ ID NO. 3, and the downstream homology arm 5'-3' is shown as SEQ ID NO. 4) to obtain recombinant bacteria.

[0094] Example 2: Construction of biotin ligase mutants

[0095] 1. Yield detection of mutant recombinant strains

[0096] The recombinant bacteria prepared in Example 1 were tested for biotin production in various mutants. These mutants presented challenges such as unchanged biotin production, low survival rates, and slow growth. Therefore, we selected recombinant strains with the highest biotin production and best growth performance at each of the 10 loci. The results are shown in Table 2.

[0097] Table 2 Biotin production of recombinant bacteria

[0098] mutation site Biotin production (mg / L) MG1655 (wild type) - MG1655-R118A 0.536 MG1655-R119V 0.217 MG1655-R121G 0.601 MG1655-F124A 0.246 MG1655-K172S 0.081 MG1655-D175V 0.662 MG1655-L176G 0.183 MG1655-N208W 0.472 MG1655-N220A 0.095 MG1655-Q221G 0.419

[0099] Note: “-” means the content is less than 0.05.

[0100] 2. Combination Mutation

[0101] The mutants in Example 1 can be divided into three parts, sites 118-124, 172-176, and 208-220. One mutant with higher biotin production was selected from each of them for combined mutation to prepare a combined mutant recombinant strain. The preparation method was consistent with that in Example 1. The biotin production of the combined mutant recombinant strain was further tested, and the results are shown in Table 3.

[0102] Table 3 Biotin production of combined mutant recombinant bacteria

[0103] mutation site Biotin production (mg / L) MG1655-R121G-D175V 0.679 MG1655-R121G-N208W 0.684 MG1655-D175V-N208W 0.218 MG1655-R121G-D175V-N208W 0.136 MG1655-R121G-D175V-Q221G 0.222 MG1655-R118A-D175V-N208W 0.652 MG1655-R118A-R121G 0.701 MG1655-R118A-R121G-D175V-N208W 0.873

[0104] As shown in Table 3, when combined mutations were made at positions 175 and 208, the biotin production of the recombinant bacteria was lower than that of the single mutations at positions 175 and 208. The reason may be that the simultaneous mutation of the two sites changed the stability of the binding pocket of BirA, resulting in a decrease in biotin production; when combined mutations were made at positions 112, 175, and 208, the biotin production was further reduced.

[0105] Considering that the biotin production was also higher when R118A and Q221G were single-point mutations, attempts were made to replace R121G with R118A and perform combined mutations with D175V and N208W; and to replace N208W with Q221G and perform combined mutations with R121G and D175V.

[0106] The results showed that when the Q221G, R121G, and D175V mutations were combined, biotin production increased compared to the N208W, R121G, and D175V mutations, but was still significantly lower than the D175V single mutation. However, when the R118A, D175V, and N208W mutations were combined, biotin production was almost the same as that of the D175V single mutation. Further testing of the combined R118A and R121G mutations revealed that biotin production was significantly better than that of the R121G single mutation.

[0107] At the same time, the combined mutations of R121G and D175V, and R121G and N208W all had good effects. Therefore, we tried to combine mutations of the four sites R118A, R121G, D175V, and N208W. The results showed that the biotin production of the recombinant bacteria MG1655-R118A-R121G-D175V-N208W with the four-site combined mutation was significantly improved, which was better than single-point mutations and other combined mutations.

[0108] The above results show that the recombinant bacteria based on the biotin ligase mutant prepared above have a certain improvement in the production of biotin.

[0109] Sequences used in this application

[0110] Biotin ligase amino acid sequence (SEQ ID NO.1):

[0111] MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEV LRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK

[0112] Biotin ligase nucleotide sequence (SEQ ID NO.2):

[0113] ATGAAGGATAACACCGTGCCACTGAAATTGATTGCCCTGTTAGCGAACGGTGAATTTCACTCTGGCGAGCAGTTGGGTGAAACGCTGGGAATGAGCCGGGCGGCTATTAATAAACACATTCAGACACTGCGTGACTGGGGCGTTGATGTCTTTACCGTTCCGGGTAAAGGATACAGCCTGCCTGAGCCTATCCAGTTACTTAATGCTAAACAGATATTGGGTCAGCTGGATGGCGGTAGTGTAGCCGTGCTGCCAGTGATTGACTCCACGAATCAGTACCTTCTTGATCGTATCGGAGAGCTTAAATCGGGCGATGCTTGCATTGCAGAATACCAGCAGGCTGGCCGTGGTCGCCGGGGTCGGAAATGGTTTTCGCCTTTTGGCGCAAACTTATATTTGTCGATGTTCTGGCGTCTGGAACAAGGCCCGGCGGCGGCGATTGGTTTAAGTCTGGTTATCGGTATCGTGATGGCGGAAGTATTACGCAAGCTGGGTGCAGATAAAGTTCGTGTTAAATGGCCTAATGACCTCTATCTGCAGGATCGCAAGCTGGCAGGCATTCTGGTGGAGCTGACTGGCAAAACTGGCGATGCGGCGCAAATAGTCATTGGAGCCGGGATCAACATGGCAATGCGCCGTGTTGAAGAGAGTGTCGTTAATCAGGGGTGGATCACGCTGCAGGAAGCGGGGATCAATCTCGATCGTAATACGTTGGCGGCCATGCTAATACGTGAATTACGTGCTGCGTTGGAACTCTTCGAACAAGAAGGATTGGCACCTTATCTGTCGCGCTGGGAAAAGCTGGATAATTTTATTAATCGCCCAGTGAAACTTATCATTGGTGATAAAGAAATATTTGGCATTTCACGCGGAATAGACAAACAGGGGGCTTTATTACTTGAGCAGGATGGAATAATAAAACCCTGGATGGGCGGTGAAATATCCCTGCGTAGTGCAGAAAAATAA

[0114] Upstream homologous arm nucleotide sequence 5'-3' (SEQ ID NO.3):

[0115] GTAGGTCTGCGTCTGCCAAAAGAGTGGCAACCTGTACTAACGTATGGTGACTTAACTCGTCTGGATCCTACAACAGTAACGCCACAGCAAGTATTTAATGCGGTGTGTCATATGCGCACCACCAAACTCCCTGATCCAAAAGTGAATGGCAATGCCGGTAGTTTCTTCAAAAACCCTGTTGTATCTGCCGAAACGGCTAAAGCATTACTGTCACAATTTCCAACAGCACCAAATTACCCCCAGGCGGATGGTTCAGTAAAACTGGCAGCAGGTTGGCTTATCGATCAGTGCCAGCTAAAAGGGATGCAAATAGGTGGGGCTGCGGTGCACCGTCAACAGGCGTTAGTTCTCATTAATGAAGACAATGCAAAAAGCGAAGATGTTGTACAGCTGGCGCATCATGTAAGACAGAAAGTTGGTGAAAAATTTAATGTCTGGCTTGAGCCTGAAGTCCGCTTTATTGGTGCATCAGGTGAAGTGAGCGCAGTGGAGACAATTTC

[0116] Downstream homologous arm nucleotide sequence 5'-3' (SEQ ID NO.4):

[0117] TTCCGATCTCAAATCCGGCGTGCCAAACGTTACAGCACCTGTTTACTCACATCTTATTTATGATGTGATCCCGGATGGAGATAAAACGGTTGTTCAGCCTGATATTTTAATTCTTGAAGGGTTAAATGTCTTACAGAGCGGGATGGATTATCCACACGATCCACATCATGTATTTGTTTCTGATTTTGTCGATTTTTCGATATATGTTGATGCACCGGAAGACTTACTTCAGACATGGTATATCAACCGT TTTCTGAAATTCCGCGAAGGGGCTTTTACCGACCCGGATTCCTATTTTCATAACTACGCGAAATTAACTAAAGAAGAAGCGATTAAGACTGCCATGACATTGTGGAAAGAGATCAACTGGCTGAACTTAAAGCAAAATATTCTACCTACTCGTGAGCGCGCCAGTTTAATCCTGACGAAAAGTGCTAATCATGCGGTAGAAGAGGTCAGACTACGCAAATAATTTGCAGGGGAGCGAATACTCCCCTTTC

[0118] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A biotin ligase mutant, characterized in that The biotin ligase mutant has one or more amino acid mutations at positions 118, 119, 121, 124, 172, 175, 176, 208, 220, and 221 based on the amino acid sequence shown in SEQ ID NO.

1.

2. The biotin ligase mutant according to claim 1, characterized in that Corresponding to the amino acid sequence shown in SEQ ID NO.1, the arginine at position 118 is mutated to alanine; or, Arginine at position 119 is mutated to valine; or Arginine at position 121 is mutated to glycine; or Phenylalanine at position 124 is mutated to alanine; or Lysine at position 172 is mutated to serine; or Aspartic acid at position 175 is mutated to valine; or Leucine at position 176 is mutated to glycine; or Asparagine at position 208 is mutated to tryptophan; or Asparagine at position 220 is mutated to alanine; or The glutamine at position 221 was mutated to glycine.

3. The biotin ligase mutant according to any one of claims 1 to 2, characterized in that Mutations also exist: Arginine at position 121 is mutated to glycine, and aspartic acid at position 175 is mutated to valine; or Arginine at position 121 is mutated to glycine, and asparagine at position 208 is mutated to tryptophan; or Arginine at position 118 is mutated to alanine, aspartic acid at position 175 is mutated to valine, and asparagine at position 208 is mutated to tryptophan; or Arginine at position 118 is mutated to alanine, and arginine at position 121 is mutated to glycine; or The arginine at position 118 was mutated to alanine, the arginine at position 121 was mutated to glycine, the aspartic acid at position 175 was mutated to valine, and the asparagine at position 208 was mutated to tryptophan.

4. A polynucleotide encoding the biotin ligase mutant according to any one of claims 1 to 3.

5. A vector carrying the polynucleotide according to claim 4, characterized in that The vectors include pPIC series vectors, pPICZ series vectors, pPICZα and pET series vectors.

6. A cell expressing the biotin ligase mutant according to any one of claims 1 to 3, characterized in that: The cells include Bacillus subtilis and Escherichia coli.

7. A genetically engineered Escherichia coli, characterized in that: The biotin ligase of the strain has one or more amino acid mutations at positions 118, 119, 121, 124, 172, 175, 176, 208, 220, and 221 based on SEQ ID NO. 1; Preferably, the arginine at position 118 is mutated to alanine; or, Arginine at position 119 is mutated to valine; or Arginine at position 121 is mutated to glycine; or Phenylalanine at position 124 is mutated to alanine; or Lysine at position 172 is mutated to serine; or Aspartic acid at position 175 is mutated to valine; or Leucine at position 176 is mutated to glycine; or Asparagine at position 208 is mutated to tryptophan; or Asparagine at position 220 is mutated to alanine; or Glutamine at position 221 is mutated to glycine; or Arginine at position 121 is mutated to glycine, and aspartic acid at position 175 is mutated to valine; or Arginine at position 121 is mutated to glycine, and asparagine at position 208 is mutated to tryptophan; or Arginine at position 118 is mutated to alanine, aspartic acid at position 175 is mutated to valine, and asparagine at position 208 is mutated to tryptophan; or Arginine at position 118 is mutated to alanine, and arginine at position 121 is mutated to glycine; or The arginine at position 118 was mutated to alanine, the arginine at position 121 was mutated to glycine, the aspartic acid at position 175 was mutated to valine, and the asparagine at position 208 was mutated to tryptophan.

8. Use of the biotin ligase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the vector of the polynucleotide according to claim 5, the cell according to claim 6, or the genetically engineered Escherichia coli according to claim 7 in the preparation of biotin.

9. A method for preparing biotin using Escherichia coli, characterized in that: Using the genetically engineered Escherichia coli bacteria of claim 7 to ferment and prepare biotin; Fermentation medium (L): glucose 10 g, CoCl2·6H2O 2 mg, KH2PO4 7.5 g, ZnSO4·7H2O 2 mg, yeast powder 3 g, CaCl2 4 mg, citric acid 1.8 g, vitamin B1 1.5 mg, MgSO4·7H2O 2 g, CuSO4 0.5 mg, FeSO4·7H2O 70 mg, MnSO4·H2O 10 mg; The fermentation conditions were 200 rpm and 37°C for 24 h.

10. Use of the biotin ligase mutant according to any one of claims 1 to 3 in protein labeling and detection or preparation of antibody-drug conjugates or cell surface marker imaging; in, Protein labeling and detection involves using the biotin ligase mutant to biotinylate proteins, including ELISA, Western blot, and immunofluorescence; The preparation of antibody-drug conjugates is to connect the drug molecule to the antibody using the biotin ligase mutant to prepare a targeted drug; Cell surface marker imaging is the combination of the biotin ligase mutant and fluorescently labeled streptavidin to achieve the visualization of proteins on the surface of living cells.

Images