Ancfns-i-48 and mutants thereof and methods for apigenin production

Abstract

The application discloses an AncFNS-I-48 and a mutant thereof and a production method of apigenin, and belongs to the technical field of enzyme engineering. The technical problems to be solved are to provide an ancestral reconstruction enzyme AncFNS-I-48 and a mutant thereof, so as to solve the technical problems of low catalytic activity, poor storage stability and low soluble expression level of FNS-I enzymes in the prior art. The technical solution points are to provide an ancestral reconstruction enzyme AncFNS-I-48, the amino acid sequence of the AncFNS-I-48 is shown in SEQ ID NO: 15, and the nucleotide sequence is shown in SEQ ID NO: 28. Meanwhile, the AncFNS-I-48 mutant is also provided, and the AncFNS-I-48 mutant is selected from any one of H261F, L186F, A169K, H261A, N297D, Q71Y, I115V, M85L, M230D, V159E and T204P.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and involves the technical field of the intersection of enzyme engineering and artificial intelligence. Specifically, it relates to AncFNS-I-48 and its mutants and a method for producing apigenin. Background Technology

[0002] Apogenin (CAS: 520-36-5,4',5,7-trihydroxyflavone) is a flavonoid compound with anti-inflammatory, antioxidant, and neuroprotective activities. Currently, apigenin is mainly produced through plant extraction and chemical synthesis. Plant extraction is limited by factors such as plant growth cycles and environmental conditions, resulting in limited yields; chemical synthesis involves cumbersome procedures and environmental pollution. Therefore, there is a need to develop a green, economical, and sustainable method for apigenin production.

[0003] like Figure 1 As shown, the enzymatic synthesis, centered on the dehydrogenation of naringenin at the C2-C3 position to apigenin catalyzed by flavonoid synthase I (FNS I or FNS-I), is a green alternative. FNS-I belongs to the 2-ketoglutarate-dependent dioxygenase (2-ODD) superfamily, consisting of approximately 350 amino acids, and is mainly distributed in the Apiaceae family. The first FNS-I was identified in parsley, and it has a 2-ODD homology (2BRT) in the PDB (Protein Data Bank), with the active site composed of a conserved His-His-Asp Fe... 2+ Coordination pocket. The FNS-I subfamily has limited crystal structures specific to it, and reported studies have only addressed in vivo functional identification within plants; research on systematic in vitro engineering of FNS-I is still lacking.

[0004] Relevant patent documents retrieved: The publication, titled "Flavone Synthases and Their Applications," is from China, publication number CN110616204A, and publication date December 27, 2019. This document discloses flavonoid synthases DcFNS derived from carrots (Daucus carota) and flavonoid synthases AgFNS derived from celery (Apium graveolens), and their applications in catalyzing the conversion of naringenin to apigenin. The document also discloses derivatives with flavonoid synthase activity formed by substituting, deleting, or adding one or more amino acid residues into a specific amino acid sequence.

[0005] Relevant non-patent literature retrieved: The document, titled "Study on Metabolic Engineering of Yersinia lipolytica for Apigenin Synthesis," by Wang Yuhui, Hunan Agricultural University, and published on May 1, 2024, discloses a method for green synthesis of apigenin using Yersinia lipolytica as the chassis cell through metabolic engineering and fermentation optimization strategies. Using Yersinia lipolytica Po1f(Δku70) as the chassis cell, engineered strains were constructed by overexpressing FNS-encoding genes for flavonoid synthases from different sources. Furthermore, the yield of apigenin was increased by extending the fermentation time and optimizing the conditions for adding exogenous precursors.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The FNS-I disclosed in CN110616204A is derived from natural plants (carrots and celery) and is a wild-type enzyme; its catalytic activity and storage stability still need improvement. While this document mentions derivatives with amino acid substitutions, deletions, or additions, it does not provide any specific mutants or their activity data. Currently, there is a lack of technical solutions for modifying FNS-I to improve its catalytic activity and storage stability. Summary of the Invention

[0007] The purpose of this invention is to provide: AncFNS-I-48 and its mutants, apigenin production methods, and related technologies are proposed to address technical problems such as low catalytic activity of FNS-I enzymes, poor storage stability, and low soluble expression levels in existing technologies, or combinations thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] Definitions of the standard terminology can be found in the references “Molecular Cloning: A Laboratory Manual (Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, 2001),” “Microbiology Tutorial (4th Edition), Higher Education Press, author: Zhou Deqing, 2020,” “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng, and Guo Hongwei, 2019-06-19,” and “Genetic Engineering, Higher Education Press, 2013-08-01.”

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as gene cloning, site-directed mutagenesis, and high-performance liquid chromatography (HPLC) detection, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0014] The term "flavonoid synthase I (FNS I)" used in this article refers to: a soluble Fe... 2+ / α-ketoglutarate dioxygenase superfamily, enzymes that catalyze the dehydrogenation of flavanones (such as naringenin) at the C2-C3 position of the C ring to produce flavonoids (such as apigenin).

[0015] As used in this article, the term "mutant" refers to a protein variant obtained by replacing one or more amino acid residues in the amino acid sequence of a parental / wild-type protein through site-directed mutagenesis.

[0016] The term "flavonoid synthase activity" as used in this article refers to the enzyme activity that catalyzes the dehydrogenation of flavanone compounds (such as naringenin) at the C2-C3 positions to generate corresponding flavonoid compounds (such as apigenin).

[0017] The term "whole-cell catalyst" as used in this article refers to a catalyst that uses intact microbial cells obtained directly from fermentation culture as a catalytic reaction tool, without the need for cell disruption and enzyme extraction.

[0018] The term “ancestral sequence reconstruction” used in this article refers to the technique of inferring and reconstructing the sequence of extinct ancestral proteins based on phylogenetic analysis of existing homologous sequences using computational biology methods.

[0019] The term "site-directed mutagenesis" as used in this article refers to the targeted alteration of specific codons at the DNA level using molecular biology methods, thereby replacing specific amino acid residues at the protein level.

[0020] The term “AncFNS-I-48” as used in this article refers to flavonoid synthase I obtained through ancestral sequence reconstruction, whose amino acid sequence is shown in SEQ ID NO:15 and has a length of 354 amino acids.

[0021] The term "Naringenin" as used in this article refers to a flavanone compound that is a direct precursor in the biosynthesis of apigenin, with CAS number 480-41-1.

[0022] The term “Apigenin” as used in this article refers to a flavonoid compound with CAS number 520-36-5.

[0023] The term “2-ketoglutarate” (2-OG) used in this article refers to one of the cofactors required for the catalytic reaction of flavonoid synthase I.

[0024] In a first aspect, the present invention provides: an ancestral reconstruction enzyme AncFNS-I-48, the amino acid sequence of which is shown in SEQ ID NO:15.

[0025] Secondly, the present invention provides: an AncFNS-I-48 mutant, wherein the AncFNS-I-48 mutant is obtained by site-directed mutagenesis using the AncFNS-I-48 described in the first aspect as a parent, and the mutant is selected from any one of the following: (a) AncFNS-I-48-H261F: Histidine at position 261 is mutated to phenylalanine; (b) AncFNS-I-48-L186F: Leucine at position 186 is mutated to phenylalanine; (c) AncFNS-I-48-A169K: Alanine at position 169 is mutated to lysine; (d) AncFNS-I-48-H261A: Histidine at position 261 is mutated to alanine; (e) AncFNS-I-48-N297D: Asparagine at position 297 is mutated to aspartic acid. (f) AncFNS-I-48-Q71Y: Glutamine at position 71 is mutated to tyrosine; (g)AncFNS-I-48-I115V: Isoleucine at position 115 is mutated to valine; (h)AncFNS-I-48-M85L: Methionine at position 85 is mutated to leucine; (i) AncFNS-I-48-M230D: Methionine at position 230 is mutated to aspartic acid; (j) AncFNS-I-48-V159E: Valine at position 159 is mutated to glutamic acid; (k)AncFNS-I-48-T204P: Threonine at position 204 is mutated to proline.

[0026] Thirdly, the present invention provides: a nucleotide molecule encoding the AncFNS-I-48 described in the first aspect or the AncFNS-I-48 mutant described in the second aspect.

[0027] According to some embodiments of the present invention, the nucleotide molecule is a nucleic acid sequence optimized by E. coli codon preference, as shown in SEQ ID NO:28-39.

[0028] The nucleotide molecule is any one of the following: (a) The nucleotide sequence is shown in SEQ ID NO:28, encoding AncFNS-I-48 (SEQ ID NO:15); (b) The nucleotide sequence is shown in SEQ ID NO:29, encoding the AncFNS-I-48 mutant AncFNS-I-48-H261F (SEQ ID NO:17). (c) The nucleotide sequence is shown in SEQ ID NO:30, encoding the AncFNS-I-48 mutant AncFNS-I-48-L186F (SEQ ID NO:18). (d) The nucleotide sequence is shown in SEQ ID NO:31, encoding the AncFNS-I-48 mutant AncFNS-I-48-A169K (SEQ ID NO:19). (e) The nucleotide sequence is shown in SEQ ID NO:32, encoding the AncFNS-I-48 mutant AncFNS-I-48-H261A (SEQ ID NO:20). (f) The nucleotide sequence is shown in SEQ ID NO:33, encoding the AncFNS-I-48 mutant AncFNS-I-48-N297D (SEQ ID NO:21). (g) The nucleotide sequence is shown in SEQ ID NO:34, encoding the AncFNS-I-48 mutant AncFNS-I-48-Q71Y (SEQ ID NO:22); (h) The nucleotide sequence is shown in SEQ ID NO:35, encoding the AncFNS-I-48 mutant AncFNS-I-48-I115V (SEQ ID NO:23). (i) The nucleotide sequence is shown in SEQ ID NO:36, encoding the AncFNS-I-48 mutant AncFNS-I-48-M85L (SEQ ID NO:24); (j) The nucleotide sequence is shown in SEQ ID NO:37, encoding the AncFNS-I-48 mutant AncFNS-I-48-M230D (SEQ ID NO:25); (k) The nucleotide sequence is shown in SEQ ID NO:38, encoding the AncFNS-I-48 mutant AncFNS-I-48-V159E (SEQ ID NO:26). (l) The nucleotide sequence is shown in SEQ ID NO:39, encoding the AncFNS-I-48 mutant AncFNS-I-48-T204P (SEQ ID NO:27).

[0029] Fourthly, the present invention provides a recombinant expression vector comprising the nucleotide molecules described in the third aspect.

[0030] The preferred backbone vector for the recombinant expression vector is pET-28a. It is understood that the backbone vector includes, but is not limited to, the aforementioned vector backbone.

[0031] The recombinant expression vector further includes at least one of a tag, an resistance selection gene, and a promoter operatively linked to the nucleotide molecule.

[0032] Fifthly, the present invention provides: a recombinant host cell, said recombinant host cell comprising the nucleotide molecule described in the third aspect or the recombinant expression vector described in the fourth aspect.

[0033] According to some embodiments of the present invention, the recombinant host cell may be Escherichia coli.

[0034] Furthermore, the recombinant host cell is preferably BL21(DE3).

[0035] Sixthly, the present invention provides: a method for producing apigenin, the method comprising: Using naringenin as a substrate, apigenin was obtained by catalytic reaction using AncFNS-I-48 as described in the first aspect, AncFNS-I-48 mutant as described in the second aspect, or recombinant host cells as described in the fifth aspect.

[0036] That is, the catalytic reaction can be catalyzed by whole cells or by using crude enzyme solution.

[0037] According to some embodiments of the present invention, the catalytic reaction is carried out in a reaction system containing NaHCO3 buffer, 2-ketoglutaric acid and DMSO.

[0038] Furthermore, in the reaction system, the concentration of the substrate naringenin is 2 g / L-5 g / L. Preferably, the concentration of the substrate naringenin is 3 g / L.

[0039] Furthermore, in the reaction system, the molar equivalent of the 2-ketoglutaric acid is 1 to 4 times that of the substrate naringenin, preferably 2 times.

[0040] Furthermore, the molar equivalent of the L-ascorbic acid sodium is 1 to 2 times that of the substrate naringenin, preferably 2 times.

[0041] Further, the concentration of FeSO4 is 100~500μM, preferably 250μM.

[0042] Furthermore, the pH of the NaHCO3 buffer solution is 7.5-8.5, preferably 8.0.

[0043] According to some embodiments of the present invention, the catalytic temperature is 20°C-30°C, and the catalytic time is 3h-10h. Exemplarily, the catalytic temperature can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or any value within the range of any two of the above values, preferably 25°C. Exemplarily, the catalytic time can be 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, or any value within the range of any two of the above values, preferably 6h.

[0044] In a seventh aspect, the present invention provides the use of the AncFNS-I-48 mutant described in the first aspect or the AncFNS-I-48 mutant described in the second aspect in the preparation of apigenin.

[0045] Eighthly, the present invention provides: a method for mining flavonoid synthases, comprising the following steps: (1) Starting with the known PcFNS-I sequence, use AlphaFold3 to perform three-dimensional structural modeling; (2) Use Foldseek to perform structural similarity search; (3) Perform ancestral sequence recovery to obtain ancestral sequence reconstruction enzyme; (4) Enzyme activity prediction, scoring, and ranking were performed using the CataPro deep learning model; (5) Heterologous expression and activity screening of candidate enzymes.

[0046] The AncFNS-I-48 mentioned in the first aspect was obtained using this flavonoid synthase mining method.

[0047] Ninthly, a method for screening flavonoid synthase mutants includes the following steps: (1) Using AncFNS-I-48 as the parent, a full-site virtual mutation scan was performed based on the AlphaFold3 structural model; (2) Mutation sites were filtered using PSSM co-evolutionary analysis and Pythia thermal stability assessment; (3) Input the candidate mutation sites into the CataPro deep learning model for catalytic activity prediction, scoring and ranking; (4) Construct mutant libraries and perform high-throughput primary screening and shake-flask secondary screening.

[0048] The AncFNS-I-48 mutant described in the second aspect was obtained using the screening method for this flavonoid synthase mutant.

[0049] Examples 1-2 of this invention at least support the protection scope of "AncFNS-I-48 and its mutants".

[0050] "AncFNS-I-48 and its mutants" are summarized from the AncFNS-I-48, AncFNS-I-48-V159E, AncFNS-I-48-I115V, AncFNS-I-48-N230D, AncFNS-I-48-N297D, AncFNS-I-48-L186F, AncFNS-I-48-H261F, AncFNS-I-48-T204P, and AncFNS-I-48-A169K mentioned above and / or in Examples 1-2. Therefore, those skilled in the art can reasonably infer that "AncFNS-I-48 and its mutants," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "AncFNS-I-48 and its mutants."

[0051] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention provides AncFNS-I-48 and its mutants, as well as a method for producing apigenin, which has better technical effects, specifically in the following aspects: 1. Compared with the prior art, the present invention significantly improves the soluble expression level of FNS-I in Escherichia coli through ancestor sequence reconstruction technology.

[0052] 2. This invention provides an ancestral sequence reconstruction enzyme, AncFNS-I-48, derived from *Pheretima aspergillum*, and its mutants. AncFNS-I-48 and its mutants exhibit higher catalytic activity. Experimental tests show that the AncFNS-I-48-V159E mutant of this invention achieves a apigenin yield of 1.59 g / L in a 20 mL reaction system over 2 hours, which is 2.0 times that of wild-type AncFNS-I-48 (0.72 g / L).

[0053] 3. This invention provides a systematic FNS-I enzyme mining and modification scheme through an AI-assisted enzyme mining and mutation design method (AlphaFold3 + Foldseek + ancestor sequence reconstruction + CataPro), which has important methodological value. Attached Figure Description

[0054] Figure 1 This is a roadmap for the use of FNS-I-based flavonoid synthases in apigenin synthesis.

[0055] Figure 2 Gel imaging images of AcFNS-I and AncFNS-I-48 proteins, from left to right: whole cells (W) of AcFNS-I, supernatant (S) of AcFNS-I, precipitate (P) of AcFNS-I, whole cells (W) of AncFNS-I-48, supernatant (S) of AncFNS-I-48, precipitate (P) of AncFNS-I-48, and marker.

[0056] Figure 3 The relative catalytic activity of the AncFNS-I-48 mutant is given. Detailed Implementation

[0057] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0059] All data processing in this invention was performed using OriginPro 2024 software. Analysis between different groups was conducted using independent samples t-tests or one-way ANOVA tests. P <0.05 indicates that the difference is statistically significant.

[0060] Example 1: Systematic mining of FNS-I enzymes and acquisition of ancestral sequence reconstruction enzyme AncFNS-I-48 Starting with parsley PcFNS-I (UniProt: Q7XZQ8), FNS-I homologs were systematically mined according to the following process: (1) AlphaFold3 structural modeling: FNS-I three-dimensional structure prediction based on 2BRT homologous templates. His232, His288, and Asp234 constitute Fe 2+ Coordination triangle, with Lys213 as the key proton acceptor; (2) Foldseek structural similarity search: Search for structurally similar sequences in the afdb50, afdb-swissprot, and afdb-proteome databases; (3) Ancestor Sequence Recovery and CataPro Activity Prediction: In the prediction process, the target protein sequence is first used as the query. Foldseek is used to search for homologous templates with E-value ≤ 1e-5 and coverage ≥ 70% in the structural database. The top 20 templates in TM-score are selected for ancestor sequence reconstruction. The maximum likelihood method (such as PAML) is used to calculate multiple ancestral candidate sequences with a posterior probability ≥ 0.70, and these are combined with the natural sequence to form a candidate set. Subsequently, all candidate sequences are input into the CataPro deep learning model, and each sequence is run 5 times for independent prediction to obtain the mean and standard deviation of the probability of catalytic activity (PCA). The scoring system comprehensively considers four dimensions: PCA mean (weight 0.6), conservation of active site residues (BLOSUM62 similarity based on Foldseek template annotation, weight 0.2), structural dynamic stability (normalized fluctuation index output by CataPro, weight 0.1), and the posterior probability of the ancestral sequence (natural sequence is counted as 1.0, weight 0.1). The overall score is calculated using the weighted summation method described above, and an elimination rule is set: sequences with a PCA score below 0.50 or a conservatism score below 0.60 are directly eliminated. Finally, sequences are sorted in descending order of their overall scores; if the score difference is ≤0.05, sequences with smaller standard deviations are prioritized to ensure robustness.

[0061] (4) Heterologous expression and initial screening of candidate enzymes: The genes of the top 16 candidate enzymes were synthesized (see Table 1 for specific sequences and information), cloned into pET-28a (N-terminal His tag, Wuhan Miaoling Biotechnology Co., Ltd., P0023), and the plasmid was transformed into Escherichia coli BL21(DE3) competent cells (recombinant host cells). Single clones (positive transformants) were picked and inoculated into 50 mL LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37℃ until OD. 600 =0.6-0.8, add IPTG to a final concentration of 0.5mM, cool to 18℃ and induce for 16 hours. Centrifuge at 4000rpm for 10min to harvest bacterial sludge. Add 10mL PBS, set the ultrasonic disruptor parameters to 40% power, 3s on, 2s off, disrupt for 10min to obtain crude enzyme solution.

[0062] Screening conditions: 10 mM naringenin substrate, 50 mM NaHCO3 (pH 8.0), 20 mM 2-ketoglutaric acid, 20 mM ascorbic acid, 250 μM FeSO4, 20% crude enzyme solution, reaction at 30℃ and 200 rpm for 2 h. HPLC quantification (HPLC conditions: C18 reversed-phase column (250×4.6 mm, 5 μm), detection wavelength 290 nm, mobile phase gradient elution of methanol-0.1% formic acid aqueous solution (0 min: 20% methanol → 20 min: 60% methanol → 25 min: 20% methanol), flow rate 1.0 mL / min, column temperature 30℃, injection volume 10 μL. A standard curve was established using apigenin standard (purity ≥98%) with external standard method for quantification, r = 0.9989).

[0063] Please refer to Table 1 again for the screening results: a total of 16 candidate enzymes were obtained. Among them, AcFNS-I (0.82 g / L), AncFNS-I-50 (0.99 g / L), and AncFNS-I-48 (1.21 g / L) showed the best activity. The amino acid sequence of AncFNS-I-48 is shown in SEQ ID NO:15, and its length is 354 amino acids.

[0064] Table 1 Sequence identifiers and product accumulation of FNS-I candidate enzymes

[0065] Among them, SEQ ID NO:1: AaFNS-I (360 aa): MAPPTTITALAQEKTLNLAFVRDEDERPKVAYNQFSNEIPIISLAGMDDDTGRRPQICRKIVEAFEDWGIFQVVDHGIDGTLISEMTRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVKDWREFVTYFSYPIDDRDYSRWPDKPQGWRSTTEVYSEKLMVLGAKLLEVLSEAMG LEKEALTKACVNMEQKVLINYYPTCPPEPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLRIREGEKAVLDEAITYAEMYKKNMTKHIEVATLKKLAKEKRLQEEKAKLETESKSAD.

[0066] SEQ ID NO:2: AcFNS-I (355 aa): MAPTTITALSQEKSLNLDFVRDEDERPKVAYNQFSNEIPIISLAGMDDDSNGRRPEICRKIVEAFEDWGIFQVVDHGIDKGLISQMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVKDWREFVTYFSYPIEDRDYSRWPEKPEGWRSTTEVYSEKLMVLGAKLLEVLSE AMGLEKEALTKACVNMEQKVLINYYPTCPEPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVAALKKLAKEKRLQDEKAKLEM。

[0067] SEQ ID NO:3:AgFNS-I(356 aa): MAPTTITALSQEKSLNLDFVRDEDERPKVAYNQFSNEIPIISLAGMDDDSNGRRPEICRKIVEAFEDWGIFQVVDHGIDKGLISQMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVKDWREFVTYFSYPIEDRDYSRWPEKPEGWRSTTEVYSEKLMVLGAKLLEVLSE AMGLEKEALTKACVNMEQKVLINYYPTCPEPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVAALKKLAKEKRLQDEKAKLEM。

[0068] SEQ ID NO:4:PcFNS-I(358 aa): MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDSDGRRPEICRKIVKACEDWGIFQVVDHGIDSGLISEMTRLSREFFALPAEEKLEYDTTGGKRGGFTISTVLQGDDAMDWREFVTYFSYPARDYSRWPKKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLE KGDLTKACVDMEQKVLINYYPTCPQPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFRNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKCMTKHIEVATRKKLAKEKRLQDEKAKLEMKSKSADENLA。

[0069] SEQ ID NO:5:CuFNS-I(365 aa): MAPTTITALAQEKTLNSDFVRDEDERKPVAYNQFSTEIPIISLAGIDDDSKGRRPEVCRKIVEAFEDWGIFQVVDHGVDSALISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHQQGDDVRDWREFVTYFSYPVDARDYSRWPEKPEGWRSVTEVYSEKLMVLGAKLLEVLSEAMGLD KGALTKACVNMEQKVLINYYPTCPEPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGVFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKPILEEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQEEKAKLETKTKSADGILA。

[0070] SEQ ID NO:6:CmFNS-I(356 aa): MAPTTITALSQEKSLNLDFVRDEDERPKVAYNQFSNEIPIISLAGMDDDSNGRRPEICRKIVEAFEDWGIFQVVDHGIDKGLISQMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVKDWREFVTYFSYPIEDDYSRWPEKPEKSEGSEWLVLVLEK AMGLEKEALTKACVNMEQKVLINYYPTCPEPDLTLGVRRHTDPGTILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHYLS NGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVAALKKLAKEKRLQDEKAKLEM。

[0071] SEQ ID NO:7:DcFNS-I(356 aa): MAPTTITALSQEKSLNLDFVRDEDERPKVAYNQFSNEIPIISLAGMDDDSNGRRPEICRKIVEAFEDWGIFQVVDHGIDKGLISQMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVKDWREFVTYFSYPIEDDYSRWPEKPEKSEGSEWLVLVLEK AMGLEKEALTKACVNMEQKVLINYYPTCPEPDLTLGVRRHTDPGTILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHYLS NGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVAALKKLAKEKRLQDEKAKLEM。

[0072] SEQ ID NO:8:CuFNS-I-v2(360 aa: MAPTTITALAQEKTLNSDFVRDEDERPKVAYNQFSTEIPIISLAGIDSKGRRPEVCRKIVEAFEDWGIFQVVDHGVDSALISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTQQGDDVRDWREFVTYFSYPVDARDYSRWPEKPEGSEWRSVLSKEVLVELVEVAMKLL GLDKGALTKACVNMEQKVLINYYPTCPEPDLTLGVRRHTDPPGTILLQDMVGGLQATRDGGKTWITVQPVEGVFVVNLGDHGHYLSNGR FKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKPILEEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQEEKAKLETKTKSA。

[0073] SEQ ID NO:9:DcFNS-I-v2(361 aa: MAPITISALSKETTLNPDFVRDEDERPKVAYNQFSNEIPVISLAGIDDDSVDTRSEVCRKIVEAFEDWGIFQAVDHGIDTTLISEMYRLSREFFALPAEEKLYDTTGGKRGGFTVSTHLKGDSAMDWREFVTYFTYPISDRDYSRWPDEKPEGWREFVTYFTYGYKLLG LEKEAIAKACVDMEQKVLINYYPTCPQPDLTGVRRHTDPGAITLLLQDQVGGLQATRDGGKTWITVKPVEGALVVNLGDYGHYLSNGRF KNADHQAVVNSTYTRMSIATFQYPSPDAIVYPLKIREGEKPIVDEAITFAEMYRRIMSKPGGEEAALKKVAKEKHLQEEKAKLEMKPKTAA。

[0074] SEQ ID NO:10:AtFNS-I(358 aa: MAPGTLTELAGESKLNSKFVRDEDERPKVAYNVFSDEIPVISLAGIDDVDGKRGEICRQIVEACENWGIFQVVDHGVDTNLVADMTRLARDFFALPPEDKLRFDMSGGKKGGFIVSSHLQGEAVQDWREIVTYFSYPVRNRDYSRWPDKPEGWVKVTEEYSERLMSLACKLLEVLSEAM GLEKESLTNACVDMDQKIVVNYYPKCPQPDLTLGLKRHTDPGTITLLLLQDQVGGLQATRDNGKTWITVQPVEGAFVVNLGDHGHFLSNGRFKNADHQAVVNSNSSRLSIATFQNPAPDATVYPLKVREGEKAILEEPITFAEMYKRKGRDLELARLKKLAKEERDHKEVDKPVDQIFA。

[0075] SEQ ID NO:11:CcFNS-I(356 aa): MAAPISLKWEEHSLHENKFVRDEDERPKVPYNTFSNEIPVISLAGIDGCRRAECDEIVKACEDWGIFQVVDHGVDTKLSDMTGLARDFFHLPTQEKLRFDMTGGKKGGFIVSSHLQGEAVQDWREIVTYFSYPIKARDYSRWPDKPNEWRAVTEEYSKVLMGLACKLLEVLSEAMG LEKEALTKACVDMDQKVVVNYYPKCPQPDLTLGLKRHTDPGTTITLLLQDQVGGLQATRDGGESWITVKPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSSTSRLSIATFQNPAPEAIVYPLKINEGEKSIMEEPMTFMEMYKKKMSDTLELARLKLAKDKQQDLEVKPIQNIFA。

[0076] SEQ ID NO:12:DcFNS-I-v3(357 aa): MAPSTITALSKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDSNGRRPEVCRKIVKACEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLVYDTTGGKKGGFTISTHLQGDDVRDWREFVVYFSYPVSARDYSRWPKKPEGDVRDWREFVVYFSYPVSARDYSRWPKKPEGDVRDWREFVVYFSYPVSARDYSRWPKKPEGWRSKEVLSEVLVLTVLVLT MGLEKEALTEACVEMEQKVLINYYPTCPQPDLTGVRRHTDPGTILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHYLSN GRFRNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATRKKLAKEKRLQNEKAKLEMKS。

[0077] SEQ ID NO:13:AgFNS-I-v2(355 aa: MAPVTITALTNEKTLNLDFVRDEDERPKVAYNQFSTEIPIISLAGVDDDSNGRRGEICKIVEAFEEWGIFQVVDHGIDTGLISEMARLSREFFALPAEEKLVYDTTGGRKGGFTISTHLQGDDVRDWREFATYFSYPISARDYSRWPKKPEGWRSEVLSEKEVLSEVLGAKLL AMGLEKEALSKACVNMEQKVLINYYPTCPEPDLTLGVRRHTDPGTILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHYLSNGRFRNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAMTYAEMYKNMTKHIEVKKKKKKKK

[0078] SEQ ID NO:14:CmFNS-I-v2(355 aa: MAPTTITALSQEKSLNLDFVRDEDERPKVAYNQFSNEIPIISLAGMDDDSNGRRPEICRKIVEAFEDWGIFQVVDHGIDKGLISQMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVKDWREFVTYFSYPIEDRDYSRWPEKPEGWRSTTEVYSEKLMVLGAKLLEVLSE AMGLEKEALTKACVNMEQKVLINYYPTCPEPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVAALKKLAKEKRLQDEKAKLEM。

[0079] SEQ ID 15:AncFNS-I-48(354 aa) MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSE AMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM。

[0080] SEQ ID NO:16:AncFNS-I-50(354 aa): MAPTTITALSKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPISARDYSRWPKKPEGWRSTTEVYSEKLMVLGAKLLEVLSE AMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0081] AcFNS-I exhibits good activity but low solubility in recombinant expression. To improve the solubility of the target protein, an ancestral sequence reconstruction strategy was introduced. Expression results showed that, compared with AcFNS-I, the proportion of soluble expression of AncFNS-I in the BL21(DE3) host was significantly increased. This is presumably because the introduction of ancestral residues enhances the protein's folding stability and conformational homogeneity, reducing inclusion body formation. Figure 2 Gel imaging images of AcFNS-I, AncFNS-I-48, and AncFNS-I-50 show that ancestral sequence remodeling enhances the solubility of AcFNS-I. W represents whole cells, S represents supernatant, and P represents precipitate.

[0082] Example 2 Construction and activity determination of AncFNS-I-48 mutant 2.1 Using AncFNS-I-48 as the parent, a mutation design was performed based on the AlphaFold3 structural model: (1) Virtual mutation scanning of all sites: Virtual mutation scanning of all amino acid sites was performed using PSSM co-evolutionary analysis and Pythia thermostability assessment; (2) Site filtering: Excluding the core region of the enzyme catalytic pocket (Fe composed of His232 / His288 / Asp234) 2+ (Coordination triangle) and residues that directly interact with iron ions; (3) CataPro activity prediction score: The candidate mutation sites were input into the CataPro deep learning model for catalytic activity prediction, scoring and ranking, consistent with the scoring criteria in Example 1. The top 11 mutants with the highest scores were then used for enzyme activity verification.

[0083] 2.2 Validation of mutant enzyme activity: The AncFNS-I-48 mutants designed by mutation are shown in Table 2 (H261F, L186F, A169K, H261A, N297D, Q71Y, I115V, M85L, M230D, V159E, T204P). By designing a pair of forward and reverse complementary primers containing the target mutant bases, and using the wild-type pET-28a target gene recombinant plasmid as a template, the point mutation was introduced by full plasmid PCR amplification with high-fidelity PCR polymerase PrimeSTAR® Max DNAPolymerase Ver.2 (TaKaRa, R047A). The amplified product was digested with DpnI enzyme (TaKaRa, 1235A) to remove the methylated original template and then directly transformed into DH5α competent cells (Shanghai Weidi Biotechnology Co., Ltd., DL1001M). The cells were plated on kanamycin plates for screening, and single clones were picked and sequenced for verification, thus obtaining the point mutant recombinant plasmid with the correct sequence. The plasmid was transformed into E. coli BL21(DE3) competent cells (Shanghai Weidi Biotechnology Co., Ltd., EC1002M). Single colonies (positive transformants) were picked and inoculated into 50 mL LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37°C until OD600. 600 =0.8, add IPTG to a final concentration of 0.5 mM, cool to 18℃ and induce for 16 hours. Centrifuge at 4000 rpm for 10 min to harvest bacterial sludge. Add 10 mL PBS, set the ultrasonic disruptor parameters to 40% power, 3 s on, 2 s off, disrupt for 10 min to obtain crude enzyme solution.

[0084] Reaction system: 20 mL system, substrate naringenin 11 mM, crude enzyme solution 20%, NaHCO3 50 mM (pH 8.0), 2-ketoglutaric acid 20 mM, sodium ascorbate 20 mM, FeSO4 250 μM, reaction at 30℃ and 200 rpm for 2 h, quantified by HPLC (HPLC quantification method and conditions are the same as "HPLC quantification" in Example 1), results are as follows. Figure 3 As shown.

[0085] The results are shown in Table 2: AncFNS-I-48-V159E, AncFNS-I-48-I115V, AncFNS-I-48-M230D, AncFNS-I-48-N297D, AncFNS-I-48-L186F, and AncFNS-I-48-H261F all showed good conversion rates (39.68%-56.90%) and relative activities (1.27-2.01).

[0086] Relative activity: The conversion rate % of AncFNS-I-48 is normalized to 1.00, which is obtained by dividing the conversion rate of all mutants by the conversion rate of AncFNS-I-48.

[0087] Table 2 Activity data of AncFNS-I-48 mutants

[0088] Wherein, SEQ ID NO:17: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDFGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0089] SEQ ID NO:18: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLEKEAFTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0090] SEQ ID NO:19: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGKKLLEVLSEAMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0091] SEQ ID NO:20: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSRSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDAGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0092] SEQ ID NO:21: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSRSRFEFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQDAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0093] SEQ ID NO:22: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFYVVDHGIDSGLISEMSRLSRSRSEFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0094] SEQ ID NO:23: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSRSREFFALPAEEKLRYDTTGGKRGGFTVSTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0095] SEQ ID NO:24: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISELSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0096] SEQ ID NO:25: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSE AMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTDPGTITILLQDDVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM。

[0097] SEQ ID NO:26: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEEYSEKLMVLGAKLLEVLSE AMGLEKEALTKACVNMEQKVLINYYPTCPQPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM。

[0098] SEQ ID NO:27: MAPTTITALAKEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDNGRRPEICRKIVEAFEDWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLRYDTTGGKRGGFTISTHLQGDDVRDWREFVTYFSYPIDARDYSRWPDKPEGWRSTTEVYSEKLMVLGAKLLEVLSEAMGLEKEALTKACVNMEQKVLINYYPPCPQPDLTLGVRRHTPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSTSSRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIEVATQKKLAKEKRLQDEKAKLEM.

[0099] SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30 SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:34 SEQ ID NO:35 SEQ ID NO:36 SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39 Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An ancestral reconstructive enzyme AncFNS-I-48, characterized in that, The amino acid sequence of AncFNS-I-48 is shown in SEQ ID NO:

15.

2. An AncFNS-I-48 mutant, characterized in that, The AncFNS-I-48 mutant is a mutant obtained by site-directed mutagenesis using AncFNS-I-48 as the parent in claim 1, and the mutant is selected from any one of the following: (a) AncFNS-I-48-H261F: Histidine at position 261 is mutated to phenylalanine; (b) AncFNS-I-48-L186F: Leucine at position 186 is mutated to phenylalanine; (c) AncFNS-I-48-A169K: Alanine at position 169 is mutated to lysine; (d) AncFNS-I-48-H261A: Histidine at position 261 is mutated to alanine; (e) AncFNS-I-48-N297D: Asparagine at position 297 is mutated to aspartic acid. (f) AncFNS-I-48-Q71Y: Glutamine at position 71 is mutated to tyrosine; (g)AncFNS-I-48-I115V: Isoleucine at position 115 is mutated to valine; (h)AncFNS-I-48-M85L: Methionine at position 85 is mutated to leucine; (i) AncFNS-I-48-M230D: Methionine at position 230 is mutated to aspartic acid; (j) AncFNS-I-48-V159E: Valine at position 159 is mutated to glutamic acid; (k)AncFNS-I-48-T204P: Threonine at position 204 is mutated to proline.

3. The AncFNS-I-48 mutant according to claim 2, characterized in that, The AncFNS-I-48 mutant is AncFNS-I-48-V159E, and the amino acid sequence of AncFNS-I-48-V159E is shown in SEQ ID NO:

26.

4. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the AncFNS-I-48 of claim 1 or the AncFNS-I-48 mutant of claim 2.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleotide molecule of claim 4.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the nucleotide molecule of claim 4, or the recombinant expression vector of claim 5.

7. A method for producing apigenin, characterized in that, The production method includes: using naringenin as a substrate, and using AncFNS-I-48 as described in claim 1, the AncFNS-I-48 mutant as described in claim 2, or the recombinant host cell as described in claim 6 to carry out a catalytic reaction to obtain apigenin.

8. The production method according to claim 7, characterized in that, The catalytic reaction was carried out in a reaction system containing NaHCO3 buffer, 2-ketoglutaric acid and DMSO.

9. The use of the AncFNS-I-48 of claim 1 and / or the AncFNS-I-48 mutant of claim 2 in the preparation of apigenin.

10. A method for mining flavonoid synthase, characterized in that, Includes the following steps: (1) Starting with the known FNS-I sequence, use AlphaFold3 to perform three-dimensional structural modeling; (2) Use Foldseek to perform structural similarity search; (3) Perform ancestral sequence recovery to obtain ancestral sequence reconstruction enzyme; (4) Enzyme activity prediction, scoring, and ranking were performed using the CataPro deep learning model; (5) Heterologous expression and activity screening of candidate enzymes.

11. A method for screening flavonoid synthase mutants, characterized in that, Includes the following steps: (1) Using AncFNS-I-48 as the parent, a full-site virtual mutation scan was performed based on the AlphaFold3 structural model; (2) Mutation sites were filtered using PSSM co-evolutionary analysis and Pythia thermal stability assessment; (3) Input the candidate mutation sites into the CataPro deep learning model for catalytic activity prediction, scoring and ranking; (4) Construct mutant libraries and perform high-throughput primary screening and shake-flask secondary screening.

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