Tryptophan synthase mutants and their use in the synthesis of chiral tryptophan analogs

Abstract

The application discloses a tryptophan synthetase and a mutant thereof, and application of the mutant of the beta subunit tryptophan synthetase as a catalyst in catalyzing synthesis of chiral tryptophan analogs. The mutant of the tryptophan synthetase has the advantages of high reactivity, high stereoselectivity, wide substrate range and the like, the chiral value of a product is greater than 99%, the purity is greater than 99%, and the highest can reach 99.9%, which is beneficial to realize low-cost production and has an industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a pyridoxal phosphate-dependent β-subunit dimer tryptophan synthase mutant with enhanced catalytic activity and its application in the catalytic synthesis of chiral tryptophan analogs. Background Technology

[0002] Tryptophan is one of the eight essential amino acids that the human body cannot synthesize on its own. As a precursor to many neurotransmitters and hormones, it is not only used in the synthesis of serotonin and melatonin during metabolism, but also participates in the uric acid pathway, producing various neuroregulatory substances such as quinolinic acid and uric acid. Therefore, it is closely related to brain health and immune regulation. Chemical modification of the indole ring on tryptophan can produce a large number of non-natural amino acids with unique biological activities. The applications of these analogues are often highly specific and have significant value in multiple fields such as medicine, chemical engineering, and agriculture.

[0003] For example, L-7-methyltryptophan (CAS 33468-36-9) is a non-natural amino acid, belonging to the 7-methyl-substituted analogues of tryptophan, and selectively inhibits indoleamine 2,3-dioxygenase 1 (IDO1). This target is widely expressed in immune cells and tumor cells, responsible for catalyzing the catabolism of tryptophan in the uric acid pathway. Overexpression and / or overactivation of IDO1 leads to excessive consumption of tryptophan and the production of immunosuppressive metabolites, thereby inhibiting T cells and inducing their apoptosis. L-7-methyltryptophan, by competitively binding to the active site of the IDO1 enzyme, can alleviate tryptophan deficiency, relieve T cell inhibition, and thus enhance the ability of immune cells to clear cancer cells. Therefore, it is often used in research related to cancer immunotherapy. In addition, L-5-fluorotryptophan (5-Fluoro-L-tryptophan, CAS 16626-02-1) is also an important immunomodulator and tumor inhibitor. It not only inhibits IDO1 but can also be misrecognized by tryptophan-tRNA synthetase and used in protein synthesis. The misrecognized L-5-fluorotryptophan accumulates in proteins, disrupting their normal three-dimensional structure, causing functional abnormalities and significant inactivation. This selectively induces apoptosis in metabolically active, rapidly proliferating tumor cells, while having a relatively smaller impact on cells with normal metabolic levels. Tryptophan analogues hold great promise for applications in immunology and antitumor therapy, and their industrial synthesis has therefore attracted considerable attention.

[0004] CN108315370A reports a method for preparing halogenated tryptophan, comprising the following steps: a halogenated indole reacts with formaldehyde and dimethylamine in a Mannich reaction to generate halogenated arundin; the halogenated arundin reacts with diethyl acetaminomalonate under sodium ethoxide catalysis to generate an intermediate diester; the intermediate diester is hydrolyzed under alkaline conditions to obtain acetylhalogenated tryptophan; and the acetylhalogenated tryptophan is enzymatically resolved to obtain halogenated tryptophan. This reaction requires formaldehyde, which has certain biotoxicity, and the reaction steps are complex. Furthermore, the final enzymatic resolution is required to obtain 5-fluorotryptophan, making it unsuitable for industrial production.

[0005] Tryptophan synthase (EC 4.2.1.20) is a pyridoxal phosphate (PLP)-dependent heterotetrameric protein widely found in plants, bacteria, and fungi, primarily involved in the catalytic activity of the last two steps in the tryptophan synthesis pathway. Its four subunits are arranged linearly in the order α-β-β'-α', and each heterodimer αβ possesses independent catalytic activity. The catalytic mechanism is as follows: indole-3-glycerophosphate is cleaved into indole in the α subunit, and then the indole passes through a space approximately 25 cm long between the α-β subunits. The α-subunit enters the β-subunit via a channel, and with the assistance of PLP, the β-subunit dehydrates and condenses indole with L-serine to synthesize L-tryptophan. Studies have shown that the affinity and selectivity of the α-subunit for indole-related derivatives, as well as the transduction efficiency of compounds in the channel between the α and β subunits, limit the preparation of tryptophan analogs containing different substituents. Although the β-subunit (TrpB) is the subunit unit that exhibits activity in the coupling of L-serine and indole, its use as a single independent enzyme remains infeasible. Upon separation from the natural complex αββ'α', TrpB loses up to 95% of its original activity and is highly susceptible to inactivation, with reduced chirality. However, the synthesis of tryptophan analogs using the β-subunit alone remains attractive for industrial production. In a 2015 study, Arnold et al. directed the evolution of a β-subunit tryptophan synthase based on wild-type enzyme proteins from the species Pyrococcus furiosus, achieving activity exceeding that of the natural complex αββ'α' (300 mM). -1 s -1 vs. 50mM -1 s -1However, the substrate concentrations of L-serine and indole are only 20 mM, which is still some distance from industrial production (Directed evolution of the tryptophansynthase β-unit for stand-alone function recapitulates allosteric activation, 2015, 112(47):14599-14604). There is an urgent need in this field for a β-subunit tryptophan synthase with high catalytic performance to realize the production of L-tryptophan from L-serine and indole, significantly reduce production costs, and thus realize the large-scale industrial production of tryptophan analogues.

[0006] CN119307565A reports an enzymatic method for synthesizing L-7-methyltryptophan. Using L-serine as a substrate, L-serine, 7-methylindole, pyridoxal 5-phosphate, water, and tryptophan synthase are added to the reaction system to obtain crude L-7-methyltryptophan. The reaction temperature is 25–40℃, the pH is 6.5–7.5, and the mass ratio of L-serine to 7-methylindole is 1:1.3–1.5. This method has a low substrate concentration (approximately 15 g / L–20 g / L), low equivalent of inexpensive L-serine, and high equivalent of 7-methylindole, resulting in low product conversion and high production costs. Using 7-methylindole as the metric, the tryptophan synthase used in this method has low efficiency, with a 7-methylindole to catalytic bacterial sludge weight ratio of 1.3:1, and a conversion rate of 96%. Insufficient conversion leads to difficulties in post-processing for substrate removal, thus affecting product quality and cost. Furthermore, its post-processing involves distillation, which is energy-intensive and difficult to control, resulting in a final product purity of 98%, lower than the 99.5% purity of this invention.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide an engineered β-subunit tryptophan synthase mutant with high stereoselectivity, high catalytic activity, and good stability, which can be used to prepare a variety of chiral tryptophan analogs, especially the asymmetric synthesis of tryptophan analogs such as L-7-methyltryptophan and L-5-fluorotryptophan. The invention also provides the gene for the engineered tryptophan synthase, a recombinant expression vector containing the gene, an engineered strain and its efficient preparation method, as well as a reaction process for preparing L-7-methyltryptophan using the engineered methyltryptophan synthase.

[0009] The first aspect of the present invention is to provide a tryptophan synthase mutant, characterized in that it is a tryptophan synthase mutant with mutations at one, two, three or more sites, including sites 15, 20, 71, 112, 169, 181, 185, 225, 236, 269, 276, 284, 294, 302 and 323, of the amino acids corresponding to sites 1-389 shown in SEQ ID No. 2.

[0010] Furthermore, the mutations are: P15L / Q; E20G / P / Q / S; I71V; Q112N; N169T; Y181T; L185V; V225I; M236V; N269T; F276L / S / T; L284C / T; T294S; D302N / S; T323A / F; More preferably, the mutation corresponding to amino acids 1-389 shown in SEQ ID No. 2 contains one of the following mutations: P15Q; E20Q; I71V; Q112N; F276T; F276S; T294S; D302S; T323A; T323F; L284T and T323A; N269T, L284C and T323A; N169 and T323F; Y181T and T323F; M236V and T323F; P15L, E20G, I71V, F276S, T294S and T323A; P1 5L, E20G, V225I, T294S and T323A; P15L, I71V, F276S, T294S and T323A; E20P, I71V, F276L and T294S; I71V, F276S, T294S and T323F; E20S, I71V, L185V, F276S, T294S and T323F; I71V, Q112N, N169T, Y181T, M236V, F276S, T294S, D302N and T323F; Further preferably, the mutation in the amino acids corresponding to positions 1-389 of SEQ ID No.2 is one of the following: P15L, E20G, I71V, F276S, T294S and T323A; T323A.

[0011] Most preferably, the amino acid sequence of the tryptophan synthase mutant is selected from the following mutant sequences: The amino acid sequence shown in SEQ ID No:4; the amino acid sequence shown in SEQ ID No:6; The amino acid sequence shown in SEQ ID No:8; the amino acid sequence shown in SEQ ID No:10; The amino acid sequence shown in SEQ ID No:12; the amino acid sequence shown in SEQ ID No:14; The amino acid sequence shown in SEQ ID No:16; the amino acid sequence shown in SEQ ID No:18; The amino acid sequence shown in SEQ ID No:20; the amino acid sequence shown in SEQ ID No:22; The amino acid sequence shown in SEQ ID No:24; the amino acid sequence shown in SEQ ID No:26; The amino acid sequence shown in SEQ ID No:28; the amino acid sequence shown in SEQ ID No:30; The amino acid sequence shown in SEQ ID No:32; the amino acid sequence shown in SEQ ID No:34; The amino acid sequence shown in SEQ ID No:36; the amino acid sequence shown in SEQ ID No:38; The amino acid sequence shown in SEQ ID No:40; the amino acid sequence shown in SEQ ID No:42; The amino acid sequence shown in SEQ ID No:44; the amino acid sequence shown in SEQ ID No:46.

[0012] Simultaneously, this invention includes an engineered β-subunit tryptophan synthase mutant, said mutant being composed of any one of the amino acid sequences shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46; the derived protein includes a derivatized enzyme protein formed by substitution, deletion, or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, and having the function of said protein; the derived protein includes the following corresponding residue positions with SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46. The amino acid sequence differing from the IDNO:2 sequence by one or more residues: X15, X20, X71, X112, X169, X181, X185, X225, X236, X269, X276, X284, X294, X302, X323. Improved tryptophan synthase mutants include amino acid sequences containing at least one of the following characteristics: P15L / Q, E20G / P / Q / S, I71V, Q112N, N169T, Y181T, L185V, V225I, M236V, N269T, F276L / S / T, L284C / T, T294S, D302N / S, T323A / F, or simultaneously, based on these differences, the insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24 or more amino acid residues.

[0013] Furthermore, the amino acid sequence of the tryptophan synthase mutant has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or higher homology with SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46, respectively. Moreover, the enzyme activity of the mutant, catalyzed by L-serine and compound II to synthesize compound I in a reaction environment of pH 7.5-9.0 and temperature 45℃-65℃, is improved compared to SEQ ID NO: 2.

[0014] A second aspect of the present invention provides a group encoding the aforementioned tryptophan synthase mutant.

[0015] More preferably, and most preferably, the gene sequence encoding the tryptophan synthase mutant is selected from the following sequences: The sequence shown in SEQ ID No:3; the sequence shown in SEQ ID No:5; The sequence shown in SEQ ID No:7; the sequence shown in SEQ ID No:9; The sequence shown in SEQ ID No:11; the sequence shown in SEQ ID No:13; The sequence shown in SEQ ID No: 15; the sequence shown in SEQ ID No: 17; The sequence shown in SEQ ID No:19; the sequence shown in SEQ ID No:21; The sequence shown in SEQ ID No:23; the sequence shown in SEQ ID No:25; The sequence shown in SEQ ID No:27; the sequence shown in SEQ ID No:29; The sequence shown in SEQ ID No:31; the sequence shown in SEQ ID No:33; The sequence shown in SEQ ID No:35; the sequence shown in SEQ ID No:37; The sequence shown in SEQ ID No:39; the sequence shown in SEQ ID No:41; The sequence shown in SEQ ID No:43; the sequence shown in SEQ ID No:45.

[0016] A third aspect of the present invention provides an expression vector containing a group of the aforementioned tryptophan synthase mutant.

[0017] A fourth aspect of the present invention provides recombinant cells containing groups of the aforementioned tryptophan synthase mutant.

[0018] Regarding the second to fourth aspects, the present invention also provides a gene encoding the above-described engineered β-subunit tryptophan synthase mutant. As those skilled in the art know, due to the degeneracy of nucleotide codons, the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 is not limited to SEQ ID No: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45. The nucleic acid sequence of the tryptophan synthase gene of the present invention can also be any other nucleic acid sequence corresponding to the protein sequences shown in SEQ ID No: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 in the coding sequence listing.

[0019] The derived nucleotide sequences also include homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or higher with the nucleotide sequences shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45, and the corresponding mutants exhibit increased enzyme activity compared to SEQ ID NO: 2 in the synthesis of compound I catalyzed by L-serine and compound II in a reaction environment of pH 7.5-9.0 and temperature 45℃-65℃.

[0020] The identity between two amino acid sequences or two nucleotide sequences can be obtained using algorithms commonly used in this field. It can be calculated using NCBI Blastp and Blastn software with default parameters, or it can be calculated using the Clustal W algorithm (Nucleic Acid Research, 22 (22): 4673-4680, 1994).

[0021] The fifth aspect of this invention provides a method for synthesizing a chiral tryptophan analog of formula I catalyzed by tryptophan synthase or a mutant thereof, the reaction formula of which is as follows:

[0022] The tryptophan synthase is derived from the amino acid sequence shown in SEQ ID NO:2, and the tryptophan synthase mutant is obtained by the aforementioned mutation. The substituent R in Formula II is selected from the following groups: , or ; The substituent R1 is selected from one of F, Cl, Br, I, -OH, -CH3, -CH2CH3 or -CH2CH2CH3; The substituent R2 is selected from one of F, Cl, Br, I, -OH, -CH3, -CH2CH3 or -CH2CH2CH3.

[0023] More preferably, the compound of formula II is selected from one of the following , , ,or The compounds produced can be L-7-methyltryptophan and L-5-fluorotryptophan, etc. Furthermore, the concentration loading of the compound of formula II is 10-110 g / L. And / or, the amount of tryptophan synthase or its mutant is 10-40 g / L, and / or, the reaction pH range is 7.0-9.0, and / or, the buffer pH range is 7.0-9.0, and / or, the conversion rate is ≥99%, and / or, the chiral ee(R) is ≥99.00%.

[0024] More preferably, the concentration loading of the compound of formula II is 90 g / L, and / or the amount of tryptophan synthase or its mutant is 25 g / L, and / or the reaction temperature is 20-90℃, preferably 60℃, and / or the reaction pH is 8.5, and the enzyme catalytic reaction time is 18 h.

[0025] In some embodiments, engineered tryptophan synthase mutants, in the form of intact cells, crude extracts, isolated mutants, or purified mutants, can be used alone or in an immobilized form (e.g., immobilized on resin). These engineered tryptophan synthase mutants can be used to produce L-7-methyltryptophan and L-5-fluorotryptophan. Therefore, in another aspect, the present invention provides a highly efficient process for the synthesis of L-7-methyltryptophan and the preparation of L-5-fluorotryptophan. Specifically: Step (1): In a 0.05-1M pH 8.0 PB or other equivalent buffer system, with a concentration of 10-110 g / L of compound II, add 1.0-2 equivalents of substrate L-serine and enzyme solution.

[0026] Preferably, in step (1), the compound of formula II can be one of 7-methylindole, 5-fluoroindole, 3-methylindole, or 5-hydroxyindole; more preferably, it can be 7-methylindole. The substrate concentration is 10-110 g / L, more preferably 10-90 g / L, and the L-serine equivalent is 1.0-2.5. The enzyme form is 5-40 g / L of engineered bacterial wet cells containing the target enzyme. Step (2): Stir the reaction at 45℃-65℃ for 12-20h. The conversion rate of compound II can reach 99% or more as tested.

[0027] Preferably, the reaction temperature in step (2) is 50-65℃; the catalytic reaction time is 18-20h.

[0028] Step (3): Post-treatment involves adjusting the pH to 3.5 or below with acid, filtering to remove proteins, and then adjusting the pH to 6-7.5 with alkali to precipitate the product. The product has a chirality value >99%, a purity greater than 99%, and can reach up to 99.9%. Preferably, the acidification process in the post-treatment can use hydrochloric acid, acetic acid, formic acid, sulfuric acid, citric acid, etc.; more preferably, hydrochloric acid and sulfuric acid. The alkali adjustment process in the post-treatment can use sodium hydroxide, potassium hydroxide, ammonia, sodium oxide, potassium oxide, etc.; more preferably, sodium hydroxide, potassium hydroxide, and ammonia. The protein removal filtration equipment can be a ceramic membrane, an ultrafiltration membrane, or a vacuum filter, plate and frame filter, etc.; more preferably, a ceramic membrane device.

[0029] The following is an explanation and description of the terminology used in this invention: Thermococcus taurensis with the amino acid sequence SEQ ID NO:2 Thermococcus kodakarensis tryptophan synthase from [source] Tk TrpB is the starting point for the improvement of this invention. The inventors screened out some mutants with significantly enhanced enzyme activity by mutating it.

[0030] As used in this invention, "wild-type tryptophan synthase" refers to a naturally occurring, unmodified tryptophan synthase whose nucleotides can be obtained through genetic engineering techniques, such as genome sequencing and polymerase chain reaction (PCR), and whose amino acid sequence can be deduced from the nucleotide sequence. The amino acid sequence of the wild-type tryptophan synthase is shown in SEQ ID NO:2.

[0031] The mutant protein of this invention, as used herein, is referred to by the terms "mutant," "tryptophan synthase mutant protein," "mutant protein," "tryptophan synthase mutant protein," "mutant protein of this invention," "tryptophan synthase mutant protein of this invention," and "tryptophan synthase mutant of this invention," which are used interchangeably and all refer to a non-naturally occurring tryptophan synthase. The mutant protein is an artificially modified protein based on the protein shown in SEQ ID NO:2, and the mutant protein of this invention has highly efficient catalytic activity to generate optically pure L-type tryptophan-derived compounds.

[0032] Using homology modeling, molecular docking was performed on the preferred substrates in the examples based on the model structure. The docking results were analyzed, and 15 residues that may affect the catalytic properties of the protein were selected. These residues include: X15, X20, X71, X112, X169, X181, X185, X225, X236, X269, X276, X284, X294, X302, and X323. These residues were subjected to saturation mutagenesis and site-directed mutagenesis using whole plasmid PCR.

[0033] As used in this article, the term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, "A28G" indicates that alanine A at position 28 is mutated to glycine G, and so on.

[0034] "Saturation mutagenesis" is a method that modifies the gene encoding a target protein to obtain mutants in a short period of time, where the target amino acid is replaced by one of 19 other amino acids. This method is not only a powerful tool for targeted protein modification but also an important means of studying protein structure-function relationships. Saturation mutagenesis often yields more desirable evolutionary variants than single-point mutagenesis. Furthermore, saturation mutagenesis excels at addressing problems that site-directed mutagenesis cannot solve. The mutants obtained through saturation mutagenesis are sequenced and identified, and their activity against different substrates and tolerance to high temperatures are tested.

[0035] Site-directed mutagenesis refers to the introduction of desired changes (usually changes indicating a favorable direction) into a target DNA fragment (which can be genomic or plasmid) using methods such as polymerase chain reaction (PCR). These changes include base addition, deletion, and point mutations. Site-directed mutagenesis can rapidly and efficiently improve the characteristics and characterization of the target protein expressed by the DNA, making it a very useful tool in gene research. The method of introducing site-directed mutagenesis using whole plasmid PCR is simple and effective, and is currently widely used. The principle is that a pair of primers (forward and reverse) containing the mutation site, along with a template plasmid, are annealed and then subjected to "cyclic extension" using polymerase. (Cyclic extension refers to the polymerase extending the primers according to the template, returning to the 5' end of the primer after one cycle, and then repeating the cycle of heating and annealing. This reaction differs from rolling circle amplification and does not form multiple tandem copies.) The extension products of the forward and reverse primers are annealed and paired to form a notched open circular plasmid. The Dpn I digestion extension product, since the original template plasmid is derived from conventional E. coli and is modified by dam methylation, is sensitive to Dpn I and is cleaved. However, the in vitro synthesized plasmid with the mutant sequence is not methylated and is not cleaved. Therefore, it can be successfully transformed in the subsequent transformation, and the clone of the mutant plasmid can be obtained.

[0036] The mutants screened from the mutant library were subjected to third-generation sequencing. Based on the sequencing results, suitable mutants were selected for amplification reaction activity testing. After multiple rounds of evolution, the inventors obtained a series of engineered β-subunit tryptophan synthase mutants. These mutants showed significantly improved activity and selectivity for a variety of indole-derived substrates. These mutants can be used for industrial production, with greatly enhanced catalytic efficiency.

[0037] In this invention, homology refers to the "sequence identity" between two amino acid sequences, that is, the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For instance, it can be determined using the BLAST program from the NCBI database. For information on the determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Primers for Sequence Analysis, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991.

[0038] The proteins described above that share 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9%) homology with the mutants shown in SEQ ID NO: 2 and possess tryptophan-like synthesis function, have active sites, active pockets, active mechanisms, and protein structures that are highly likely to be the same as the proteins provided in (a) of the corresponding proteins.

[0039] "Amino acid residues" can be represented by three-letter or one-letter amino acid codes according to standards known and agreed upon in the art. In this invention, the abbreviations for amino acid residues are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0040] Conservative amino acid substitutions or replacements are well known in the art; for example, the following group of conservative amino acid substitutions is preferred (1). (5) One amino acid residue is replaced by another amino acid in the same group: (1) Smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (2) Residues with negative polar charge and their (uncharged) amides: Asp, Asn, Glu and Gln; (3) Residues with positive polar charge: His, Arg and Lys; (4) Larger aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and (5) Aromatic residues: Phe, Tyr and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by lysine; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; and Val is replaced by Ile or Leu.

[0041] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "Blosum62 scoring matrix" in the prior art.

[0042] The improved engineered tryptophan synthase mutant provided by this invention has better activity and / or stability compared with the wild type corresponding to SEQ ID NO:2, and can prepare L-type tryptophan derivatives asymmetrically with higher stereoselectivity, especially L-7-methyltryptophan with higher efficiency.

[0043] Besides L-7-methyltryptophan, as exemplified above, the engineered tryptophan synthase mutants of this invention can also catalyze the generation of various tryptophan analogs from a variety of indole analogs and benzene ring compounds (such as 5-fluoroindole, 3-methyl-2-indolone, and phenol). The engineered tryptophan synthase mutants disclosed in this invention, SEQ ID Nos: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46, are capable of converting substrates of formula II into products of formula I with superior performance (including higher activity, stereoselectivity, or stability) compared to SEQ ID NO: 2.

[0044] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0045] Beneficial technical effects of the present invention: 1. Through experimental verification, the inventors discovered that a wild-type tryptophan synthase derived from *Thermococcus kodakarensis* can exist relatively stably in the homodimeric ββ' state and exhibits catalytic activity for L-7-methyltryptophan. With the assistance of the coenzyme pyridoxal phosphate, this enzyme can asymmetricly catalyze the conversion of 7-methylindole and serine to L-7-methyltryptophan. The amino acid sequence of this wild-type enzyme is shown in SEQ ID NO:2. However, in the homodimeric state, the catalytic efficiency and stereoselectivity of this wild-type enzyme are reduced to varying degrees. Its catalytic activity and product chiral purity do not meet the requirements of reaction processes in industrial production, and its performance needs further improvement. In order to obtain a highly active and selective tryptophan synthase for industrial synthesis of chiral tryptophan analogs, this invention uses the enzyme-catalyzed synthesis of L-7-methyltryptophan from 7-methylindole and L-serine as a reference application. Using directed evolution technology, SEQ ID NO:2 was engineered to develop a series of engineered tryptophan synthase mutants with high stability, high activity, high stereoselectivity, and a broader substrate spectrum.

[0046] 2. The enzyme of this invention has high efficiency, high substrate and product tolerance, can catalyze reactions at pH 7.5-9, and high temperature tolerance, can catalyze reactions at 45℃-65℃, can complete the conversion with 10g-110g / L indole analogs, the conversion time is about 12-18h, the conversion rate is >99%, which is convenient for commercial application and has high economic benefits.

[0047] 3. The product of this invention has a high chirality value, exceeding 99.0%. The process yield is >84.0%, and the product purity is >99.0%. The L-serine:indole analog molar ratio of 1.0-1.5:1 effectively reduces the consumption of indole analogs, making it more suitable for industrial scale-up. During the reaction process, organic acids such as acetic acid, formic acid, p-toluenesulfonic acid, and citric acid, as well as inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, can be used for acid adjustment; the application range is wide. The reaction and post-processing conditions are mild, without involving hazardous reactions or highly toxic reagents. The process is simple to operate and has a high tolerance for error. It is safe and environmentally friendly for production personnel. Attached Figure Description

[0048] Figure 1 Chiral chromatogram of L-7-methyltryptophan synthesis; Figure 2Chromatogram of the purity of L-7-methyltryptophan synthesis; Figure 3 Chiral chromatogram of L-5-fluorotryptophan synthesis; Figure 4 Chromatogram of the purity of L-5-fluorotryptophan synthesis; Figure 5 Chiral chromatogram of L-tyrosine synthesis; Figure 6 Chromatogram of the purity of L-tyrosine synthesis; Detailed Implementation

[0049] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.

[0050] A single colony of *E. coli* BL21(DE3) containing the target engineered enzyme peptide expression plasmid (SEQ ID NO. 20) was inoculated into LB medium containing chloramphenicol (5.0 g / L Yeast Extract, 10 g / L Tryptone, 10 g / L Sodium Chloride). The culture was incubated at 30°C with shaking at 250 rpm for 16 h. When the OD600 of the culture was 3.5 to 5.0, the culture was removed from the shaker.

[0051] A 1.0L fermenter containing 0.6L of basal fermentation medium was sterilized in an autoclave at 121°C for 30 minutes. The shake-flask culture medium described above was then inoculated into the fermenter. The fermentation temperature was maintained at 37°C, and the agitator speed was controlled within the range of 200-1000 rpm. Air was supplied to the fermentation vessel at a rate of 0.4-0.8 L / min to maintain a dissolved oxygen level above 35%. The pH of the culture medium was maintained at 7.0 by adding 25-28% v / v ammonium hydroxide. Microbial growth was maintained by feeding a feed solution primarily containing 500 g / L dextrose monohydrate. When the OD600 reached 25±5, the culture temperature was lowered and maintained at 30°C. Then, α-lactose monohydrate was added to induce the expression of the target enzyme. Fermentation continued for approximately 16 hours before the fermentation broth was collected. The fermentation broth was centrifuged at 8000 rpm for 10 minutes at 4°C using a Thermo Multifuge X3R centrifuge to collect the wet cells. The collected wet bacterial cells can be used directly in the following post-processing steps, or they can be frozen and stored at -20°C until use. Example 1: Screening of improved engineered tryptophan synthase Table 1 below illustrates the catalytic performance of the engineered tryptophan synthase and its mutants for L-7-methyltryptophan developed in this invention. Each row provides the nucleotide and amino acid sequence numbers of a specific engineered tryptophan synthase mutant, along with the residue differences compared to SEQ ID No:2. The catalytic performance of each example engineered tryptophan synthase mutant is expressed as the fold increase in activity compared to SEQ ID No:2 and its chiral selectivity ee (%). Specific screening conditions were: 7-methylindole 2 g / L, L-serine 2 g / L, PLP 0.12 g / L, DMSO 20%, 0.1 M PB pH 9.0, temperature 60 °C, and time activity - 18 h selectivity (18 h).

[0052] Table 1. Screening of catalytic activity ; As can be seen, SEQ ID NO.34 showed the highest activity enhancement rate of 3.48, with an ee% of 99.76%.

[0053] Example 2: Engineered tryptophan synthase mutant SEQ ID NO.34 catalyzes the synthesis of tryptophan analogs. Add 10 mL of 0.1 M pH 8.0 PB, 0.01 g PLP, 0.3 g wet bacterial cells (SEQ ID NO. 34), 0.1 g L-serine, and 0.1 g substrate to a 20 mL glass bottle. Maintain the temperature at 55 °C and stir the reaction for 15-18 h. After the reaction is completed, take a sample and perform HPLC to detect the formation of product 2a-2d. The conversion rate and chirality are shown in Table 2 below.

[0054] Table 2 Catalytic asymmetric synthesis of tryptophan analogues ; Example 3: Synthesis of L-7-methyltryptophan (SEQ ID NO.34) Add 1L of 0.1M pH 8.0 PB, 1.33g PLP, 300g wet bacterial cells (SEQ ID NO. 34), 100g L-serine, and 100g 7-methylindole substrate to a 2L reaction flask. Maintain the temperature at 55℃ and stir for 15-18h. After the reaction, take a sample and perform HPLC to detect the formation of L-7-methyltryptophan and calculate the conversion rate. Adjust the pH of the reaction solution to 1 with concentrated hydrochloric acid, dissolve the enzyme, filter, and adjust the pH of the filtrate to 6.9 with sodium hydroxide. Stir for 3h, and filter to obtain 149.4g of white solid. After further processing and separation, the yield is 90%. The chirality value is 99.85%, and the purity is 99.59%. See attached figure. Figure 1 and Figure 2 .

[0055] Example 4: Synthesis of L-5-Fluorottryptophan (SEQ ID NO.20) In a 2L reaction flask, add 1L of 0.1M pH 8.0 PB, 0.12g PLP, 35g of wet bacterial cells (SEQ ID NO. 20), 90g of L-serine, and 90g of 5-fluoroindole. Maintain the temperature at 60℃ and stir for 15-18h. After the reaction, take a sample for HPLC analysis; the conversion rate is 99%. Adjust the pH of the reaction solution to 1 with 2M sulfuric acid, dissolve the enzyme, filter, and adjust the pH of the filtrate to 7.1 with sodium hydroxide. Stir for 3h, and filter to obtain 121.36g of off-white solid. Chirality value 99.96%, purity 99.58%. Figure 3 and Figure 4 The yield was 82.1%.

[0056] Example 5: L-Tyrosine Synthesis (SEQ ID NO.34) Add 500ml of PB (pH 8.0), 0.12g of PLP, 10g of L-serine, 5g of phenol, and 25g of wet bacterial cells (SEQ ID NO. 34) to a 1L reaction flask. Maintain the temperature at 55℃-60℃ and stir for 18h. After the reaction, take a sample for HPLC analysis; the conversion rate was 30%. Adjust the pH of the reaction solution to 1 with concentrated hydrochloric acid, dissolve the enzyme, filter, concentrate the filtrate to 20ml, adjust the pH to 6.7 with sodium hydroxide, stir for 3h, and filter to obtain 1.5g of off-white solid L-tyrosine. Chirality value 99.98%, purity 99.96%. Figure 5 , Figure 6 ).

[0057] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A mutant of tryptophan synthase, characterized in that, Tryptophan synthase mutants with mutations at one, two, three, or more sites, including sites 15, 20, 71, 112, 169, 181, 185, 225, 236, 269, 276, 284, 294, 302, and 323, corresponding to amino acids 1-389 shown in SEQ ID No.

2.

2. The tryptophan synthase mutant according to claim 1, characterized in that, The mutations are: P15L / Q; E20G / P / Q / S; I71V; Q112N; N169T; Y181T; L185V; V225I; M236V; N269T; F276L / S / T; L284C / T; T294S; D302N / S; T323A / F.

3. The tryptophan synthase mutant according to claim 2, characterized in that, The mutation corresponding to amino acids 1-389 of SEQ ID No. 2 contains one of the following mutations: P15Q; E20Q; I71V; Q112N; F276T; F276S; T294S; D302S; T323A; T323F; L284T and T323A; N269T, L284C and T323A; N169 and T323F; Y181T and T323F; M236V and T323F; P15L, E20G, I71V, F276S, T294S and T323A; P1 5L, E20G, V225I, T294S and T323A; P15L, I71V, F276S, T294S and T323A; E20P, I71V, F276L and T294S; I71V, F276S, T294S and T323F; E20S, I71V, L185V, F276S, T294S and T323F; I71V, Q112N, N169T, Y181T, M236V, F276S, T294S, D302N and T323F.

4. The tryptophan synthase mutant according to claim 3, characterized in that, The mutation corresponding to amino acids 1-389 shown in SEQ ID No.2 is one of the following: P15L, E20G, I71V, F276S, T294S and T323A; T323A.

5. The tryptophan synthase mutant according to any one of claims 1-4, characterized in that, The amino acid sequence of the tryptophan synthase mutant is selected from the following mutant sequences: The amino acid sequence shown in SEQ ID No:4; the amino acid sequence shown in SEQ ID No:6; The amino acid sequence shown in SEQ ID No:8; the amino acid sequence shown in SEQ ID No:10; The amino acid sequence shown in SEQ ID No:12; the amino acid sequence shown in SEQ ID No:14; The amino acid sequence shown in SEQ ID No:16; the amino acid sequence shown in SEQ ID No:18; The amino acid sequence shown in SEQ ID No:20; the amino acid sequence shown in SEQ ID No:22; The amino acid sequence shown in SEQ ID No:24; the amino acid sequence shown in SEQ ID No:26; The amino acid sequence shown in SEQ ID No:28; the amino acid sequence shown in SEQ ID No:30; The amino acid sequence shown in SEQ ID No:32; the amino acid sequence shown in SEQ ID No:34; The amino acid sequence shown in SEQ ID No:36; the amino acid sequence shown in SEQ ID No:38; The amino acid sequence shown in SEQ ID No:40; the amino acid sequence shown in SEQ ID No:42; The amino acid sequence shown in SEQ ID No:44; the amino acid sequence shown in SEQ ID No:

46.

6. A group encoding a mutant of tryptophan synthase according to any one of claims 1-4.

7. An expression vector containing a group of the tryptophan synthase mutant according to any one of claims 1-4.

8. A recombinant cell containing a group of the tryptophan synthase mutant according to any one of claims 1-4.

9. A method for synthesizing a chiral tryptophan analog of formula I catalyzed by a tryptophan synthase or a mutant thereof, wherein the reaction formula is as follows: ； in, The tryptophan synthase is derived from the amino acid sequence shown in SEQ ID NO:2, and the mutant of the tryptophan synthase is obtained by any one of the mutations described in claims 1-4. The substituent R in Formula II is selected from the following groups: , or ; The substituent R1 is selected from one of F, Cl, Br, I, -OH, -CH3, -CH2CH3 or -CH2CH2CH3; The substituent R2 is selected from one of F, Cl, Br, I, -OH, -CH3, -CH2CH3 or -CH2CH2CH3.

10. The method according to claim 9, characterized in that: The compound of formula II is selected from one of the following: , , ,or .

11. The method according to claim 9, characterized in that: The concentration loading of the compound of formula II is 10-110 g / L. And / or, the dosage of tryptophan synthase or its mutant is 10-40 g / L. And / or, the reaction pH range is 7.0-9.

0. And / or, the buffer solution pH range is 7.0-9.

0. And / or, conversion rate ≥99%, And / or, chiral ee(R) ≥ 99.00%.

12. The method according to claim 11, characterized in that: The concentration loading of the compound of formula II is 90 g / L. And / or, the dosage of tryptophan synthase or its mutant is 25 g / L. And / or, the reaction temperature is 20-90℃, preferably 60℃. And / or, the reaction pH is 8.5, and the enzyme-catalyzed reaction time is 18 hours.

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