Directed evolution systems and methods for biocatalysts, screened biocatalyst mutants, synthetic unnatural amino acids therefor, and applications thereof

Abstract

The application discloses a biological catalyst directional evolution system and method, a screened biological catalyst mutant, a synthesized unnatural amino acid and application. The directional evolution system comprises a first carrier coding an antibiotic resistance protein and an orthogonal tRNA, and a second carrier coding a biological catalyst mutant to be screened and an aminoacyl-tRNA synthetase mutant homologous to the orthogonal tRNA; the target biological catalyst mutant can catalyze a substrate to generate an unnatural amino acid; a termination codon is arranged in the coding sequence of the antibiotic resistance protein; when the unnatural amino acid is absent, the termination codon interrupts the expression of the antibiotic resistance protein, and the antibiotic activity is lost; when the unnatural amino acid is present, the antibiotic resistance protein is completely expressed, and the antibiotic activity is reserved. Based on this, the application further screens to obtain the biological catalyst mutant, and uses the same as a core biological catalyst to drive the biosynthesis of the unnatural amino acid, so that the preparation of the unnatural amino acid in a kilogram scale is realized.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and synthetic biology, specifically to a directed evolution system and method for biocatalysts, a biocatalyst mutant obtained through screening, a method for synthesizing non-natural amino acids using the biocatalyst mutant, the synthesized non-natural amino acids, and the applications of the non-natural amino acids. Background Technology

[0002] Biocatalysts, with their unique technological advantages such as high efficiency, stereoselectivity, mild reaction conditions, and environmental friendliness, have been widely applied in many cutting-edge fields, including the synthesis of pharmaceutical intermediates, the preparation of industrial raw materials, environmental remediation, and biomedicine, becoming a core technological support for the development of modern biotechnology and synthetic biology. Among them, tyrosine phenol lyase (TPL), as a key biocatalyst, can specifically catalyze the condensation reaction between phenolic compounds and pyruvate to generate the corresponding tyrosine derivatives, playing an irreplaceable role in the biosynthesis of non-natural amino acids.

[0003] Directed evolution is a core technology for modifying the function of biocatalysts. It simulates natural evolution by artificially introducing gene mutations (including random and site-directed mutations), constructing mutant libraries, and then using efficient screening strategies to obtain mutants with the target function. Since Frances Arnold proposed the concept of directed evolution in the 1990s, this technology has been widely used to improve and modify the function of various proteins. Frances Arnold, George Smith, and Gregory Winter were awarded the 2018 Nobel Prize in Chemistry for their pioneering work in directed evolution and related fields.

[0004] However, in directed evolution studies of biocatalysts, researchers often need to screen mutants one by one for activity. This screening-based evolutionary strategy has a significant drawback: low throughput. For a protein composed of 100 amino acids, the potential number of mutants is approximately 20. 100 In contrast, in screening-based directed evolution, only a few hundred to a few thousand mutants are typically tested. However, the target mutants with the best activity in the mutation space are often rare, and traditional screening methods struggle to efficiently capture low-abundance beneficial mutations from a large library, which severely limits the depth of exploration into the function of new enzymes.

[0005] To significantly increase coverage of the mutation space, a high-throughput selection strategy suitable for biocatalyst mutant libraries is needed, and this strategy should be designed as an in vivo evolution method. Such an evolutionary method does not require knowledge of the enzyme activity information of each mutant; instead, it sets uniform criteria to select from the library, retaining mutant genes that meet the requirements and removing those that do not. In this way, hundreds of millions of mutant libraries can be selected experimentally in a single run, thereby greatly increasing the screening throughput.

[0006] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a directed evolution system and method for biocatalysts, screened biocatalyst mutants and their synthetic non-natural amino acids and applications. It aims to solve the problems of the lack of high-throughput in vivo directed evolution systems that can be directly applied to biocatalysts, the low throughput and poor efficiency of traditional screening methods, the difficulty in efficiently capturing low-abundance beneficial mutations from a large mutant library, and the lack of biocatalysts for the biosynthesis of non-natural amino acids.

[0008] The technical solution of the present invention is as follows: Firstly, a directed evolution system for biocatalysts is provided, comprising: a first carrier and a second carrier; The first vector encodes an antibiotic resistance protein and orthogonal tRNA; The second vector encodes the biocatalyst mutant to be screened and the aminoacyl-tRNA synthetase mutant homologous to the orthogonal tRNA; in, When the biocatalyst mutant to be screened is the target biocatalyst mutant, it can catalyze the substrate to generate non-natural amino acids; The coding sequence of the antibiotic resistance protein contains a stop codon; When the non-natural amino acid is absent, the stop codon interrupts the expression of the antibiotic resistance protein, resulting in the loss of antibiotic activity. When the non-natural amino acid is present, the orthogonal tRNA and aminoacyl-tRNA synthetase mutant incorporates the non-natural amino acid into the stop codon site, thereby enabling the complete expression of the antibiotic resistance protein and preserving the antibiotic activity.

[0009] Specifically, this invention develops a directed evolution system for biocatalysts based on genetic code extension technology. Figure 1The core design of this system involves designing the enzymatic product of the biocatalyst to be a non-natural amino acid (UAA). An orthogonal aminoacyl-tRNA synthetase / tRNA system is used to associate the encoding of the UAA with the expression of antibiotic resistance proteins, thus achieving a precise coupling between enzyme catalytic activity and host cell survival. After the system is transferred into host cells, the host cells carry the gene encoding the antibiotic resistance protein, but this gene contains a stop codon (such as TAG), preventing its full-length expression. When certain biocatalyst mutants can catalyze the substrate to produce the required UAA (i.e., when the biocatalyst mutant to be screened is the target biocatalyst mutant), the UAA can be recognized by the aminoacyl-tRNA synthetase mutant and introduced into the stop codon site, allowing the antibiotic resistance protein encoding gene to be read and expressed in full length, thus enabling the host cell to survive. However, when other biocatalyst mutants cannot catalyze the substrate to produce the required UAA (i.e., when the biocatalyst mutant to be screened is not the target biocatalyst mutant), the lack of the required UAA prevents the full-length expression of the antibiotic resistance protein, leading to the death of the host cell under the influence of antibiotics.

[0010] In a preferred embodiment, the antibiotic resistance protein is selected from one or more of TEM-1 β-lactamase, aminoglycoside phosphotransferase, tetracycline efflux MFS transporter, and chloramphenicol acetyltransferase; The amino acid sequence of the TEM-1 β-lactamase before the introduction of the stop codon is shown in SEQ ID NO.1. After the introduction of the stop codon, the codons at positions K71 and / or A148 in SEQ ID NO.1 are replaced with the stop codon. The amino acid sequence of the aminoglycoside phosphotransferase before the introduction of the stop codon is shown in SEQ ID NO.2. After the introduction of the stop codon, the codons at positions N32 and / or F147 in SEQ ID NO.2 are replaced with the stop codon. The amino acid sequence of the tetracycline efflux MFS transporter before the introduction of the stop codon is shown in SEQ ID NO.3. After the introduction of the stop codon, the codons at positions R69 and / or F197 in SEQ ID NO.3 are replaced with the stop codon. The amino acid sequence of the chloramphenicol acetyltransferase before the introduction of the stop codon is shown in SEQ ID NO.4. After the introduction of the stop codon, the codons at positions D112 and / or Y127 in SEQ ID NO.4 are replaced with the stop codon.

[0011] In a further preferred embodiment, the antibiotic resistance protein is selected from TEM-1 β-lactamase and / or chloramphenicol acetyltransferase; The amino acid sequence of the TEM-1 β-lactamase before the introduction of the stop codon is shown in SEQ ID NO.1. After the introduction of the stop codon, the codon at position A148 in SEQ ID NO.1 is replaced with the stop codon. The amino acid sequence of the chloramphenicol acetyltransferase before the introduction of the stop codon is shown in SEQ ID NO.4. After the introduction of the stop codon, the codons at positions D112 and / or Y127 in SEQ ID NO.4 are replaced with the stop codon. Specifically, the present invention has found through testing that the incorporation of non-natural amino acids at position A148 of TEM-1 β-lactamase and positions D112 and / or Y127 of chloramphenicol acetyltransferase can successfully restore the growth of host cells in the presence of the corresponding antibiotics.

[0012] In a further preferred embodiment, the antibiotic resistance protein is chloramphenicol acetyltransferase, and the amino acid sequence of the chloramphenicol acetyltransferase before the introduction of the stop codon is shown in SEQ ID NO.4. After the introduction of the stop codon, the codons at positions D112 and / or Y127 in SEQ ID NO.4 are replaced with the stop codon.

[0013] Specifically, given that chloramphenicol acetyltransferase may include two stop codon substitution sites (i.e., non-natural amino acid incorporation sites), and one or two substitution sites can be selected as needed to achieve regulation of selection pressure, it is the preferred technical solution.

[0014] In a preferred embodiment, the stop codon is the TAG stop codon, but it is not limited thereto.

[0015] In a preferred embodiment, the biocatalyst mutant is a tyrosine phenol lyase (TPL) mutant; The aminoacyl-tRNA synthetase mutant is a chimeric pyrrolidone-lysyl-tRNA synthetase mutant (chPylRS), and the amino acid sequence of its parent (i.e., the amino acid sequence of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant before the introduction of the mutation site) is shown in SEQ ID NO.5. The substrate, non-natural amino acid, and chimeric pyrrolidyl-tRNA synthetase mutant are selected from one or more of the following combinations: (1) The substrate is 2-chlorophenol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, N311C, C313W, Y349F, V366T and W382T; (2) The substrate is 2-bromophenol, the non-natural amino acid is (S)-2-amino-3-(3-bromo-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, N311C, C313W, Y349F, V366T and W382T; (3) The substrate is 2-iodophenol, the non-natural amino acid is (S)-2-amino-3-(4-hydroxy-3-iodophenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, N311C, C313W, Y349F, V366T and W382S; (4) The substrate is 2-nitrophenol, the non-natural amino acid is (S)-2-amino-3-(4-hydroxy-3-nitrophenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are M265T, L270I, L274G, D278G, N311G, C313R and Y349F; (5) The substrate is 2,3-dihydro-1H-indene-4-ol, the non-natural amino acid is (S)-2-amino-3-(7-hydroxy-2,3-dihydro-1H-indene-4-yl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are I253V, F260L, L270I, Y271F, Y273F, L274G, Y294F, N311G, C313W, T329A, Y349F and W382T; (6) The substrate is 2-chloro-6-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-5-fluoro-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are I253V, L270I, Y271F, Y273F, L274G, Y294F, N311G, C313W, Y349F and W382T; (7) The substrate is 3-chloro-5-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(2-chloro-6-fluoro-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, E302V, N311C, C313W, Y349F, A365G, V366H and W382T; (8) The substrate is 3-bromo-5-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(2-bromo-6-fluoro-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, N311C, C313W, D344G, Y349F, A365G, V366H and W382T; (9) The substrate is 2-fluorophenyl-1,3-diol, the non-natural amino acid is (S)-2-amino-3-(3-fluoro-2,4-dihydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are F260S, L270I, L274G, E302V, N311C, C313W, Y349F, A365G, V366H and W382T; (10) The substrate is 2-chlorophenyl-1,3-diol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-2,4-dihydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, E302V, N311C, C313W, Y349F, A365G, V366H and W382T.

[0016] Specifically, this invention screened tyrosine lyase mutants and discovered the above-mentioned combination of substrate, non-natural amino acid, and chimeric pyrrolidone-lysyl-tRNA synthetase mutant. When the biocatalyst mutant to be screened (tyrosine lyase mutant) can catalyze the substrate to generate the non-natural amino acid, the chimeric pyrrolidone-lysyl-tRNA synthetase mutant can recognize and introduce the non-natural amino acid to the stop codon site, enabling the antibiotic resistance protein coding gene to be read through and expressed in full length, thus allowing the host cell to survive.

[0017] In a preferred embodiment, the nucleotide sequence of the orthogonal tRNA is shown in SEQ ID NO.10; and the nucleotide sequence of the antibiotic resistance protein is shown in SEQ ID NO.11.

[0018] In a preferred embodiment, the first vector and / or the second vector are plasmids, but not limited thereto.

[0019] Secondly, a method for directed evolution of biocatalysts is provided, including the following steps: The biocatalyst directed evolution system described in the first aspect is transformed into a host cell, a substrate and an antibiotic corresponding to the antibiotic resistance protein are added, and stress screening is performed. The surviving host cells contain a target biocatalyst mutant that can catalyze the substrate to generate non-natural amino acids.

[0020] Specifically, when the antibiotic resistance protein is TEM-1 β-lactamase, the corresponding antibiotic is ampicillin (Amp); when the antibiotic resistance protein is aminoglycoside phosphotransferase, the corresponding antibiotic is kanamycin (Kan); when the antibiotic resistance protein is tetracycline efflux MFS transporter, the corresponding antibiotic is tetracycline (Tet); and when the antibiotic resistance protein is chloramphenicol acetyltransferase, the corresponding antibiotic is chloramphenicol (Cm).

[0021] In a preferred embodiment, the concentration of the substrate is 200 μM-1000 μM, more preferably 400 μM-600 μM, and most preferably 500 μM, but is not limited thereto.

[0022] In a preferred embodiment, the concentration of the antibiotic is 10 μg / mL to 40 μg / mL, more preferably 10 μg / mL to 30 μg / mL, and most preferably 20 μg / mL, but is not limited thereto.

[0023] In a preferred embodiment, the host cell is selected from one or more of Escherichia coli, yeast, and mammalian cells, more preferably Escherichia coli, but not limited thereto.

[0024] The preferred technical solution, the directed evolution method for biocatalysts, specifically includes: Provides a library of biocatalyst mutants; A second vector was constructed using the aforementioned biocatalyst mutant library; The first and second vectors are co-transformed into host cells, enriched in a culture medium containing the substrate and an antibiotic corresponding to the antibiotic resistance protein, and subjected to stress screening. The surviving host cells contain a biocatalyst mutant whose enzymatic product is the non-natural amino acid.

[0025] A further preferred technical solution is that the method for constructing the biocatalyst mutant library includes: focused saturation mutation and random mutation.

[0026] Thirdly, a biocatalyst mutant is provided, obtained by screening using the biocatalyst directed evolution system described in the first aspect or the biocatalyst directed evolution method described in the second aspect.

[0027] In a preferred embodiment, the biocatalyst mutant is a tyrosine phenol lyase mutant, the parental amino acid sequence of which (i.e., the amino acid sequence of the tyrosine phenol lyase mutant before the introduction of the mutation site) is shown in SEQ ID NO.8, and the mutation site is selected from one of the following: (1) S12A and M379V; (2) S12V and M379V; (3) F36P; (4) R82E; (5) M379C; (6) M379P; (7) M379A; (8) M379V; (9) M379T; (10) F36C and M379C; (11) F36C and M379V; (12) F36T and M379C; (13) V283G, M288S and M379V; (14) M288S and M379V; (15) M288F and M379S; (16) F448C; (17) F36L and M379V; (18) V283T, M288G and M379S.

[0028] Fourthly, a method for synthesizing non-natural amino acids is provided, in which a biocatalyst mutant obtained by screening using the biocatalyst directed evolution system described in the first aspect or the biocatalyst directed evolution method described in the second aspect is used to catalyze the synthesis of substrates into non-natural amino acids in cells.

[0029] In a preferred embodiment, the biocatalyst mutant is a tyrosine phenol lyase mutant, and the amino acid sequence of its parent (i.e., the amino acid sequence of the tyrosine phenol lyase mutant before the introduction of the mutation site) is shown in SEQ ID NO.8. The substrate, non-natural amino acids, and tyrosine phenol lyase mutant are selected from one or more of the following combinations: (1) The substrate is 2-chlorophenol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) M288S and M379V, (b) M379C, (c) M379P; (2) The substrate is 2-bromophenol, the non-natural amino acid is (S)-2-amino-3-(3-bromo-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) F36C and M379C, (b) M379A, (c) F36C and M379V; (3) The substrate is 2-iodophenol, the non-natural amino acid is (S)-2-amino-3-(4-hydroxy-3-iodophenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) S12A and M379V, (b) F36P, (c) F36T and M379C; (4) The substrate is 2-nitrophenol, the non-natural amino acid is (S)-2-amino-3-(4-hydroxy-3-nitrophenyl)propionic acid, and the mutation sites of the tyrosine phenol lyase mutant are F36L and M379V; (5) The substrate is 2,3-dihydro-1H-indene-4-ol, the non-natural amino acid is (S)-2-amino-3-(7-hydroxy-2,3-dihydro-1H-indene-4-yl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is M379P; (6) The substrate is 3-chloro-5-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(2-chloro-6-fluoro-4-hydroxyphenyl)propionic acid, and the mutation sites of the tyrosine phenol lyase mutant are S12V and M379V; (7) The substrate is 2,6-difluorophenol, the non-natural amino acid is (S)-2-amino-3-(3,5-difluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) R82E, (b) M379C, (c) M379V; (8) The substrate is 2-chloro-6-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-5-fluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) S12A and M379V, (b) M379T, (c) M379C; (9) The substrate is 3,5-difluorophenol, the non-natural amino acid is (S)-2-amino-3-(2,6-difluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) F448C, (b) R82E, (c) M288S and M379V; (10) The substrate is 3-bromo-5-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(2-bromo-6-fluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) V283T, M288G and M379S, (b) M379P, (c) M379L and T406A; (11) The substrate is 2,3-difluorophenol, the non-natural amino acid is (S)-2-amino-3-(2,3-difluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) F36T and M379C, (b) V283G, M288S and M379V, (c) M288S and M379V; (12) The substrate is 2,3,5,6-tetrafluorophenol, the non-natural amino acid is (S)-2-amino-3-(2,3,5,6-tetrafluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) M288F and M379S, (b) M379C, (c) M379T; (13) The substrate is 2-fluoro-1,3-benzenediol, the non-natural amino acid is (S)-2-amino-3-(3-fluoro-2,4-dihydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) M379T, (b) S12A and M379V, (c) S12V and M379V; (14) The substrate is 2-chloro-1,3-benzenediol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-2,4-dihydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) V283T, M288G and M379S, (b) M379C, (c) M379V and T406A.

[0030] Specifically, this invention investigated the substrates and unnatural amino acids corresponding to different biocatalyst mutants (tyrosine phenol lyase mutants), and discovered the above-mentioned combinations of substrates, unnatural amino acids, and tyrosine phenol lyase mutants. In each combination, the tyrosine phenol lyase mutant can catalyze the corresponding substrate to generate the corresponding unnatural amino acid. The specific structures of the above substrates and corresponding unnatural amino acids are shown in Table 1.

[0031] Table 1. Names and structures of the substrates and corresponding non-natural amino acids involved in this invention.

[0032] Fifthly, the provision of non-natural amino acids, said non-natural amino acids being selected from one or more of the following structures: .

[0033] In a preferred embodiment, the non-natural amino acids are prepared using the non-natural amino acid synthesis method described in the fourth aspect.

[0034] Sixthly, the application of the non-natural amino acids as described in the fifth aspect is provided, wherein the non-natural amino acids are used for site-specific incorporation into green fluorescent protein to optimize its fluorescence luminescence properties and thermal stability.

[0035] A preferred technical solution is a method for synthesizing the green fluorescent protein, comprising: introducing a stop codon into the coding sequence of the green fluorescent protein to incorporate the non-natural amino acid through the stop codon. The amino acid sequence of the green fluorescent protein before the introduction of the stop codon is shown in SEQ ID NO.7. After the stop codon is introduced, the codon at position D190 in SEQ ID NO.7 is replaced with the stop codon.

[0036] Beneficial Effects: This invention establishes for the first time a directed evolution system for biocatalysts based on genetic code extension technology. This system precisely couples enzyme catalytic activity with host cell survival, achieving ultra-high throughput library screening capabilities. It overcomes the challenges of low throughput and poor efficiency in traditional screening methods, making it difficult to efficiently capture low-abundance beneficial mutations from large mutant libraries. Based on this directed evolution system, this invention successfully obtained several chimeric pyrrolidone-lysyl-tRNA synthetase mutants that recognize novel non-natural amino acids and tyrosine phenol lyase mutants with novel catalytic activities. The obtained tyrosine phenol lyase mutants were applied to the gram-scale synthesis of non-natural amino acids, and the fluorescence properties of green fluorescent protein were optimized using non-natural amino acids, demonstrating its feasibility and value in industrial applications. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of a directed evolution system for biocatalysts based on genetic code extension; it illustrates the core design principle of the system: the enzymatic reaction products of the biocatalyst are designed as non-natural amino acids (UAA), and the encoding of non-natural amino acids is linked to the expression of antibiotic resistance genes through an orthogonal aminoacyl-tRNA synthetase / tRNA system, thereby achieving a precise coupling between enzyme catalytic activity and host cell survival.

[0038] Figure 2 It is a flowchart of the in vivo directed evolution of biocatalysts; it shows the complete directed evolution process, including key steps such as mutant library construction, survival pressure selection, and enrichment of active mutants.

[0039] Figure 3 It is a schematic diagram of the protein structure and TAG site of the candidate selection tag; it shows the crystal structure of four antibiotic resistance proteins, namely TEM-1 β-lactamase, aminoglycoside phosphotransferase, chloramphenicol acetyltransferase and tetracycline efflux MFS transporter, as well as the specific sites for introducing the amber codon.

[0040] Figure 4 The document presents the process and results of label selection; it also shows the experimental results of restoring bacterial growth after incorporating CbzK into the A148 site of TEM-1 β-lactamase and the D112 and Y127 sites of chloramphenicol acetyltransferase.

[0041] Figure 5 This is an experimental result diagram of adjustable pressure selection tags; it shows the growth of strains with different numbers and positions of TAG insertions under different chloramphenicol pressures, demonstrating that survival pressure can be finely adjusted through codon position combinations.

[0042] Figure 6 This is a flowchart of the chPylRS directed evolution system; it demonstrates a triple selection system: negative selection, survival pressure selection, and fluorescence selection.

[0043] Figure 7 The diagram shows the chPylRS mutant obtained through evolution and its results; it also demonstrates the recognition efficiency of the chPylRS-B2 and chPylRS-B4 mutants for different non-natural amino acids.

[0044] Figure 8 This is a diagram showing the construction and testing results of the TPL directed evolution system; it illustrates the construction of the dual plasmid system and the recovery of bacterial growth in the presence of chloramphenicol.

[0045] Figure 9 This is a dose-dependent graph showing the growth of host cells on the catalytic substrate; it illustrates the relationship between bacterial growth and the concentrations of TPL substrate and chloramphenicol.

[0046] Figure 10 The figure shows the enrichment efficiency of TPL-active mutants; it illustrates the enrichment process of TPL*-active mutants under different initial ratios, demonstrating the ability of directed evolution systems to efficiently enrich low-abundance active mutants from highly diverse libraries.

[0047] Figure 11 It serves as the construction and gene information carrier for the TrpB directed evolution system, demonstrating the scalability of directed evolution systems.

[0048] Figure 12 This is a dose-dependent graph showing the growth of host cells on the catalytic substrate; it illustrates the relationship between bacterial growth and the concentrations of TrpB substrate and chloramphenicol.

[0049] Figure 13 The figure shows the enrichment efficiency of the TrpB active mutant; it demonstrates the enrichment process of the TrpB*-active mutant under the condition of an initial ratio of 1:1000, proving the ability of directed evolution systems to efficiently enrich low-abundance active mutants from highly diverse libraries.

[0050] Figure 14 This diagram shows the results of TPL achieving highly efficient catalysis of 2-halophenols through directed evolution; it demonstrates the catalytic activity of the evolved mutants for 2-chlorophenol, 2-bromophenol, and 2-iodophenol.

[0051] Figure 15 This is a simulation verification result of co-selective directed evolution; it shows the feasibility verification results of the dual-selection directed evolution system simultaneously evolving TPL and chPylRS.

[0052] Figure 16 This is a flowchart of the directed evolution of TPL and chPylRS using a co-directed evolution system; it demonstrates the co-directed evolution of TPL and chPylRS under mixed substrate conditions.

[0053] Figure 17 This is a diagram showing the results of simultaneous directed evolution of TPL and chPylRS using a co-directed evolution system; it illustrates the co-evolutionary functional TPL-chPylRS pairs obtained for different substrates.

[0054] Figure 18 This is the substrate spectrum of TPL after directed evolution; it demonstrates the catalytic activity of the evolved TPL mutant for a variety of phenol analogs.

[0055] Figure 19 This is a graph showing the gram-scale synthesis results of tyrosine analogs; it illustrates the experimental results of gram-scale synthesis using the directed-evolution TPL mutant.

[0056] Figure 20 This is a novel non-natural amino acid structural formula; it shows the chemical structures of some novel non-natural amino acids developed in this invention, wherein nY1 is 2,3-trimethylene-L-tyrosine, nY2 is 3-chloro-5-fluoro-L-tyrosine, nY3 is 2-chloro-6-fluoro-L-tyrosine, nY4 is 2-bromo-6-fluoro-L-tyrosine, nY5 is 2-hydroxy-3-fluoro-L-tyrosine, and nY6 is 2-hydroxy-3-chloro-L-tyrosine.

[0057] Figure 21 This invention identifies a specific chimeric pyrrolidone-lysyl-tRNA synthetase (chPylRS) mutant that recognizes six novel non-natural amino acids.

[0058] Figure 22 The results show the effects of novel non-natural amino acids on the optical properties and thermal stability of green fluorescent protein; they demonstrate the significant improvement in the relative fluorescence intensity of green fluorescent protein after site-specific incorporation of non-natural amino acids and the enhanced fluorescence retention rate (thermal stability) under heat treatment conditions. Detailed Implementation

[0059] This invention provides a directed evolution system and method for biocatalysts, screened biocatalyst mutants, and their synthetic non-natural amino acids and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0060] Linking the function of a target protein to host survival and designing selection-based in vivo evolution methods can significantly increase the coverage of mutant libraries. Protein evolution methods based on survival selection or survival pressure directly link enzyme activity to host survival or proliferation. By setting uniform criteria, only mutants meeting the conditions are retained, thereby achieving the goal of evolution by screening massive libraries. A representative example is phage-assisted continuous evolution (PACE). Although PACE has demonstrated its power in directed protein evolution, this system is currently difficult to use for the evolution of biocatalysts. This is because effectively linking the chemical conversion efficiency of enzymatic reactions to the expression of biological macromolecules (such as phage proteins) and constructing logically rigorous biochemical circuits is extremely difficult.

[0061] In practice, the directed evolution of biocatalysts is often difficult to design in vivo selection assays. In most cases, researchers need to screen mutants one by one for activity. Genetic Code Expansion (GCE) technology is a groundbreaking biotechnology approach. By designing and modifying specific aminoacyl-tRNA synthetases (aaRS) and their corresponding tRNA pairs, cells can precisely incorporate unnatural amino acids (UAAs) into designated sites during protein translation. This links the function of the target protein to the host's survival, achieving a selection-based in vivo evolution method. Currently, over 400 structurally diverse unnatural amino acids can be recognized and inserted into proteins by orthogonal aminoacyl-tRNA synthetase / tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS) is a class of orthogonal aminoacyl-tRNA synthetases that specifically recognize pyrrolysine and its analogues and load them onto the corresponding orthogonal tRNAs (tRNAs). CUA On the ), the readout expression of the amber stop codon (TAG) is mediated.

[0062] Based on this, this invention develops a highly efficient in vivo directed evolution system that links biocatalyst activity to host cell survival through genetic code expansion (GCE). In this system, the enzymatic product is designed as a non-natural amino acid (UAA). Subsequently, the encoding of the UAA by an aminoacyl-tRNA synthetase (such as PylRS) promotes the readthrough of stop codons (such as TAGs) on genes encoding antibiotic resistance proteins (such as chloramphenicol resistance proteins), thereby enabling host bacteria carrying effective biocatalyst mutants to survive under antibiotic stress. Figure 2 ).

[0063] The specific development strategy and steps for the biocatalyst directed evolution system are as follows: (1) Development and optimization of selection tags. This invention first focuses on the development of selection tags to construct an efficient biocatalyst evolution system. Four commonly used antibiotics and their corresponding resistance genes were initially selected as candidate selection tags: TEM-1 β-lactamase / ampicillin (Amp), aminoglycoside phosphotransferase / kanamycin (Kan), tetracycline efflux MFS transporter / tetracycline (Tet), and chloramphenicol acetyltransferase / chloramphenicol (Cm). In order to construct a directed evolution system regulated by unnatural amino acids (UAA), this invention analyzed the crystal structure of these antibiotic resistance proteins and determined the specific sites for introducing the amber codon (TAG). Among them, the incorporation of N-ε-benzyloxycarbonyl-L-lysine (CbzK) at the A148 site of β-lactamase and at the D112 and Y127 sites of chloramphenicol acetyltransferase (CmR) successfully restored bacterial growth in the presence of the corresponding antibiotics. Given that the chloramphenicol resistance (CmR) tag provides two orthogonal UAA incorporation sites, enabling the regulation of selection pressure, this tag was selected as the primary selection tag.

[0064] (2) Directed evolution of chimeric pyrrolidone-lysyl-tRNA synthetase (chPylRS) mutants. In order to obtain chPylRS mutants that recognize target non-natural amino acids, this invention establishes a triple screening system: negative screening—using the toxic protein Barnase for negative screening. Barnase contains the amber codon, and chPylRS mutants that can recognize natural amino acids will cause host cell death; survival pressure selection—using chloramphenicol acetyltransferase (CmR) for selection. CmR contains the amber codon, and only chPylRS mutants that can recognize the target UAA can survive under chloramphenicol pressure; fluorescence screening—using green fluorescent protein (GFP) for final screening, and quantitatively analyzing the efficiency of chPylRS mutants incorporating non-natural amino acids. After three rounds of screening, the chPylRS mutant B2 (L270I / L274G / N311C / C313W / Y349F / V366T / W382T) which can efficiently recognize 3-chloro-L-tyrosine and 3-bromo-L-tyrosine, and the chPylRS mutant B4 (L270I / L274G / N311C / C313W / Y349F / V366T / W382S) which specifically recognizes 3-iodo-L-tyrosine were obtained.

[0065] (3) Establishment of the TPL directed evolution system. This invention uses tyrosine phenol lyase (TPL) as a model biocatalyst to construct an in vivo directed evolution system for biocatalysts. TPL can catalyze the conversion of phenol analogs into tyrosine derivatives. These products are then recognized as substrates by chimeric pyrrolidone-lysyl-tRNA synthetase (chPylRS) and integrated into a select tag, thereby effectively coupling the enzyme activity of TPL with bacterial growth. This invention constructs a dual plasmid system: one plasmid carries the CmR-112TAG reporter gene and its homologous orthogonal tRNA, and the other plasmid encodes TPL-M379V and chPylRS-B2. To verify this system, this invention co-transforms the above dual plasmids into *E. coli*. BL21(AI) The results showed that this dual-plasmid system successfully coupled TPL enzyme activity with bacterial survival. In the presence of chloramphenicol (Cm), both direct addition of the product 3-chloro-L-tyrosine and addition of the precursor 2-chlorophenol (which is converted to 3-chloro-L-tyrosine in vivo) significantly restored bacterial growth.

[0066] (4) Validation of the TPL directed evolution system. To establish an effective time window for selecting survival pressure, an inactivating mutant TPL-K257A (TPL*-dead) was constructed as a negative control. Under defined survival pressures of 20 μg / mL chloramphenicol and 500 μM 2-chlorophenol, the growth kinetics of bacteria carrying TPL*-active or TPL*-dead were monitored. The results showed that the growth of TPL*-active was strictly dependent on the presence of the substrate, while TPL*-dead could not grow under the given conditions, confirming the rigor and reliability of the directed evolution system.

[0067] To further evaluate the enrichment screening capability of the directed evolution system, this invention designed bacterial mixtures containing the directed evolution system and either TPL*-active or TPL*-dead at initial ratios of 1:10, 1:100, and 1:1000, and conducted three consecutive rounds of enrichment screening. Particularly in the 1:1000 group simulating a low-abundance mutant scenario, the proportion of TPL*-active significantly increased from the initial 0.1% to 94%, with an enrichment fold of over 15,000 times (the initial ratio of TPL*-active to TPL*-dead in the mixture was 0.1:100, a difference of 1000 times; after enrichment, the ratio of TPL*-active to TPL*-dead was 94:6, a difference of 15.7 times).

[0068] (5) Directed Evolution of TPL. Based on the successfully established efficient directed evolution system, this system was applied to the actual directed evolution study of TPL. Initial characterization showed that the wild type of TPL (TPL-WT) had weak activity against 2-chlorophenol, but no detectable activity against 2-bromophenol or 2-iodophenol. Therefore, this invention selected these three ortho-halophenols as target substrates to develop new activities in TPL. Based on the structure of the TPL-phenol complex and its catalytic mechanism, this invention rationally designed and selected residues located at the edge of the enzyme's active pocket to construct a focused saturation mutant library. After a round of evolution, several TPL mutants with new catalytic activities were obtained: for 2-chlorophenol substrate, the M379P and M379C mutants achieved a catalytic yield of 99% within 6 hours, which is 20 times higher than that of wild-type TPL; for 2-bromophenol substrate, the F36C / M379V mutant achieved a catalytic yield of 97% within 6 hours; and for 2-iodophenol substrate, the F36T / M379C mutant achieved a catalytic yield of 99% within 6 hours.

[0069] (6) Co-evolutionary Strategy. A fundamental challenge in expanding the substrate range of TPL in this system is the inherent interdependence between enzyme evolution and UAA recognition: evolving TPL to accept new substrates requires a pre-existing chPylRS capable of recognizing the resulting novel UAA. To address this circular dependency, this invention tested the feasibility of simultaneously evolving TPL and chPylRS through a dual-directed evolutionary system. This invention first designed a model system to simulate co-evolution, with an initial ratio of 0.01% dual-active mutants, 10% each of TPL-dead or chPylRS-dead variants, and 80% dual-inactivation mutants. After three rounds of survival selection, the dual-active population enriched from 0.01% to over 80%, achieving a 40,000-fold enrichment (the initial ratio of dual-active mutants to other mutants in the mixture was 0.1:1000, i.e., a 10,000-fold difference; after enrichment, the ratio of dual-active mutants to other mutants was 80:20, i.e., a 4-fold difference), confirming the co-evolutionary capability of the system.

[0070] (7) Gram-scale synthesis of non-natural amino acids. A key application of biocatalysts is the large-scale industrial synthesis of high-value-added chemicals. To demonstrate the industrial potential of the evolved TPL mutant of this invention, gram-scale synthesis experiments were conducted using the F36T / M379C mutant with 2-iodophenol as a substrate—a substrate for which TPL activity had not been previously reported. By employing a stepwise addition strategy of 2-iodophenol, a final titer of 24.9 g / L of 3-iodo-L-tyrosine was achieved. In addition, gram-scale synthesis experiments of 4-indanol were conducted using the M379P mutant, ultimately achieving a titer of 34.0 g / L of 2,3-trimethylene-L-tyrosine. By employing a stepwise addition strategy to treat 2-fluoro-1,3-benzenediol, the product was successfully accumulated gradually, ultimately reaching a concentration of 36.8 g / L of 2-hydroxy-3-fluoro-L-tyrosine. Based on this system, this invention obtained several TPL mutants with novel catalytic activities, which can catalyze the conversion reactions of various novel substrates, realize the gram-scale synthesis of non-natural amino acids, and achieve product concentrations of 24.9-36.8 g / L, which have industrial application potential.

[0071] The present invention will be further described below through specific embodiments.

[0072] Example 1: Development and Optimization of the Selection and Marking System This embodiment first focuses on the development of selection tags to construct an efficient directed evolution system for biocatalysts. Four commonly used antibiotics and their corresponding resistance genes were initially selected as candidate selection tags: TEM-1 β-lactamase / ampicillin (Amp), aminoglycoside phosphotransferase / kanamycin (Kan), tetracycline efflux MFS transporter / tetracycline (Tet), and chloramphenicol acetyltransferase / chloramphenicol (Cm).

[0073] To construct a directed evolution system regulated by unnatural amino acids (UAAs), this embodiment identified specific sites for introducing amber stop codons (TAGs) by analyzing the crystal structures of these antibiotic resistance proteins. The screening criteria aimed to ensure that translational truncation at this site would eliminate antibiotic resistance, while successful incorporation of the unnatural amino acid would restore protein function. For each selected tag protein, two such sites were screened and tested using orthogonal UAA-PylRS pairs.

[0074] Based on structural analysis, this embodiment selected the following sites for amber codon mutation in four resistance genes ( Figure 3 TEM-1 β-lactamase (AmpR, PDB: 1ZG4, SEQ ID NO.1): K71 and A148 sites; Aminoglycoside phosphotransferase (KanR, PDB: 4EJ7, SEQ ID NO.2): N32 and F147 sites; Chloramphenicolacetyltransferase (CmR, PDB: 1PD5, SEQ ID NO.3): D112 and Y127 sites; Tetracycline efflux MFS repressor (TetR, UniProt: A0A845I2J6, SEQ ID NO.4): R69 and F197 sites.

[0075] To introduce an amber stop codon at a key site, this embodiment employs a chimeric pyrrololysyl-tRNA synthetase mutant (Y271A / Y349F) that specifically recognizes N-ε-benzyloxycarbonyl-L-lysine (CbzK). Figure 4 As shown, testing revealed that incorporation of CbzK at the A148 site of β-lactamase and the D112 and Y127 sites of chloramphenicol acetyltransferase (CmR) successfully restored bacterial growth in the presence of the corresponding antibiotics. Given that the chloramphenicol resistance tag provides two orthogonal UAA incorporation sites, enabling regulation of selection pressure, this tag was selected as the primary selection tag.

[0076] Further site characterization confirmed that these sites can function independently or synergistically. Under chloramphenicol selection pressure, the strain containing the double TAG insertion (CmR-112TAG.127TAG) grew the slowest, followed by the single-site Y127TAG mutant strain, while the D112TAG strain grew the fastest. Figure 5 This result establishes a strategy for finely modulating survival pressure through codon position combinations. Furthermore, a clear dose-dependent relationship was observed between bacterial growth and the concentrations of CbzK and antibiotics, establishing a robust correlation between UAA supply and cellular fitness.

[0077] Example 2 Directed evolution of chPylRS mutant The aminoacyl-tRNA synthetase backbone used in this embodiment is a chimeric pyrrololysyl-tRNA synthetase (chPylRS). To improve its activity, four mutations (V31I, T56P, H62Y, A100E) were introduced into its tRNA binding domain in this embodiment to create the parental variant chPylRS-IPYE (SEQ ID NO.5).

[0078] To obtain chPylRS mutants that recognize target non-natural amino acids, this embodiment establishes a directed evolution system for chPylRS ( Figure 6 ),include: Negative selection: Negative selection is performed using the toxic protein Barnase. Amber codons (TAGs) are introduced at the Q3 and D45 sites of Barnase (SEQ ID NO.6), which can recognize variants of natural amino acids that lead to host cell death. Survival stress selection: Survival stress selection was performed using chloramphenicol acetyltransferase (CmR). An amber codon (TAG) was introduced at the D112 site of CmR. Only mutants that could recognize the target UAA could survive under chloramphenicol stress. Fluorescence screening: Green fluorescent protein (GFP) was used for secondary screening, and the efficiency of non-natural amino acid incorporation in the chPylRS mutant was quantitatively analyzed by unit fluorescence intensity. In the experiment, a tag sequence (HHHHHH) was added to the C-terminus of GFP to form GFP-6×His (SEQ ID NO.7), and an amber codon (TAG) was introduced at its D190 site.

[0079] After three rounds of evolution, the chPylRS mutant B2 (or chPylRS-B2), capable of efficiently recognizing 3-chloro-L-tyrosine and 3-bromo-L-tyrosine, was obtained. Its mutations are: L270I / L274G / N311C / C313W / Y349F / V366T / W382T. The B2 mutant exhibits high recognition efficiency for both 3-chloro-L-tyrosine and 3-bromo-L-tyrosine, effectively integrating these non-natural amino acids into the TAG sites of target proteins in vivo. Simultaneously, the chPylRS mutant B4 (or chPylRS-B4), specifically recognizing 3-iodo-L-tyrosine, was obtained. Its mutations are: L270I / L274G / N311C / C313W / Y349F / V366T / W382S. The B4 mutant shows high specificity for 3-iodo-L-tyrosine. Specific results are as follows: Figure 7 As shown.

[0080] Example 3: Establishment of the TPL Directed Evolution System In this embodiment, tyrosine phenol lyase (TPL) was used as a model biocatalyst to construct an in vivo directed evolution system. Wild-type TPL (SEQ ID NO.8) catalyzes the conversion of phenol analogs into tyrosine derivatives. These products are then recognized as substrates by chimeric pyrrolidone-lysyl-tRNA synthetase (chPylRS) and integrated into a select tag, thereby effectively coupling the enzymatic activity of TPL with bacterial growth. In the experiment, a tag sequence (MGSSHHHHHH) was added to the N-terminus of wild-type TPL to obtain 6×His-TPL (SEQ ID NO.9).

[0081] Given that the TPL-M379V mutant (hereinafter referred to as TPL*-active) can convert 2-chlorophenol into 3-chloro-L-tyrosine (3-Cl-Tyr), and that the chPylRS-B2 mutant can respond to the amber codon at position 112 of the chloramphenicol acetyltransferase gene (CmR-112TAG) to integrate the generated 3-chloro-L-tyrosine into the resistance protein, this embodiment uses this enzyme-substrate pair as a model to evaluate the effectiveness of the directed evolution system.

[0082] To this end, this embodiment constructs a dual-plasmid system: one plasmid carries a CmR-112TAG select tag (SEQ ID NO.10) and an orthogonal tRNA (SEQ ID NO.11) paired with chimeric pyrrolidone-lysyl-tRNA synthetase (chPylRS), while the other plasmid encodes TPL*-active and chPylRS-B2. The functions of each component in this genetic pathway have been independently verified through experiments.

[0083] To verify the feasibility of using this dual-plasmid system for directed evolution of biocatalysts, this embodiment co-transformed the above-mentioned dual plasmids into *E. coli*. BL21(AI) The results showed that this dual-plasmid system successfully coupled TPL enzyme activity with bacterial survival. Notably, in the presence of chloramphenicol (Cm), both direct addition of the product 3-chloro-L-tyrosine and addition of the precursor 2-chlorophenol (which is converted to 3-chloro-L-tyrosine in vivo) significantly restored bacterial growth. This growth recovery phenomenon was observed in both liquid (LB broth) and solid (LB agar) media, fully demonstrating the versatility of this directed evolution system in future screening and evolutionary applications. Specific results are as follows... Figure 8 As shown.

[0084] Subsequently, a dose-dependent matrix experiment was conducted in this embodiment. Figure 9 (a) demonstrates that bacterial growth is well controlled by the concentration of the TPL substrate (2-chlorophenol) and the survival pressure (chloramphenicol, Cm). Based on these findings, this example selected a moderate phenol substrate concentration (500 μM) and a moderate selection pressure (20 μg / mL Cm) as the standard conditions for subsequent experiments.

[0085] To establish an effective selection time window, an inactivating mutant, TPL-K257A (hereinafter referred to as TPL*-dead), was constructed as a negative control in this embodiment. Under a defined selection pressure of 20 μg / mL chloramphenicol and 500 μM 2-chlorophenol, the growth kinetics of bacteria carrying TPL*-active or TPL*-dead were monitored. The results showed that the growth of TPL*-active was strictly dependent on the presence of the substrate, while TPL*-dead could not grow under the selection conditions, confirming the rigor and reliability of the directed evolution system. Furthermore, no escape mutants were observed during the 24-hour culture period of the TPL*-dead group, verifying the fidelity and reliability of the system. Figure 9 (b)

[0086] To further evaluate the enrichment capacity of the directed evolution system for active mutants, this embodiment designed a bacterial mixture containing a selection tag and either TPL*-active or TPL*-dead. The initial ratios of TPL*-active or TPL*-dead were 1:10, 1:100, and 1:1000, respectively, and three consecutive rounds of selection were performed. The proportion of the active mutant TPL*-active in the mixed bacterial community increased with each screening round. Particularly in the 1:1000 group simulating a low-abundance mutant scenario, the proportion of TPL*-active significantly increased from the initial 0.1% to 94%, with an enrichment fold of over 15,000 times. Figure 10These results fully demonstrate that the directed evolution system constructed in this embodiment possesses the outstanding ability to efficiently and accurately enrich low-abundance active mutants from highly diverse libraries.

[0087] Example 4: Establishment of the TrpB Directed Evolution System To verify the scalability of the directed evolution system, this embodiment adopts... Thermotoga maritima The β subunit of tryptophan synthase (TmTrpB, hereinafter referred to as TrpB) was used as a second model. This example first demonstrates that the TrpB mutant (TmAzul, hereinafter referred to as TrpB*-active, which, in the experiment, is tagged with a sequence (HHHHHH) to form TrpB-TmAzul-6×His (SEQ ID NO.12)) can catalyze the conversion of 7-cyanoindole (7-CN-indole) to 7-cyano-L-tryptophan (7-CN-Trp) (…). Figure 11 (a). In addition, the chimeric phenylalanine-tRNA synthetase (chPheRS) mutant 1-B2 (chPheRS-IPYE-1-B2, SEQ ID NO.13) has been shown to recognize 7-CN-Trp for amber stop codon reading.

[0088] Therefore, following the logic of the TPL directed evolution system, TPL was replaced with TrpB, and the PylRS / tRNA pairs were replaced with orthogonal chPheRS / tRNA pairs. The reconfigured directed evolution system was then validated. Figure 11 (b) In this embodiment, an inactivated mutant TrpB-K83A (TrpB*-dead) was constructed as a negative control. Consistent with the TPL directed evolution system, the growth of cells carrying the active TrpB mutant (TrpB*-active) was strictly dependent on 7-cyanoyindole. Conversely, the TrpB*-dead mutant could not grow under the selection conditions due to the dual growth pressure of 7-cyanoyindole and chloramphenicol. Figure 12 ).

[0089] To quantify enrichment efficiency, a competitive growth experiment was conducted in this embodiment, transforming host cells with a mixture of plasmids containing TrpB*-active and TrpB*-dead at initial ratios of 1:10, 1:100, and 1:1000. These mixtures were then subjected to three consecutive rounds of enrichment. It was observed that the proportion of TrpB*-active gradually increased with each round of selection. Notably, in the 1:1000 group, the proportion of TrpB*-active mutants increased from 0.1% to 90%, achieving an enrichment of over 9,000-fold. Figure 13 ).

[0090] Example 5 Directed Evolution of TPL Mutants Based on the successfully established efficient in vivo directed evolution system, this embodiment applies it to the actual directed evolution study of TPL. Initial characterization showed that TPL-WT has weak activity against 2-chlorophenol, and no detectable activity against 2-bromophenol or 2-iodophenol. Therefore, this embodiment selects these three ortho-halophenols as target substrates to develop new activities in TPL-WT.

[0091] Based on the structure and catalytic mechanism of the TPL-phenol complex, this embodiment rationally designed and selected residues located at the edge of the enzyme's active pocket to construct a focused saturation mutant library, including N34 / F36 / M379 (library L1), T49 / S51 (library L2), Q98 / R100 (library L3), F123 / T124 / T125 (library L4), V283 / M288 / M379 (library L5), M379 / R381 (library L6), and M379 / L446 / F448 (library L7). In addition, a random mutant library (library L8) was generated using error-prone PCR.

[0092] In this embodiment, a standard survival pressure selection procedure was performed for each library and each substrate. After one round of evolution, the enriched mutants were characterized for enzyme activity and sequenced. The results showed that the evolved mutants exhibited significantly enhanced catalytic activity towards the target substrates: For the 2-chlorophenol substrate, the M379P and M379C mutants achieved a catalytic yield of 99% within 6 hours, a 20-fold improvement compared to the wild-type enzyme. These mutants were able to efficiently catalyze the conversion of 2-chlorophenol to 3-chloro-L-tyrosine at 25°C and pH 8.0.

[0093] For the 2-bromophenol substrate, the F36C / M379V mutant achieved a catalytic yield of 97% within 6 hours. Previous studies had not reported any catalytic activity of the TPL mutant for 2-bromophenol, and preliminary experimental results also confirmed that the wild-type enzyme had no catalytic activity for 2-bromophenol.

[0094] For the 2-iodophenol substrate, the F36T / M379C mutant achieved a catalytic yield of 99% within 6 hours. This is the first report of the catalytic activity of TPL for the iodophenol substrate.

[0095] Specific results are as follows Figure 14 As shown.

[0096] The process of directed evolution is as follows: The TPL mutant library was transformed into competent cells carrying a select tag and plated onto agar plates containing 50 μg / mL ampicillin, 25 μg / mL kanamycin, and an inducer (final concentration of 0.2% arabinose) and incubated overnight at 30°C. The next day, all colonies on the plates were collected, and the bacterial suspension was diluted to OD200. 600 The culture medium was diluted 1:100 with ampicillin (2.0), and the first round of enrichment culture was performed. This enrichment medium contained 50 μg / mL ampicillin, 25 μg / mL kanamycin, 20 μg / mL chloramphenicol, 0.5 mM phenol analogues (2-chlorophenol, 2-bromophenol, or 2-iodophenol), 0.2% arabinose, 50 μM pyridoxal phosphate (PLP), 5 mM sodium pyruvate, and 1 / 5 volume of KPI buffer (50 mM, pH 8.0, containing 100 mM ammonium chloride and 40 mM ammonium sulfate). After incubation at 37°C for 12 hours, a portion of the bacterial culture was plated onto solid LB agar plates. The next day, all colonies on the plates were collected for plasmid extraction, and the plasmid pool was retransformed into selective competent cells. The transformants were then plated onto solid LB agar plates and incubated overnight at 37°C to isolate single colonies for subsequent mutant screening.

[0097] Single colonies were picked and inoculated into 96-well plates and incubated overnight at 37°C. The seed culture was diluted 1:100 to fresh medium and incubated at 37°C for 2 hours. Then, 0.2% arabinose was added for induction, and the culture was continued at 25°C for 6 hours. The induced culture was diluted 1:100 to the same selective medium as the enrichment culture and incubated at 37°C for 12 hours. Finally, the OD of each well was measured. 600 Value; vigorous bacterial growth (OD) 600 A higher value indicates that the corresponding TPL mutant has catalytic activity against the target phenol analogue.

[0098] The methods for synthesizing non-natural amino acids are as follows: The biocatalytic reaction system consisted of whole cells (60 g / L), sodium pyruvate (30 g / L), ammonium acetate (30 g / L), pyridoxal phosphate (50 μM), potassium chloride (50 mM), Triton X-100 (0.1 g / L), and a phenol analog (20 mM). After the reaction system was prepared, the pH was adjusted to 8.0-8.5 using ammonia, and then the whole-cell catalyst (60 g / L, wet weight) was added. The whole-cell catalyst was prepared by transforming the TPL mutant corresponding to the target phenol analog into bacteria for expression and collecting the bacterial sludge. The reaction was carried out at 25°C, with sodium pyruvate (14 g / L), ammonium acetate (6.4 g / L), and the phenol analog (20 mM) added every 6 hours. The phenol analog was 2-chlorophenol, 2-bromophenol, or 2-iodophenol.

[0099] After the reaction was complete, the pH was adjusted to 2–3 using 6 M HCl and maintained for 2 hours to terminate the reaction. The mixture was centrifuged to remove the precipitate, and the resulting supernatant constituted the aqueous phase, which was extracted three times with dichloromethane (50 mL). The aqueous phase was then concentrated by rotary evaporation to remove water. The crude tyrosine analogue (10 mmol) was dissolved in methanol (25 mL), and triethylamine (60 mmol), 4-dimethylaminopyridine (DMAP, 0.01 g), and di-tert-butyl dicarbonate (Boc₂O, 12 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 6 hours, then acidified to pH 2–3 with 6 M HCl and extracted with ethyl acetate. The combined organic phases were washed with saturated NaCl solution (50 mL), dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure to obtain the Boc-protected intermediate.

[0100] Subsequently, the intermediate was treated with a mixture of trifluoroacetic acid (TFA) and dichloromethane (DCM) (volume ratio 1:5, total 50 mL) at room temperature for 6 hours to remove the Boc group. The reaction mixture was filtered, and the solid residue was washed with DCM (20 mL × 3) and dried to obtain the purified tyrosine analogue.

[0101] Example 6: Co-evolutionary Strategy A fundamental challenge in expanding the substrate range of TPL in the TPL-directed evolution system of Example 3 is the inherent interdependence between enzyme evolution and UAA recognition: evolving TPL to accept new substrates requires a pre-existing chPylRS capable of recognizing the resulting novel UAA. To address this circular dependency, this example tested the feasibility of simultaneously evolving TPL and chPylRS using a dual-directed evolution system.

[0102] This embodiment first designed a model system to simulate co-evolution. TPL-M379V (TPL*-active) and chPylRS-B2 (recognizing 3-Cl-Tyr) were defined as active mutants, while TPL-K257A (TPL*-dead) and chPylRS-R391A (an ATP-binding defective mutant) were defined as their inactivating mutants. Four combinations were constructed: TPL-M379V-chPylRS-B2 (dual-active mutant), TPL-K257A-chPylRS-B2 (single-inactivating mutant, TPL-dead), TPL-M379V-chPylRS-R391A (single-inactivating mutant, chPylRS-dead), and TPL-K257A-chPylRS-R391A (dual-inactivating mutant). The initial artificial library consisted of 0.01% dual-active mutants, 10% each of TPL-dead or chPylRS-dead mutants, and 80% dual-inactivation mutants. After three rounds of survival selection, the dual-active population increased from 0.01% to over 80%, representing a 40,000-fold enrichment, confirming the co-evolutionary ability of this system. Figure 15 ).

[0103] To obtain a co-evolutionary biocatalyst for recognizing and utilizing novel UAAs, this embodiment constructed a combined mutant library comprising TPL and chPylRS. In the library design, site-directed mutagenesis was performed on key functional residues. In chPylRS, two pairs of previously shown synergistic sites (311 / 313 and 366 / 382) were combined. In TPL, three residues (12, 36, and 379) identified from previous directed evolution were targeted as key sites for phenol analog recognition.

[0104] The screening of this library was based on a rigorous co-directed evolution system. Cells carrying the combined library were plated on a medium containing a mixture of three phenol analogs and chloramphenicol. In this co-directed evolution system, cell survival depends on the synergistic occurrence of two biochemical events. First, the intracellularly expressed TPL mutant must be catalytically active, capable of converting at least one phenol analog to the corresponding UAA in vivo. Second, the co-expressed chPylRS mutant in the same cell must specifically recognize this newly synthesized UAA and catalyze its aminoacylation to orthogonal tRNA. Only when both conditions are met can the full-length functional chloramphenicol resistance protein be expressed, allowing the cells to survive under chloramphenicol selection.

[0105] After a round of survival selection, an enriched population was obtained. To determine the specificity of individual clones for each phenol analog, a sorting strategy was employed in this embodiment. The enriched population was divided into three equal parts, and then a secondary selection was performed in a medium supplemented with a single phenol analog and chloramphenicol. Clones that survived under specific conditions indicated that their TPL mutants could catalyze the specific substrate, and their chPylRS mutants could recognize the corresponding UAA product (…). Figure 16 Using this method, co-evolutionary functional TPL-chPylRS pairs were successfully isolated and identified. Figure 17 ): For the substrate 2-nitrophenol (2-NO2-phenol): the TPL mutant F36L / M379V showed a relative catalytic efficiency of 43%, and the paired chPylRS mutants M265T / L270I / L274G / D278G / N311G / C313R / Y349F specifically recognized 3-nitro-L-tyrosine (3-NO2-Tyr).

[0106] For the substrate 4-indenol: the TPL mutant M379P showed a high relative catalytic efficiency of 99%, and the paired chPylRS mutants were I253V / F260L / L270I / Y271F / Y273F / L274G / Y294F / N311G / C313W / T329A / Y349F / W382T, which specifically recognized 2,3-trimethylene-L-tyrosine (3-(4-hydroxyindan-5-yl)-L-alanine).

[0107] For the substrate 3-chloro-5-fluorophenol (3-Cl-5-F-phenol): the TPL variant S12V / M379V achieves a relative catalytic efficiency of 99%, and the paired chPylRS variants L270I / L274G / E302V / N311C / C313W / Y349F / A365G / V366H / W382T specifically recognize 2-chloro-6-fluoro-L-tyrosine (2-Cl-6-F-Tyr).

[0108] Example 7: Further Expansion of the Substrate Range This embodiment further expands the substrate range of TPL. A library containing 72 TPL mutants (isolated from previous evolutionary work) was screened against a range of phenol analogs. The reaction process was monitored by liquid chromatography-mass spectrometry (LC-MS) to identify active substrate-enzyme pairs.

[0109] These advantageous mutants significantly expanded the substrate range of TPL. Notably, the TPL mutant S12V / M379V achieved an excellent conversion rate of 99% for 2-fluoro-1,3-benzenediol. Meanwhile, the TPL mutant M288S / M379V achieved biocatalytic efficiencies exceeding 95% for 2,3-difluorophenol, and the TPL mutant M379C achieved biocatalytic efficiencies exceeding 95% for 2-chloro-6-fluorophenol. Furthermore, the evolved TPL mutants exhibited high catalytic activity for 2,6-difluorophenol, 3,5-difluorophenol, 3-bromo-5-fluorophenol, 2,3,5,6-tetrafluorophenol, and 2-chloro-1,3-benzenediol. Figure 18 ).

[0110] Example 8 Gram-scale synthesis verification Biocatalysis technology has significant application value in large-scale industrial synthesis. To evaluate the industrial application potential of the TPL mutant screened by directed evolution in this invention, gram-scale synthesis experiments were conducted using the F36T / M379C mutant. This experiment used 2-iodophenol as a substrate, for which there have been no previous reports of TPL catalytic activity.

[0111] The biocatalytic reaction system consisted of whole cells (60 g / L), sodium pyruvate (30 g / L), ammonium acetate (30 g / L), pyridoxal phosphate (50 μM), potassium chloride (50 mM), Triton X-100 (0.1 g / L), and a phenol analog (20 mM). After the reaction system was prepared, the pH was adjusted to 8.0-8.5 using ammonia, and then the whole-cell catalyst (60 g / L, wet weight) was added. The whole-cell catalyst was prepared by expressing the TPL mutant corresponding to the target phenol analog in bacteria and collecting the bacterial sludge. The reaction was carried out at 25°C, with sodium pyruvate (14 g / L), ammonium acetate (6.4 g / L), and the phenol analog (20 mM) added every 6 hours. The phenol analog was 2-iodophenol, 4-indanol, or 2-fluoro-1,3-benzenediol.

[0112] By employing a stepwise fed-batch strategy to add 2-iodophenol, the product was gradually accumulated, ultimately achieving a 3-iodo-L-tyrosine titer of 24.9 g / L. Furthermore, gram-scale catalysis of 4-indanol using the M379P variant resulted in a 2,3-trimethylene-L-tyrosine titer of 34.0 g / L. For the 2-fluoro-1,3-phenylene glycol substrate, a similar stepwise addition strategy successfully achieved efficient product accumulation, ultimately reaching a 2-hydroxy-3-fluoro-L-tyrosine concentration of 36.8 g / L. Specific results are as follows... Figure 19 As shown.

[0113] Furthermore, novel non-natural amino acids were synthesized at gram scale from the TPL mutants screened using the method of this invention. The chemical structures of the novel non-natural amino acids are as follows: Figure 20 As shown.

[0114] The amino acid sequence of the TPL mutant parent is shown in SEQ ID NO.8. The combination of substrate, non-natural amino acid, and TPL mutant is as follows: (1) The substrate was 4-indanol, the non-natural amino acid was 2,3-trimethylene-L-tyrosine, and the mutation site of the TPL mutant was M379P. 2,3-trimethylene-L-tyrosine was denoted as nY1, and its high-resolution mass spectrometry (ESI-HRMS) results showed a molecular ion peak [M+H]. + The measured value is m / z = 222.1125, which is in excellent agreement with the theoretically calculated value of m / z = 222.1085, confirming its molecular formula. Further NMR spectroscopy... 1 The characteristic peak of H NMR (400 MHz, D2O) is δ[6.65 (d, J = 8.1 Hz, 1H), 6.25 (d, J = 8.1 Hz, 1H), 3.24 (dd, J = 8.2, 5.4 Hz, 1H), 2.74(dd, J = 13.8, 5.4 Hz, 1H), 2.68 (t, J = 7.4 Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H), 2.45 (dd, J = 13.8, 8.3 Hz, 1H), 1.82 (t, J = 7.1 Hz, 2H)]; Carbon NMR spectrum 13 C NMR (101 MHz, D2O) showed corresponding carbon skeleton signals at δ [183.09, 161.10, 144.74, 132.54, 128.95, 119.88, 116.26, 56.95, 38.05, 31.59, 29.75, 24.28], fully confirming the structure of this indene-substituted propionic acid.

[0115] (2) The substrate was 2-chloro-6-fluorophenol, and the non-natural amino acid was 3-chloro-5-fluoro-L-tyrosine. The mutation sites of the TPL mutant were: (a) S12A and M379V, (b) M379T, or (c) M379C. 3-chloro-5-fluoro-L-tyrosine was designated nY2, and its high-resolution mass spectrometry (ESI-HRMS) results showed that the molecular ion peak was [M+H]. + The measured value is m / z = 234.0328, which is in excellent agreement with the theoretically calculated value of m / z = 234.0289, confirming its molecular formula. Further analysis using hydrogen nuclear magnetic resonance (NMR) spectra... 1 The characteristic peaks of H NMR (400MHz, D2O) are δ[6.93 (s, 1H), 6.81 (d, 1H), and δ[6.81 (d, 1H)]. J = 12.1 Hz, 1H), 3.43 (t, 1H), 2.80 (dd, J = 13.8, 5.4 Hz, 1H), 2.64 (dd, J = 13.7, 7.3 Hz, 1H)]; Carbon NMR spectrum 13 C NMR (101 MHz, D2O) showed the corresponding carbon skeleton signal at δ [182.46, 156.11, 153.76, 149.53, 125.18, 123.68, 122.23, 114.81, 114.61, 57.37, 39.56]; in addition, the NMR fluorine spectrum... 19 The characteristic peaks of F NMR (377 MHz, D2O) show δ [-75.55, -134.51], which fully confirms the structure of this halotyrosine.

[0116] (3) The substrate was 3-chloro-5-fluorophenol, and the non-natural amino acid was 2-chloro-6-fluoro-L-tyrosine. The mutation sites of the TPL mutant were S12V and M379V. 2-chloro-6-fluoro-L-tyrosine was designated nY3, and its high-resolution mass spectrometry (ESI-HRMS) results showed that the molecular ion peak [M+H]... + The measured value is m / z = 234.0321, which is in excellent agreement with the theoretically calculated value of m / z = 234.0289, confirming its molecular formula. Further NMR spectroscopy... 1 The characteristic peaks of H NMR (400 MHz, D2O) are δ[6.39 (s, 1H), 6.18 (dd, J = 13.1, 2.5 Hz, 1H), 3.36 (dd, J = 8.5, 6.0 Hz, 1H), 2.90 (dd, J= 13.9, 5.6 Hz, 1H), 2.72 (dd, J = 13.6, 8.9 Hz, 1H)]; Carbon NMR spectrum 13 C NMR (101 MHz, D2O) showed corresponding carbon skeleton signals at δ [182.81, 166.67, 163.83, 161.42, 134.75, 115.37, 109.12, 104.18, 103.97, 56.27, 31.29]; in addition, the NMR fluorine spectrum... 19 The characteristic peaks of F NMR (377 MHz, D2O) show δ [-75.61, -114.63], which fully confirms the structure of this halotyrosine.

[0117] (4) The substrate was 3-bromo-5-fluorophenol, and the non-natural amino acid was 2-bromo-6-fluoro-L-tyrosine. The mutation sites of the TPL mutant were: (a) V283T, M288G, and M379S, (b) M379P, or (c) M379L and T406A. 2-bromo-6-fluoro-L-tyrosine was designated nY4, and its high-resolution mass spectrometry (ESI-HRMS) results showed that the molecular ion peak was [M+H]. + The measured value is m / z = 277.9816, which is in excellent agreement with the theoretically calculated value m / z = 277.9783, confirming its molecular formula. Further analysis using hydrogen nuclear magnetic resonance (NMR) spectra... 1 The characteristic peak of H NMR (400 MHz, D2O) is δ[6.65 (d, J = 1.4 Hz, 1H), 6.27 (d, J =13.3 Hz, 1H), 3.48 (t, 1H), 2.97 (dd, J = 14.5, 7.4 Hz, 1H), 2.78 (dd, J =13.5, 10.0 Hz, 1H)]; Carbon NMR spectrum 13 C NMR (101 MHz, D2O) showed the corresponding carbon skeleton signal at δ [182.78, 167.16, 163.58, 161.16, 125.01, 118.76, 110.77, 104.80, 104.59, 56.37, 33.72]; in addition, the NMR fluorine spectrum... 19 The characteristic peaks of F NMR (377 MHz, D2O) show δ [-75.56, -113.29.], which fully confirms the structure of this halotyrosine.

[0118] (5) The substrate was 2-fluoro-1,3-benzenediol, and the non-natural amino acid was 2-hydroxy-3-fluoro-L-tyrosine. The mutation sites of the TPL mutant were: (a) M379T, (b) S12A and M379V, or (c) S12V and M379V. 2-hydroxy-3-fluoro-L-tyrosine was designated nY5, and its high-resolution mass spectrometry (ESI-HRMS) results showed that the molecular ion peak [M+H]... + The measured value is m / z = 216.0661, which is in excellent agreement with the theoretically calculated value m / z = 216.0627, confirming its molecular formula. Further analysis using hydrogen nuclear magnetic resonance (NMR) spectra... 1 The characteristic peak of H NMR (400 MHz, D2O) is δ[6.48 (d, J = 9.0 Hz, 1H), 5.86 (t, J =8.5 Hz, 1H), 3.36 (dd, J = 9.5, 4.4 Hz, 1H), 2.89 (dd, J = 13.7, 4.4 Hz, 1H), 2.38 (dd, J = 13.7, 9.6 Hz, 1H)]; Carbon NMR spectrum 13 C NMR (101 MHz, D2O) showed the corresponding carbon skeleton signal at δ [183.41, 153.91, 153.04, 152.91, 149.14, 146.93, 123.95, 115.59, 105.42, 56.77, 36.15]; in addition, the NMR fluorine spectrum... 19 The characteristic peaks of F NMR (377 MHz, D2O) showed δ [-75.56, -161.78], which fully confirmed the structure of this hydroxyfluorotyrosine.

[0119] (6) The substrate is 2-chloro-1,3-benzenediol, and the non-natural amino acid is 2-hydroxy-3-chloro-L-tyrosine. The mutation sites of the TPL mutant are: (a) V283T, M288G, and M379S, (b) M379C, or (c) M379V and T406A. 2-hydroxy-3-chloro-L-tyrosine is designated nY6, and its high-resolution mass spectrometry (ESI-HRMS) results show that the molecular ion peak [M+H]... + The measured value is m / z = 232.0365, which is in excellent agreement with the theoretically calculated value of m / z = 232.0332, confirming its molecular formula. Further analysis using hydrogen nuclear magnetic resonance (NMR) spectra... 1The characteristic peaks of H NMR (400 MHz, D2O) are δ[6.62 (s, 1H), 3.37 (dd, 1H), and 3.37 (dd, 1H). J =9.6, 4.2 Hz, 1H), 2.92 (dd, J = 13.7, 4.2 Hz, 1H), 2.34 (dd, J = 13.6, 9.7 Hz, 1H)]; Carbon NMR spectrum 13 C NMR (101 MHz, D2O) showed corresponding carbon skeleton signals at δ [183.46, 161.95, 161.66, 161.61, 128.04, 127.93, 114.58, 114.48, 104.50, 56.56, 37.16], fully confirming the structure of this hydroxychlorotyrosine.

[0120] Example 9: Application of novel non-natural amino acids in optimizing the optical properties of green fluorescent protein To further verify the practical application potential of the six novel non-natural amino acids (nY1-nY6) screened in this invention in protein engineering, this embodiment explores the effects of these novel non-natural amino acids on the optical properties of green fluorescent protein (GFP).

[0121] The luminescence of wild-type GFP depends on the stable presence of its internal chromophore. The D190 site of GFP is located near the surface of its β-barrel structure. Studies have shown that by using an orthogonal translation system to precisely introduce halogenated non-natural amino acids (such as tyrosine derivatives containing fluorine, chlorine, or bromine) containing specific electron-withdrawing groups or larger atomic radii at this site, a local halogen bond network can be formed between the halogen atom and neighboring amino acid residues or water molecules. This non-covalent interaction significantly enhances the local rigidity of the β-barrel structure of GFP, thereby limiting conformational fluctuations of the chromophore in the excited state, reducing non-radiative energy dissipation, and ultimately improving the fluorescence intensity and thermal stability of GFP.

[0122] Using evolutionary systems to screen for specific recognition Figure 20Six novel non-natural amino acid-based chimeric pyrrolidone-lysyl-tRNA synthetase (chPylRS) mutants were induced to express in *E. coli* containing the GFP-D190TAG gene (SEQ ID NO.7) by adding these six novel non-natural amino acids. After cell collection, the GFP mutant proteins were purified by Ni-NTA affinity chromatography, and all protein samples were uniformly diluted to the same concentration using a BCA protein concentration assay kit. The normalized proteins were divided into an untreated group and a heat-treated group. The relative fluorescence intensity of each mutant protein in the untreated group was measured using a multi-mode microplate reader (excitation wavelength 488 nm, emission wavelength 509 nm). Simultaneously, the heat-treated proteins were heated at 80°C for 15 minutes and then rapidly cooled in an ice bath, and their residual fluorescence intensity was measured. The ratio of fluorescence intensity between the heat-treated group and the untreated group was calculated as the fluorescence retention rate.

[0123] The results are as follows Figure 21 and Figure 22 As shown. Figure 21 This study demonstrates the recognition and efficient incorporation mechanism of novel non-natural amino acids by specific chPylRS mutants. Figure 22 Relative fluorescence intensity tests showed that, under equal concentration conditions, GFP mutants incorporating polyhalogenated non-natural amino acids (especially those incorporating 2-chloro-6-fluoro-L-tyrosine and 2-bromo-6-fluoro-L-tyrosine) exhibited significantly enhanced single-molecule fluorescence intensity compared to the wild-type (WT) GFP, with their chromophores protected by a more rigid network. Thermal stability tests further demonstrated that the wild-type GFP retained only 37% of its fluorescence under 80°C heat stress, indicating severe thermal defolding of its β-barrel structure; while the introduction of novel polyhalogenated amino acids significantly improved the fluorescence retention of the mutants (exceeding 50%). These results fully demonstrate that the strong halogen bond network formed by the novel polyhalogenated non-natural amino acids significantly enhances the microscopic rigidity of the protein, effectively resisting thermal denaturation. This not only confirms the success of the biocatalyst directed evolution system of this invention but also showcases the enormous industrial value of novel non-natural amino acids in protein engineering.

[0124] In summary, the results of the embodiments fully demonstrate the feasibility and value of the biocatalyst directed evolution system of the present invention in industrial applications.

[0125] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

[0126] The nucleotide and amino acid sequences involved in this invention are specifically as follows: The amino acid sequence of TEM-1 β-lactamase (AmpR) (SEQ ID NO.1, underlined portion is the insertion site of TAG / non-natural amino acid): MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTF K VLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELT A FLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*.

[0127] Amino acid sequence of aminoglycoside phosphotransferase (KanR) (SEQ ID NO.2, underlined portion is the insertion site of TAG / non-natural amino acid): MSHIQRETSCSRPRLNSNMDADLYGYKWARD N VGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRV F RLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*.

[0128] The amino acid sequence of the tetracycline efflux MFS transporter (TetR) (SEQ ID NO.3, underlined portion is the insertion site of TAG / non-natural amino acid): MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHYGVLLALYALMQFLCAPVLGALSD RFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRIVAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLCGCFLMQESHKGERRPMPLRA F NPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*.

[0129] The amino acid sequence of chloramphenicol acetyltransferase (CmR) (SEQ ID NO.4, underlined portion is the insertion site of TAG / non-natural amino acid): MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFLKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSSLWSEYHD D FRQFLHIYSQDVAC Y GENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA*.

[0130] The amino acid sequence (SEQ ID NO. 5) of the parent of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant (chPylRS-IPYE, including the mutation sites (underlined parts): V31I, T56P, H62Y and A100E): MDKKPLDVLISATGLWMSRTGTLHKIKHYE I SRSKIYIEMACGDHLVVNNSRSCR P ARAFR Y HKYRKTCKRCRVSDEDINNFLTRSTEGKTSVKVKVVS EPKVKKAMPKSVSRAPKPLENPVSAKASTDTSRSVPSPAKSTPNSPVPTSASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNF CLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*.

[0131] The amino acid sequence of the toxic protein Barnase (SEQ ID NO.6, underlined portion indicates the insertion site of a TAG / non-natural amino acid): MA Q VINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLA D VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR*.

[0132] The amino acid sequence of green fluorescent protein GFP-6×His (SEQ ID NO.7, underlined portion is the insertion site of TAG / non-natural amino acid): MGKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIG D GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGPHHHHHH*.

[0133] The amino acid sequence of the parent of the tyrosine phenol lyase (TPL) mutant (SEQ ID NO.8): MNYPAEPFRIKSVETVSMIPRDERLKKMQEAGYNTFLLNSKDIYIDLLTDSGTNAMSDKQWAGMMMGDEAYAGSENFYHLERTVQELFGFKHIVPTHQGRGAENLLSQLAIKPGQYVAGNMYFTTTRYHQEKNGAVFVDIVRDEAHDAGLNIAFKGDIDLKKLQKLIDEKGAENIAYICLAVTVNLAGGQPVSMANMRAVRELTEAHGIKVFYDATRCVENAYFIKEQEQGFENKSIAEIVHEMFSYADGCTMSGKKDCLVNIGGFLCMNDDEMFSSAKELVVVYEGMPSYGGLAGRDMEAMAIGLREAMQYEYIEHRVKQVRYLGDKLKAAGVPIVEPVGGHAVFLDARRFCEHLTQDEFPAQSLAASIYVETGVRSMERGIISAGRNNVTGEHHRPKLETVRLTIPRRVYTYAHMDVVADGIIKLYQHKEDIRGLKFIYEPKQLRFFTARFDYI*.

[0134] Amino acid sequence of 6×His-TPL (SEQ ID NO. 9): MGSSHHHHHHSSGLVPRGSHMNYPAEPFRIKSVETVSMIPRDERLKKMQEAGYNTFLLNSKDIYIDLLTDSGTNAMSDKQWAGMMMGDEAYAGSENFYHLERTVQELFGFKHIVPTHQGRGAENLLSQLAIKPGQYVAGNMYFTTTRYHQEKNGAVFVDIVRDEAHDAGLNIAFKGDIDLKKLQKLIDEKGAENIAYICLAVTVNLAGGQPVSMANMRAVRELTEAHGIKVFYDATRCVENAYFIKEQEQGFENKSIAEIVHEMFSYADGCTMSGKKDCLVNIGGFLCMNDDEMFSSAKELVVVYEGMPSYGGLAGRDMEAMAIGLREAMQYEYIEHRVKQVRYLGDKLKAAGVPIVEPVGGHAVFLDARRFCEHLTQDEFPAQSLAASIYVETGVRSMERGIISAGRNNVTGEHHRPKLETVRLTIPRRVYTYAHMDVVADGIIKLYQHKEDIRGLKFIYEPKQLRFFTARFDYI*.

[0135] The nucleotide sequence of the CmR-112TAG selection tag (SEQ ID NO. 10): atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggagttccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacTAGttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcactatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtctgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaa。

[0136] Nucleotide sequence of orthogonal tRNA (SEQ ID NO. 11): CTTTTCCAGCATGACATCATCTCGATTCCTGATAATAGGCAAGCCCAACTAAGGGCCCCAAAGGCCGGT。

[0137] Amino acid sequence of TrpB-TmAzul-6×His (SEQ ID NO. 12): MKGYFGPYGGQYVPEILMGALEELEAAYEGIMKDESFWKEFNDLLRDYAGRPTPLYFARRLSEKYGARVYLKREDLLHTGAHKINNAIGQVLLAKLMGKTRIIAETGAGQHGVATATAAALFGMECVIYMGEEDTIRQKLNVERMKLLGAKVVPVKSGSRTLKDAIDEALRDWITNLQTTYYVSGSVVGPHPYPIIVR NFQKVIGEETKKQIPEKEGRLPDYIVACVSGGSNAAGIFYPFIDSGVKLIGVEAGGEGLETGKHAASLLKGKIGYLHGSKTFVLQDDRGQVQVSHSVSA GLDYSGVGPEHAYWRETGKVLYDAVTDEEALDAFIELSRLEGIIPALESSHALAYLKKINIKGKVVVVNLSGRGDKDLESVLNHPYVRERIRHHHHHH*.

[0138] The amino acid sequence of the chimeric phenylalanine-tRNA synthetase mutant (chPheRS-IPYE-1-B2, including the mutation sites (underlined): V31I, T56P, H62Y, and A100E) (SEQ ID NO.13): MDKKPLDVLISATGLWMSRTGTLHKIKHYE I SRSKIYIEMACGDHLVVNNSRSCR P ARAFR Y HKYRKTCKRCRVSDEDINNFLTRSTEGKTSVKVKVVS EPKVKKAMPKSVSRAPKPLENPVSAKASTDTSRSVPSPPAKSTPNSVPTSASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEEREGGSGGGSGGGSGGGSGGGSGGGSQAWGSRPPAAECATQRAPGSVVELLGKSYPQDDHSNLTRKVLTRVGRNLHNQQHHPLWLIKERVKEHFYKQYVGRFGTPLFSVYDNLSPVVTTWQNFDSLLIPADHPCRKKGDNYYLNRTHMLRAHTSAHQWDLLHAGLDAFLVVGDVYRRDQIDSQHYPIFHQLDAVRLFSKHELFAGIKDGESLQLFEQSSRSAHKQETHTMEAVKLVEFDLKQTLTRLMAHLFGDELEIRWVDCYVPFGHPSFEMEINFHGEWLEVLGCGVMEQQLVNSAGAQDRIGWGFGLGLERLAMILYDIPDIRLFWCEDERFLKQFCVSNINQKVKFQPLSK*.

Claims

1. A directed evolution system for biocatalysts, characterized in that, include: First carrier and second carrier; The first vector encodes an antibiotic resistance protein and orthogonal tRNA; The second vector encodes the biocatalyst mutant to be screened and the aminoacyl-tRNA synthetase mutant homologous to the orthogonal tRNA; in, When the biocatalyst mutant to be screened is the target biocatalyst mutant, it can catalyze the substrate to generate non-natural amino acids; The coding sequence of the antibiotic resistance protein contains a stop codon; When the non-natural amino acid is absent, the stop codon interrupts the expression of the antibiotic resistance protein, resulting in the loss of antibiotic activity. When the non-natural amino acid is present, the orthogonal tRNA and aminoacyl-tRNA synthetase mutant incorporates the non-natural amino acid into the stop codon site, thereby enabling the complete expression of the antibiotic resistance protein and preserving the antibiotic activity.

2. The directed evolution system for biocatalysts according to claim 1, characterized in that, The antibiotic resistance protein is selected from one or more of TEM-1 β-lactamase, aminoglycoside phosphotransferase, tetracycline efflux MFS transporter and chloramphenicol acetyltransferase; The amino acid sequence of the TEM-1 β-lactamase before the introduction of the stop codon is shown in SEQ ID NO.

1. After the introduction of the stop codon, the codons at positions K71 and / or A148 in SEQ ID NO.1 are replaced with the stop codon. The amino acid sequence of the aminoglycoside phosphotransferase before the introduction of the stop codon is shown in SEQ ID NO.

2. After the introduction of the stop codon, the codons at positions N32 and / or F147 in SEQ ID NO.2 are replaced with the stop codon. The amino acid sequence of the tetracycline efflux MFS transporter before the introduction of the stop codon is shown in SEQ ID NO.

3. After the introduction of the stop codon, the codons at positions R69 and / or F197 in SEQ ID NO.3 are replaced with the stop codon. The amino acid sequence of the chloramphenicol acetyltransferase before the introduction of the stop codon is shown in SEQ ID NO.

4. After the introduction of the stop codon, the codons at positions D112 and / or Y127 in SEQ ID NO.4 are replaced with the stop codon. And / or, the stop codon is the TAG stop codon.

3. The directed evolution system for biocatalysts according to claim 1, characterized in that, The biocatalyst mutant is a tyrosine phenol lyase mutant; The aminoacyl-tRNA synthetase mutant is a chimeric pyrrololysyl-tRNA synthetase mutant, and the amino acid sequence of its parent is shown in SEQ ID NO.5; The substrate, non-natural amino acid, and chimeric pyrrolidyl-tRNA synthetase mutant are selected from one or more of the following combinations: (1) The substrate is 2-chlorophenol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, N311C, C313W, Y349F, V366T and W382T; (2) The substrate is 2-bromophenol, the non-natural amino acid is (S)-2-amino-3-(3-bromo-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, N311C, C313W, Y349F, V366T and W382T; (3) The substrate is 2-iodophenol, the non-natural amino acid is (S)-2-amino-3-(4-hydroxy-3-iodophenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, N311C, C313W, Y349F, V366T and W382S; (4) The substrate is 2-nitrophenol, the non-natural amino acid is (S)-2-amino-3-(4-hydroxy-3-nitrophenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are M265T, L270I, L274G, D278G, N311G, C313R and Y349F; (5) The substrate is 2,3-dihydro-1H-indene-4-ol, the non-natural amino acid is (S)-2-amino-3-(7-hydroxy-2,3-dihydro-1H-indene-4-yl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are I253V, F260L, L270I, Y271F, Y273F, L274G, Y294F, N311G, C313W, T329A, Y349F and W382T; (6) The substrate is 2-chloro-6-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-5-fluoro-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are I253V, L270I, Y271F, Y273F, L274G, Y294F, N311G, C313W, Y349F and W382T; (7) The substrate is 3-chloro-5-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(2-chloro-6-fluoro-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, E302V, N311C, C313W, Y349F, A365G, V366H and W382T; (8) The substrate is 3-bromo-5-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(2-bromo-6-fluoro-4-hydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, N311C, C313W, D344G, Y349F, A365G, V366H and W382T; (9) The substrate is 2-fluorophenyl-1,3-diol, the non-natural amino acid is (S)-2-amino-3-(3-fluoro-2,4-dihydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are F260S, L270I, L274G, E302V, N311C, C313W, Y349F, A365G, V366H and W382T; (10) The substrate is 2-chlorophenyl-1,3-diol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-2,4-dihydroxyphenyl)propionic acid, and the mutation sites of the chimeric pyrrolidone-lysyl-tRNA synthetase mutant are L270I, L274G, E302V, N311C, C313W, Y349F, A365G, V366H and W382T.

4. The directed evolution system for biocatalysts according to claim 1, characterized in that, The nucleotide sequence of the orthogonal tRNA is shown in SEQ ID NO.10; the nucleotide sequence of the antibiotic resistance protein is shown in SEQ ID NO.

11.

5. A method for directed evolution of biocatalysts, characterized in that, Including the following steps: The directed evolution system of the biocatalyst according to any one of claims 1-4 is transformed into a host cell, a substrate and an antibiotic corresponding to the antibiotic resistance protein are added, and stress screening is performed. The surviving host cells contain the target biocatalyst mutant that can catalyze the substrate to generate non-natural amino acids.

6. A biocatalyst mutant, characterized in that, The biocatalyst was screened using the directed evolution system of any one of claims 1-4 or the directed evolution method of claim 5. Preferably, the biocatalyst mutant is a tyrosine phenol lyase mutant, the parent amino acid sequence of which is shown in SEQ ID NO.8, and the mutation site is selected from one of the following: (1) S12A and M379V; (2) S12V and M379V; (3) F36P; (4) R82E; (5) M379C; (6) M379P; (7) M379A; (8) M379V; (9) M379T; (10) F36C and M379C; (11) F36C and M379V; (12) F36T and M379C; (13) V283G, M288S and M379V; (14) M288S and M379V; (15) M288F and M379S; (16) F448C; (17) F36L and M379V; (18) V283T, M288G and M379S.

7. A method for synthesizing non-natural amino acids, characterized in that, The biocatalyst mutants obtained by screening using the biocatalyst directed evolution system according to any one of claims 1-4 or the biocatalyst directed evolution method according to claim 5 can catalyze the synthesis of substrates into non-natural amino acids in cells.

8. The method for synthesizing non-natural amino acids according to claim 7, characterized in that, The biocatalyst mutant is a tyrosine phenol lyase mutant, and the amino acid sequence of its parent is shown in SEQ ID NO.8; The substrate, non-natural amino acids, and tyrosine phenol lyase mutant are selected from one or more of the following combinations: (1) The substrate is 2-chlorophenol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) M288S and M379V, (b) M379C, (c) M379P; (2) The substrate is 2-bromophenol, the non-natural amino acid is (S)-2-amino-3-(3-bromo-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) F36C and M379C, (b) M379A, (c) F36C and M379V; (3) The substrate is 2-iodophenol, the non-natural amino acid is (S)-2-amino-3-(4-hydroxy-3-iodophenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) S12A and M379V, (b) F36P, (c) F36T and M379C; (4) The substrate is 2-nitrophenol, the non-natural amino acid is (S)-2-amino-3-(4-hydroxy-3-nitrophenyl)propionic acid, and the mutation sites of the tyrosine phenol lyase mutant are F36L and M379V; (5) The substrate is 2,3-dihydro-1H-indene-4-ol, the non-natural amino acid is (S)-2-amino-3-(7-hydroxy-2,3-dihydro-1H-indene-4-yl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is M379P; (6) The substrate is 3-chloro-5-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(2-chloro-6-fluoro-4-hydroxyphenyl)propionic acid, and the mutation sites of the tyrosine phenol lyase mutant are S12V and M379V; (7) The substrate is 2,6-difluorophenol, the non-natural amino acid is (S)-2-amino-3-(3,5-difluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) R82E, (b) M379C, (c) M379V; (8) The substrate is 2-chloro-6-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-5-fluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) S12A and M379V, (b) M379T, (c) M379C; (9) The substrate is 3,5-difluorophenol, the non-natural amino acid is (S)-2-amino-3-(2,6-difluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) F448C, (b) R82E, (c) M288S and M379V; (10) The substrate is 3-bromo-5-fluorophenol, the non-natural amino acid is (S)-2-amino-3-(2-bromo-6-fluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) V283T, M288G and M379S, (b) M379P, (c) M379L and T406A; (11) The substrate is 2,3-difluorophenol, the non-natural amino acid is (S)-2-amino-3-(2,3-difluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) F36T and M379C, (b) V283G, M288S and M379V, (c) M288S and M379V; (12) The substrate is 2,3,5,6-tetrafluorophenol, the non-natural amino acid is (S)-2-amino-3-(2,3,5,6-tetrafluoro-4-hydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) M288F and M379S, (b) M379C, (c) M379T; (13) The substrate is 2-fluoro-1,3-benzenediol, the non-natural amino acid is (S)-2-amino-3-(3-fluoro-2,4-dihydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) M379T, (b) S12A and M379V, (c) S12V and M379V; (14) The substrate is 2-chloro-1,3-benzenediol, the non-natural amino acid is (S)-2-amino-3-(3-chloro-2,4-dihydroxyphenyl)propionic acid, and the mutation site of the tyrosine phenol lyase mutant is selected from one of the following: (a) V283T, M288G and M379S, (b) M379C, (c) M379V and T406A.

9. Non-natural amino acids, characterized in that, The non-natural amino acid is selected from one or more of the following structures: 。 10. The application of the non-natural amino acid as described in claim 9, characterized in that, The non-natural amino acids were used for site-specific incorporation into green fluorescent protein to optimize its fluorescence luminescence properties and thermal stability.

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