Biotin ligase mutant and use thereof

Abstract

The application discloses a biotin ligase mutant and application thereof, and belongs to the technical field of genetic engineering. The biotin ligase mutant is prepared by performing multiple mutations on 10 amino acids with a binding force action site which are screened through biotin ligase and substrate molecule docking. On this basis, the biotin ligase mutant is connected to Escherichia coli by using a homologous recombination technology, and a series of Escherichia coli recombinant bacteria with high biotin yield are obtained. The biotin yield of the recombinant bacteria reaches 0.873 mg / L after 24 h fermentation.

Description

Technical Field

[0001] This invention relates to a biotin ligase mutant and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] Biotin, also known as vitamin B7 or coenzyme R, is a water-soluble vitamin belonging to the B vitamin group. Its molecular structure contains a fused thiazolidinyl ring and a furan ring, making it an important coenzyme for many carboxylases and widely involved in basic metabolic processes such as fatty acid synthesis, amino acid metabolism, and glucose production. Due to its important roles in cell proliferation, gene expression regulation, and immune function maintenance, biotin is widely used in food additives, nutritional supplements, animal feed, and cosmetics, particularly showing significant effects on skin health, hair growth, and nail strengthening. Furthermore, biotin is also used in molecular biology, commonly for labeling proteins and nucleic acids, playing a crucial role in affinity purification and bioassay techniques.

[0003] Industrially, biotin production primarily relies on two pathways: microbial fermentation and chemical synthesis. Microbial fermentation typically employs strains such as *Escherichia coli* and *Pichia pastoris*, using genetic engineering to enhance their biotin synthesis capabilities, thereby achieving efficient production. BirA is a protein with dual catalytic and transcriptional regulatory functions. On one hand, it acts as a biotin ligase, catalyzing and mediating the biotinylation reaction of proteins, linking biotin to specific proteins. On the other hand, BirA is also a biotin-inducible inhibitor, acting as a regulator of biotin synthesis, negatively regulating biotin synthesis.

[0004] Escherichia coli (E. coli) has significant advantages in biotin production. It possesses a complete biotin biosynthesis pathway, which can be directly enhanced through metabolic engineering. Furthermore, its genetic manipulation system is highly mature, offering a rich toolbox of synthetic biology tools for efficient gene editing, pathway optimization, and expression regulation. In addition, E. coli grows rapidly, utilizes inexpensive carbon sources, and adapts to high-density cultivation, significantly reducing industrial production costs. In industrial applications, E. coli is a Generally Recognized As Safe (GRAS) strain, with abundant fermentation processes and regulatory data available, facilitating large-scale and compliant production. Patent CN118207171B discloses a biotin ligase mutant; E. coli expressing this mutant has the ability to produce biotin at a yield of 0.156 mg / L, but the biotin yield of this method remains relatively low.

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

[0006] To address the aforementioned problems, this invention involves docking biotin ligase with its substrate molecules and repeatedly mutating 10 amino acids with binding sites to prepare a series of biotin ligase mutants. Based on this, homologous recombination technology is used to ligate the biotin ligase mutants into *E. coli*, resulting in a series of recombinant *E. coli* strains with high biotin production.

[0007] The first objective of this invention is to provide a biotin ligase mutant having 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] The arginine at position 119 is mutated to valine; or,

[0010] The arginine at position 121 is mutated to glycine; or,

[0011] The phenylalanine at position 124 is mutated to alanine; or,

[0012] The lysine residue at position 172 is mutated to a serine residue; or,

[0013] The aspartic acid at position 175 is mutated to valine; or,

[0014] The leucine at position 176 is mutated to glycine; or,

[0015] The asparagine at position 208 is mutated to tryptophan; or,

[0016] The asparagine at position 220 is mutated to alanine; or,

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

[0018] In one implementation, mutations also exist:

[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] Arginine at position 118 is mutated to alanine, arginine at position 121 is mutated to glycine, aspartic acid at position 175 is mutated to valine, and asparagine at position 208 is mutated to tryptophan.

[0024] A second objective of this invention is to provide a polynucleotide encoding any of the aforementioned biotin ligase mutants.

[0025] A third objective of this invention is to provide a carrier for carrying the aforementioned polynucleotides.

[0026] In one embodiment, the vector includes 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, said cells including Bacillus subtilis and Escherichia coli.

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

[0029] The 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 the structure shown in SEQ ID NO.1.

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

[0031] The arginine at position 119 is mutated to valine; or,

[0032] The arginine at position 121 is mutated to glycine; or,

[0033] The phenylalanine at position 124 is mutated to alanine; or,

[0034] The lysine residue at position 172 is mutated to a serine residue; or,

[0035] The aspartic acid at position 175 is mutated to valine; or,

[0036] The leucine at position 176 is mutated to glycine; or,

[0037] The asparagine at position 208 is mutated to tryptophan; or,

[0038] The asparagine at position 220 is mutated to alanine; or,

[0039] The 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] Arginine at position 118 is mutated to alanine, arginine at position 121 is mutated to glycine, aspartic acid at position 175 is mutated to valine, and asparagine at position 208 is mutated to tryptophan.

[0045] A sixth object of the present invention is to provide the 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 Escherichia coli genetically engineered bacteria in the preparation of biotin.

[0046] The seventh objective of this invention is to provide a method for preparing biotin using Escherichia coli, wherein biotin is prepared by fermentation using the aforementioned genetically engineered Escherichia coli.

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

[0048] Fermentation conditions were 200 rpm and 37°C for 24 hours.

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

[0050] Among them, protein labeling and detection involves using the biotin ligase mutant for biotinylated proteins, including ELISA, Western blot, and immunofluorescence.

[0051] The preparation of antibody-drug conjugates involves linking drug molecules to antibodies using the biotin ligase mutant to prepare targeted drugs.

[0052] Cell surface labeling imaging involves the biotin ligase mutant binding to fluorescently labeled streptavidin to visualize proteins on the surface of living cells.

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

[0054] Beneficial effects of the present invention

[0055] This invention involves docking biotin ligase with its substrate molecules, screening 10 amino acids with binding sites, and then repeatedly mutating them to prepare a series of biotin ligase mutants. Based on this, homologous recombination technology is used to ligate the biotin ligase mutants into *E. coli*, resulting in a series of recombinant *E. coli* strains with high biotin production.

[0056] Specifically,

[0057] (1) The recombinant Escherichia coli prepared in this application, when subjected to a single-point mutation of biotin ligase, achieved a maximum biotin concentration of 0.662 mg / L after 24 hours of fermentation;

[0058] (2) When the biotin ligase combination of the recombinant Escherichia coli prepared in this application is mutated, the biotin concentration reaches a maximum of 0.873 mg / L after 24 hours of fermentation. Attached Figure Description

[0059] Figure 1 This is a schematic diagram of the simulation results of biotin ligase BirA and ATP molecules; red represents amino acid sites with binding force. Detailed Implementation

[0060] The preferred embodiments of the present invention are described below. 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] This invention uses molecular docking software to analyze the binding site of biotin ligase BirA (amino acid sequence shown in SEQ ID NO.1) with ATP. By mutating the amino acid at the binding site to a hydrophilic or less dimer amino acid, 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 selected sites are numbered 118, 119, 121, 124, 172, 175, 176, 208, 220, and 221.

[0063] In one embodiment of the present invention, the hydrophilic or non-dimeric amino acid 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 are performed on the selected 10 sites, 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 K172E. 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, the above-mentioned biotin ligase mutations result in strains with low survival rates and slow growth, such as recombinant strains expressing mutants R118V, R119Y, R119T, N208E, etc.; there are also recombinant strains whose yields are not significantly increased, such as recombinant strains expressing mutants R118W, K172A, K172E, K172T, Q221Y, Q221A, etc.

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

[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, a mutant with a high biotin production is selected sequentially from three regions for combined mutation to prepare a combined mutant 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 was significantly increased after 24 hours of fermentation, which was superior to that of the unmutated recombinant bacteria.

[0070] In one embodiment of the present invention, the binding affinity of the above-mentioned biotin ligase mutant to ATP and bioin-5'-AMP is weakened, the release of bioin-5'-AMP is increased, and it is no longer specifically modified, so that it can label neighboring proteins, thereby improving the labeling efficiency and range of the organism.

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

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

[0073] In one embodiment of the present invention, protein labeling and detection involves using the biotin ligase mutant to biotinylate proteins, including ELISA, Western blot, and immunofluorescence.

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

[0075] In one embodiment of the present invention, cell surface labeling imaging is achieved by the biotin ligase mutant combining with fluorescently labeled streptavidin to visualize proteins on the surface of living cells.

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

[0077] Raw materials used in the examples:

[0078] The nucleotide sequence was synthesized by Genscript Biotech Inc.

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

[0080] Test method:

[0081] Seed culture medium preparation (L): 10g tryptone, 5g yeast extract, 10g NaCl.

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

[0083] Biotin detection method:

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

[0085] Example 1: Construction of a biotin ligase mutant

[0086] 1. Screening of active sites

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

[0088] 2. Mutation at active sites

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

[0090] Table 1 Mutation sites

[0091]

[0092]

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

[0094] Example 2: Construction of biotin ligase mutant

[0095] 1. Detection of yield of mutant recombinant strains

[0096] The recombinant bacteria prepared in Example 1 were used to test the biotin production of different mutant recombinant bacteria. The mutants exhibited problems such as unchanged recombinant bacterial production, low survival rate, and slow growth. Therefore, the recombinant strains with the highest production and best growth performance at each of the 10 sites were selected, and the results are shown in Table 2.

[0097] Table 2 Biotin Production from 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: "-" indicates a content of less than 0.05.

[0100] 2. Combinatorial mutation

[0101] The mutant in 1 can be divided into three parts: sites 118–124, 172–176, and 208–220. One mutant with a higher biotin production was selected from these sites for combined mutation to prepare a combined mutant recombinant strain. The preparation method was the same as 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 by 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 the combined mutations at positions 175 and 208 were performed, the biotin production of the recombinant bacteria was lower than that of the single-point mutations at positions 175 and 208. This may be because the simultaneous mutations at two points changed the stability of the BirA binding pocket, resulting in a decrease in biotin production. When the combined mutations at positions 112, 175, and 208 were performed, the biotin production decreased further.

[0105] Considering that biotin production is also high when R118A and Q221G are mutated at a single point, we tried replacing R121G with R118A and performing a combined mutation with D175V and N208W; and replacing N208W with Q221G and performing a combined mutation with R121G and D175V.

[0106] The results showed that when the Q221G, R121G, and D175V combined mutations were present, biotin production was higher than that of the N208W, R121G, and D175V combined mutations, but still much lower than that of the D175V single-point mutation. However, when the R118A, D175V, and N208W combined mutations were present, biotin production was almost the same as that of the D175V single-point mutation. Further examination of the R118A and R121G combined mutations revealed that biotin production was significantly better than that of the R121G single mutation.

[0107] Meanwhile, the combined mutations of R121G with D175V and R121G with N208W showed good results. Therefore, we tried combined mutations of four sites: R118A, R121G, D175V, and N208W. The results showed that the biotin production of the four-site combined mutant recombinant strain MG1655-R118A-R121G-D175V-N208W was significantly improved, which was better than single-site mutations and other combined mutations.

[0108] The results in summary indicate that the recombinant bacteria based on biotin ligase mutants prepared above all showed a certain increase in biotin production.

[0109] The sequence 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 with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A biotin ligase mutant, characterized in that, The biotin ligase mutant is based on the biotin ligase with the amino acid sequence shown in SEQ ID NO.1, with the following mutations: arginine at position 118 is mutated to alanine, arginine at position 121 is mutated to glycine, aspartic acid at position 175 is mutated to valine, and asparagine at position 208 is mutated to tryptophan.

2. A polynucleotide encoding the biotin ligase mutant of claim 1.

3. A vector carrying the polynucleotide of claim 2, characterized in that, The carriers include pPIC series carriers or pET series carriers.

4. A cell expressing the mutant biotin ligase of claim 1, characterized in that, The cells include Bacillus subtilis.

5. A genetically engineered Escherichia coli bacterium, characterized in that, The biotin ligase of the genetically engineered Escherichia coli is based on the biotin ligase shown in SEQ ID NO.1, with the following mutations: arginine at position 118 is mutated to alanine, arginine at position 121 is mutated to glycine, aspartic acid at position 175 is mutated to valine, and asparagine at position 208 is mutated to tryptophan.

6. The use of the biotin ligase mutant of claim 1, the polynucleotide of claim 2, the vector of claim 3, the cell of claim 4, or the Escherichia coli genetically engineered bacterium of claim 5 in the preparation of biotin.

7. A method for preparing biotin using Escherichia coli, characterized in that, Biotin was prepared by fermentation using the genetically engineered Escherichia coli strain described in claim 5; Each liter of fermentation medium contains: 10 g glucose, 2 mg CoCl2·6H2O, 7.5 g KH2PO4, 2 mg ZnSO4·7H2O, 3 g yeast extract, 4 mg CaCl2, 1.8 g citric acid, 1.5 mg vitamin B1, 2 g MgSO4·7H2O, 0.5 mg CuSO4, 70 mg FeSO4·7H2O, and 10 mg MnSO4·H2O; The fermentation conditions were 200 rpm and 37°C for 24 h.

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