A biotin synthetase mutant

Abstract

The application provides a biotin synthetase mutant, and specifically comprises the following contents: a biotin synthetase mutant library is constructed, and finally a mutant with improved catalytic activity is screened. Compared with a wild type, the mutant provided by the application improves the efficiency of synthesizing biotin from desulfo-biotin by 42.3%.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and specifically relates to a biotin synthase mutant with enhanced catalytic activity. Background Technology

[0002] Biotin, with the molecular formula C 10 H 16 N₂O₃S, with a molecular weight of 244.31 g / mol, has an imidazole ring, a sulfur-containing tetrahydrothiophene ring, and a valeric acid side chain. Biotin, as an essential cofactor for cellular life activities, participates in many enzymatic reactions involving carboxylation, decarboxylation, and transcarboxylation. These biotin-activated carboxylases participate in the three major metabolic reactions in the cellular center: gluconeogenesis, fatty acid synthesis, and amino acid metabolism. Furthermore, biotin plays a crucial role in a range of life activities, including group silencing, cell proliferation, DNA repair, and apoptosis. Biotin is widely used in animal feed, food additives, cosmetics, diagnostics, fermentation, and biomedicine. As a key feed additive, biotin promotes the growth and development of livestock and poultry, positively impacting weight gain, feed conversion efficiency, meat and fur quality, and egg production. As of 2023, the global biotin market was valued at $1.6 billion and is projected to grow to nearly $2 billion by 2030. To date, commercially available biotin has been synthesized chemically, a reaction pioneered by Sternbach and Goldberg, which laid the foundation for subsequent industrial biotin synthesis. Biosynthesis of biotin is more environmentally friendly, safer, and simpler, aligning with the economic needs of sustainable development. However, current microbial synthesis methods are limited by low yields. For example, wild-type *E. coli* K-12 can only produce approximately 10-15 ng / L of biotin. To effectively compete with chemical synthesis, biosynthesis needs to achieve biotin production exceeding 1 g / L. Initially, researchers used strain mutagenesis to identify dominant biotin producers, but this method proved unpredictable and labor-intensive. In recent years, with a deeper understanding of biotin synthesis and regulation in microorganisms, research focus has shifted to precise genetic engineering methods, such as using advanced tools like CRISPR / Cas9 to redesign the metabolic pathways of microbial strains to increase biotin production. Strategies include overexpressing biotin synthesis genes, reducing negative feedback inhibition, and improving the availability of cofactors. Optimizing the fermentation process is also crucial. For example, modifications in Escherichia coli increased its biotin production to 208.7 mg / L for 46 hours, and similar genetic engineering increased the production of Pseudomonas mutagenesis to 271.88 mg / L.

[0003] The final step in biotin synthesis is catalyzed by biotin synthase BioB, which is also the rate-limiting step in biotin synthesis. This enzyme inserts a sulfur atom between the 6th and 9th carbon atoms of the desulfobiotin molecule, thereby forming a tetrathione ring. This enzymatic reaction depends on S-adenosylmethionine (SAM) and the iron-sulfur ([FeS]) cluster as cofactors and exhibits a slow catalytic rate (kJ / kJ). cat =0.002s -1 This reaction is limited by the regeneration of the [FeS] clusters generated by the Isc and Suf systems. In vitro, the number of turnovers per subunit of BioB is limited, contrasting with its efficiency in vivo, where up to 20 turnovers can be achieved. Farrar and Jarrett mutated Asp at position 155 of BioB to Asn and Asn at position 153 to Ala, respectively, but this did not improve the catalytic efficiency of BioB. Computer software programs can be used for in-depth analysis and computational assistance of key residues in the catalytically active pocket to improve the catalytic efficiency of BioB. Summary of the Invention

[0004] The purpose of this invention is to provide a biotin synthase mutant with enhanced catalytic activity.

[0005] In a first aspect, the present invention provides a biotin synthase mutant, wherein the mutant corresponds to a mutation at amino acid residue 232 of the amino acid sequence of biotin synthase derived from wild-type Pseudomonas mutabilis, wherein the amino acid sequence number is SEQ ID NO.1.

[0006] In a second aspect, the present invention provides a biotin synthase mutant, wherein the 232nd amino acid of the mutant is mutated from lysine to arginine compared with SEQ ID NO.1.

[0007] In a third aspect, the present invention provides a recombinant nucleic acid expression vector having a nucleic acid sequence encoding the biotin synthase mutant described in the second aspect of the present invention.

[0008] In a fourth aspect, the present invention provides a recombinant genetically engineered strain containing the recombinant nucleic acid expression vector described in the third aspect.

[0009] In a fifth aspect, the present invention provides a method for biotin synthesis, comprising fermenting and culturing the recombinant genetically engineered strain described in the fourth aspect of the invention for converting desulfurized biotin into biotin. The specific process includes shake-flask fermentation of the engineered strain described in the fourth aspect of the invention. The culture temperature is 37°C, the culture time is 48 hours, the culture medium is LB medium supplemented with 1 g / L desulfurized biotin and 50 mg / L kanamycin resistance, and the shaking speed is 220 rpm.

[0010] In a sixth aspect, the present invention provides a method for screening biotin synthase mutants as described in the second aspect of the present invention, comprising the following steps:

[0011] (1) BioB synthase derived from wild-type Pseudomonas aeruginosa and its ligand S-adenosyl-L-methionin (SAM) were modeled using iron-sulfur cluster homology to obtain a BioB complex model. The BioB complex model was then coupled with the model of the substrate desulfurized biotin to the active pocket of protein catalysis, and molecular dynamics simulations were used to screen out potential amino acid sites that could enhance catalytic activity.

[0012] (2) The selected amino acid sites were mutated to alanine, and the key mutated amino acid sites for catalytic activity were selected again.

[0013] (3) Construct the plasmid expression vector library of each amino acid site saturated mutation selected in step (2), and import it into the constructed chassis strain E.coli BL21ΔyigMΔbioB to express biotin synthesis; use biotin detection indicator strain Lactobacillus plantarum to detect the biotin synthesis yield of each mutant strain and control strain.

[0014] Furthermore, the specific steps include:

[0015] (1) Using the SWISS-MODEL server, the crystal structure of biotin synthase BioB from *E. coli* (PDBID: 1R30) was used as a template. The amino acid sequence SEQ ID NO.1 was input to generate a three-dimensional model of biotin synthase BioB from *Pseudomonas aeruginosa*, its ligand SAM, and the iron-sulfur cluster. The three-dimensional model of the complex was docked with dethiobiotin using AutoDock. The composite model was selected based on the optimal energy and docking posture. The structural stability of the obtained composite model at 301 K was further evaluated by Discovery Studio molecular dynamics simulation. The changes in the interaction forces between biotin synthase BioB and SAM and dethiobiotin were studied. Residues D62, D68, N159, D161, R174, V231, and N232 were selected as mutation sites.

[0016] (2) Mutate the seven selected sites to alanine, and select the amino acids at positions 62, 68, 159, 161, 174, 231 and 232 of the biotin synthase from wild-type mutagenic pseudomonocytes for subsequent site-directed saturation mutagenesis.

[0017] (3) A recombinant expression vector library containing saturated mutations at these 7 sites was constructed and introduced into the chassis strain *E. coli* BL21ΔyigMΔbioB. For the control recombinant nucleic acid vector, unmutated *BioB* was integrated into the same expression vector and introduced into the chassis strain. The vectors were plated on LB agar plates containing kanamycin and incubated overnight at 37°C. Single colonies of the mutant library and the control strain were picked and cultured in 96-well plates for 48 hours. 2.5 μL of the fermentation supernatant was centrifuged and transferred to 96-well clear plates containing biotinylate-based culture medium containing *Lactobacillus plantarum* at the corresponding positions. Cell concentration was measured every 2 hours using a microplate reader. The cell concentration of *Lactobacillus plantarum* was correlated with biotin concentration; strains with higher concentrations than the control group were selected.

[0018] This invention, based on the amino acid sequence shown in SEQ ID NO.1, utilizes computer-aided rational design to reduce the size of the mutant library, decreasing the screening workload (160 mutants) and increasing the hit rate of positive mutants. Through high-throughput screening of biotin synthase mutants, this invention identified three mutants with enhanced activity. Compared to the wild-type enzyme, the mutant 232Lys->Arg, with the highest transformation efficiency, showed a 42.3% increase in biotin synthesis in the chassis strain. Attached image description:

[0019] Figure 1 A three-dimensional model of biotin synthase BioB and its ligands from *Pseudomonas aeruginosa*.

[0020] Figure 2 The results of Laplace plot analysis for the BioB complex model;

[0021] Figure 3 The growth of *Lactobacillus plantarum* in culture media with different concentrations of biotin was observed.

[0022] Figure 4 The growth of control strain E. coli BL21ΔyigMΔbioB / pUCPK-BioB and mutant strain in 96-well plates;

[0023] Figure 5 The biotin yield of the mutant strain during shake-flask fermentation. Detailed Implementation

[0024] The following embodiments, in conjunction with the accompanying drawings, further illustrate and describe the technical solution of the present invention:

[0025] Unless otherwise specified in the following examples, molecular biology procedures should be performed in accordance with the kit instructions or the "Molecular Cloning: A Laboratory Manual".

[0026] Example 1: Construction of a 3D model of a wild-type biotin synthase complex derived from *Pseudomonas mutabilis*

[0027] The amino acid sequence of BioB is SEQ ID NO.1 (source: Pseudomonas mutabilis ATCC31014).

[0028] MSASTTATTRHDWSLAEVKALFQQPFNDLLFQAQTVHRAHFDPNRVQVSTLLSIKTGACPEDCKYCPQSGHYNTGLEKQKLMEVQKVLEEAARAKAIGSTRFCMGAAWKHPSAKDMPYVLEMVKGVKAMGLETCMTLGKLDQDQTLALAQAGLDYYNHNLDTSPEFYGSIITTRTY SERLQTLAYVRDAGMKICSGGILGMGESLDDRAGLLIQLANLPEHPESVPINMLVKVAGTPLAEEEDVDPFDFIRMLAVARILMPKSHVRLSAGREQMNEQMQALAFMAGANSIFYGEKLLTTANPQADKDMQLFARLGIKPEAREEHADEVHQAAIEQALVEQCSSEMFYNAASA*

[0029] BLAST analysis of the amino acid sequence SEQ ID NO.1 in the National Center for Biotechnology Information (NCBI) database revealed homologous structures in the PDB database, with the biotin synthase BioB from *E. coli* showing a similarity of over 70%. Inputting the SEQ ID NO.1 sequence into the SWISS-MODEL website yielded over 30 usable templates. Ultimately, the crystal structure of *E. coli*-derived BioB (PDB ID: 1R30) was selected as the template to generate a three-dimensional model of *BioBacillus mutabilis*-derived BioB. Figure 1 The spherical model represents iron-sulfur clusters, and the stick-shaped model represents SAM.

[0030] The next step is to evaluate the complex model based on the Laplace conformation. Figure 2 It can be seen that 100% of the amino acid residues are within the reasonable range (89.8% in the optimal range, 8.8% in the acceptable range, and 0.7% in the generally permissible range), indicating that the constructed model has high reliability and can be used as a template for subsequent studies. The three-dimensional model of the BioB complex was docked with the substrate desulfobiotin using LibDock software, and the composite model was selected based on the optimal energy and docking posture.

[0031] Example 2: Screening for mutant amino acids using molecular dynamics simulations

[0032] Using Discovery Studio software, the BioB complex model obtained through molecular dynamics simulations was analyzed, and the changes in the interaction forces between BioB and SAM and DTB were investigated. First, structural fluctuation (ΔRMSF) analysis was performed. As the temperature increased from 50 K, the number of intermediate residues (60-80), 150-180, and 220-240), as well as the C-terminal residues, significantly increased. Residues A62, D161, R174, V231, and N159 frequently formed hydrogen bonds with SAM at 301 K (probabilities of 97%, 79%, 72%, 67%, and 65%, respectively), while D68 formed hydrogen bonds with DTB at a higher frequency (75%) at this temperature. Similarly, analysis revealed frequent hydrophobic interactions between N232, D62, and D161 and SAM and DTB (probabilities of conformational interaction were 100%, 79%, and 78%, respectively). 301K is the optimal growth temperature for the biotin synthase BioB in *Pseudomonas mutabilis*. It is speculated that at this temperature, the affinity of the aforementioned amino acids for the substrates DTB and SAM is related. The ΔRMSF of these amino acid residues is also very large, indicating their high flexibility (plasticity) and suitability for modification and research.

[0033] Example 3: Replace the amino acid at each mutation site with alanine.

[0034] The original amino acids were mutated to alanine at positions 62, 68, 159, 161, 174, 231, and 232 of the bioB gene in plasmid pUCPK-BioB. The resulting seven plasmids, pUCPK-BioB62Ala, pUCPK-BioB68Ala, pUCPK-BioB159Ala, pUCPK-BioB161Ala, pUCPK-BioB174Ala, pUCPK-BioB231Ala, and pUCPK-BioB232Ala, were then electroporated into the chassis strain *E. coli* BL21ΔyigMΔbioB. The substrate strain *E. coli* BL21ΔyigMΔbioB containing the plasmid pUCPK-B, the substrate strain without the plasmid (as a control group), and the substrate strain containing these seven mutant plasmids were cultured in 50 mL LB shake flasks containing 0.5 g / L DTB for 48 h, with three replicates for each strain. It can be seen that only the fermentation broth of strain *E. coli* BL21ΔyigMΔbioB / pUCPK-B showed detectable biotin; the other strains did not. Therefore, the amino acids at these seven sites play a normal role in the catalytic reaction of biotin synthase BioB, and changes at these sites may enhance the catalytic activity of BioB.

[0035] Example 4: A method for constructing a high-throughput screening method for biotin synthases with enhanced catalytic activity.

[0036] Lactobacillus plantarum ATCC 8014 preserved in glycerol tubes was streaked onto an MRS plate and incubated overnight at 37°C. Single colonies were then picked and inoculated into test tubes containing 5 mL of MRS liquid medium. After incubation at 37°C and 200 rpm for 18 hours, the inoculum was transferred at a rate of 1% to shake flasks containing 50 mL of MRS liquid medium and incubated at 37°C and 200 rpm until the cell density reached OD0.05. 600 When the temperature reaches approximately 2.0, take 1 mL of bacterial suspension, resuspend the bacterial suspension in 1 mL of sterile water, centrifuge at 8000g for 3 min, discard the supernatant, wash twice more with sterile water, and add 50 μL of sterile water to resuspend the bacterial suspension.

[0037] Weigh 2g of biotin-based culture medium into a shake flask, add 50mL of pure water, and sterilize at 105℃ for 10min. Once the medium has cooled to room temperature, add the prepared *Lactobacillus plantarum*, shake well, and pour into a sterile pipette. Use a multi-pipette to transfer 200μL of the mixed medium into a sterile 96-well clear agar plate.

[0038] Weigh 200 mg of biotin standard into a 1 L volumetric flask, add 1 L of purified water to dilute to volume, and prepare a biotin standard stock solution. Serially dilute the stock solution to 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / L. Weigh 50 mg of desulfurized biotin, dissolve it in a 50 mL volumetric flask, dilute to volume, and then serially dilute to 0.5 mg / L.

[0039] Pipe 2 μL of diluted biotin and desulfobiotin standards into a prepared 96-well plate. No reagents are added to the control group. Incubate at 37°C. Observation temperature (OD) of *Lactobacillus plantarum* cells is measured every two hours. 600 ( Figure 3 ).

[0040] Subsequent experiments selected OD values ​​from bacterial cultures at 10, 12, and 14 hours of culture. 600 To determine the biotin content.

[0041] Example 5: High-throughput screening of biotin-producing mutants

[0042] The strain *E. coli* BL21ΔyigMΔbioB / pUCPK-BioB was selected as the control, and the strain *E. coli* BL21ΔyigMΔbioB / pUCPK-BioB62Ala was selected as the experimental group. Spots were picked into 96-well plates and incubated at 37°C. Figure 4It can be seen that the strain entered the logarithmic phase at the 4th hour. At the 10th hour, 20 μL of the bacterial culture was taken and transferred to a new 96-well plate for further culture.

[0043] The mutant plasmid library for each site was electrotransferred into competent *E. coli* BL21ΔyigMΔbioB, and after recovery, plated on Kanamycin-resistant LB agar plates. Ninety-three spots from each mutant site were selected and inoculated into 96-well plates, with the remaining three spots serving as the control group (*E. coli* BL21ΔyigMΔbioB / pUCPK-BioB). After 10 hours of incubation, 20 μL of the bacterial culture was transferred to 96-well plates and incubated for another 48 hours. The 96-well plates were centrifuged, and 3 μL of the supernatant was used to detect biotin production. The eight selected strains—62F4, H2; 231E6, F8, G6; 232G6, C11, C12—were preserved and streaked onto LB agar plates. Three spots from each strain were selected as parallel inoculations into 96-well plates and incubated on LB medium at 37°C for 10 hours. 20 μL of the bacterial culture was then transferred to LB medium containing 0.5 g / L DTB in a deep-well plate. Sequencing of the eight strains obtained from the secondary screening revealed that they were 232Lys->Arg, 62Asp->Gly, and 62Asp->Asn.

[0044] Example 6: High-yield biotin mutant strain fermented in shake flasks

[0045] Single-mutant strains and combined-mutant strains were fermented together, with three replicates for each strain. The concentration of DTB added was 1 g / L. Figure 5 As shown, the 232Lys->Arg strain exhibited the strongest biotin synthesis capacity at 0.686 mg / L, compared to only 0.482 mg / L in the non-mutated strain. This indicates that the mutant strain's ability to convert desulfurized biotin into biotin was increased by 42.3%.

Claims

1. A biotin synthase mutant with enhanced catalytic activity, characterized in that, The biotin synthase mutant is obtained by mutating the 232nd amino acid from lysine to arginine based on the wild-type biotin synthase shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that, The recombinant expression vector contains a nucleic acid sequence encoding the biotin synthase mutant of claim 1.

3. A recombinant genetically engineered strain, characterized in that, It contains the recombinant expression vector as described in claim 2.

4. A method for biotin synthesis, characterized in that, The recombinant genetically engineered strain described in claim 3 is fermented and cultured for the conversion of desulfurized biotin into biotin.

Images