Application of mutation site as target spot in preparation of fluorine-containing hydroxytryptophan

Abstract

The invention relates to the field of bioengineering, in particular to application of a mutation site as a target spot in preparation of fluorine-containing hydroxytryptophan. The invention provides application of a mutation site as a target spot in preparation of fluorine-containing hydroxytryptophan. The mutation sites comprise one or more of the 312th site, the 357th site, the 364th site and the 379th site of the amino acid sequence of the wild tryptophan hydroxylase. According to the invention, TPH145 is modified by using a CAST / ISM enzyme semi-rational directed evolution strategy, a mutant M5 (G379S / P312I / F364M / C357I) is successfully obtained, 4F-5HTP can be specifically obtained, the selectivity is 99.9%, the conversion rate is 79%, and the reaction yield is 61%.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to the application of mutation sites as targets in the preparation of fluorinated hydroxytryptophan. Background Art

[0002] 5-Hydroxytryptophan (5HTP) is the precursor of serotonin and melatonin in animals. They play an important role in regulating emotions and behaviors, as well as physiological functions such as sleep, pain, and body temperature. They have been successfully used to treat depression, insomnia, and migraine. Currently, the synthesis of 5HTP uses tryptophan hydroxylase (TPH145) to catalyze the hydroxylation reaction of L-tryptophan, in which Fe 2+ and tetrahydrobiopterin (BH4) as cofactors, O 2 The introduction of fluorine atoms into 5HTP often enhances the efficacy of the drug, but studies have found that TPH145 catalyzes 4F-Trp (1a) to break the CF bond, producing 4-fluoro-5-hydroxytryptophan (4F-5HTP, 2a) and 4-hydroxytryptophan (4HTP, 3), respectively, making it impossible to efficiently introduce fluorine atoms into 5HTP. Summary of the invention

[0003] In view of this, the present invention provides the use of mutation sites as targets in the preparation of fluorinated hydroxytryptophan. The present invention uses the CAST / ISM enzyme semi-rational directed evolution strategy to transform TPH145 and successfully obtains a mutant M5 (G379S / P312I / F364M / C357I), which can specifically obtain 4F-5HTP with a selectivity of 99.9%, a conversion rate of 79%, and a reaction yield of 61%.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides the use of mutation sites as targets in the preparation of fluorinated hydroxytryptophan;

[0006] The mutation sites include: one or more of positions 312, 357, 364 and 379 in the amino acid sequence of the wild-type tryptophan hydroxylase.

[0007] The present invention also provides a mutant, which has one or more of the following amino acid site mutations based on the wild-type tryptophan hydroxylase: position 312, position 357, position 364 and position 379, and the amino acid sequence of the wild-type tryptophan hydroxylase is shown in SEQ ID NO: 1.

[0008] In some embodiments of the present invention, in the above mutant, the mutation at position 312 includes: any one of P312G, P312A, P312I, P312V or P312S;

[0009] The mutation at position 357 includes any one of C357L, C357I, C357S, C357T, C357H or C357P;

[0010] The mutation at position 364 includes any one of F364M, F364V, F364G, F364S, F364T or F364C;

[0011] The mutation at position 379 includes any one of G379S, G379T or G379A.

[0012] In some embodiments of the present invention, in the above mutant, the mutation at position 312 is: P312I;

[0013] The mutation at position 357 is: C357I;

[0014] The mutation at position 364 is: F364M;

[0015] The mutation at position 379 is: G379S.

[0016] In some embodiments of the present invention, the mutants described above have:

[0017] (1), the amino acid sequence shown in SEQ ID NO: 2; or

[0018] (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or

[0019] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0020] The present invention also provides a nucleic acid molecule encoding the above mutant, which has:

[0021] (4) the nucleotide sequence shown in SEQ ID NO: 3; or

[0022] (5) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (4); or

[0023] (6) a sequence having at least 80% homology with the nucleotide sequence described in (4) or (5); or

[0024] (7) A complementary sequence of the nucleotide sequence described in (4), (5) or (6).

[0025] The present invention also provides an expression vector, comprising: the above nucleic acid molecule.

[0026] The present invention also provides a host for transforming and / or transfecting the above expression vector.

[0027] The present invention also provides a product, comprising: the mutant, the nucleic acid molecule, the expression vector and / or the host.

[0028] The present invention also provides the use of the mutant, the nucleic acid molecule, the expression vector, the host and / or the product in the preparation of fluorine-containing hydroxytryptophan.

[0029] In some embodiments of the present invention, in the above application, the fluorinated hydroxytryptophan includes one or more of 4-fluoro-5-hydroxytryptophan, 6-fluoro-5-hydroxytryptophan, 7-fluoro-5-hydroxytryptophan and 4,6-2-fluoro-5-hydroxytryptophan.

[0030] The present invention also provides a method for preparing fluorinated hydroxytryptophan, which uses fluorinated tryptophan as a substrate and undergoes a catalytic reaction with the mutant to obtain the fluorinated hydroxytryptophan.

[0031] In some embodiments of the present invention, in the above preparation method, the catalytic reaction includes: 1% to 1.5% (v / v) glycerol, hydrated ferrous sulfate with a final concentration of 10 μM, and 50 mM PBS Buffer with a pH of 7.4.

[0032] In some embodiments of the present invention, in the above preparation method, the final concentration of the substrate is 10 mM.

[0033] In some embodiments of the present invention, in the above preparation method, the reaction conditions of the catalytic reaction are: 30° C., 220 rpm, and shaking for 1 to 2 days.

[0034] In some embodiments of the present invention, in the above preparation method, the fluorine-containing tryptophan includes: one or more of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, 4,5-2-fluorotryptophan, 4,6-2-fluorotryptophan, 5,6-2-fluorotryptophan and 4,5,6,7-4-fluorotryptophan.

[0035] In some embodiments of the present invention, in the above-mentioned preparation method, the preparation method of the fluorinated tryptophan comprises the following steps: using fluorinated indole as a substrate, and obtaining the fluorinated tryptophan after a tryptophan synthase-catalyzed reaction.

[0036] In some embodiments of the present invention, in the above preparation method, the fluorine-containing indole includes one or more of 4-fluoroindole, 5-fluoroindole, 6-fluoroindole, 7-fluoroindole, 4,5-2-fluoroindole, 4,6-2-fluoroindole, 5,6-2-fluoroindole and 4,5,6,7-4-fluoroindole.

[0037] In some embodiments of the present invention, in the above-mentioned preparation method, the tryptophan synthetase includes: a wild type tryptophan synthetase or a mutant of tryptophan synthetase; the point mutant of the tryptophan synthetase is a point mutant of the wild type tryptophan synthetase in which position 145 is mutated from M to T and position 167 is mutated from N to D.

[0038] In some embodiments of the present invention, in the above preparation method, the reaction conditions of the catalytic reaction are: 75° C., 220 rpm, shaking reaction for 1 to 2 days; the reaction solution is: PBS Buffer (50 mM, pH 7.4).

[0039] The present invention utilizes the CAST / ISM enzyme semirational directed evolution strategy to transform TPH145 and successfully obtains a mutant M5 (G379S / P312I / F364M / C357I), which can specifically obtain 4F-5HTP with a selectivity of 99.9%, a conversion rate of 79% and a reaction yield of 61%. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0041] Figure 1 The difference diagram of the amino acid sequence of TPH145 and TPH2 is shown; wherein: TPH145 (NΔ145 / CΔ24ofTPH2, PDB: 4VO6). Red represents the 145 amino acids truncated at the N-terminus, and blue represents the 24 amino acids truncated at the C-terminus;

[0042] Figure 2 The schematic diagram of the multi-enzyme cascade pathway of tryptophan hydroxylase TPH145 in the whole cell is shown; it includes three parts: Module 1 is the 5HTP synthesis pathway; Module 2 is the BH4 generation pathway; Module 3 is the BH4 regeneration pathway;

[0043] Figure 3 The schematic diagram of the TPH145 whole-cell catalytic dual-plasmid system is shown; wherein: A is the TPH145 gene cloned on the plasmid pRSFDuet-1; B is the 5 genes involved in the BH4 production and recycling system cloned on the plasmid pACYCDuet-1;

[0044] Figure 4 The reaction formula of TPH145 catalyzing 4F-Trp to synthesize 4F-5HTP and 4HTP is shown;

[0045] Figure 5 The liquid phase diagram showing the synthesis of 4F-5HTP and 4HTP from 4F-Trp catalyzed by TPH145;

[0046] Figure 6 The key amino acid site selection of the catalytic pocket of TPH145 enzyme is shown;

[0047] Figure 7 Schematic diagram of primer design for single-site saturation mutagenesis (SM) of TPH145 using degenerate codons NDT / VMA / ATG / TGG; wherein: A is a schematic diagram of degenerate primer design of NDT / VMA / ATG / TGG; B is a schematic diagram of degenerate codons of NDT / VMA / ATG / TGG representing different amino acids;

[0048] Figure 8 The present invention shows the design of combinatorial library primers for TPH145 regional selectivity screening using the TCSM strategy; wherein: serine (S), isoleucine (I) and methionine (M) are used as building blocks for the G379 / P312 / F364 site;

[0049] Fig. 9 The figure shows the use of DYA / KGC codon degeneracy to design primers and create a single-point small steric hindrance amino acid mutation library for TPH145; wherein: A shows a schematic diagram of the design of degenerate primers for DYA and KGC; B shows a schematic diagram of the degenerate codons of DYA and KGC representing different amino acids;

[0050] Fig.10 This shows the universality of fluorinated 5-hydroxytryptophan;

[0051] Fig.11 The hydrogen spectrum, fluorine spectrum and carbon spectrum of 4F-5HTP (2a) are shown; wherein: A 1 HNMR (500MHz, CD 4 O); B means 19 FNMR (470MHz, CD 4 O); C shows 13 C NMR (126MHz, CD 4 O). DETAILED DESCRIPTION

[0052] The invention discloses the application of a mutation site as a target in the preparation of fluorine-containing hydroxytryptophan.

[0053] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0054] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0055] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0056] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0057] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0058] In Examples 1 to 4 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0059] The present invention will be further described below in conjunction with embodiments:

[0060] Example 1 Construction of recombinant plasmids and strains required for the experiment

[0061] TPH145 is obtained by truncating 145 amino acids at the N-terminus and 24 amino acids at the C-terminus of TPH2 (PDB: 4VO6). The solubility and catalytic activity of the truncated TPH145 are significantly improved (e.g. Figure 1). TPH145 is a non-heme-ferrous and tetrahydropterin (BH4)-dependent hydroxylase that can hydroxylate L-tryptophan to 5-hydroxytryptophan (5-HTP) in the presence of oxygen and ferrous ions. The tetrahydropterin BH4 cofactor synthesis and recycling pathway contains five exogenous enzymes introduced from mammals. The process must be carried out in Escherichia coli cells. The synthesis of BH4 uses glycerol as a carbon source and starts with GTP. It is catalyzed by cyclohydrolase I (GCHI), 6-pyruvoyl tetrahydropterin synthase (PTPS), and sepiapterin reductase (SPR). BH4 will be oxidized to 4α-hydroxy-H4-biopterin (4α-OH-BH4) during the hydroxylase reaction. Under the action of pterin-4α-carbinolamine dehydratase (PCD), a molecule of water will be removed to generate quinonoid dihydrobiopterin (qBH2). Under the action of dihydrobiopterin reductase (DHPR), qBH2 will generate BH4 with NADH as electron donor, thus completing the coenzyme BH4 cycle. The whole reaction pathway is as follows: Figure 2 The TPH145 whole-cell catalytic system uses a dual-plasmid system. The TPH145 gene is cloned on the plasmid pRSFDuet-1, and the five genes involved in the BH4 production and recycling system are cloned on the plasmid pACYCDuet-1 (such as Figure 3 ). Specific experimental operation process: The recombinant plasmid was constructed using Novozan's one-step cloning kit. All enzymes used in the experiment are shown in Table 2.

[0062] Table 1 TPH145 whole cell catalytic pathway plasmid and strain construction.

[0063]

[0064] Table 2 Amino acid sequences and partial nucleic acid sequences of genes used

[0065]

[0066]

[0067]

[0068] Example 2 Study on the reaction of 4F-Trp catalyzed by TPH145

[0069] Experiments have found that tryptophan hydroxylase TPH145 catalyzes the CF bond cleavage of 4F-Trp (1a) to produce 4-fluoro-5-hydroxytryptophan (4F-5HTP, 2a) and 4-hydroxytryptophan (4HTP, 3), respectively. Figure 4 Specific reaction system: substrate 4F-Trp (final concentration 10mM), glycerol (reaction volume ratio about 1%-1.5%), ferrous sulfate heptahydrate (final concentration 10μM), TPH145 double plasmid whole cell catalytic system (bacterial concentration 0.5g / mL), reaction liquid PBS Buffer (50mM, pH7.4), the volume of the entire reaction system varies from 10mL to 1L. Reaction conditions: 30℃, 220rpm, shaker reaction for 1 to 2 days. Liquid phase detection conditions: AgilentSB-Aq (4.6mm)×250mm×5μM), detection wavelength 275nm; detection temperature 35℃; injection volume 1μL; detection time 20min; mobile phase A: pure water (0.1% formic acid); mobile phase B: acetonitrile (0.1% formic acid), mobile phase A: mobile phase ratio 93:7, peak time: 1a:14.5min, 2a:7.9min, 3:7.2min. Reaction condition control experimental design, i.TPH145, oxygen, substrate 1a, BH4, Fe 2+ ii. TPH145, oxygen, substrate 1a, Fe 2+ ; iii.TPH145, oxygen, substrate 1a, BH4; iv.TPH145, oxygen, BH4, Fe 2+ Finally, it can be known that the cofactor BH4, ferrous ions and free oxygen in the air in the reaction system are all necessary for the reaction to occur. Figure 5 .

[0070] Example 3

[0071] TPH145 uses free oxygen in the air as its oxygen source, and mainly relies on the catalytic triad H318-H323-Q363 to achieve the C(sp2)-H bond oxidation reaction of the indole benzene ring of tryptophan. The catalytic triad H318-H323-Q363 is located inside the catalytic pocket of the enzyme. Through molecular docking and fingerprint analysis, ten amino acids F287, E313D, P312, P314, E319, Y358, I362, F364, G379, and I412 near the catalytic triad inside the catalytic pocket of the enzyme were selected as key sites that may affect regional selectivity. The degenerate codons NDT / VMA / ATG / TGG were used to construct a single-point saturation mutation library (such as Figure 6 , Figure 7, Table 3). The experiment found that the three amino acid sites G379, P312, and F364 had a greater impact on selectivity and catalytic activity. Therefore, based on the above experimental results, the three sites G379 / P312 / F364 were selected using the triple codon saturation mutation strategy (TCSM) and the library was constructed using the "SIM" ( Figure 8 , Table 4), the screening amount is 192 mutants, and 4F-5HTP can be obtained in batches efficiently. Among them, M4 (G379S / P312I / F364M) can achieve regional selectivity changes, and the product only generates 4F-5HTP. Furthermore, through molecular docking and RossetaDesign and other means, four amino acids Y281, C357, F359, and T414 located outside the catalytic pocket were selected as key sites that may affect catalytic activity, and M4 was used as a template for iterative evolution. Drawing on the focused rational iterative site-directed mutagenesis (FRISM) strategy, DYA / KGC degenerate primers were designed to cover small steric amino acids, including glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), serine (S), threonine (T) and cysteine ​​(C) to construct a single-point saturation mutation library (SM) for screening. The screening amount was reduced to 36 (compared to the 96 mutants in the single-point saturation mutation library) Fig. 9 , Table 5). Finally, C357I was iterated to obtain the quadruple mutant M5 (G379S / P312I / F364M / C357I) with improved activity. The conversion rate and selectivity of the dominant mutants of TP145 are shown in Table 6.

[0072] Specific experimental operation process: The single-point saturation mutation library of TPH145 was constructed using the large primer method of PrimeSTAR DNA polymerase. The PCR program was: 95°C for 5 minutes, followed by 32 cycles of amplification (95°C for 30s, 56°C for 30s, 72°C for 40s), and extension at 72°C for 10 minutes. For large fragment PCR: 95°C for 5 minutes, followed by 30 cycles of amplification (95°C for 30s, 60°C for 30s, 72°C for 4min), and extension at 72°C for 10 minutes. The PCR product was then digested with 0.5μL DpnI at 37°C for 3 hours. Each digested PCR mixture was then transformed into electrocompetent Escherichia coli BL21 (DE3). Residues F287, E313D, P312, P314, E319, Y358, I362, F364, G379, and I412 were replaced with 19 other canonical amino acids by the Tang method and sequenced by scraper to identify the quality of the library construction.

[0073] Table 3. List of region-selective primers used to screen the TPH145 SM library for substrate 1a.

[0074]

[0075]

[0076]

[0077] Note: NDT refers to asparagine (N), serine (S), isoleucine (I), histidine (H), arginine (R), leucine (L), tyrosine (Y), cysteine ​​(C), phenylalanine (F), aspartic acid (D), glycine (G), and valine (V). VMA refers to glutamic acid (E), alanine (A), glutamine (Q), proline (P), lysine (K), and threonine (T). ATG refers to methionine (M). TGG refers to tryptophan (W). Table 4 Primer list for TPH145 combinatorial library of substrate 1a. Serine (S), isoleucine (I), and methionine (M) were used as G379 / P312 / F364 a Building blocks of sites

[0078]

[0079] Note: The model enzyme corresponding to substrate 1a is TPH145 (WT). The codons and corresponding amino acids are indicated by the upstream primer design, CCG: proline (P), AGC: serine (S), ATK: ATT (isoleucine, I) and ATG (methionine, M), TTT: phenylalanine (F). The codons and corresponding amino acids are indicated by the downstream primer design, reverse complementary codons: AAA: phenylalanine (F), GCT: serine (S), MAT: AAT (isoleucine, I) and CAT (methionine, M), GCY: GCT (serine, S) and GCC (glycine, G).

[0080] Table 5. List of primers used to screen TPH145 DYA / KGC library activity against substrate 1a.

[0081]

[0082] Note: The model enzyme corresponding to substrate 1a is TPH145 (WT). The codons and corresponding amino acids are indicated by the upstream primer design, TTT: phenylalanine (F), GAT: aspartic acid (D), AAA: lysine (K), CTG: leucine (L), RAA: GAA (glutamic acid, E) and AAA (lysine, K).

[0083] The codons and corresponding amino acids were indicated by the downstream primer design, with the reverse complement codons: AAT: isoleucine (I), ATC: aspartic acid (D), TTT: lysine (K), CAG: leucine (L), TTY: GAA (glutamic acid, E) and AAA (lysine, K).

[0084] Table 6 Conversion rate and selectivity of 4F-Trp catalyzed by TPH145 dominant mutants

[0085]

[0086]

[0087] Example 4

[0088] The successfully modified TPH145 mutant M5 was tested on the fluorinated substrates 4F-Trp (1a), 6F-Trp (1b), 7F-Trp (1c), 4,6-2F-Trp (1d), 5,6,7-3F-Trp (1e), 4,5,6,7-4F-Trp (1f) according to the reaction conditions of Example 3. As a result, fluorinated 5-hydroxytryptophan products were obtained, namely 4F-5HTP (2a), 6F-5HTP (2b), 7F-5HTP (2c), 4,6-2F-5HTP (2d), 5,6,7-3F-5HTP (2e), 4,5,6,7-4F-5HTP (2f), and the fluorine atoms did not break (such as Fig.10 ). The NMR spectrum of 4F-5HTP (2a) is as follows ( Fig.11 ). Specific experimental process: Induce expression of the BL21 (DE3) / Δtna recombinant strain containing the dual plasmids pRSF-TPH145 and pACYC-BH4. Shake flask screening process: Draw the target strain on the resistance LB plate and culture it in a constant temperature incubator at 37°C for 24 hours. Pick a single colony and incubate it overnight in 5mL LB (25μg / mL chloramphenicol, 50μg / mL kanamycin) at 37°C and 220rpm. Take 1mL of the overnight bacterial solution and place it in 45mL TB medium. Add 5mL PBS (pH7.4, 50mM) buffer, as well as 25μg / mL chloramphenicol and 50μg / mL kanamycin. Incubate at 37°C and 220rpm for about 2 to 3 hours until the OD 600Reach 0.6-0.8. Add IPTG at a final concentration of 0.1mM and incubate at 30°C and 220rpm for enzyme protein expression for 24h-48h. Collect the bacteria by centrifugation and resuspend them in PBS (pH7.4, 50mM) buffer to 0.5g / L bacterial solution for catalytic reaction. Substrate 4F-Trp (final concentration 10mM), glycerol (reaction volume ratio about 1%-1.5%), ferrous sulfate heptahydrate (final concentration 10μM), TPH145 double plasmid whole cell catalytic system (bacterial concentration 0.5g / mL), reaction solution PBS Buffer (50mM, pH7.4), the volume of the entire reaction system varies from 10mL to 1L.

[0089] Reaction conditions: 30°C, 220 rpm, shaker reaction for 1 to 2 days.

[0090]

[0091] 4-Fluoro-5-hydroxytryptophan(2a)

[0092] 1 H NMR (500MHz, CD 4 O)δ7.00(s,1H),6.89(d,J=8.7Hz,1H),6.68(t,J=8.4Hz,1H),3.84(dd,J=1 0.1,3.9Hz,1H),3.53(dd,J=14.8,3.4Hz,1H),3.00(dd,J=15.1,9.9Hz,1H). 19 F NMR (470MHz, CD 4 O)δ-152.96. 13 C NMR (126MHz, CD 4 O) δ145.40,143.52,136.01(d,J=11.6Hz),133.65(d,J=10.1Hz),124.98,116.47 (d,J=17.5Hz),113.55,106.79(d,J=4.1Hz),106.20(d,J=2.2Hz),55.74,28.19.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of mutation sites as targets in the preparation of fluorinated hydroxytryptophan; The mutation sites include: One or more of positions 312, 357, 364 and 379 of the amino acid sequence of wild-type tryptophan hydroxylase.

2. A mutant, characterized in that Based on the wild-type tryptophan hydroxylase, it has the following one or more amino acid site mutations: 312th, 357th, 364th and 379th. The amino acid sequence of the wild-type tryptophan hydroxylase is shown in SEQ ID NO:

1.

3. The mutant according to claim 2, characterized in that The mutation at position 312 includes: any one of P312G, P312A, P312I, P312V or P312S; The mutation at position 357 includes any one of C357L, C357I, C357S, C357T, C357H or C357P; The mutation at position 364 includes any one of F364M, F364V, F364G, F364S, F364T or F364C; The mutation at position 379 includes any one of G379S, G379T or G379A.

4. The mutant according to claim 2 or 3, characterized in that It has: (1), the amino acid sequence shown in SEQ ID NO: 2; or (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

5. A nucleic acid molecule encoding a mutant according to any one of claims 2 to 4, characterized in that: It has: (4) the nucleotide sequence shown in SEQ ID NO: 3; or (5) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (4); or (6) a sequence having at least 80% homology with the nucleotide sequence described in (4) or (5); or (7) A complementary sequence of the nucleotide sequence described in (4), (5) or (6).

6. An expression vector, characterized in that include: The nucleic acid molecule according to claim 5.

7. A host, characterized in that Transformation and / or transfection with the expression vector according to claim 6.

8. A product, characterized in that include: The mutant according to any one of claims 2 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6 and / or the host according to claim 7.

9. Use of the mutant according to any one of claims 2 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host according to claim 7 and / or the product according to claim 8 in the preparation of fluorinated hydroxytryptophan.

10. A method for preparing fluorinated hydroxytryptophan, characterized in that: The fluorinated hydroxytryptophan is obtained by using fluorinated tryptophan as a substrate and reacting with the mutant according to any one of claims 2 to 4.

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