Fusion enzyme mutant and application thereof in fluororesveratrol synthesis

Abstract

The invention relates to the field of bioengineering, in particular to a fusion enzyme mutant and application thereof in fluororesveratrol synthesis. The invention provides a mutant composition. The mutant composition comprises a mutant of p-coumaroyl-CoA ligase and a mutant of stilbene synthase, the mutant of the p-coumaroyl-coenzyme A ligase is characterized in that on the basis of wild type p-coumaroyl-coenzyme A ligase, the mutant has mutation at the 113th site and / or the 537th site; the mutant of the stilbene synthase comprises mutation at the 170th site on the basis of wild type stilbene synthase. According to the invention, the fusion enzyme is subjected to directed evolution by applying a semi-rational CAST / ISM strategy and an irrational error-prone PCR strategy for the first time, a fusion enzyme mutant At4CL (F537G / F113G)-VvSTS (V170A) with improved catalytic activity is obtained, and finally, a series of fluorine-containing resveratrol is efficiently synthesized.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a fusion enzyme mutant and an application thereof in the synthesis of fluororesveratrol. Background Art

[0002] Resveratrol (3,5,4'-trihydroxy-trans-1,2-stilbene) is a member of the polyphenolic compounds and is found in various food and beverage production plants, such as grapes, peanuts, etc. Several physiological functions of resveratrol have been found to be beneficial to human health, including anti-cancer chemoprevention, cardiovascular disease, antioxidant and anti-inflammatory activities. The introduction of fluorine atoms into the above drugs often enhances metabolic stability and lipophilicity. However, the overall synthesis efficiency is low, which restricts industrial production. Summary of the invention

[0003] In view of this, the present invention provides a fusion enzyme mutant and its application in the synthesis of fluoro-resveratrol. The present invention provides a whole-cell multi-enzyme cascade pathway of fluoro-resveratrol, wherein p-coumaric acid is the main rate-limiting enzyme through coumaroyl-CoA ligase (4CL) and stilbene synthase (STS), the latter of which belongs to a type III polyketide synthase (PKSs), which sequentially condenses three units of malonyl-CoA with one molecule of p-coumaryl-CoA synthesized by 4CL, and finally cyclizes to form resveratrol. The two enzyme cascades can convert tyrosine into resveratrol. Due to the strong correlation and specificity of the two enzymes, the catalytic activity can be improved by combining them into a fusion enzyme At4CL::VvSTS with a Gly-Ser-Gly linker. For the first time, the fusion enzyme was directed evolved using a semi-rational CAST / ISM strategy and an irrational error-prone PCR strategy, and the fusion enzyme mutant At4CL(F537G / F113G)-VvSTS(V170A) with improved catalytic activity was obtained, and finally a series of fluorinated resveratrol were efficiently synthesized.

[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 a mutant composition, comprising: a mutant of p-coumaroyl-CoA ligase and a mutant of stilbene synthase;

[0006] The mutant of p-coumaroyl-CoA ligase includes: based on the wild-type p-coumaroyl-CoA ligase, having mutations at position 113 and / or position 537;

[0007] The mutant of the stilbene synthase includes: based on the wild-type stilbene synthase, a mutation at position 170 is present.

[0008] In some embodiments of the present invention, in the mutant composition, the mutation at position 113 in the mutant of p-coumaroyl-CoA ligase comprises: any one of F113A, F113G, F113I, F113P or F113M; and / or

[0009] The mutation at position 537 in the mutant of p-coumaryl-CoA ligase comprises any one of F537G, F537A, F537T, F537H, F537H or F537S; and / or

[0010] The mutation at position 170 in the mutant of the stilbene synthase includes any one of V170A, V170G, V170T, V170I or V170P.

[0011] In some embodiments of the present invention, in the mutant composition, the mutation at position 113 in the mutant of p-coumaroyl-CoA ligase is: F113G; and

[0012] The mutant of p-coumaroyl-CoA ligase has a mutation at position 537: F537G; and

[0013] The 170th mutation in the mutant of the stilbene synthase includes: V170A.

[0014] In some embodiments of the present invention, in the mutant composition, the mutant of p-coumaroyl-CoA ligase has:

[0015] (1), the amino acid sequence shown in SEQ ID NO: 1; or

[0016] (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

[0017] (3) an amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2);

[0018] The mutant of the stilbene synthase has:

[0019] (4) the amino acid sequence shown in SEQ ID NO: 2; or

[0020] (5) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (4), and having the same or similar function as the amino acid sequence shown in (4); or

[0021] (6) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5).

[0022] The present invention also provides a nucleic acid molecule encoding the mutant composition, wherein the nucleic acid molecule encoding the mutant of p-coumaroyl-CoA ligase has:

[0023] (7), the nucleotide sequence shown in SEQ ID NO: 3; or

[0024] (8) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence described in (7); or

[0025] (9) a sequence having at least 80% homology to the nucleotide sequence described in (7) or (8); or

[0026] (10) A complementary sequence of the nucleotide sequence described in (7), (8) or (9);

[0027] The nucleic acid molecule encoding the mutant of the stilbene synthase has:

[0028] (11), the nucleotide sequence shown in SEQ ID NO: 4; or

[0029] (12) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence described in (11); or

[0030] (13) a sequence having at least 80% homology with the nucleotide sequence described in (11) or (12); or

[0031] (14) A complementary sequence of the nucleotide sequence described in (11), (12) or (13).

[0032] The present invention also provides a fusion enzyme, comprising: the mutant composition and a linker; the linker comprises: any one of Gly-Ser-Gly, Ser-Ser-Gly and Gly-Gly-Gly.

[0033] In some embodiments of the present invention, in the above fusion enzyme, the linker is: Gly-Ser-Gly.

[0034] In some embodiments of the invention, it has:

[0035] (15), the amino acid sequence shown in SEQ ID NO: 5; or

[0036] (16) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (15), and having the same or similar function as the amino acid sequence shown in (15); or

[0037] (17) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (15) or (16).

[0038] In some embodiments of the present invention, the above fusion enzyme further comprises: tyrosine ammonia lyase derived from Rhodobactersphaeroides.

[0039] The present invention also provides a nucleic acid molecule encoding the above fusion enzyme, which has:

[0040] (18), the nucleotide sequence shown in SEQ ID NO: 6; or

[0041] (19) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence described in (18); or

[0042] (20) a sequence having at least 80% homology to the nucleotide sequence described in (18) or (19); or

[0043] (21) A complementary sequence of the nucleotide sequence described in (18), (19) or (20).

[0044] The present invention also provides a host for transforming and / or transfecting the above nucleic acid molecule.

[0045] The present invention also provides the use of the mutant composition, the nucleic acid molecule, the fusion enzyme, the nucleic acid molecule and / or the host in the preparation of fluorine-containing resveratrol.

[0046] In some embodiments of the present invention, in the above application, the fluorine-containing resveratrol includes: one or more of 2-fluoro-resveratrol, 3-fluoro-resveratrol, 3,5-2-fluoro-resveratrol and 2,3-2-fluoro-resveratrol.

[0047] The present invention also provides a method for preparing fluorine-containing resveratrol, which uses fluorine-containing tyrosine as a substrate and undergoes a catalytic reaction using the above fusion enzyme to obtain the fluorine-containing resveratrol.

[0048] In some embodiments of the present invention, in the above preparation method, the final concentration of the fluorotyrosine is 10 mM.

[0049] In some embodiments of the present invention, in the above preparation method, the conditions of the catalytic reaction are: 30° C., 220 rpm, and shaking reaction for 1 to 2 days.

[0050] In some embodiments of the present invention, in the above preparation method, the catalytic reaction uses 50 mM, pH 7.4 PBS Buffer.

[0051] In some embodiments of the present invention, in the above-mentioned preparation method, the preparation method of the fluorinated tyrosine comprises the following steps: using fluorinated phenol as a substrate, and subjecting the reaction to catalysis by tyrosine phenol lyase to obtain the fluorinated tyrosine.

[0052] In some embodiments of the present invention, in the above preparation method, the fluorinated phenol includes: one or more of 2-fluorophenol, 3-fluorophenol, 2,3-2-fluorophenol and 3,5-2-fluorophenol.

[0053] In some embodiments of the present invention, in the above preparation method, the conditions of the catalytic reaction are: 30° C., static reaction for 2 days.

[0054] In some embodiments of the present invention, in the above preparation method, the catalytic reaction uses: 50mM, pH8.0 PBS Buffer.

[0055] The present invention is the first to use the semi-rational CAST / ISM strategy and the irrational error-prone PCR strategy to carry out directed evolution of the fusion enzyme, and obtain the fusion enzyme mutant At4CL (F537G / F113G)-VvSTS (V170A) with improved catalytic activity, and finally efficiently synthesize a series of fluorinated resveratrol. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] 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.

[0057] Figure 1 Schematic diagram of the multi-enzyme cascade pathway of fluorinated resveratrol;

[0058] Figure 2 The performance characterization of the fluorinated resveratrol biosensor is shown; wherein: A shows a diagram of the mechanism of action of the fluorinated resveratrol biosensor; B shows a full wavelength scan of the excitation light and the emission light; C shows a control experiment of the transcription factor TtgRmut3 and fluorinated resveratrol; D shows the linear relationship between the concentration of fluorinated resveratrol and fluorescence; E shows the detection of the fluorescence background noise of the reaction system; F shows the fitting test of the actual whole-cell reaction system; G shows the actual bacterial red protein fluorescence expression level of different concentrations of fluorinated resveratrol.

[0059] Figure 3 The key amino acid site selection of the fusion enzyme At4CL::VvSTS is shown;

[0060] Figure 4Schematic diagram of primer design for constructing a single-site saturation mutation library of At4CL::VvSTS using degenerate codons; 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;

[0061] Figure 5 The primer design for constructing a combinatorial mutation library with enhanced catalytic activity using the degenerate codon NDT is shown;

[0062] Figure 6 The synthesis of a series of fluorinated resveratrol was demonstrated;

[0063] Figure 7 The hydrogen, fluorine and carbon spectra of fluorinated resveratrol 5a are shown 1 H, 13 C and 19 F spectra of 5a; where A indicates 1 HNMR; B 19 F NMR; C 13 CNMR. DETAILED DESCRIPTION

[0064] The invention discloses a fusion enzyme mutant and application thereof in the synthesis of fluororesveratrol.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In Examples 1 to 5 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0071] The present invention will be further described below in conjunction with embodiments:

[0072] Example 1 Construction of recombinant plasmids and strains required for the experiment

[0073] The first step of the multi-enzyme cascade pathway of fluorinated resveratrol is fluorinated phenol as the initial substrate, and the model substrate is ortho-monofluorophenol. The corresponding series of fluorinated tyrosines are catalyzed by tyrosine phenol lyase (CfTPL) from Citrobacterfreundii, which has the highest reported activity so far. The second step is that tyrosine ammonia lyase (RsTAL) from Rhodobactersphaeroides can catalyze the synthesis of fluorinated tyrosine into the corresponding fluorinated p-coumaric acid. The third step is completed by two enzymes, p-coumaryl-CoA ligase (At4CL) from Arabidopsis thaliana and stilbene synthase (VvSTS) from grapes. This step must be carried out in cells, and finally the corresponding fluorinated resveratrol is synthesized from fluorinated p-coumaric acid. The catalytic activity of the fusion enzyme At4CL::VvSTS can be further improved by combining it with a Gly-Ser-Gly linker. Specific experimental operation process: The recombinant plasmid was constructed using Novozan's one-step cloning kit.

[0074] Table 1 Plasmids and strains used in the synthesis of fluorinated resveratrol via multienzyme cascade pathway.

[0075]

[0076] Example 2 Construction of a high-throughput screening method based on a fluorinated resveratrol biosensor

[0077] The bottleneck of irrational directed evolution lies in screening, and it is necessary to construct a high-throughput screening method for fluorinated resveratrol. Based on the specific recognition of resveratrol biosensor reported in the literature by Professor Tang Shuangyan, this experiment discovered for the first time that this biosensor is also suitable for fluorinated resveratrol. The well plate screening system was constructed, characterized and optimized for the biosensor. It can be seen from the performance characterization that the optimal excitation wavelength Ex is 553nm, the emission wavelength Em is 587nm, the sensitivity can reach the micromolar level, the substrate specificity is strong, the background noise is low and stable, the fitting degree is above 0.8, and the well plate screening effect is good (such as Figure 2 Specific operation process: Take 100 μL of the shaken bacterial suspension to be tested or the supernatant after centrifugation and place it in the fluorescent enzyme labeling plate, and perform fluorescence detection under the conditions of the best excitation light and emission light on the microplate reader, Ex: 553nm, Em: 587nm and gain 70, and take the average of three times.

[0078] Example 3 Construction and screening of a single-site saturation mutation library for the key rate-limiting fusion enzyme At4CL::VvSTS

[0079] According to the experimental results, the large accumulation of the intermediate p-coumaric acid indicates that the conversion rate of At4CL (PDB: 3TSY) is low, that is, At4CL:: VvSTS is the rate-limiting enzyme in this pathway. By constructing a high-throughput screening method that specifically recognizes fluorinated resveratrol biosensors, key amino acid sites in the catalytic pocket of the rate-limiting fusion enzyme At4CL:: VvSTS were selected. Nine sites S112, S212, I251, P256, F258, I260, G352, T358, and F537 were selected for the initial screening of At4CL, and three sites H161, C164, and V170 were selected for the initial screening of VvSTS for single-site saturation mutation library construction and screening (as shown in Table 2, Figure 3 and Figure 4 ).

[0080] Specific experimental operation process: The single-point saturation mutation library of At4CL::VvSTS 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 0.5 μL DpnI at 37°C for 3 hours. Each digested PCR mixture was then transformed into electrocompetent Escherichia coli BL21 (DE3). Residues S112, S212, I251, P256, F258, I260, G352, T358, and F537 were replaced with 19 other canonical amino acids by the Tang method and sequenced by scraper to identify the quality of the library construction.

[0081] Table 2 Primer design for constructing a single-site saturation mutation library using the fusion enzyme At4CL::VvSTS

[0082]

[0083]

[0084]

[0085] 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 glutamate (E), alanine (A), glutamine (Q), proline (P), lysine (K) and threonine (T). ATG refers to methionine (M). TGG refers to tryptophan (W). Example 4 Construction and screening of the key rate-limiting fusion enzyme At4CL::VvSTS through combinatorial mutation library and irrational mutation library

[0086] Mutants with increased activity were found at the amino acid sites S112 and F537 of At4CL and the amino acid site V170 of VvSTS. Subsequently, a combinatorial saturation mutation library of these three sites was constructed using the CAST / ISM strategy, and the M2:At4CL(F537G)-VvSTS(V170A) mutant showed the highest activity increase (e.g. Figure 5, Table 3 and Table 4). In addition, using M2 as a template, the fusion enzyme At4CL::VvSTS was directed evolution using error-prone PCR technology. Finally, the At4CL (F113G) site was iterated, among which M3:At4CL (F537G / F113G)-VvSTS (V170A) had the highest activity, and its k cat The / Km value increased by about 1.6 times compared with the wild type (as shown in Table 5 and Table 6). Specific experimental operation process: the same as Example 3. The amino acid sequences of all enzymes used in the present invention are shown in Table 7.

[0087] Table 3 Primer list for screening the activity of the fusion enzyme At4CL::VvSTS combinatorial library of substrate 2a

[0088]

[0089] Table 4 List of error-prone PCR primers for screening the fusion enzyme At4CL::VvSTS of substrate 2a

[0090]

[0091] Table 5 Activity screening results of the fusion enzyme At4CL::VvSTS combinatorial library of substrate 2a

[0092]

[0093] Table 6 Activity screening results of fusion enzyme At4CL::VvSTS epPCR library on substrate 2a

[0094]

[0095] Table 7 Amino acid sequences of all enzymes used and nucleic acid sequence of anti-fabD

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Note: The bold part indicates the linker.

[0103] Table 8 Conversion rate of mutants of fusion enzyme At4CL::VvSTS

[0104]

[0105]

[0106] Example 5 Whole-cell multi-enzyme cascade catalytic synthesis of a series of fluorinated resveratrol

[0107] Using fluorinated tyrosine 2F-Tyr (2a), 3F-Tyr (2b), 3,5-2F-Tyr (2c) and 2,3-2F-Tyr (2d) as substrates, the corresponding series of fluorinated resveratrol 5a, 5b, 5c, 5d were finally obtained through a three-step enzyme cascade, such as Figure 6 . Specific operation process: Induce expression of the BL21 (DE3) recombinant strain containing the recombinant plasmid pRSF-RsTAL_VvSTS::At4CL-anti fab. Shake flask screening process: Streak 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 (100μg / mL ampicillin, 50μg / mL kanamycin) at 37°C and 220rpm. Take 1mL of the overnight bacterial solution and place it in 45mL TB medium (pre-added with 2g / L F-tyrosine), and add 5mL PBS (pH7.4, 50mM) buffer, as well as 100μg / mL ampicillin and 50μg / mL kanamycin. Incubate at 37°C and 220rpm for about 2 to 3 hours until the OD 600 Reach 0.6-0.8. Add IPTG at a final concentration of 0.25 mM and incubate at 30°C and 220 rpm to express the enzyme protein and catalyze the reaction. Take samples for detection after 24 hours. HPLC liquid phase detection conditions: chromatographic column Agilent C18 SB-Aq column (4.6×250 mm; 5 μm), eluted with water (0.1% (v / v) formic acid) / acetonitrile (0.1% (v / v) formic acid) = 97:3 at a flow rate of 1 mL / min, ultraviolet absorption wavelength λ: 280 nm, detection time: t = 8.8 min. The NMR spectrum of the compound with the structure shown in Formula 5a is shown in Figure 7 , the data is as follows:

[0108] 1 H NMR(500MHz,DMSO-d6)δ7.46(t,J=8.7Hz,1H),7.06(d,J=16.4Hz,1H),6.88(d,J=16.3Hz,1H),6.61 (dd,J=8.6,2.5Hz,1H),6.51(dd,J=12.6,2.4Hz,1H),6.45(d,J=2.3Hz,2H),6.18(t,J=2.2Hz,1H). 19F NMR (470MHz, DMSO-d6) δ-119.04 (dd, J = 12.4, 8.8Hz). 13 C NMR (126MHz, DMSO-d6) δ161.1 (d, J = 247.5Hz), 158.4 (d, J = 11.8Hz), 158.3, 139.8, 127.9 (d, J = 4.8Hz), 127.5 (d, J = 5.6Hz), 120.2(d,J=3.3Hz),116.3(d,J=12.4Hz),111.5(d,J=2.8Hz),104.5,102.3(d,J=25.0Hz),101.6.HRMS(ESI)m / zcalcd.for C 14 H 11 FO 3 + ([M+H] + ):246.0723,found 246.0728.

[0109] 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. A mutant composition, characterized in that include: mutants of p-coumaroyl-CoA ligase and mutants of stilbene synthase; The mutant of p-coumaroyl-CoA ligase includes: based on the wild-type p-coumaroyl-CoA ligase, having mutations at position 113 and / or position 537; The mutant of the stilbene synthase includes: based on the wild-type stilbene synthase, a mutation at position 170 is present.

2. The mutant composition according to claim 1, characterized in that The mutation at position 113 in the mutant of p-coumaroyl-CoA ligase comprises any one of F113A, F113G, F113I, F113P or F113M; and / or The mutation at position 537 in the mutant of p-coumaryl-CoA ligase comprises any one of F537G, F537A, F537T, F537H, F537H or F537S; and / or The mutation at position 170 in the mutant of the stilbene synthase includes any one of V170A, V170G, V170T, V170I or V170P.

3. The mutant composition according to claim 1 or 2, characterized in that: The mutant of p-coumaroyl-CoA ligase has: (1), the amino acid sequence shown in SEQ ID NO: 1; 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); The mutant of the stilbene synthase has: (4) the amino acid sequence shown in SEQ ID NO: 2; or (5) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (4), and having the same or similar function as the amino acid sequence shown in (4); or (6) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5).

4. A nucleic acid molecule encoding a mutant composition according to any one of claims 1 to 3, characterized in that: The nucleic acid molecule encoding the mutant of p-coumaroyl-CoA ligase has: (7), the nucleotide sequence shown in SEQ ID NO: 3; or (8) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (7); or (9) a sequence having at least 80% homology to the nucleotide sequence described in (7) or (8); or (10) A complementary sequence of the nucleotide sequence described in (7), (8) or (9); The nucleic acid molecule encoding the mutant of the stilbene synthase has: (11), the nucleotide sequence shown in SEQ ID NO: 4; or (12) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence described in (11); or (13) a sequence having at least 80% homology with the nucleotide sequence described in (11) or (12); or (14) A complementary sequence of the nucleotide sequence described in (11), (12) or (13).

5. A fusion enzyme, characterized in that include: The mutant composition and linker according to any one of claims 1 to 3; the linker comprises any one of Gly-Ser-Gly, Ser-Ser-Gly and Gly-Gly-Gly.

6. The fusion enzyme according to claim 5, characterized in that It has: (15), the amino acid sequence shown in SEQ ID NO: 5; or (16) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (15), and having the same or similar function as the amino acid sequence shown in (15); or (17) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (15) or (16).

7. The fusion enzyme according to claim 5 or 6, characterized in that Also includes: Tyrosine ammonia lyase from Rhodobactersphaeroides.

8. A nucleic acid molecule encoding the fusion enzyme according to claim 5 or 6, characterized in that: It has: (18), the nucleotide sequence shown in SEQ ID NO: 6; or (19) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence described in (18); or (20) a sequence having at least 80% homology to the nucleotide sequence described in (18) or (19); or (21) A complementary sequence of the nucleotide sequence described in (18), (19) or (20).

9. Use of the mutant composition according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the fusion enzyme according to any one of claims 5 to 7 and / or the nucleic acid molecule according to claim 8 in the preparation of fluorine-containing resveratrol.

10. A method for preparing fluorinated resveratrol, characterized in that: The fluorinated resveratrol is obtained by using fluorinated tyrosine as a substrate and catalyzing the reaction using the fusion enzyme as claimed in claim 7.

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