Desthiobiotin synthetase mutant and its application in biotin production

Abstract

The present invention discloses a mutant of desulfurization biotin synthetase of Escherichia coli and its application in biotin production. Saturation mutation is performed on the 142nd amino acid of desulfurization biotin synthetase, and it is found that when the 142nd amino acid of desulfurization biotin synthetase is mutated to serine, asparagine, glutamine, leucine, alanine, histidine, threonine or isoleucine, the enzyme activity is improved and the biotin yield is also significantly improved. The mutant can be applied to the biological production of biotin.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a desulfurization biotin synthetase mutant and application thereof in biotin production. Background Art

[0002] Biotin, also known as vitamin H, vitamin B7, and coenzyme R, plays an important role in biochemical reaction pathways such as fat synthesis and gluconeogenesis, and is one of the most important cofactors involved in the central metabolism of eukaryotic and prokaryotic organisms (lipid synthesis, amino acid metabolism, and sugar metabolism). It plays an important role in maintaining the normal growth and development of humans and animals, bone marrow health, and skin maintenance. It is widely used in many fields such as food additives, medicine, cosmetics, and animal husbandry, and the market demand is increasing year by year.

[0003] Currently, most commercially available biotin is synthesized through a multi-step chemical process developed by Goldberg and Sternbach. However, chemical synthesis of biotin has problems such as complex reactions, high energy consumption, high costs, and environmental unfriendliness. Therefore, a green and cost-effective biosynthetic method for producing biotin is extremely important.

[0004] The biotin synthesis pathway of E. coli cells is divided into two stages: the synthesis of pimelic acid thioester and the synthesis of the bicyclic ring. The synthesis of the bicyclic ring begins with the synthesis of pimelic acid thioester, which is catalyzed by the key enzymes BioF, BioA, BioD, and BioB in sequence, and finally synthesizes biotin (see Appendix Figure 1 ).

[0005] BioD is a dethiobiotin synthase, which participates in the penultimate step of biotin synthesis. With the participation of CO2 and ATP, it catalyzes the substrate 7,8-diaminopelargonic acid (DAPA) to produce dethiobiotin (DTB). DTB is a precursor of biotin and directly affects the amount of biotin synthesis. However, the catalytic efficiency of BioD in Escherichia coli is very low, only 0.06 s -1 The DTB synthesis reaction catalyzed by it becomes the rate-limiting step in biotin synthesis in Escherichia coli.

[0006] At present, the research on desulfurization biotin synthase BioD at home and abroad focuses on the screening and development of Mycobacterium tuberculosis desulfurization biotin synthase as an anti-tuberculosis drug, and there are almost no reports on the modification of BioD and its application in the industrial production of biotin. Escherichia colidethiobiotin synthetase: effects of mutations on enzyme catalytic and structural properties.[J]. Biochemistry, 1997, 36(16): 4751-4760. DOI:10.1021 / bi9631677.) Through the crystal structure study of Escherichia coli dethiobiotin synthetase, the importance of some active site residues (Thr11, Glu12, Lys15, Lys37, Ser41, Asn175) was revealed, but the relationship between them and DTB synthesis activity and biotin production was not clarified. Up to now, the relevant research results have not been able to find the key sites that significantly improve the activity of dethiobiotin synthetase, which makes it difficult to improve the production of biotin by rationally modifying BioD. Therefore, it is of great significance to explore the key targets that affect the activity of BioD enzyme and conduct in-depth and comprehensive research on this basis to improve the production of biotin. Summary of the invention

[0007] In order to increase the production of biotin, the present invention first induced mutagenesis in Escherichia coli, screened out mutants with significantly increased biotin production, and sequenced them to find that when the valine at position 142 of desulfurization biotin synthase (BioD) was mutated to serine, the biotin production was significantly increased. In order to further study the effect of the 142nd amino acid on BioD, the present invention carried out amino acid saturation mutation on the site and measured the change in biotin production after mutation.

[0008] The present invention provides an Escherichia coli ( Escherichia coli ) A dethiobiotin synthetase mutant and its application in biotin production, wherein the mutant increases the biotin yield without affecting the growth of the strain.

[0009] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides an Escherichia coli desulfurization biotin synthetase mutant, wherein the mutant comprises or consists of an amino acid sequence selected from the following:

[0010] 1) A mutation from V to A in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142A );

[0011] 2) A mutation from V to R in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142R );

[0012] 3) A mutation from V to N in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142N );

[0013] 4) A mutation from V to D in the amino acid sequence of the desulfurization biotin synthase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142D );

[0014] 5) A mutation from V to C in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142C );

[0015] 6) A mutation from V to Q in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142Q );

[0016] 7) A mutation from V to E in the amino acid sequence of the desulfurization biotin synthase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142E );

[0017] 8) A mutation from V to G in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142G );

[0018] 9) A mutation from V to H in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142H );

[0019] 10) A mutation from V to I in the amino acid sequence of the desulfurization biotin synthase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142I );

[0020] 11) A mutation from V to L in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142L );

[0021] 12) A mutation from V to K in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142K );

[0022] 13) A mutation from V to M in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142M );

[0023] 14) A mutation from V to F in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142F );

[0024] 15) A mutation from V to P in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142P );

[0025] 16) A mutation from V to S in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142S );

[0026] 17) A mutation from V to T in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142T );

[0027] 18) A mutation from V to W in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142W );

[0028] 19) A mutation from V to Y in the amino acid 142 of the desulfurization biotin synthetase from Escherichia coli as shown in SEQ ID NO: 1 (BioD V142Y ).

[0029] In a second aspect, the present invention provides a nucleic acid molecule encoding the dethiobiotin synthetase mutant.

[0030] In a third aspect, the present invention provides biological materials containing the nucleic acid molecule, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineered bacteria.

[0031] In a fourth aspect, the present invention provides a recombinant microorganism comprising the desulfurization biotin synthetase mutant or a nucleic acid molecule encoding the desulfurization biotin synthetase mutant.

[0032] In a fifth aspect, the present invention provides use of the dethiobiotin synthetase mutant, the nucleic acid molecule, the biological material, or the recombinant microorganism in biotin production.

[0033] In a sixth aspect, the present invention provides the use of the dethiobiotin synthetase mutant, the nucleic acid molecule, the biological material, or the recombinant microorganism in improving biological production.

[0034] In one embodiment of the present invention, the mutant is based on the dethiobiotin synthetase shown in SEQ ID NO: 1, and a mutation is performed on the following amino acid sites:

[0035] The valine at position 142 was mutated to serine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0036] Preferably, the valine at position 142 is mutated to serine, asparagine, glutamine, leucine, alanine, histidine, threonine or isoleucine, respectively. More preferably, the valine at position 142 is mutated to alanine, histidine, threonine or isoleucine, respectively.

[0037] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0038] The present invention obtains a mutant BioD having improved desulfurization biotin synthase activity by mutating the wild-type Escherichia coli desulfurization biotin synthase BioD. V142S 、BioD V142N 、BioD V142Q 、BioD V142L 、BioD V142A 、BioD V142H 、BioD V142T 、BioD V142I , especially BioD V142I 、BioD V142T 、BioD V142H 、BioD V142A , effectively increasing the production of biotin. Especially the mutant BioD V142I The enzyme activity is 2.3 times that of the wild-type dethiobiotin synthase, and the biotin production is increased by 36%, which is of great significance for the large-scale production of biotin. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the biotin bicyclic synthesis pathway

[0040] Figure 2 Relative enzyme activity of dethiobiotin synthetase mutants

[0041] Figure 3The yield of biotin produced by recombinant Escherichia coli expressing desulfobiotin synthase mutant was detected using ΔbioFbioCbioDbioB plates

[0042] Figure 4 Results of HPLC-MS detection of biotin production by recombinant Escherichia coli expressing dethiobiotin synthase mutants

[0043] Figure 5 Growth curve of recombinant Escherichia coli expressing dethiobiotin synthetase mutant DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific examples. Where specific experimental conditions are not indicated, they are all based on conventional conditions well known to those skilled in the art.

[0045] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), etc., or according to the conditions recommended by the manufacturer's instructions.

[0046] Example 1: Screening of mutation points in desulfurization biotin synthetase

[0047] 1. Mutagenesis screening of strains with improved biotin production

[0048] (1) NTG mutagenesis

[0049] The wild-type strain of Escherichia coli BW25113 was used as the base strain, inoculated into 5 mL LB liquid medium for activation, and cultured overnight at 37°C and 220 rpm. 1 mL of the activated bacterial solution was transferred into 100 mL LB liquid medium and cultured at 37°C and 220 rpm until the OD 600 Reach 1.0. Collect the cells by centrifugation at 6000 rpm for 5 min, wash three times with 0.9% saline, and remove the supernatant by centrifugation at 6000 rpm. Resuspend the cells with 20 mL of 0.9% saline, add NTG (nitrosoguanidine) to a final concentration of 0.1~0.5 g / L, and perform mutagenesis for 20 min. Dilute the treated bacterial suspension to an appropriate concentration, apply 100 µL to an LB plate, and culture at 37°C overnight.

[0050] (2) Deep well plate initial screening

[0051] ① Inoculate the single colonies on the LB plate into the seed plate and culture at 37°C and 1000 rpm for 16 h to obtain activated seed solution. The seed plate is a 96-deep-well plate with 1 mL of liquid seed culture medium in each well.

[0052] ② Transfer the activated seed solution to the corresponding fermentation plate at an inoculation volume of 5% (V / V), culture at 37°C, 1000rpm for 24 hours, and store the seed plate in a refrigerator at 4°C. The fermentation plate is a 96-deep-well plate with 1 mL of liquid fermentation medium in each well.

[0053] ③ Place the cultured fermentation plate in a centrifuge and centrifuge for 10 min. Take the supernatant and dilute it appropriately. Drop it onto the biotin detection plate. Culture it at 37°C for 16 h and measure the diameter of the growth circle.

[0054] Preparation of seed culture medium (L): 10 g tryptone, 5 g yeast extract, 10 g NaCl.

[0055] Preparation of 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, CuSO40.5 mg, FeSO4·7H2O 70 mg, MnSO4·H2O 10 mg.

[0056] The biotin detection plate is prepared by the preparation method of the "biotin detection plate" described in Examples 2-3 of the specification of patent CN118207171A.

[0057] (3) Shake flask fermentation rescreening

[0058] Transformants with a growth circle diameter that increased by more than 1.5 times compared with the wild-type control strain BW25113 were selected for shake flask fermentation rescreening. The transformants were inoculated into 5 mL of seed culture medium and cultured overnight at 37°C and 220 rpm to obtain seed culture. The inoculation amount was transferred to 20 mL of fermentation medium at 1% (V / V), and cultured at 37°C and 220 rpm for 24 hours to obtain fermentation broth samples. The fermentation broth samples were treated at 95°C for 5 minutes, centrifuged at 12000 rpm for 5 minutes, and then dropped onto the biotin detection plate. They were cultured at 37°C for 16 hours, and the growth circle diameter was measured to preliminarily judge the biotin production capacity of the strain.

[0059] 2. Genome sequencing

[0060] In order to explore the high-production mechanism of biotin in the mutant strain, the genome of the mutant strain with the strongest biotin production ability was sequenced.

[0061] Analysis of the sequencing results showed that the gene encoding the desulfurization biotin synthase BioD of the Escherichia coli strain with the strongest biotin production capacity ( bioD ) mutated, with the valine at position 142 mutated to serine.

[0062] Example 2: Construction of desulfurization biotin synthetase mutants

[0063] In order to further study whether the 142nd amino acid of dethiobiotin synthetase is a key site affecting enzyme activity and biotin production, amino acid saturation mutation was performed on it. In addition to the original valine, mutants with the remaining 19 common amino acid mutations at this site were constructed.

[0064] (1) Using the BW25113 genome sequence as a template, PCR amplification was performed using primers bioD-F / bioD-R to obtain bioD The gene fragment encoded the BioD amino acid sequence as shown in SEQ ID NO: 1. Using the pBAD / HisA plasmid as a template, the backbone fragment was amplified using primers pBAD-F / pBAD-R, and the Gibson seamless cloning technique was used to clone the bioD The fragment was connected to the pBAD / HisA vector to obtain the vector pBAD-bioD. The plasmid was transformed into competent E. coli BW25113 to obtain the recombinant strain BW25113-BioD.

[0065] (2) Using the vector pBAD-bioD as a template, use 19 pairs of mutation primers (see Table 1) to bioD The gene was subjected to point mutation and the PCR amplification product was used Dpn After 2 h of digestion with enzyme I, the vector was transferred into competent E. coli DH5α cells and plated on LB plates containing ampicillin (50 μg / mL). After culturing at 37°C for 12 h, the positive clones were identified using primers pBAD-JD-F / pBAD-JD-R, and 19 vectors pBAD-bioD* containing BioD mutations were finally obtained (see Table 2). The vectors were transferred into competent E. coli BW25113 cells to obtain 19 mutant recombinant strains (see Table 3).

[0066] Table 1 bioD Primers used for constructing mutation vector

[0067]

[0068] Table 2 Plasmids used in the present invention

[0069]

[0070] Table 3 Strains used in the present invention

[0071]

[0072] Example 3: In vitro enzyme activity test of desulfurization biotin synthetase mutants

[0073] 1. Induced expression of recombinant protein

[0074] The 19 mutant recombinant strains constructed in Example 2 and BW25113-BioD as a control were inoculated into LB medium containing kanamycin (50 μg / mL) and cultured overnight at 37°C 220 rpm to obtain seed cultures. The seed cultures were transferred to 50 mL TB medium at a 1% (V / V) inoculation rate and cultured at 37°C 220 rpm until OD 600 When the p-value reached 0.8, L-arabinose with a final concentration of 4 g / L was added for induction, and the culture was continued at 30°C, 220 rpm for 20 h. The bacteria were then collected by centrifugation at 4°C, 12,000 rpm for 10 min.

[0075] Preparation of LB medium (L): 10 g tryptone, 5 g yeast extract, 10 g NaCl.

[0076] Preparation of TB medium (L): glycerol 4 g, yeast extract 24 g, tryptone 12 g, KH2PO4 2.31 g, K2HPO4 16.43 g, pH 7.5.

[0077] 2. Recombinant protein purification

[0078] Add sterile water to resuspend the collected bacteria and centrifuge at 4°C to remove the supernatant, repeat twice. Add pre-cooled lysis buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.5) to resuspend the bacteria, break the cells by high-pressure homogenization, centrifuge at 4°C 10000 rpm for 10 min, and collect the supernatant.

[0079] Protein purification was performed using Mag-Beads His-Tag protein purification beads. Load the sample into a centrifuge tube containing magnetic beads, invert to mix, and incubate at 4°C for 30 min. Place the centrifuge tube on a magnetic separator, and after the solution becomes clear, remove the supernatant with a pipette. Add 10 mL of wash buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 7.5) to the centrifuge tube to wash the impurities. Repeat the wash more than 3 times. Add 10 mL of elution buffer (20 mMTris-HCl, 500 mM NaCl, 500 mM imidazole, 5% glycerol, pH 7.5) to the centrifuge tube, gently pipette 3-5 times, mix, place the centrifuge tube on a magnetic separator, and after the solution becomes clear, use a pipette to aspirate the supernatant, which is the target protein fraction. The purified protein was transferred to an ultrafiltration tube with a molecular weight cutoff of 10 kDa for concentration and the buffer was replaced with a reaction buffer (20 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 1 mM DTT, pH 8.0). The expression of the target protein was detected by SDS-PAGE.

[0080] 3. Desulfhiobiotin synthetase mutant enzyme activity test

[0081] The above purified protein was used for catalytic test. The enzymatic activity of dethiobiotin synthetase was determined by the increase in the amount of product DTB per unit time. The test reaction solution contained 50 mM Tris-HCl, 150 μM NaCl, 5 mM MgCl2, 10 mMNaHCO3, 300 μM ATP, 100 μM DAPA, pH 7.5. 0.25 to 3.0 mM purified protein was added to the above reaction solution and reacted at 37°C for 45 min. The catalytic reaction was terminated by adding 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 250 mM EDTA and incubating at 37°C for 15 minutes (see reference Salaemae W, and, et al. Nucleotidetriphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase[J]. Tuberculosis, 2015. DOI:10.1016 / j.tube.2015.02.046. Page 261, left column 2.5). The synthesized DTB product was detected by HPLC-MS, and the wild-type dethiobiotin synthetase BioD was used as a control. The relative enzyme activity of dethiobiotin synthetase and its mutants was calculated by measuring the amount of product DTB produced per unit time.

[0082] The results are as follows Figure 2 As shown, compared with the wild-type dethiobiotin synthase BioD, 19 mutants showed a V142S 、BioD V142N 、BioD V142Q 、BioD V142L 、BioD V142A 、BioD V142H 、BioD V142T 、BioD V142I All have higher enzyme activity (see Figure 2 ), among which BioD V142A 、BioD V142H 、BioD V142T 、BioD V142I The enzyme activities of BioD were 1.7, 1.9, 1.8 and 2.3 times higher than those of the wild type, especially when valine was mutated to isoleucine. V142I The above results show that the 142nd amino acid of BioD is very important for its enzyme activity in catalyzing the formation of DTB, and when it mutates to different amino acids, its activity is affected differently.

[0083] Example 4: Plate detection of biotin production by desulfurization biotin synthetase mutants

[0084] (1) The strain BW25113-BioD expressing the four mutants with the highest desulfurization biotin synthase activity was V142A 、BW25113-BioD V142H 、BW25113-BioD V142T 、BW25113-BioD V142I and control strains BW25113-BioD and BW25113 were inoculated into 5 mL of seed culture medium, respectively, and cultured overnight at 37°C and 220 rpm to obtain seed cultures.

[0085] (2) The seed culture of step (1) was transferred to 20 mL of fermentation medium at an inoculum rate of 1% (V / V), and cultured at 37°C and 220 rpm for 24 h to obtain a fermentation broth sample.

[0086] (3) Take the fermentation broth samples, treat them at 95°C for 5 min, centrifuge them at 12,000 rpm for 5 min, dilute them 2-fold, drop them onto the biotin detection plate, and incubate them at 37°C for 16 h.

[0087] Biotin detection plate results show (see Figure 3 ), the mutant strain BW25113-BioD was added V142A 、BW25113-BioDV142H 、BW25113-BioD V142T 、BW25113-BioD V142I The detection plates of fermentation broth samples of the wild-type strain BW25113 and the control strain BW25113-BioD showed obvious growth circles, and the growth circles of the mutants were significantly larger than those of the control strains, while no growth circles were observed on the detection plates of fermentation broth samples of the wild-type strain BW25113. The above results show that compared with the control strain BW25113-BioD expressing unmutated BioD, the mutant strain BW25113-BioD V142A 、BW25113-BioD V142H 、BW25113-BioD V142T 、BW25113-BioD V142I The biotin synthesis was significantly increased.

[0088] Through measurement, we know (see Table 4), Figure 3 Medium mutant BW25113-BioD V142A 、BW25113-BioD V142T 、BW25113-BioD V142H 、BW25113-BioD V142I The diameter of the growth circle of BW25113-BioD was larger than that of BW25113-BioD and BW25113 as the control. V142I The growth circle of BioD was the largest, which was consistent with the results of in vitro enzyme activity test. This also showed that when the 142nd mutation of BioD was introduced, the expression mutant BioD V142A 、BioD V142T 、BioD V142H 、BioD V142I strains have higher biotin synthesis capacity.

[0089] Table 4 Growth circle size of recombinant strains after fermentation

[0090]

[0091] Example 5: Quantitative determination of biotin production

[0092] The fermentation broth samples obtained in step (2) of Example 4 were separately taken, treated at 95° C. for 5 min, and then centrifuged at 12,000 rpm for 10 min. The supernatant was taken into a new centrifuge tube, and the biotin content in the fermentation broth was further quantitatively determined using HPLC-MS.

[0093] The results of HPLC-MS showed that (see Figure 4The biotin production of the four strains with the BioD mutation was significantly improved compared with the control strain without the BioD mutation (BW25113-BioD), including BioD V142I The mutant strain had the highest biotin production, reaching 136% of the control strain. This result fully verified that the 142nd amino acid of desulfurization biotin synthetase BioD is crucial for the synthesis of biotin. When it mutates from valine to a specific amino acid, it can significantly increase the amount of biotin synthesis, among which the mutant obtained by mutation to isoleucine has the most significant improvement in biotin synthesis ability.

[0094] Example 6: Detection of the effect of desulfurization biotin synthetase mutants on strain growth

[0095] When some proteins are overexpressed, they will produce certain toxicity to cells, thereby affecting cell growth. In order to verify whether overexpression of BioD mutants affects the growth of strains, the seed culture activated in Example 4 was transferred to 1 mL of fermentation medium at an inoculum volume of 1% (V / V) and mixed evenly. 100 μL was pipetted into a shallow well plate and cultured at 37°C and 800 rpm for 24 h using a microbial growth curve analyzer to monitor the growth of each strain.

[0096] From the growth curve results, it can be seen that (see Figure 5 ), compared with the wild-type control strain BW25113, there was no significant difference in the growth of strains expressing unmutated BioD or strains expressing BioD mutants. This shows that overexpression of desulfurization biotin synthase BioD or its mutants does not produce cytotoxicity. When the valine acid at position 142 of BioD was mutated to isoleucine, alanine, histidine and threonine, it not only increased the production of biotin, but also did not affect the growth of the strain, thereby improving the production efficiency of the strain.

[0097] The invention effectively solves the problem of low biotin yield caused by low enzyme activity of desulfurization biotin synthetase, obtains a desulfurization biotin synthetase mutant with high biotin yield, and has important significance for high-yield production of biotin.

[0098] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A desulfurization biotin synthetase mutant, characterized in that: The mutant is based on the amino acid sequence shown in SEQ ID NO: 1, and the valine at position 142 is mutated to serine, asparagine, glutamine, leucine, alanine, histidine, threonine or isoleucine.

2. The desulfurization biotin synthetase mutant according to claim 1, characterized in that The valine at position 142 was mutated to alanine, histidine, threonine or isoleucine.

3. A nucleic acid molecule encoding the dethiobiotin synthetase mutant according to any one of claims 1 to 2.

4. A plasmid vector containing the nucleic acid molecule according to claim 3.

5. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli comprises the desulfurization biotin synthetase mutant according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, or the plasmid vector according to claim 4.

6. Use of the desulfurization biotin synthetase mutant according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, the plasmid vector according to claim 4 or the recombinant Escherichia coli according to claim 5 in biotin production.

7. A method for producing biotin, characterized in that: Use the desulfurization biotin synthetase mutant according to any one of claims 1-2, the nucleic acid molecule according to claim 3, the plasmid vector according to claim 4 or the recombinant Escherichia coli according to claim 5.

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