Tryptophan hydroxylase mutant and its application

Abstract

The mutant provided by the present invention has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and a gene sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4. The mutant is obtained by multi-site directed mutagenesis of the wild-type tryptophan 5-hydroxylase of the sponge symbiotic bacteria. The relative enzyme activity of the mutant is up to 206% of that of the wild type. In the test of catalyzing tryptophan to produce 5-HTP, the 10-min tryptophan conversion rate of the mutant can reach 49.82%, which is 2.6 times that of the wild type. The tryptophan 5-hydroxylase mutant provided by the present invention significantly improves the enzyme activity and can be fully used for industrial enzyme catalysis and efficient synthesis of 5-HTP.

Description

Technical field:

[0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a tryptophan hydroxylase mutant with improved activity and application thereof. Background technology:

[0002] 5-Hydroxytryptophan (5-HTP) is a tryptophan derivative, formed by replacing the 5′ hydrogen atom on the benzene ring of tryptophan with a hydroxyl group. 5-HTP is a non-protein amino acid that, in mammals, serves as a biosynthetic precursor for the neurotransmitter serotonin and the amine hormone melatonin. In healthcare, 5-HTP is widely used as a psychotropic agent in clinical treatments, including relieving headaches, promoting sleep, regulating appetite, and treating depression. It has demonstrated significant therapeutic potential and possesses extremely high healthcare value, garnering widespread attention.

[0003] Currently, natural product extraction is the main method for commercially producing 5-HTP. However, the raw material supply of this method is severely restricted by the production area and season, resulting in limited production capacity and difficulty in meeting the growing market demand. The chemical synthesis of 5-HTP substrates is costly, the process route is complex, involves numerous reactions and harsh conditions, and requires the use of large amounts of organic solvents, which increases the environmental burden. Therefore, it is not suitable for large-scale production of 5-HTP. Microbial fermentation has the advantages of easy raw material availability, high efficiency, low cost and sustainable production, making it undoubtedly a more feasible option for the preparation of 5-HTP. However, when expressed in microorganisms, tryptophan hydroxylase (TPH) in the 5-HTP biosynthesis pathway suffers from low activity, poor solubility and insufficient supply of cofactor BH4. Although most current research focuses on the discovery and modification of highly active TPH to improve enzyme activity and solubility, the enzymes in these studies are limited to the aromatic amino acid hydroxylase (AAAH) family. The enzymatic production of 5-HTP requires oxygen as a co-substrate, tetrahydropterin (BH4) and Fe 2+ As a cofactor, the catalytic reaction involves a cyclic regeneration process of the cofactor BH4. During the reaction, TPH first catalyzes BH4, oxygen molecules, and ferrous ions to form an active hydroxylation intermediate, and then inserts one of the oxygen atoms into the aromatic ring of L-tryptophan to synthesize 5-HTP. However, the intermediate product quinone-type dihydropterin in the BH4 cycle is poorly stable and can be converted into a BH4 structural analogue that cannot participate in the tryptophan hydroxylation reaction through non-enzymatic reactions. This reduces the catalytic activity of the enzyme and prevents the efficient synthesis of the target product 5-HTP. Summary of the invention:

[0004] In view of the above-mentioned deficiencies in the prior art, the main objectives of the present invention are to obtain a tryptophan 5-hydroxylase (KERL) mutant using site-directed mutagenesis and protein engineering techniques, to provide a recombinant plasmid or engineered strain expressing the tryptophan 5-hydroxylase mutant, and to provide applications of the tryptophan 5-hydroxylase mutant.

[0005] The following definitions are used in the description of the technical solution of the present invention:

[0006] 1. Amino acid residues are represented using the generally accepted IUPAC nomenclature, using either the three-letter abbreviation amino acid or the single-letter symbol. DNA sequences are represented using the generally accepted IUPAC nomenclature.

[0007] 2. Mutant Identification: The mutated amino acid in the tryptophan hydroxylase mutant is indicated by "amino acid substituted at the original amino acid position." For example, I77A indicates that the amino acid at position 77 of the parent tryptophan 5-hydroxylase has mutated from isoleucine Ile to alanine Ala. Another example is I226A / P229C, which indicates that the amino acid at position 226 of the parent has mutated from isoleucine Ile to alanine Ala, and the amino acid at position 229 has mutated from proline Pro to cysteine ​​Cys.

[0008] In the present invention, R275V / S282P and R275V / S282P / T379E represent two tryptophan 5-hydroxylase mutants obtained from wild-type tryptophan 5-hydroxylase (KERL), and lowercase italic kerl represents the coding gene of wild-type KERL.

[0009] The technical solution of the present invention is summarized as follows:

[0010] Through site-directed mutagenesis, with the goal of expanding the substrate pocket of tryptophan 5-hydroxylase and enhancing its activity in producing 5-HTP, a semi-rational design was conducted using wild-type tryptophan 5-hydroxylase from the sponge symbiotic bacteria (Entotheonella factor) as the parent, and amino acid mutations were performed using site-directed mutagenesis to obtain mutants. The details are as follows:

[0011] A tryptophan 5-hydroxylase mutant, characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] Among them, the mutant type corresponding to SEQ ID NO:1 is R275V / S282P, which is obtained by mutating the arginine Arg at position 275 to valine Val and the serine Ser at position 282 to proline Pro in the wild-type KERL derived from the sponge symbiotic bacteria (Entotheonella factor) as the parent.

[0013] The mutant type corresponding to SEQ ID NO: 2 is R275V / S282P / T379E, which is also obtained by using the above-mentioned wild-type KERL as the parent and further introducing a mutation at amino acid position 379 (threonine Thr is mutated to glutamic acid Glu) based on the R275V / S282P mutation shown in SEQ ID NO: 1.

[0014] The present invention also provides the gene sequence of the tryptophan 5-hydroxylase mutant, as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0015] The present invention also provides an expression vector carrying the gene sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0016] Preferably, the above expression vector is pQE-80L, pET-22b or pMA5.

[0017] The present invention also provides an engineered strain containing the above expression vector.

[0018] Preferably, the engineered strain is Escherichia coli, Bacillus subtilis or Pichia pastoris.

[0019] More preferably, the engineered strain is Escherichia coli K-12MG1655.

[0020] Application of the above tryptophan 5-hydroxylase mutant in the production of 5-HTP.

[0021] The use of the above tryptophan-5-hydroxylase mutant in the reaction process of catalyzing tryptophan to produce 5-HTP.

[0022] Application of the above expression vector or engineered strain in the production of 5-HTP.

[0023] The experimental steps for obtaining the tryptophan 5-hydroxylase mutant of the present invention are summarized as follows:

[0024] 1. Using the wild-type tryptophan 5-hydroxylase gene ker1 from the sponge symbiotic bacteria (Entotheonella factor) and the recombinant plasmid pQE-80L-ker1 constructed from the plasmid pQE-80L as templates, we designed site-directed mutagenesis primers and performed site-directed mutagenesis PCR amplification reactions.

[0025] 2. The site-directed mutagenesis PCR product is digested with Dpn I, self-circularized, and then transferred into the host bacteria for culture. The positive clones obtained are screened with antibiotics and induced for expression. Mutants with high activity are screened by converting tryptophan.

[0026] 3. The selected high-activity mutants were sequenced and identified. The sequencing results were compared with the wild type to obtain two tryptophan 5-hydroxylase mutants with enhanced activity, R275V / S282P and R275V / S282P / T379E.

[0027] 4. Purify and determine the catalytic activity of the mutant;

[0028] 5. Use the mutant to catalyze the production of 5-HTP from tryptophan.

[0029] Beneficial effects of the present invention:

[0030] The present invention obtains tryptophan-5-hydroxylase mutants R275V / S282P and R275V / S282P / T379E through site-directed mutagenesis. The relative enzyme activities of the mutants R275V / S282P and R275V / S282P / T379E are 185% and 206% of the wild-type KERL, respectively. In a test of catalyzing tryptophan to produce 5-HTP, the 10-minute tryptophan conversion rates of the mutants R275V / S282P and R275V / S282P / T379E reached 35.76% and 49.82%, respectively, which are 1.8 times and 2.6 times that of the wild-type, respectively. The tryptophan-5-hydroxylase mutants provided by the present invention have significantly improved enzyme activity and are fully applicable to industrial enzyme-catalyzed efficient synthesis of 5-HTP. Description of the drawings:

[0031] Figure 1 : HPLC identification results of the catalytic products. In the figure, “sample” corresponds to the product sample after the mutant R275V / S282P catalyzed the tryptophan reaction.

[0032] Figure 2 : Comparison of relative enzyme activities between the wild type and mutants; in the figure, “WT” represents wild type KERL.

[0033] Figure 3 : Comparison of the conversion rate of tryptophan to 5-HTP catalyzed by the wild type and mutants; in the figure, "WT" represents the wild type KERL. Specific implementation method:

[0034] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art.

[0035] Example 1: Construction of recombinant plasmid

[0036] The ker1 gene sequence (as shown in SEQ ID NO: 6) derived from the sponge animal symbiotic bacteria (Entotheonella factor) was optimized against the preference of the Escherichia coli host codon, and then the gene sequence was synthesized. The gene sequence was connected to the vector pQE-80L through two restriction enzyme sites, BamHI and HindIII. The ligation product was transferred to the cloning host E.coli JM109, coated on LB solid medium for cultivation, and then the plasmid in the transformant was extracted for PCR and double enzyme digestion verification, and the successful sequencing verification was verified. After correct verification, the pQE-80L-ker1 recombinant plasmid was obtained.

[0037] Example 2: Construction of tryptophan 5-hydroxylase mutants

[0038] Using the pQE-80L-ker1 recombinant plasmid constructed in Example 1 as a template, mutation primers were designed, and site-directed mutagenesis PCR amplification reaction was performed. Taking the introduction of mutations at the three amino acid sites R275V / S282P / T379E as an example, the corresponding mutation primers included the following three pairs of primers: R275V-F / R, S282P-F / R, and T379E-F / R; wherein, primer (5'-3') R275V-F and primer (5'-3') R275V-R are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; primer (5'-3') S282P-F and primer (5'-3') S282P-R are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; primer (5'-3') T379E-F and primer (5'-3') T379E-R are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Shown in NO:12.

[0039] Add the reagents listed in Table 1 in sequence to a 0.2 mL EP tube to perform PCR amplification reaction.

[0040] Table 1 PCR amplification reaction system

[0041]

[0042]

[0043] The PCR reaction conditions and cycling process are shown in Table 2.

[0044] Table 2 PCR reaction conditions and cycles

[0045]

[0046] After the reaction, the product was demethylated. 0.35 μL Dpn I was added to the PCR product and reacted at 37°C for 1 h. The demethylated product was verified by agarose gel electrophoresis. The gene length was approximately 6000 bp. After successful verification, the next step of the reaction was carried out.

[0047] For the self-cyclization of the PCR product, the reagents listed in Table 3 were added sequentially in a 0.2 mL EP tube to perform the cyclization reaction.

[0048] Table 3 PCR product cyclization reaction system

[0049]

[0050] The ligation product after the cyclization reaction was added to E. coli K-12MG1655 competent cells and the following steps were performed: gently mix, incubate on ice for 30 minutes; heat shock at 42°C for 90 seconds; immediately incubate on ice for 5 minutes; add 600 μL of LB resuscitation solution and incubate at 37°C at 220 rpm for 40-60 minutes; centrifuge at 4000 rpm for 5 minutes; resuspend the pellet in 200 μL of LB medium, spread the mixture on a plate containing Amp resistance. After the bacterial suspension is fully absorbed, incubate inverted at 37°C for 12-16 hours until a single colony appears; pick a single colony for activity detection. The above methods were used to obtain engineered strains containing the mutant R275V / S282P and R275V / S282P / T379E genes, respectively. Among them, the amino acid sequence and gene sequence of the mutant R275V / S282P are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively; the amino acid sequence and gene sequence of the mutant R275V / S282P / T379E are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0051] The detection peaks of samples and standards are as follows Figure 1 shown.

[0052] Example 3: Purification of mutant proteins

[0053] Protein purification: Resuspend the engineered bacterial cells containing the mutant gene in Lysis Buffer (50 mM NaH2PO4, 10 mM imidazole, 300 mM NaCl, pH 8.0), mix thoroughly, add 200 μL lysozyme and 120 μL PMSF, mix thoroughly, and place in a beaker on ice for 20 minutes. Disrupt the cell suspension by ultrasonication for 15 minutes (2.5 seconds with 3.0 seconds intervals). The disrupted solution was centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was combined with Ni-NTA superflow eluted with Lysis Buffer for 1 h. The combined solution was passed through a purification column and contaminants were eluted with Wash Buffer (50 mM NaH2PO4, 50 mM Mimidazole, 300 mM NaCl, pH = 8.0). The target protein was then eluted with Elution Buffer (50 mM NaH2PO4, 300 mM Mimidazole, 300 mM NaCl, pH = 8.0). The Elution Buffer was then dialyzed against 50 mM HEPES (pH = 8.0, containing 375 mM NaCl). The mutant enzyme solution was concentrated by ultrafiltration. Protein concentration was determined using a Nanodrop 2000. The activity of the mutants was assayed using the following catalytic reaction system: 3 mM tryptophan, 3 mM H₂O₂, and 20 μmol of enzyme dissolved in 1 mL of 50 mM HEPES (pH 8.0). The reaction was incubated at 30°C for 10 minutes and terminated by boiling for 10 minutes. After centrifugation, the supernatant was diluted to an appropriate concentration and filtered through a 0.22 μm aqueous filter into a chromatographic injection vial. The product was identified and analyzed by HPLC. HPLC assay conditions were as follows:

[0054] Chromatograph: Agilent 1260;

[0055] Detector: UV detector (Alltech Chrom, ELSD6000)

[0056] Injection: Agilent autosampler; injection volume 10 μL;

[0057] Chromatographic column: Diamonsil C18 (5 μm, 4.6 × 250 mm, DiKMA, Beijing); column temperature 33 °C;

[0058] Mobile phase: 10% acetonitrile in water; flow rate: 1 mL / min.

[0059] The test results showed that both mutants could efficiently catalyze tryptophan to produce 5-HTP. Figure 1 shown.

[0060] Example 4: Determination of relative enzyme activity of mutants

[0061] The enzyme activity of the present invention is defined as follows: Under standard reaction conditions, 1 mL of 50 mM HEPES (pH = 8.0), 3 mM tryptophan, 3 mM H2O2, and 20 μM of pure enzyme solution of the wild type or mutant are uniformly mixed. The reaction mixture is incubated at 30°C for 10 minutes and the reaction is terminated by boiling for 10 minutes. One unit (U) of enzyme is defined as the amount of 1 μM tryptophan consumed per minute under the above conditions. The relative enzyme activity of the mutant is based on the wild type enzyme activity as 100%. The relative enzyme activity of the purified mutant and wild type was determined with reference to the enzyme reaction system of Example 3. The results are shown in FIG. Figure 2 As shown, the relative enzyme activity of R275V / S282P is 185% of that of the wild type, and the relative enzyme activity of R275V / S282P / T379E is 206% of that of the wild type. Both mutants significantly improved their catalytic activity towards tryptophan compared to the wild type.

[0062] Example 5: Production of 5-HTP using mutants

[0063] In a 1L Erlenmeyer flask, HEPES buffer (50mM, pH 8.0) containing 3mM tryptophan and 20μM wild-type or mutant pure enzyme solution was added and reacted at 30°C for 10min. After the reaction, the mixture was boiled for 10min and centrifuged at 12,000rpm for 5min to terminate the reaction. The 5-HTP content in the supernatant was determined by HPLC, and the conversion rate of the wild-type and mutant to 5-HTP was compared under the same reaction time. The results are shown in Figure 2. Figure 3 As shown: After 10 minutes of reaction, the tryptophan conversion rate catalyzed by the wild-type KERL can reach 19.33%, the tryptophan conversion rate of the mutant R275V / S282P can reach 35.76%, which is 1.8 times that of the wild-type, and the tryptophan conversion rate of the mutant R275V / S282P / T379E can reach 49.82%, which is 2.6 times that of the wild-type.

Claims

1. A tryptophan 5-hydroxylase mutant, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The tryptophan 5-hydroxylase mutant according to claim 1, characterized in that The gene sequence of the mutant is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

3. An expression vector, characterized in that The expression vector carries the gene sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

4. The expression vector according to claim 3, characterized in that: The type of the expression vector is pQE-80L, pET-22b or pMA5.

5. An engineered strain, characterized in that The engineered strain contains the expression vector according to claim 3.

6. The engineered strain according to claim 5, characterized in that: The engineered strain is Escherichia coli, Bacillus subtilis or Pichia pastoris.

7. The engineered strain according to claim 5, characterized in that: The engineering strain is Escherichia coli E. coli K-12MG1655, and the expression vector contained therein is pQE-80L.

8. Use of the tryptophan 5-hydroxylase mutant according to claim 1 or 2 in producing 5-hydroxytryptophan.

9. Use of the expression vector according to claim 3 or 4 in producing 5-hydroxytryptophan.

10. Use of the engineered strain according to any one of claims 5 to 7 in the production of 5-hydroxytryptophan.

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