CN118956783B - Desulfur biotin synthetase mutant and its application in biotin production - Google Patents

Abstract

The invention discloses an escherichia coli desulfurization biotin synthetase mutant and application thereof in biotin production. Saturation mutation of amino acid 142 of desulphated biotin synthetase shows that when mutated into serine, asparagine, glutamine, leucine, alanine, histidine, threonine or isoleucine, the enzymatic activity is improved and the biotin yield is also significantly improved. The mutant can be applied to biological production of biotin.

Description

Desulfur biotin synthetase mutant and its application in biotin production

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a desulfur biotin synthetase mutant and application thereof in biotin production.

Background

Biotin (biotin), also known as vitamin H, vitamin B7, coenzyme R, plays an important role in biochemical reaction pathways such as fat synthesis, glycogenesis, etc., and is one of the most important cofactors involved in central metabolism (lipid synthesis, amino acid metabolism, glycometabolism) of eukaryotic and prokaryotic organisms. Plays an important role in maintaining normal growth and development of human and animals, bone marrow health and skin, and is widely applied to various fields such as food additives, medicines, cosmetics, animal husbandry and the like, and the market demand is increasing year by year.

At present, most of the commercial biotin is synthesized through a multi-step chemical process developed by Goldberg and Sternbach, however, the chemical synthesis of biotin has the problems of complex reaction, high energy consumption, high cost, unfriendly environment and the like, so that the green, economical and efficient biosynthesis method for producing the biotin has extremely important significance.

The synthesis of biotin by E.coli cells is divided into two stages, namely, the synthesis of pimelate thioester and the double-loop synthesis, wherein the double-loop synthesis starts from pimelate thioester, is catalyzed by key enzyme BioF, bioA, bioD, bioB in turn, and finally biotin is synthesized (see FIG. 1).

BioD is a desulphated biotin synthase, involved in the penultimate step of biotin synthesis, which catalyzes the production of desulphated biotin (Dethiobiotin, DTB) from the substrate 7, 8-diaminononanoic acid (7, 8-diaminopelargonic acid, DAPA) in the presence of CO ₂ and ATP. DTB is a precursor of biotin and directly affects the synthesis amount of biotin. However, the catalytic efficiency of BioD in E.coli is very low, only 0.06 s ^-1, and the catalytic synthesis reaction of DTB becomes the rate limiting step for the synthesis of E.coli biotin.

At present, research on desulfurization biotin synthetase BioD at home and abroad is focused on screening development direction of mycobacterium tuberculosis desulfurization biotin synthetase as antitubercular drugs, and reports of modifying BioD and applying it to biotin industrial production are hardly seen. Yang G et al （Yang G, and, et al. Active site mutants of Escherichia coli dethiobiotin synthetase: effects of mutations on enzyme catalytic and structural properties.[J]. Biochemistry, 1997, 36(16): 4751-4760. DOI:10.1021/bi9631677.） revealed the importance of part of the active site residues (Thr 11, glu12, lys15, lys37, ser41, asn 175) by crystal structure studies of E.coli desulphated biotin synthetases, but failed to elucidate the relationship between DTB synthesis activity and biotin production. Up to the present, the related research has not found a key site for significantly improving the enzymatic activity of desulphated biotin synthase, which makes it difficult to break through the dilemma of improving the yield of biotin by rational modification of BioD. Therefore, the key target points affecting the activity of the BioD enzyme are mined, and the deep and comprehensive research is carried out on the key target points on the basis of the key target points, so that the key target points have important significance in improving the biotin yield.

Disclosure of Invention

In order to improve the biotin yield, the invention firstly carries out mutagenesis on escherichia coli, screens mutant strains with obviously improved biotin yield, and discovers that after sequencing, when valine at position 142 of desulfurated biotin synthetase (BioD) is mutated into serine, the biotin yield is obviously improved. To further investigate the effect of amino acid 142 on BioD, amino acid saturation mutations were made at this site and the changes in biotin production after mutation were determined.

The invention provides a desulfur biotin synthetase mutant of escherichia coli (ESCHERICHIA COLI) and application thereof in biotin production, wherein the mutant improves the biotin yield and does not influence the strain growth.

To achieve the object of the present invention, in a first aspect, the present invention provides a mutant of a desulphated biotin synthase of E.coli, said mutant comprising or consisting of an amino acid sequence selected from the group consisting of:

1) Mutation of amino acid 142 from V to A in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142A);

2) Mutation of amino acid 142 from V to R in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142R);

3) Mutation of amino acid 142 from V to N in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142N);

4) Mutation of amino acid 142 from V to D in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142D);

5) Mutation of amino acid 142 from V to C in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142C);

6) Mutation of amino acid 142 from V to Q in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142Q);

7) Mutation of amino acid 142 from V to E in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142E);

8) Mutation of amino acid 142 from V to G in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142G);

9) Mutation of amino acid 142 from V to H in the amino acid sequence shown in SEQ ID NO. 1 of a desulphated biotin synthase from E.coli (BioD ^V142H);

10 Mutation of amino acid 142 from V to I in the amino acid sequence shown in SEQ ID NO:1 of a desulphated biotin synthase from E.coli (BioD ^V142I);

11 Mutation of amino acid 142 from V to L in the amino acid sequence shown in SEQ ID NO.1 of the desulphated biotin synthetase derived from E.coli (BioD ^V142L);

12 Mutation of amino acid 142 from V to K in the amino acid sequence shown in SEQ ID NO:1 of a desulphated biotin synthase from E.coli (BioD ^V142K);

13 Mutation of amino acid 142 from V to M in the amino acid sequence shown in SEQ ID NO. 1 of the desulphated biotin synthetase derived from E.coli (BioD ^V142M);

14 Mutation of amino acid 142 from V to F in the amino acid sequence shown in SEQ ID NO:1 of a desulphated biotin synthase from E.coli (BioD ^V142F);

15 Mutation of amino acid 142 from V to P in the amino acid sequence shown in SEQ ID NO. 1 (BioD ^V142P) of a desulphated biotin synthetase derived from E.coli;

16 Mutation of amino acid 142 from V to S in the amino acid sequence shown in SEQ ID NO:1 of a desulphated biotin synthase from E.coli (BioD ^V142S);

17 Mutation of amino acid 142 from V to T in the amino acid sequence shown in SEQ ID NO. 1 (BioD ^V142T) of a desulphated biotin synthetase derived from E.coli;

18 Mutation of amino acid 142 from V to W in the amino acid sequence shown in SEQ ID NO:1 of a desulphated biotin synthase from E.coli (BioD ^V142W);

19 Mutation of amino acid 142 from V to Y in the amino acid sequence shown in SEQ ID NO. 1 (BioD ^V142Y) from Escherichia coli desulphation biotin synthetase.

In a second aspect, the invention provides nucleic acid molecules encoding the desulphated biotin synthase mutants.

In a third aspect, the invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineering bacteria.

In a fourth aspect, the present invention provides a recombinant microorganism comprising the desulphated biotin synthase mutant or a nucleic acid molecule encoding the desulphated biotin synthase mutant.

In a fifth aspect, the present invention provides the use of said desulphated biotin synthase mutant or said nucleic acid molecule or said biological material or said recombinant microorganism in the production of biotin.

In a sixth aspect, the invention provides the use of said desulphated biotin synthase mutant or said nucleic acid molecule or said biological material or said recombinant microorganism to increase biological yield.

In one embodiment of the present invention, the mutant is a mutant having an amino acid site selected from the group consisting of:

Valine at position 142 is mutated to serine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, respectively.

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

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

According to the invention, mutant BioD^V142S、BioD^V142N、BioD^V142Q、BioD^V142L、BioD^V142A、BioD^V142H、BioD^V142T、BioD^V142I, with improved activity of the desulphated biotin synthase, especially BioD ^V142I、BioD^V142T、BioD^V142H、BioD^V142A, is obtained by carrying out mutation on the wild escherichia coli desulphated biotin synthase, so that the biotin yield is effectively improved. In particular, the mutant BioD ^V142I has 2.3 times of the enzyme activity of the wild type desulphated biotin synthetase, and the biotin yield is improved by 36 percent, thus having important significance for large-scale production of biotin.

Drawings

FIG. 1 schematic representation of the bicyclic synthetic pathway of biotin

FIG. 2 relative enzyme activities of desulphated biotin synthase mutants

FIG. 3 results of fermentation production of biotin by recombinant E.coli expressing a mutant of desulphated biotin synthase using Delta bioFbioCbioDbioB plate assay

FIG. 4 results of fermentation production of biotin by recombinant E.coli expressing a mutant of desulphated biotin synthase using HPLC-MS

FIG. 5 growth curves of recombinant E.coli expressing desulphated biotin synthase mutants

Detailed Description

The invention will be further illustrated in detail with reference to specific examples. The specific experimental conditions are not specified and are conventional conditions well known to those skilled in the art.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecularCloning: aLaboratoryManual, 2001), and the like, or according to the manufacturer's instructions.

Example 1 screening of desulphated Biotin synthase mutation points

1. Mutant screening for strains with increased biotin production

(1) NTG mutagenesis

The escherichia coli wild-type strain BW25113 is taken as a basic strain, inoculated in a liquid medium containing 5mL LB for activation, and cultured at 37 ℃ and 220 rpm overnight. The activated bacterial liquid 1mL is transferred into a 100mL LB liquid culture medium, and is cultured at 37 ℃ and 220 rpm until the OD ₆₀₀ reaches 1.0. 6000 The cells were collected by centrifugation at 5min rpm, washed 3 times with 0.9% physiological saline, and the supernatant was removed by centrifugation at 6000, 6000 rpm. After the cells were resuspended in 20mL of 0.9% physiological saline, NTG (nitrosoguanidine) was added to a final concentration of 0.1 to 0.5 g/L, and the resulting mixture was subjected to mutagenesis treatment of 20 min. The treated bacterial suspension was diluted to the appropriate concentration, 100. Mu.L was pipetted onto LB plates and incubated overnight at 37 ℃.

(2) Deep hole plate primary screen

① Single colonies on LB plates were inoculated into seed plates, respectively, and 16: 16 h were cultured at 37℃and 1000 rpm to obtain activated seed solutions. The seed plate is a 96-deep well plate with 1 mL liquid seed medium per well.

② The activated seed liquid was transferred to the corresponding fermentation plate at an inoculum size of 5% (V/V), and cultured at 37 ℃ and 1000 rpm for 24 h, while the seed plate was stored in a 4 ℃ refrigerator. The fermentation plate is a 96-deep plate with 1 mL liquid fermentation medium in each hole.

③ The cultured fermentation plate was placed in a centrifuge, centrifuged for 10 min, the supernatant was diluted appropriately, dropped onto a biotin detection plate, cultured at 37℃for 16: 16 h, and the diameter of the growth ring was measured.

Preparation of seed culture medium (L) tryptone 10 g, yeast extract 5g, naCl 10 g.

Preparation of fermentation Medium (L) glucose 10 g, coCl ₂·6H₂O 2 mg、KH₂PO₄ 7.5 g、ZnSO₄·7H₂ O2 mg, yeast powder 3 g, caCl ₂ 4 mg, citric acid 1.8 g, vitamins B1 1.5 mg、MgSO₄·7H₂O 2 g、CuSO₄ 0.5 mg、FeSO₄·7H₂O 70 mg、MnSO₄·H₂O 10 mg.

The biotin detection plate was prepared by the method for preparing a "biotin detection plate" described in examples 2 to 3 of the specification of patent CN 118207171A.

(3) Shaking bottle fermentation re-screening device

Transformants with growth circle diameters increased by more than 1.5 times relative to the wild-type control strain BW25113 are selected for shake flask fermentation re-screening. Transformants were inoculated into 5mL seed medium, respectively, and cultured overnight at 37 ℃ under 220: 220 rpm to obtain seed cultures. The culture was transferred to 20mL fermentation medium at an inoculum size of 1% (V/V) and cultured at 37℃for 24 hours at 220 rpm to obtain a fermentation broth sample. Respectively taking fermentation liquor samples, processing for 5min at 95 ℃, centrifuging for 5min at 12000 rpm, dripping the fermentation liquor samples onto a biotin detection plate, culturing for 16 h at 37 ℃, measuring the diameter of a growth ring, and primarily judging the biotin production capacity of the strain.

2. Genome sequencing

In order to explore the biotin high-yield mechanism of the mutant strain, genome sequencing is carried out on the mutant strain with the strongest biotin production capacity obtained by screening.

Analysis of the sequencing result revealed that the gene encoding BioD, a desulfurization biotin synthase of E.coli, which had the highest biotin-producing ability, was mutated (bioD) and valine at position 142 was mutated to serine.

EXAMPLE 2 construction of desulphated Biotin synthase mutants

In order to further study whether the 142 th amino acid of the desulphated biotin synthetase is a key site affecting the enzyme activity and biotin yield, amino acid saturation mutation is carried out on the desulphated biotin synthetase, and mutants of the rest 19 common amino acid mutations at the site are constructed except the original valine.

(1) PCR amplification is carried out by taking BW25113 genome sequence as a template and utilizing a primer bioD-F/bioD-R to obtain a bioD gene fragment, and the coded bioD amino acid sequence is shown as SEQ ID NO. 1. The pBAD/HisA plasmid is used as a template, a primer pBAD-F/pBAD-R is used for amplifying a framework fragment, and a Gibson seamless cloning technology is used for connecting a bioD fragment to a pBAD/HisA vector to obtain a vector pBAD-bioD. The plasmid was transformed into E.coli BW25113 competent to obtain recombinant strain BW25113-BioD.

(2) The bioD gene was subjected to point mutation using the vector pBAD-bioD as a template and 19 pairs of mutation primers (see Table 1), the PCR amplified product was digested with DpnI 2h and transferred into E.coli DH 5. Alpha. Competent cells, spread on LB plates containing ampicillin (50. Mu.g/mL), and positive clone identification was performed using the primers pBAD-JD-F/pBAD-JD-R after culturing at 37℃for 12h, finally obtaining 19 vectors pBAD-bioD containing BioD mutations (see Table 2). This was transferred into E.coli BW25113 competent cells to obtain 19 mutant recombinant strains (see Table 3).

Table 1 bioD primers for construction of mutant vectors

TABLE 2 plasmids used in the present invention

TABLE 3 strains used in the invention

Example 3 in vitro enzyme Activity test of desulphated Biotin synthase mutant

1. Recombinant protein induced expression

The 19 mutant recombinant strains constructed in example 2 and BW25113-BioD as a control were inoculated into LB medium containing kanamycin (50. Mu.g/mL), and cultured overnight at 37℃220 rpm, to give seed cultures. The seed culture was transferred to a 50 mL TB medium at an inoculum size of 1% (V/V), cultured at 37℃220 rpm until OD ₆₀₀ reached 0.8, induced by adding L-arabinose at a final concentration of 4 g/L, and after continuing to culture at 30℃220 rpm for 20 h, cells were collected by centrifugation at 4℃12000 rpm for 10 min.

Preparation of LB Medium (L) tryptone 10 g, yeast extract 5g, naCl 10 g.

Preparation of TB Medium (L) Glycerol 4 g, yeast extract 24 g, tryptone 12 g, KH ₂PO₄ 2.31 g、K₂HPO₄ 16.43.43 g, pH 7.5.

2. Recombinant protein purification

The collected cells were resuspended in sterile water and the supernatant removed by centrifugation at 4℃and repeated 2 times. The cells were resuspended in pre-chilled lysis buffer (20 mM Tris-HCl,500 mM NaCl,pH 7.5), disrupted by high pressure homogenization, centrifuged at 4℃10000 rpm for 10 min and the supernatant collected.

Protein purification was performed using Mag-beams His-Tag protein purification Beads. The samples were loaded into centrifuge tubes containing magnetic beads, mixed upside down and incubated at 4 ℃ for 30 min. The centrifuge tube was placed in a magnetic separator and after the solution became clear, the supernatant was removed with a pipette. The mixed protein was washed by adding 10 mL wash buffer (20 mM Tris-HCl,500 mM NaCl,50 mM imidazole, pH 7.5) to the centrifuge tube. Repeated washing for more than 3 times. Adding 10 mL elution buffer (20 mM Tris-HCl,500 mM NaCl,500 mM imidazole, 5% glycerol, pH 7.5) into a centrifuge tube, lightly blowing for 3-5 times by using a liquid-transfering device, uniformly mixing, placing the centrifuge tube on a magnetic separator, and sucking the supernatant by using the liquid-transfering device after the solution becomes clear, thus obtaining the target protein component. The purified protein was concentrated by transfer to a ultrafiltration tube with a molecular weight cut-off of 10 kDa and the buffer was replaced with reaction buffer (20 mM Tris-HCl,500 mM NaCl,10% glycerol, 1mM DTT, pH 8.0). Expression of the target protein was detected by SDS-PAGE.

3. Enzyme activity test of desulphated biotin synthetase mutant

Catalytic testing was performed using the purified proteins described above. The enzyme activity of the desulphated biotin synthase was determined as the increase in product DTB per unit time. The test reaction contained 50 mM Tris-HCl, 150. Mu.M NaCl, 5mM MgCl ₂,10 mM NaHCO₃, 300. Mu.M ATP, 100. Mu.M DAPA, pH 7.5. 0.25 to 3.0 mM purified protein was added to the above reaction solution and reacted at 37℃with 45 min. The catalytic reaction was terminated by addition of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl,250 mM EDTA, incubation at 37 ℃ for 15 minutes (see page 261, left column 2.5 of document Salaemae W, and, et al. Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase[J]. Tuberculosis, 2015. DOI:10.1016/j.tube.2015.02.046. ). The synthesized DTB product was detected by HPLC-MS, and the relative enzyme activities of the desulphated biotin synthase and its mutants were calculated by measuring the amount of product DTB produced per unit time, using the wild type desulphated biotin synthase BioD as a control.

As a result, as shown in FIG. 2, ,BioD^V142S、BioD^V142N、BioD^V142Q、BioD^V142L、BioD^V142A、BioD^V142H、BioD^V142T、BioD^V142I of the 19 mutants had higher enzyme activities than the wild-type desulphated biotin synthase BioD (see FIG. 2), wherein the enzyme activities of BioD ^V142A、BioD^V142H、BioD^V142T、BioD^V142I were more than 1.7, 1.9, 1.8 and 2.3 times that of the wild-type mutant, and particularly, the mutant BioD ^V142I obtained when valine was mutated to isoleucine, had more than 2 times that of the wild-type mutant. The above results indicate that amino acid 142 of BioD is important for its enzymatic activity for catalyzing the production of DTB, and that its activity is affected differently when it is mutated to a different amino acid.

Example 4 Flat plate detection of the production of Biotin by desulphated Biotin synthase mutant

(1) The strain BW25113-BioD ^V142A、BW25113-BioD^V142H、BW25113-BioD^V142T、BW25113-BioD^V142I expressing the four mutants with the highest activity of the desulphated biotin synthase and the control strains BW25113-BioD and BW25113 are respectively inoculated into a 5mL seed culture medium and cultured overnight at 37 ℃ and 220 rpm to obtain seed cultures.

(2) The seed culture of the step (1) is transferred to a 20 mL fermentation medium according to the inoculum size of 1% (V/V), and is cultured at 37 ℃ and 220: 220 rpm for 24: 24h, so as to obtain a fermentation broth sample.

(3) The fermentation broth samples were separately taken and subjected to 95℃treatment for 5min, 12000 rpm centrifugation for 5min, and after 2-fold dilution, were dropped onto a biotin detection plate and incubated at 37℃for 16 h.

The biotin action-test plate results showed (see FIG. 3) that the detection plates for the broth samples to which the mutant strain BW25113-BioD ^V142A、BW25113-BioD^V142H、BW25113-BioD^V142T、BW25113-BioD^V142I and the control strain BW25113-BioD were added were all clear growths, and that the mutant growths were all significantly larger than the control strain, whereas the detection plates for the broth samples to which the wild-type strain BW25113 was added were no growths observed. The above results indicate that the biotin synthesis was significantly improved in both mutant strains BW25113-BioD ^V142A、BW25113-BioD^V142H、BW25113-BioD^V142T、BW25113-BioD^V142I compared to the control strain BW25113-BioD expressing unmutated BioD.

As can be seen from the measurement (see Table 4), the growth circle diameter of the mutant BW25113-BioD ^V142A、BW25113-BioD^V142T、BW25113-BioD^V142H、BW25113-BioD^V142I in FIG. 3 was larger than that of BW25113-BioD and BW25113 as a control, and the growth circle of BW25113-BioD ^V142I was the largest, which is consistent with the in vitro enzyme activity test result, and it was also revealed that the strain expressing mutant BioD ^V142A、BioD^V142T、BioD^V142H、BioD^V142I obtained when the mutation at position 142 of BioD was introduced had a higher biotin-synthesizing ability.

TABLE 4 growth circle size after fermentation of recombinant strains

Example 5 quantitative determination of Biotin production

The fermentation broth samples obtained in the step (2) of example 4 were separately taken, subjected to 95 ℃ treatment for 5min ℃ and centrifuged at 12000 rpm for 10min, and the supernatants were taken into new centrifuge tubes and further quantitatively determined for biotin content in the fermentation broth by HPLC-MS.

The HPLC-MS test results show that the biotin production of the 4 strains into which the BioD mutation was introduced was significantly improved compared to the BioD non-mutated control strain (BW 25113-BioD), wherein the biotin production of the strain containing the BioD ^V142I mutation was highest and reached 136% of the control strain. This result fully verifies that the 142 th amino acid of the desulphated biotin synthase BioD is critical for biotin synthesis, and when the desulphated biotin synthase BioD is mutated from valine to a specific amino acid, the biotin synthesis can be significantly increased, wherein the mutant biotin obtained by mutation to isoleucine is most significantly improved in synthesis capacity.

Example 6 detection of the Effect of desulphated Biotin synthase mutants on Strain growth

Certain proteins, when overexpressed, can cause some toxicity to cells, thereby affecting cell growth. To verify whether the over-expressed BioD mutant affected the growth of the strain, the seed culture activated in example 4 was transferred to 1 mL fermentation medium at 1% (V/V) and mixed well, 100. Mu.L was pipetted into a shallow well plate, and each strain was grown by culturing 24 h at 37℃and 800 rpm using a microbial growth curve analyzer.

From the results of the growth curves, it can be seen (see fig. 5) that there was no significant difference in growth compared to the wild-type control strain BW25113, both the strain expressing the unmutated BioD and the strain expressing the BioD mutant. This suggests that overexpression of the desulphated biotin synthase BioD or mutants thereof does not produce cytotoxicity. After the 142 th valyl acid of the BioD is mutated into isoleucine, alanine, histidine and threonine, the yield of biotin is improved, the growth of the strain is not influenced, and the production efficiency of the strain is improved.

The invention effectively solves the problem of low biotin yield caused by low enzyme activity of the desulphurized biotin synthetase, obtains the desulphurized biotin synthetase mutant with high biotin yield, and has important significance on high biotin yield.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (7)

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

2. The desthiobiotin synthase mutant of claim 1 in which valine at position 142 is mutated to alanine, histidine, threonine or isoleucine.

3. A nucleic acid molecule encoding the desulphated biotin synthase mutant according to any one of claims 1-2.

4. A plasmid vector comprising the nucleic acid molecule of claim 3.

5. Recombinant escherichia coli, characterized in that it comprises a desulphated biotin synthase mutant according to any one of claims 1-2, a nucleic acid molecule according to claim 3 or a plasmid vector according to claim 4.

6. Use of a desulphated biotin synthase mutant according to any of claims 1-2, a nucleic acid molecule according to claim 3, a plasmid vector according to claim 4 or a recombinant e.coli according to claim 5 in biotin production.

7. A method for producing biotin, characterized in that the desulphated biotin synthase 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 is used.