Biotin synthetase mutant

Abstract

The invention provides a biotin synthase mutant, which specifically comprises the following steps: constructing a biotin synthase mutation library, and finally screening a mutant with improved catalytic activity. Compared with a wild type, the efficiency of synthesizing biotin by catalyzing desulfurized biotin through the mutant is improved by 42.3%.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a biotin synthetase mutant with improved catalytic activity. Background Art

[0002] Biotin, molecular formula is C 10 H 16 N2O3S, with a molecular weight of 244.31 g / mol, has an imidazole ring and a sulfur-containing tetrahydrothiophene ring structure and a valeric acid side chain. Biotin, as an essential cofactor for cell life activities, participates in many enzymatic reactions of carboxylation, decarboxylation and transcarboxylation. These carboxylases activated by biotin participate in the three major metabolic reactions in the cell center: gluconeogenesis, fatty acid synthesis and amino acid metabolism. In addition, biotin plays an important role in a series of life activities such as radical silencing, cell proliferation, DNA repair or apoptosis. Biotin is widely used in many fields such as animal feed, food additives, cosmetics, diagnosis, fermentation and biomedicine. As a key feed additive, biotin can promote the growth and development of livestock and poultry, and has a positive impact on weight gain, feed conversion efficiency, meat and fur quality, and egg production. As of 2023, the global biotin market is worth $1.6 billion and is expected to grow to nearly $2 billion by 2030. To date, commercial biotin is synthesized by chemical methods. This reaction was pioneered by Sternbach and Goldberg, laying the foundation for the subsequent industrial synthesis route of biotin. The biological synthesis of biotin is more environmentally friendly, the operation process is safer and simpler, and it meets the economic needs of sustainable development. However, current microbial synthesis methods are limited by low yields. For example, wild-type Escherichia coli K-12 can only produce about 10-15ng / L of biotin. In order to effectively compete with chemical synthesis, biosynthesis needs to achieve biotin production of more than 1g / L. Initially, researchers used strain mutagenesis to find biotin dominant producers, but this method proved to be unpredictable and labor-intensive. In recent years, with the deepening of the understanding of biotin synthesis and regulation in microorganisms, the research focus has shifted to precise genetic engineering methods, such as using advanced tools such as 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 key. For example, the modification in Escherichia coli increased its biotin production to 208.7 mg / L for 46 hours, and similar genetic engineering operations increased the production of Pseudomonas aeruginosa to 271.88 mg / L.

[0003] The final step of biotin synthesis is catalyzed by biotin synthase BioB and is the rate-limiting step of biotin synthesis. This enzyme inserts a sulfur atom between the 6th and 9th carbon atoms of the desthiobiotin molecule to form a tetrathiaanisyl ring. This enzymatic reaction depends on S-adenosylmethionine (SAM) and an iron-sulfur ([FeS]) cluster as cofactors and exhibits a slow catalytic rate (k cat =0.002s -1 ). This reaction is limited by the regeneration of the [FeS] cluster generated by the Isc and Suf systems. In vitro, each subunit of BioB has a limited number of turnovers, in contrast to its efficiency in vivo, where up to 20 turnovers can be achieved. Farrar and Jarrett mutated Asp 155 to Asn and Asn 153 to Ala, respectively, in BioB, but this did not improve the catalytic efficiency of BioB. In-depth analysis and computational assistance of key residues in the catalytic active pocket can be used using computer software programs to improve the catalytic efficiency of BioB. Summary of the invention

[0004] The object of the present invention is to provide a biotin synthetase mutant with improved catalytic activity.

[0005] In a first aspect of the present invention, a biotin synthetase mutant is provided, wherein the mutant has a mutation in the 232nd amino acid residue of the amino acid sequence corresponding to the wild-type biotin synthetase derived from Pseudomonas mutans, wherein the amino acid sequence is numbered as SEQ ID NO.1.

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

[0007] The third aspect of the present invention provides a recombinant nucleic acid expression vector, which has a nucleic acid sequence encoding the biotin synthetase mutant described in the second aspect of the present invention.

[0008] The fourth aspect of the present invention provides a recombinant genetic engineering strain containing the recombinant nucleic acid expression vector described in the third aspect.

[0009] The fifth aspect of the present invention provides a method for synthesizing biotin, wherein the recombinant genetic engineering strain described in the fourth aspect of the invention is fermented and cultured to transform desulfobiotin to synthesize biotin. The specific process includes shaking flask fermentation and culture of the engineering strain described in the fourth aspect of the present invention. The culture temperature is 37°C, the culture time is 48h, the culture medium is LB culture medium supplemented with 1g / L desulfobiotin and 50mg / L kanamycin resistance, and the shaking speed is 220rpm.

[0010] In a sixth aspect, the present invention provides a method for screening the biotin synthetase mutant according to the second aspect of the present invention, comprising the following steps:

[0011] (1) The biotin synthase BioB from wild-type Pseudomonas mutagenes and its ligand S-adenosylmethionine (SAM) and iron-sulfur cluster were homologously modeled to obtain the BioB complex model; the BioB complex model and the model of substrate dethiobiotin were docked into the active pocket of protein catalysis, and molecular dynamics simulation was used to screen out potential amino acid sites that can improve catalytic activity;

[0012] (2) Mutating the selected amino acid sites to alanine respectively, and selecting the mutant amino acid sites that are critical to catalytic activity again;

[0013] (3) constructing a plasmid expression vector library with saturated mutations at each amino acid site selected in step (2), and introducing the constructed chassis strain E. coli BL21ΔyigMΔbioB to express synthetic biotin; using the biotin detection indicator strain Lactobacillus plantarum to detect the production of synthetic biotin in each mutant strain and the control strain.

[0014] Furthermore, the method specifically includes the following steps:

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

[0016] (2) The seven selected sites were mutated to alanine respectively, and the amino acids at positions 62, 68, 159, 161, 174, 231, and 232 of the wild-type Pseudomonas mutagenesis-derived biotin synthetase were selected for subsequent site-directed saturation mutagenesis.

[0017] (3) Construct a recombinant expression vector library containing saturated mutations of these 7 sites, and introduce it into the chassis strain E. coli BL21ΔyigMΔbioB. Control the recombinant nucleic acid vector, integrate the unmutated BioB into the same expression vector, and introduce it into the chassis strain. Spread it on an LB plate containing kanamycin resistance and culture it at 37°C overnight. Pick the strains in the mutant library and the single colony of the control strain to a 96-deep-well plate and culture it for 48 hours. Centrifuge and aspirate 2.5 μL of the fermentation supernatant, transfer it to a 96-well transparent plate containing biotin detection medium of Lactobacillus plantarum according to the corresponding position, and culture it every 2 hours with an enzyme reader. The cell concentration of Lactobacillus plantarum is correlated with the biotin concentration, and those with higher concentrations than the control group are screened out.

[0018] The present invention reduces the capacity of the mutation library through computer-assisted rational design based on the amino acid sequence shown in SEQ ID NO.1, reduces the screening workload (160 mutants), and improves the hit rate of screening forward mutations. The present invention screens out three mutants with improved activity through high-throughput screening of biotin synthetase mutants. Compared with the wild-type enzyme, the mutant 232Lys->Arg with the highest conversion efficiency has an ability to synthesize biotin in the chassis strain that is 42.3% higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The three-dimensional model of biotin synthetase BioB and its ligand from Pseudomonas mutans;

[0020] Figure 2 This is the Ramachandran plot analysis result of the BioB complex model;

[0021] Figure 3 The growth of Lactobacillus plantarum in the test medium with different biotin concentrations;

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

[0023] Figure 5 Biotin production of the mutant strain in shake flask fermentation. DETAILED DESCRIPTION

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

[0025] The molecular biology operations not specifically described in the following examples were performed according to the instructions of the kits or the Molecular Cloning Experiment Guide.

[0026] Example 1: Construction of a 3D model of the wild-type biotin synthetase complex from Pseudomonas mutans

[0027] Amino acid sequence of BioB SEQ ID NO.1 (from Pseudomonas mutabilis ATCC31014)

[0028] MSASTTATTRHDWSLAEVKALFQQPFNDLLFQAQTVHRAHFDPNRVQVSTLLSIKTGACPEDCKYCPQSGHYNTGLEKQKLMEVQKVLEEAARAKAIGSTRFCMGAAWKHPSAKDMPYVLEMVKGVKAMGLETCMTLGKLDQDQTLALAQAGLDYYNHNLDTSPEFYGSIITTRTY SERLQTLAYVRDAGMKICSGGILGMGESLDDRAGLLIQLANLPEHPESVPINMLVKVAGTPLAEEEDVDPFDFIRMLAVARILMPKSHVRLSAGREQMNEQMQALAFMAGANSIFYGEKLLTTANPQADKDMQLFARLGIKPEAREEHADEVHQAAIEQALVEQCSSEMFYNAASA*

[0029] The amino acid sequence SEQ ID NO.1 was analyzed and compared with the National Center for Biotechnology Information (NCBI) database by BLAST, and it was found that there were homologous structures in the PDB database, among which the biotin synthetase BioB from Escherichia coli had a similarity of more than 70%. The sequence of SEQ ID NO.1 was input into the SWISS-MODEL website, and more than 30 templates that could be used were obtained. Finally, the crystal structure of BioB from Escherichia coli (PDB ID: 1R30) was selected as the template to generate the three-dimensional model of BioB from Pseudomonas vulgaris ( Figure 1 ). The spherical model represents the iron-sulfur cluster and the stick model represents the SAM.

[0030] Next, the complex model was evaluated by Ramachandran's conformational Figure 2 It can be seen that 100% of the amino acid residues are in the reasonable area (89.8% in the optimal area, 8.8% in the acceptable area, and 0.7% in the generally allowed area). The constructed model has high reliability and can be used as a template for subsequent research. The three-dimensional model of the BioB complex was docked with the substrate dethiobiotin using LibDock software, and the composite model was selected in the results based on the optimal energy and docking posture.

[0031] Example 2: Molecular dynamics simulation to screen out mutant amino acids

[0032] The BioB complex model obtained by molecular dynamics simulation was analyzed using Discovery studio software, and the changes in the interaction forces between BioB and SAM and DTB were studied. First, the structural fluctuation (ΔRMSF) analysis was performed. When the temperature increased from 50K, the intermediate residues and C-terminal residues of BioB 60-80, 150-180, 220-240 increased significantly. Among them, residues A62, D161, R174, V231, and N159 frequently formed hydrogen bonds with SAM at a temperature of 301K (the probabilities were 97%, 79%, 72%, 67%, and 65%, respectively), while D68 had a higher frequency of hydrogen bonding with DTB at this temperature, which was 75%; similarly, the analysis showed that N232, D62, and D161 frequently had hydrophobic interactions with SAM and DTB (the probability of conformational formation interactions was 100%, 79%, and 78%, respectively). 301K is the optimal growth temperature of the biotin synthase BioB of Pseudomonas mutagenesis. It is speculated that at this temperature, the above amino acids are related to the affinity of the substrates DTB and SAM. The ΔRMSF of these amino acid residues is also very large, indicating that they are flexible (high plasticity) and suitable for transformation and exploration.

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

[0034] The original amino acids were mutated to alanine at sites 62, 68, 159, 161, 174, 231, and 232 of the bioB gene of plasmid pUCPK-BioB. The seven plasmids pUCPK-BioB62Ala, pUCPK-BioB68Ala, pUCPK-BioB159Ala, pUCPK-BioB161Ala, pUCPK-BioB174Ala, pUCPK-BioB231Ala, and pUCPK-BioB232Ala were electroporated into the chassis strain E. coli BL21ΔyigMΔbioB. The chassis strain E.coli BL21ΔyigMΔbioB containing plasmid pUCPK-BioB and the chassis strain without plasmid as the control group were cultured in a 50mL LB shake flask containing 0.5g / L DTB for 48h, and 3 parallel samples were made for each strain. It can be seen that biotin was detected only in the fermentation broth of strain E.coli BL21ΔyigMΔbioB / pUCPK-BioB, while the other strains did not. Therefore, the amino acids at these 7 sites play a role in the normal catalytic reaction of biotin synthase BioB, and changes in these sites may improve the catalytic activity of BioB.

[0035] Example 4: Construction of a method for high-throughput screening of biotin synthases with improved catalytic activity

[0036] The Lactobacillus plantarum ATCC 8014 stored in glycerol tubes was streaked on MRS plates, cultured in a 37°C incubator overnight, and then a single colony was picked and inoculated into a test tube containing 5 mL of MRS liquid medium. After culturing at 37°C and 200 rpm for 18 h, the inoculum was transferred to a shake flask containing 50 mL of MRS liquid medium at a 1% inoculum volume and cultured at 37°C and 200 rpm until the cell density reached OD 600 When the pH reaches about 2.0, aspirate 1 mL of bacterial solution, resuspend the solution with 1 mL of sterile water, centrifuge at 8000 g for 3 min, discard the supernatant, wash twice with sterile water, and resuspend the solution with 50 μL of sterile water;

[0037] Weigh 2g of biotin detection medium into a shake flask, add 50mL of pure water, and sterilize at 105℃ for 10min. Wait until the temperature of the medium drops to room temperature, add the prepared Lactobacillus plantarum, shake evenly, and pour into a sterile pipette trough. Use a pipette gun to draw 200μL of the mixed medium into a sterile 96-well transparent detection plate.

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

[0039] Pipette 2 μL of diluted biotin standard and desulphobiotin standard into the prepared 96-well plate. No reagents were added to the control group. The plate was incubated at 37°C and the cell growth of Lactobacillus plantarum was detected every two hours. 600 ( Figure 3 ).

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

[0041] Example 5: High-throughput screening of biotin high-yielding 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, and the cells were spotted into a 96-well plate and cultured at 37°C. Figure 4It can be seen that the strain entered the logarithmic phase at the 4th hour, and 20 μL of the bacterial solution was transferred to a new 96-deep-well plate for continued cultivation at the 10th hour.

[0043] The mutant plasmid library of each site was electroporated into competent E.coli BL21ΔyigMΔbioB, and then spread on the kana resistance LB plate after recovery. 93 spots of each mutant site were selected into a 96-deep-well plate, and the remaining 3 spots were used as the control group E.coli BL21ΔyigMΔbioB / pUCPK-BioB. After 10 hours of culture, 20μL of bacterial solution was transferred to a 96-deep-well plate and continued to be cultured for 48h. The 96-deep-well plate was centrifuged, and 3μL of the supernatant was aspirated to detect the production of biotin. The 8 strains selected were: 62F4, H2; 231E6, F8, G6; 232G6, C11, C12. The seeds were preserved and streaked on the LB plate. Three spots of each strain were selected as parallel inoculations into a 96-deep-well plate, cultured in LB medium at 37℃ for 10h, and 20μL of bacterial solution was aspirated and transferred to a deep-well plate containing LB medium containing 0.5g / L DTB. The eight strains obtained from the rescreening were sequenced and found to be 232Lys->Arg, 62Asp->Gly, and 62Asp->Asn, respectively.

[0044] Example 6: Shake flask fermentation of biotin high-yielding mutant strain

[0045] The single mutant strain and the combined mutant strain were fermented together, with 3 parallel samples for each strain, and the added concentration of DTB was 1 g / L. Figure 5 As shown, 232Lys->Arg has the strongest ability to synthesize biotin, with 0.686 mg / L, compared with the unmutated strain, which has only 0.482 mg / L. The mutant's ability to convert desthiobiotin to synthesize biotin has increased by 42.3%.

Claims

1. A biotin synthetase mutant with improved catalytic activity, characterized in that: The biotin synthetase mutant has a mutation in the amino acid at position 232 of the amino acid sequence of the wild-type Pseudomonas variabilis biotin synthetase, and the amino acid sequence is shown in SEQ ID NO.

1.

2. The biotin synthetase mutant according to claim 1, characterized in that In the biotin synthetase mutant, the 232nd amino acid is mutated from lysine to arginine.

3. A recombinant nucleic acid expression vector, characterized in that: The recombinant expression vector has a nucleic acid sequence encoding the biotin synthetase mutant according to claim 2.

4. A recombinant genetically engineered strain, characterized in that: A recombinant nucleic acid expression vector comprising the recombinant nucleic acid expression vector of claim 3.

5. A method for synthesizing biotin, characterized in that: The recombinant genetic engineering strain according to claim 4 is fermented and cultured for converting desulphobiotin into biotin.

6. A method for screening the biotin synthetase mutant according to claim 2, characterized in that: The following steps are involved: (1) The biotin synthase BioB from wild-type Pseudomonas mutagenes was homologously modeled with the ligand S-adenosyl-L-methionin (SAM) and the iron-sulfur cluster to obtain the BioB complex model; the BioB complex model and the substrate desthiobiotin (DTB) model were docked into the active pocket of protein catalysis, and molecular dynamics simulation was used to screen out potential amino acid sites that can improve catalytic activity; (2) Mutating the selected amino acid sites to alanine respectively, and selecting the mutant amino acid sites that are critical to catalytic activity again; (3) constructing a plasmid expression vector library with saturated mutations at each amino acid site selected in step (2), and introducing the constructed chassis strain E. coli BL21ΔyigMΔbioB to express synthetic biotin; using the biotin detection indicator strain Lactobacillus plantarum to detect the production of synthetic biotin in each mutant strain and the control strain.

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