BioB protein mutant and application thereof in increasing vitamin yield

Abstract

The invention discloses a BioB protein mutant and application of the BioB protein mutant in improving the conversion rate of desulfurized biotin to biotin, and relates to the technical field of genetic engineering. According to the method disclosed by the invention, the BioB is analyzed by using paver software, key amino acid sites influencing reactants / products to enter and exit from a protein binding cavity in the BioB are optimized, and amino acid residues with relatively large steric hindrance are replaced by amino acid residues with relatively small steric hindrance; according to the present invention, the BioB protein activity is changed through site-specific mutagenesis, and ribosome binding site optimization is performed on the BioB protein, such that the conversion rate of the desulfurization biotin to the biotin is substantially improved so as to obtain the mutant strain with the high biotin yield, and the high application value is provided.

Description

Technical Field

[0001] This invention relates to a BioB protein mutant and its application in increasing biotin production, belonging to the field of genetic engineering. Background Technology

[0002] Biotin, also known as vitamin B7, vitamin H, and coenzyme R, has a structure consisting of an imidazolone ring, a thiophene ring, and a valeric acid side chain. It contains three chiral carbon atoms and has eight isomers, but only the all-cis D-biotin possesses physiological activity; therefore, D-biotin is the focus of this research. Biotin is an essential vitamin for maintaining normal human physiology, primarily participating in decarboxylation and carboxylation reactions in the body through protein modification, involving a series of important metabolic processes including sugars, proteins, and fatty acids. Currently, the main synthetic method for industrially producing D-biotin is chemical synthesis. In 1949, Goldberg and Sternbach first proposed a relatively complete synthetic route for D-biotin, known as the Sternbach route, which enabled large-scale industrial production of D-biotin. Although chemical synthesis is mature, its process routes are long and complex, the synthesis time is long, the reaction requirements are high, production is difficult, and it is prone to environmental pollution. Therefore, the biosynthesis of D-biotin has significant research value and promising prospects.

[0003] Significant progress has been made in the study of biotin synthase (BioB) in recent years. As the rate-limiting enzyme in the biotin synthesis pathway, it exhibits very low catalytic efficiency, with a kcat value of 5 × 10⁻⁶. -7 s -1 Researchers have conducted in-depth explorations of its structure and function, revealing its catalytic mechanism and substrate specificity, providing important clues for understanding the molecular basis of biotin biosynthesis. In recent years, research on BioB has focused on simply expressing heterozyme genes to improve biotin synthesis efficiency. However, because BioB expression has a certain inhibitory effect on bacterial growth, its effect on increasing biotin yield is limited. The BioB reaction involves the supply of the cofactor S-adenosylmethionine (SAM) and the sulfur donor [2Fe-2S] cluster. Simultaneous expression of SAM and [2Fe-2S] synthesis-related genes does not significantly improve biotin production. Considering that two molecules of SAM are consumed in one reaction cycle, and the [2Fe-2S] cluster is also consumed, the cofactor, substrate, and product need to enter and exit the BioB reaction chamber multiple times. Identifying and rationally designing strategies to improve the rate of reaction / product entry and exit from the protein-binding chamber is a novel strategy to enhance BioB catalytic efficiency, and it is expected to effectively improve the conversion efficiency of desulfurized biotin to biotin. Summary of the Invention

[0004] The technical problem to be solved by this invention is the rational modification and regulation of BioB protein to construct a mutant strain that improves the conversion of desulfurized biotin to biotin, so as to meet the demand for increased biotin production.

[0005] The purpose of this invention is to address the problem of low biotin yield in current biosynthetic methods. This invention provides a BioB protein mutant and its application in improving biotin yield. By performing targeted mutation on the BioB protein and optimizing RBS, a mutant strain is obtained. The mutant strain can improve the biotin synthesis ability of Bacillus subtilis.

[0006] The present invention achieves the above objectives through the following technical solutions:

[0007] This invention provides a BioB mutant that enhances the catalytic activity of biotin synthase, which is based on the wild-type BioB protein sequence with one or more substitution mutations at the following sites: T133, E172, R168A, V227, P61, T293, P294, V225, Q267, A263, R95, Y149, Q41; the wild-type BioB protein sequence is shown in SEQ ID NO.1.

[0008] Specifically, it involves one or more substitution mutations at the following sites based on the wild-type BioB protein sequence:

[0009] (1) Replace T at position 133 of SEQ ID NO.1 with G;

[0010] (2) Replace E in position 172 of SEQ ID NO.1 with A;

[0011] (3) Replace R in position 168 of SEQ ID NO.1 with A;

[0012] (4) The V at position 227 of SEQ ID NO.1 is replaced with G;

[0013] (5) The V at position 227 of SEQ ID NO.1 is replaced with A;

[0014] (6) Replace P at position 61 of SEQ ID NO.1 with G;

[0015] (7) Replace T at position 293 of SEQ ID NO.1 with G;

[0016] (8) Replace T at position 293 of SEQ ID NO.1 with A;

[0017] (9) Replace P at position 294 of SEQ ID NO.1 with G;

[0018] (10) The V at position 225 of SEQ ID NO.1 is replaced with G;

[0019] (11) The Q in position 267 of SEQ ID NO.1 is replaced with S;

[0020] (12) The Q at position 267 in SEQ ID NO.1 is replaced with A;

[0021] (13) The A in position 263 of SEQ ID NO.1 is replaced with G;

[0022] (14) The R in the 95th position of SEQ ID NO.1 is replaced with A;

[0023] (15) Replace Y at position 149 of SEQ ID NO.1 with A;

[0024] (16) The Q in the 41st position of SEQ ID NO.1 is replaced with A.

[0025] The present invention provides the encoding gene of the BioB mutant.

[0026] The present invention provides a recombinant vector containing the coding gene of the BioB mutant, specifically, the starting vector is a plasmid vector pP43nmk.

[0027] The present invention provides a recombinant bacterium containing the coding gene of the BioB mutant described above.

[0028] Specifically, it is Escherichia coli; and the coding gene of the BioB mutant is controlled by a promoter for expression.

[0029] Preferably, the required RBS for the expression is selected from 2159 (SEQ ID NO.2), 9001 (SEQ ID NO.3), 91026 (SEQ ID NO.4), 1082545 (SEQ ID NO.5), and UTR12 (SEQ ID NO.6).

[0030] The present invention also provides the use of the BioB mutant, or its encoding gene, or the recombinant bacteria in the preparation of desulfobiotin and / or biotin.

[0031] The present invention provides a method for preparing biotin and / or desulfurized biotin, which involves fermenting the recombinant bacteria to produce biotin and / or desulfurized biotin, optionally including the step of separating the produced biotin and / or desulfurized biotin.

[0032] Specifically, the fermentation medium contains the following essential components: glycerol, soybean peptone, acid-hydrolyzed casein (without vitamins), KH₂PO₄, KCl, MgSO₄·7H₂O, FeSO₄·7H₂O, pirimic acid, and maltose. The culture conditions are 35-42℃ and 100-300 rpm.

[0033] The beneficial effects of this invention are as follows: The BioB channel design provided by this invention can improve the conversion efficiency of desulfurized biotin to biotin, enhance the biotin synthesis capacity of strains, and has good application prospects. Attached Figure Description

[0034] The accompanying drawings, which constitute a part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 Schematic diagram of .pP43nmk-bioB.

[0036] Figure 2 Schematic diagram of protein channels for the desulfurized biotin / biotin / SAM / [2Fe-2S] cluster to enter and exit the BioB binding cavity.

[0037] Figure 3 Results of shake-flask fermentation of four protein channel site-directed mutant strains.

[0038] Figure 4 Different RBS expressions for BioB T133G Schematic diagram.

[0039] Figure 5 Different RBS strains express BioB T133G Results of shake-flask fermentation. Detailed Implementation

[0040] The present invention will now be described in detail with reference to specific accompanying drawings and embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0041] Example 1: Design and rational modification of BioB protein channels using Caver software.

[0042] This invention used Caver software to analyze BioB (amino acid sequence as shown in SEQ ID NO: 1), identifying four channels through which substrate / product / SAM / [2Fe-2S] may enter and exit the binding cavity: channel 1, channel 2, channel 3, and channel 4. Figure 2 As shown, different colors represent different protein channels, and the analysis focuses on key amino acid residues that may affect the entry and exit of reactants / products from the protein-binding cavity. This invention assessed the bottlenecks of these channels and analyzed the steric hindrance of relevant amino acid residues affecting the patency of the entire channel. Through rational modification, key amino acid sites in BioB were optimized, replacing amino acid residues with larger steric hindrance with those with smaller steric hindrance. Using site-directed mutagenesis, recombinant plasmids expressing 16 BioB mutants were constructed using the expression vector pP43nmk. The site-directed mutagenesis results for the four channels are shown in Table 1.

[0043] Table 1. Site-specific mutation sites in the four-channel design

[0044]

[0045] Example 2: Construction of mutant plasmids

[0046] Using the E. coli genome as a template, primers were designed to amplify the bioB fragment with adapter; using the pP43nmk plasmid as a template, primers were designed to amplify the plasmid backbone pP43nmk with adapter; the amplified fragment and backbone were then ligated using Gibson ligation. The ligation product was transformed into E. coli DH5α, and positive clones were selected for PCR and sequencing to verify successful plasmid construction, yielding the BioB expression plasmid pP43nmk-EcbioB.

[0047] Using pP43nmk plasmid as a template, corresponding primers were designed for site-directed mutagenesis to obtain BioB. T133G mutants, BioB E172A mutants, BioB R168A mutants, BioB V227G mutants, BioB V227A mutants, BioB P61G mutants, BioB T293G mutants, BioB T293A mutants, BioB P294G mutants, BioB V225G mutants, BioB Q267S mutants, BioB Q267A mutants, BioB A263G mutants, BioB R95A mutants, BioB Y149A mutants, BioBQ41A The mutant plasmid was transformed into E. coli DH5α, and positive clones were selected for PCR and sequencing to verify successful plasmid construction, yielding pP43nmk-bioB. T133G pP43nmk-bioB E172A pP43nmk-bioB R168A pP43nmk-bioB V227G pP43nmk-bioB V227A pP43nmk-bioB P61G pP43nmk-bioB T293G pP43nmk-bioB T293A pP43nmk-bioB P294G pP43nmk-bioB V225G pP43nmk-bioB Q267S pP43nmk-bioB Q267A pP43nmk-bioB A263G pP43nmk-bioB R95A pP43nmk-bioB Y149A pP43nmk-bioB Q41A Plasmid.

[0048] Example 3: Application of BioB protein mutants in increasing biotin production

[0049] The constructed pP43nmk-bioB T133G pP43nmk-bioB E172A pP43nmk-bioB R168A pP43nmk-bioB V227G pP43nmk-bioB V227A pP43nmk-bioB P61G pP43nmk-bioB T293G pP43nmk-bioB T293A pP43nmk-bioB P294G pP43nmk-bioB V225G pP43nmk-bioB Q267S pP43nmk-bioB Q267A pP43nmk-bioB A263G pP43nmk-bioB R95A pP43nmk-bioB Y149A pP43nmk-bioB Q41APlasmids were transformed into wild-type Bacillus subtilis. Colonies were picked and cultured in 5 mL of LB medium containing kanamycin sulfate (50 mg / L) at 37 °C and 200 rpm for 12 h to obtain seed culture. The seed culture was then transferred to an Erlenmeyer flask containing 30 mL of M-1 fermentation medium to ensure initial OD. 600 The sample was incubated at 40℃ and 200 rpm with shaking for 120 hours. 1 mL of the fermentation broth was centrifuged at 12000 rpm for 1 minute. The supernatant was aspirated with a 1 mL syringe and filtered through a sterile inorganic filter membrane. The filtered sample was then diluted for analysis. After sample processing, the OD of the fermentation product was measured. 600 The production of biotin and desulfurized biotin was detected using a microbial method.

[0050] The results are as follows Figure 3 As shown, compared to the control strain DH-14 (biotin production of 1.01 mg / L), strains with increased biotin production in channel 1 included DH-15, DH-16, and DH-17, with production rates of 2.43 mg / L, 1.14 mg / L, and 1.85 mg / L, respectively. In channel 2, strain DH-19 showed increased biotin production at 2.04 mg / L. In channel 3, strains DH-22, DH-23, DH-24, DH-25, DH-26, and DH-27 showed increased biotin production at 2.01 mg / L, 2.15 mg / L, 1.38 mg / L, 1.21 mg / L, 2.48 mg / L, and 1.14 mg / L, respectively. In channel 4, strain DH-30 showed increased biotin production at 2.2 mg / L. Several recombinant strains showed significantly increased biotin production, but DH-17 achieved a desulfurized biotin production of 18.2 mg / L, significantly higher than other recombinant strains, i.e., overexpression of pP43nmk-bioB T133G The recombinant strain had the highest total production of desulfobiotin and biotin.

[0051] M-1 fermentation medium: 40g glycerol, 50g soybean peptone, 5g acid-hydrolyzed casein (vitamin-free), 1g KH₂PO₄, 0.5g KCl, 0.5g MgSO₄·7H₂O, 10mg FeSO₄·7H₂O, 1g Pimelic acid, 3% maltose. Adjust pH to 7.2, add ddH₂O to a final volume of 1L, and autoclave at 121℃ for 20min. Prepare a stock solution using MgSO₄·7H₂O, FeSO₄·7H₂O, and maltose, and sterilize by filtration.

[0052] Example 4: BioB T133G RBS optimization design

[0053] To further improve the conversion of desulfurized biotin to biotin, this invention optimized the RBS to optimize the translation rate of BioBT133G. Using these designed or reported RBSs, the RBS intensities were 2159 (SEQ ID NO.2), 9001 (SEQ ID NO.3), 91026 (SEQ ID NO.4), 1082545 (SEQ ID NO.5), and UTR12 (SEQ ID NO.6), respectively. Plasmids pP43nmk-BioB-RBS2159, pP43nmk-BioB-RBS9001, pP43nmk-BioB-RBS91026, pP43nmk-BioB-RBS1082545, and pP43nmk-BioB-UTR12 were constructed, respectively, following the same plasmid construction procedure as in Example 2. The RBS sequence was introduced using designed primers.

[0054] Example 5: BioB with different RBS expression T133G Application in increasing biotin production

[0055] The constructed plasmids pP43nmk-BioB-RBS2159, pP43nmk-BioB-RBS9001, pP43nmk-BioB-RBS91026, pP43nmk-BioB-RBS1082545, and pP43nmk-BioB-UTR12 were transformed into Bacillus subtilis DH-1, yielding strains DH-31, DH-32, DH-33, DH-34, and DH-35. Colonies were picked and cultured in 5 mL of LB medium containing kanamycin sulfate (50 mg / L) at 37°C and 200 rpm for 12 h to obtain the seed culture. This seed culture was then transferred to Erlenmeyer flasks containing 30 mL of M-1 fermentation medium, ensuring initial OD... 600 The sample was incubated at 40℃ and 200 rpm in a shaker. Samples were taken after 120 h, and the OD of the fermentation product was measured after sample processing. 600 The yields of biotin and desulfurized biotin were as follows. Figure 5 As shown, compared with the control strain DH-13, which produced 1.08 mg / L of biotin, the strains DH-33 and DH-35 showed significantly higher biotin production, with production of 1.9 mg / L and 3.55 mg / L, respectively.

[0056] 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 BioB mutant that enhances the catalytic activity of biotin synthase, characterized in that, It has one or more substitution mutations at the following sites based on the wild-type BioB protein sequence: T133, E172, R168A, V227, P61, T293, P294, V225, Q267, A263, R95, Y149, Q41; the wild-type BioB protein sequence is shown in SEQ ID NO.

1.

2. The BioB mutant as described in claim 1, characterized in that, It has one or more substitution mutations at the following sites based on the wild-type BioB protein sequence: (1) The T in position 133 of SEQ ID NO. 1 is replaced with G; (2) Replace E at position 172 of SEQ ID NO. 1 with A; (3) The R in position 168 of SEQ ID NO. 1 is replaced with A; (4) The V at position 227 of SEQ ID NO. 1 is replaced with G; (5) The V in position 227 of SEQ ID NO. 1 is replaced with A; (6) The P in position 61 of SEQ ID NO. 1 is replaced with G; (7) The T at position 293 of SEQ ID NO. 1 is replaced with G; (8) Replace T at position 293 of SEQ ID NO. 1 with A; (9) The P at position 294 of SEQ ID NO. 1 is replaced with G; (10) The V at position 225 of SEQ ID NO. 1 is replaced with G; (11) The Q in position 267 of SEQ ID NO. 1 is replaced with S; (12) The Q in position 267 of SEQ ID NO. 1 is replaced with A; (13) The A in position 263 of SEQ ID NO. 1 is replaced with G; (14) The R in the 95th position of SEQ ID NO. 1 is replaced with A; (15) Replace Y at position 149 of SEQ ID NO. 1 with A; (16) The Q in the 41st position of SEQ ID NO .1 is replaced with A.

3. The encoding gene of the BioB mutant as described in claim 1.

4. A recombinant vector containing the coding gene of the BioB mutant as described in claim 3, specifically, a starting vector being a plasmid vector being pP43nmk.

5. A recombinant bacterium containing the encoding gene of the BioB mutant as described in claim 3.

6. The recombinant bacteria as described in claim 5, characterized in that, It is Escherichia coli; and the coding gene of the BioB mutant is controlled by a promoter for expression.

7. The recombinant bacteria as described in claim 6, characterized in that, The required RBS for the expression is selected from 2159 (SEQ ID NO. 2), 9001 (SEQ ID NO. 3), 91026 (SEQ ID NO. 4), 1082545 (SEQ ID NO. 5), and UTR12 (SEQ ID NO. 6).

8. The use of the BioB mutant as described in claim 1 or 2, or its encoding gene, or the recombinant bacteria as described in any one of claims 5 to 7, in the preparation of desulfurized biotin and / or biotin.

9. A method for preparing biotin and / or desulfurized biotin, characterized in that, The recombinant bacteria as described in any one of claims 5 to 7 are fermented to produce biotin and / or desulfurized biotin, optionally including the step of isolating the produced biotin and / or desulfurized biotin.

10. The method as described in claim 9, characterized in that, The fermentation medium consists of: glycerol, soybean peptone, acid-hydrolyzed casein (without vitamins), KH2PO4, KCl, MgSO4·7H2O, FeSO4·7H2O, Pimetlic acid, and maltose. The culture conditions are 35-42℃ and 100-300 rpm.

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