A plp-dependent decarboxylating aldolase mutant and its use in preparing unnatural amino acids

Abstract

The application discloses a PLP-dependent decarboxylating aldolase mutant and application thereof in preparation of unnatural amino acids containing gamma-hydroxyl groups. The mutant contains any one or more of G128V, G128T, G128I, L392M and M393K, based on the sequence of the amino acid shown in SEQ ID NO. 1. Compared with the parent sequence shown in SEQ ID NO. 1, the enzyme activity of the mutant is improved by 7 times, and the enzyme activity is improved by 3-7 times when the mutant catalyzes the reaction of the substrate L-aspartic acid and benzaldehyde, so the mutant has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a PLP-dependent decarboxylalloses mutant and its application in the preparation of non-natural amino acids. Background Technology

[0002] Non-natural amino acids (ncAAs) are a class of artificially designed or modified amino acids that overcome the structural limitations of natural amino acids, endowing proteins with new physicochemical properties and biological activities. Their unique structural designability and functional diversity have also led to many innovative applications in fields such as biomedicine. Within the molecular spectrum of ncAAs, γ-hydroxy non-natural amino acids (γ-OH ncAAs), due to their unique four-carbon skeleton and chiral hydroxyl groups, have become core building blocks for neuropharmaceuticals and advanced biomaterials. For example, 4-hydroxyisoleucine has glucose-dependent functions such as promoting insulin secretion and lipid metabolism, as well as lowering blood lipids, making it an ideal drug for treating diabetes and obesity. Trans-4-hydroxy-L-proline is a collagen cross-linking enhancer and also possesses antioxidant, hydroxyl free radical scavenging, and bone healing-promoting abilities, and is now widely used in medicine, chemical engineering, and cosmetic fields.

[0003] However, the biosynthesis of these high-value molecules faces severe challenges. Few catalysts in the natural enzyme library can react to obtain these ncAAs. Currently, most synthesis methods are based on chemical methods, but these methods suffer from low stereoselectivity of hydroxyl groups, insufficient yields, and demanding reaction conditions, severely limiting the large-scale application of these ncAAs. In recent years, a PLP-dependent enzyme, UstD, capable of synthesizing γ-hydroxy non-natural amino acids, has been discovered. This enzyme has the ability to activate C-C bonds to undergo aldol condensation reactions. It exhibits broad substrate inclusiveness, showing catalytic activity towards aliphatic aldehydes (C3-C8), aromatic aldehydes, heterocyclic aldehydes, and ketones, playing a crucial role in the synthesis of these substances.

[0004] Wild-type ApUstD was found to catalyze the condensation reaction between L-aspartic acid and aldehyde substrates. However, the wild-type enzyme exhibits low activity, and its fungal origin severely reduces its soluble expression in E. coli. Therefore, researchers have addressed these shortcomings, resulting in the mutant UstD, which demonstrates improved solubility and activity against a variety of aldehyde substrates. v2.0 .

[0005] However, even using evolved mutants, the catalytic efficiency remains low, and there is still a significant gap before industrialization. Therefore, there is an urgent need for a highly efficient decarboxylalloses for synthesizing non-natural amino acids containing γ-hydroxyl groups. Summary of the Invention

[0006] This invention is in UstDv2.0 Based on this, multi-site enzyme modification was carried out for the reaction of L-aspartic acid and benzaldehyde, providing a PLP-dependent decarboxylallocondensate mutant and its application in the preparation of non-natural amino acids, in order to address the problem of low catalytic activity of this enzyme for such reactions in existing technologies.

[0007] This invention develops a high-throughput screening method to improve the screening efficiency of mutants. Using the protein language models ESM2 and Evolvepro, one potential distal mutation site was identified. At the same time, two pocket mutation sites were identified by combining molecular dynamics simulations and protein structure. Subsequently, the beneficial mutations were identified by high-throughput screening. Then, the iterative saturation mutagenesis strategy was used to identify multiple single-point and multi-point mutants that showed improved responses to L-aspartic acid and benzaldehyde.

[0008] The first aspect of the present invention protects a PLP-dependent decarboxylalloproteinase mutant based on the amino acid sequence shown in SEQ ID NO.1, wherein the mutant comprises any one or more of the following: G128V, G128T, G128I, L392M, M393K.

[0009] Parent UstD v2.0 The amino acid sequence contains what is shown in SEQ ID NO.1:

[0010] MKSVATSSLDDVDKDSVPLGSSINGTAQAETPLENVIDVESVRSHFPVLGGETAAFNNASGTVVLKEAIESTSNFMYSFPFPPGVDAKSMEAITAYTGNKGKVAAFINALPDEITFGQSTTALFRLLGLSLKPMLNNDCEIVCSTLCHEAAASAWIHLSRELGITIKWWSPTTTPNSPDDPVLTTDSLKPLLSPKTRLVTCNHVSNVVGTIHPIREIADV VHTIPGAMLIVDGVASVPHRPVDVKELDVDFYCFSWYKLFGPHLGTLYASRKAQDRYMTSINHYFVSSSSLDGKLALGMPSFELQLMCSPIVSYLQDTVGWDRIVRQETV LVTILLEYLLSKPSVYRVFGRRNSDPSQRVAIVTFEVVGRSSGDVAMRVNTRNRFRITSGTLMAPRPTWDVLKPKSSDGLVRVSFVHYNTVEEVRAFCSELDEIVTRDTLE

[0011] SEQ ID NO.2:

[0012]

[0013] In some embodiments, the mutant is a substitution at one amino acid site.

[0014] In some embodiments, the mutant contains a substitution for G128V. (Compared to the parent UstD) v2.0 In comparison, the transformation efficiency of the mutant containing G128V increased by 26.1%.

[0015] In some embodiments, the mutant contains a substitution for G128T. (Compared to the parent UstD) v2.0 In comparison, the transformation rate of the mutant containing G128T increased by 26.1%.

[0016] In some embodiments, the mutant contains a substitution for G128I. (Compared to the parent UstD) v2.0 In comparison, the transformation efficiency of the mutant containing G128I increased by 10.9%.

[0017] In some embodiments, the mutant contains a substitution of L392M. (Compared to the parent UstD) v2.0 In comparison, the transformation efficiency of the mutant containing L392M increased by 19.6%.

[0018] In some embodiments, the mutant contains a substitution for M393K.

[0019] In some embodiments, the mutant is a substitution of multiple amino acid sites.

[0020] In some implementations, the multipoint mutation comprises substitutions for G128V and L392M. (Compared to the parent UstD) v2.0 In comparison, the transformation rate of mutants containing G128V and L392M increased by 50%.

[0021] In some implementations, the multipoint mutation comprises substitutions for G128V and M393Y. (Compared to the parent UstD) v2.0 In comparison, the transformation rate of mutants containing G128V and M393Y increased by 13%.

[0022] In some embodiments, the multipoint mutations include substitutions for G128V, L392M, and M393H. (Compared to the parent UstD) v2.0 In comparison, the transformation efficiency of mutants containing G128V, L392M, and M393H increased by 30.43%.

[0023] In some embodiments, the multipoint mutations include substitutions for G128V, L392M, and M393K. (Compared to the parent UstD) v2.0In comparison, the transformation efficiency of mutants containing G128V, L392M, and M393K was increased by 89.1%.

[0024] A first aspect of the present invention protects an isolated polynucleotide encoding a mutant as described above.

[0025] A second aspect of the present invention protects a nucleic acid construct comprising the polynucleotides described above.

[0026] A third aspect of the present invention protects a genetically engineered bacterium comprising a nucleic acid construct as described above or having the polynucleotides as described above integrated into its genome.

[0027] The fourth aspect of this invention protects the use of the mutants, polynucleotides, nucleic acid constructs, or host cells described above in the preparation of non-natural amino acids.

[0028] The fifth aspect of the present invention protects a method for preparing non-natural amino acids, using mutants, polynucleotides, nucleic acid constructs, or host cell catalytic substrates as described above, wherein the substrates comprise aldehydes and aspartic acid.

[0029] In some embodiments, the aldehydes are selected from formaldehyde, acetaldehyde, and derivatives of formaldehyde or acetaldehyde obtained by substituting alkyl, alkoxy, halogen, or nitro groups.

[0030] In some embodiments, the aldehydes are selected from benzaldehyde, naphthaldehyde, pyridine formaldehyde, furanaldehyde, thiophene formaldehyde, and their substituted derivatives.

[0031] In some specific embodiments, the aldehyde is selected from benzaldehyde.

[0032] In some embodiments, the non-natural amino acid is a non-natural amino acid containing a γ-hydroxyl group.

[0033] In some specific embodiments, the γ-hydroxy-containing non-natural amino acid is selected from one or more of 4-hydroxy-4-phenyl-2-aminobutyric acid, 4-hydroxy-4-(4'-nitrophenyl)-2-aminobutyric acid, and 4-hydroxy-4-(1H-imidazol-4-yl)-2-aminobutyric acid. Preferably, it is 4-hydroxy-4-phenyl-2-aminobutyric acid.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The PLP-dependent decarboxylalloses mutant constructed in this invention exhibits higher activity than UstD in catalyzing the reaction of substrates L-aspartic acid and benzaldehyde. v2.0The parental mutation showed a 3-7 fold increase in activity. Furthermore, this invention discovered a distal site where the single-point mutant activity was 2-fold increased, and this activity enhancement was maintained even when combined with two residues at the pocket position. Compared to UstD, the mutant obtained in this invention... v2.0 The parental activity was increased by 7.27 times, which helps to promote the synthesis of this type of non-natural amino acid containing γ-hydroxyl groups. Attached Figure Description

[0036] Figure 1 This is the reaction formula for the decarboxylation and aldolization process of the PLP-dependent decarboxylation aldolase UstD using benzaldehyde and L-aspartic acid as substrates.

[0037] Figure 2 The relative positions of the enzyme modification sites G128, L392, and M393 in the protein in Example 1 are shown.

[0038] Figure 3 The relative activity of mutants with full saturation mutations of G128, L392, and M393 in Example 2 is given.

[0039] Figure 4 The relative activity of the mutant with the combination of G128, L392, and M393 points in Example 3 is shown.

[0040] Figure 5 The relative activity of the mutant with the combination of G128, L392, and M393 in Example 3. Detailed Implementation

[0041] To enable those skilled in the art to fully understand the essence of the present invention and implement it, the technical solution provided by the present invention will be described in more detail below with reference to the accompanying drawings and a series of specific embodiments. It should be noted that the embodiments are merely examples and should not be construed as limiting the core scope of protection of the present invention in any way.

[0042] Based on the embodiments of the present invention, any other obvious technical variations, equivalent substitutions or further improvements that can be conceived by those skilled in the art without creative effort should be considered within the scope of protection of the present invention.

[0043] Unless otherwise specified, all technical and scientific terms used in this invention are consistent with the common understanding of one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0044] Used in the embodiments of the present invention E. coli BL21(DE3) The plasmid pET28a was purchased from Novagen; the gene synthesis of UstD was completed by Jiutian Biopharmaceutical (Shanghai) Co., Ltd.; the primer synthesis and sequence sequencing required for the construction of the UstD mutant were completed by Qingke Biotechnology Co., Ltd.

[0045] The reagents used in the catalytic reaction were: sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate, which were purchased from Sinopharm Chemical Reagent Co., Ltd.; pyridoxal phosphate (PLP) and benzaldehyde, which were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and L-aspartic acid and phenol red, which were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0046] In the following embodiments, the parent UstD v2.0 The amino acid sequence is shown in SEQ ID NO.1, and its coding sequence is shown in SEQ ID NO.2.

[0047] Example 1: Selection of Enzyme Modification Sites

[0048] A synergistic strategy combining artificial intelligence prediction and structural biology analysis is employed to identify key mutation sites. This includes the following:

[0049] Using the protein language models ESM2 and Evolvepro to study the PLP-dependent decarboxylallocondensate UstD v2.0 (SEQ ID NO.1) was predicted, and then some mutations with high predicted tag values ​​were tested for activity, thereby identifying a potentially evolutionarily plastic site G128, most of which have a positive effect on enzyme activity.

[0050] It is worth noting that G128 is located at the distal end of the active center ( Figure 2 This suggests that it may regulate the enzyme's dynamic structure or catalytic network through long-range interactions, rather than directly participating in substrate binding. Furthermore, analysis of the enzyme's three-dimensional structure (PDBID: 7MKV) and molecular dynamics revealed two sites, L392 and M393, located near the substrate and within a flexible loop region. Figure 2 These two sites may affect the degree of binding between key catalytic residues and substrates, thereby affecting the occurrence of aldol condensation reactions.

[0051] Therefore, intervention at this distal critical site (G128), combined with local mutations in the substrate binding pocket (L392 / L393), may produce a more profound and synergistic optimization effect on enzyme catalytic performance.

[0052] Example 2: Fully Saturated Mutations at Potential Activity-Enhancing Sites

[0053] As predicted in Example 1, single-point saturation mutagenesis was performed at sites G128, L392, and M393, and the crude enzyme activity was characterized using the phenol red method.

[0054] 2.1 Construction of mutant strains

[0055] 2.1.1 Single-point mutation

[0056] Design primers, on the one hand, with UstD v2.0 Using the template, site-directed mutagenesis of the remaining 19 amino acids was performed at the three sites of G128, L392, and M393. Fifty-seven mutant fragments were obtained by PCR amplification. The primers used for constructing single-point mutants are shown in Table 1.

[0057] The specific method is as follows:

[0058] 1) Whole plasmid PCR

[0059] pET28a-UstD v2.0 Using plasmids as templates, upstream and downstream primers covering the mutation sites were designed (Tables 1 and 2) for whole plasmid PCR.

[0060] 2) PCR amplification system:

[0061] 25 μL of DNA polymerase

[0062] Upstream primer (10 μM) 1 μL,

[0063] 1 μL of downstream primer (10 μM),

[0064] Template (5 ng / μL) 1 μL,

[0065] ddH2O2 2 μL.

[0066] PCR amplification conditions:

[0067] ①. Pre-denaturation: 98℃ for 3 min;

[0068] ②. Denaturation: 98℃ for 10 s; Annealing: 60℃ for 15 s; Extension: 72℃ for 1 min 10 s; 33 cycles in total;

[0069] ③. Post-extension: 72℃ for 5 min;

[0070] ④.Save at 4℃.

[0071] 3) Thermal shock conversion

[0072] The transformation was performed using a chemical heat shock method. The specific process involved adding the PCR product to... E. coli BL21(DE3)Competent cells were placed on ice for 30 minutes, then heat-shocked at 42°C for 90 seconds, and rapidly cooled on ice for 2-3 minutes. They were then revived at 37°C for 1 hour in antibiotic-free LB medium. Finally, they were spread on plates containing the appropriate antibiotics and incubated upside down at 37°C for 10-12 hours to obtain single colonies for subsequent screening.

[0073] Crude enzyme preparation method: Single colonies of the mutant strain were picked and cultured in 5 mL LB liquid medium (containing 50 μg / mL kanamycin) at 37℃ and 200 rpm for 10 h with shaking to obtain seed culture. This seed culture was then transferred to 50 mL TB liquid medium (containing 50 μg / mL kanamycin) at a 1% inoculation rate and cultured at 18℃ with shaking for 18-20 h. After culture, the cells were collected by centrifugation at 4000 rpm and stored at -20℃ overnight. The next day, the frozen cells were thawed and resuspended in 50 mM pH 7.4 phosphate buffer (containing 300 mM sodium chloride), and sonicated in an ice-water bath for 5 min. Subsequently, the cells were centrifuged at 12000 rpm and 4℃ for 20 min to separate cell debris; the supernatant was the crude enzyme solution.

[0074] 2.2 Enzyme activity assay

[0075] When determining enzyme activity, the crude enzyme activity was first measured using the phenol red detection method to obtain the high activity site. Then, the enzyme was purified, and the purified enzyme was measured again using liquid chromatography.

[0076] The specific detection method is as follows: In a 200 μL reaction system, there are 40 μL L-aspartic acid (50 mM), 10 μL benzaldehyde (25 mM), 2 μL PLP (0.1 mM), 20 μL phenol red (0.028 mM), 10 μL crude enzyme solution obtained in step 2.1, and 118 μL phosphate buffer solution (2 mM, pH 8.0).

[0077] Using a multi-functional microplate reader, the absorbance of the reaction solution at 560 nm was measured every 30 minutes at room temperature.

[0078] At the initial reaction rate, the relative enzyme activity (%) of the mutant relative to the parent can be expressed as: under the same reaction conditions, the rate of change of absorbance of the reaction system per unit time (i.e., the initial reaction rate) is measured, and the ratio of the initial reaction rate of the mutant to the initial reaction rate of the parent is the relative enzyme activity. Some beneficial mutants such as G128V, G128I, L392M, and M393H were obtained through initial screening.

[0079] For mutants with significantly enhanced activity, cell culture was repeated, and the protein was purified. The purified enzyme was then retested using high-performance liquid chromatography (HPLC). Results are shown below. Figure 3 .

[0080] The specific methods for protein expression and purification are as follows: The crude enzyme solution obtained in step 2.1 was purified by affinity chromatography. Impurities were washed away with washing buffer (50 mM phosphate buffer, 300 mM sodium chloride, 100 mM imidazole, pH 7.4). The target protein was eluted with elution buffer (50 mM phosphate buffer, 300 mM sodium chloride, 250 mM imidazole, pH 7.4). The separated target protein was placed in an ultrafiltration centrifuge tube for desalting and concentration to obtain pure target protein.

[0081] The reaction system contained 40 μL L-aspartic acid (50 mM), 10 μL benzaldehyde (25 mM), 2 μL PLP (0.1 mM), 20 μL purified protein (125 μg / mL) of single-site, double-site, and triple-site mutants obtained in step 2.1, and 128 μL phosphate buffer solution (50 mM, pH 7.4). The reaction was carried out at 37 °C for 20 minutes, and the relative enzyme activity was calculated.

[0082] High-performance liquid chromatography (HPLC) was used to characterize the mutant activity: Samples were filtered through a membrane and analyzed using a C18 reversed-phase column (5 μm, 250 × 4.6 mm). Chromatographic conditions were as follows: column temperature 30℃, detection wavelength 210 nm, mobile phase water-methanol, flow rate 0.8 mL / min. The elution gradient was as follows: methanol content was 15% at 0 min, linearly increasing to 60% within 15 min and maintaining this level for 5 min; then, it was restored to the initial ratio (15% methanol) within 0.5 min and re-equilibrated for 5 min. The relative enzyme activity detected by HPLC was the ratio of the peak area of ​​the wild-type product to the peak area of ​​the mutant product.

[0083] Table 1 Primers used for constructing some single-point mutants

[0084]

[0085] Table 2. Relative enzyme activities of different single-point mutants

[0086]

[0087] As shown in Table 2, among the single-point mutations of G128, L392, and M393, the three mutants with the highest relative activity are G128V, L392M, and G128I.

[0088] Example 3 Iterative Saturation Mutation

[0089] After obtaining single-point saturation mutation data at three sites, G128, L392, and M393, these three sites were further combined to evaluate the compatibility and synergistic potential of each mutation, especially the distal site G128, during the combination process.

[0090] 3.1 Fully Saturated Mutation

[0091] Therefore, we selected the G128V mutation (which showed the greatest activity enhancement among the single-point saturation mutations of G128, with an activity 1.9 times that of the wild type) as the starting point for our study to investigate whether mutations at this distal site would weaken the beneficial effects of mutations at other sites by fine-tuning the overall conformation or dynamics of the protein.

[0092] By combining G128V with full-saturation mutations at L392 and M393 sites, 38 combined mutants were constructed.

[0093] UstD v2.0 Using G128V as a template, single-point saturation mutations of L392 and M393 were combined to obtain two-point mutant fragments. The primers used are those listed in Table 1. See step 2.1 for details.

[0094] These two-point combination mutations were purified, and their activity was detected using high-performance liquid chromatography (HPLC). Specific steps are detailed in section 2.2, and the results are shown in Table 3. Figure 4 As shown.

[0095] Table 3. Relative enzyme activities of different two-point mutants

[0096]

[0097] As shown in Table 3, G128V exhibits an additive effect on activity when combined with the advantageous mutations of the two pocket sites. The activity of both sites is highest when G128V is combined with L392M, which has the highest activity enhancement. This result indicates that the mutation of the distal site G128 has little effect on the catalytic pocket and does not significantly weaken the original activity enhancement effect when combined with the pocket site.

[0098] 3.2 Saturation Iteration

[0099] Next, based on the two-point combination, we will continue to select the two mutations G128V / L392M that provide the highest activity enhancement as the starting point for the combination, in order to examine whether the mutation at this distal site will weaken the beneficial effects of mutations at other sites by fine-tuning the overall conformation or dynamics of the protein.

[0100] The combination of G128V / L392M and M393 sites was used for the next round of saturation iteration, resulting in 19 combined mutants.

[0101] UstDv2.0 Using G128V-L392M as a template, a single-point saturation mutation of M393 was combined (primers are shown in Table 4) to obtain three-point combined mutations. The activity of these three-point combined mutations was detected by high-performance liquid chromatography (HPLC), and the results are shown in Table 5. Figure 5 As shown.

[0102] Table 4 Primers used for constructing partially perfect iterative saturation mutations

[0103]

[0104] Table 5. Relative enzyme activities of different three-point mutants

[0105]

[0106] As shown in Table 5, the three-point mutant G128V / L392M / M393K with significantly enhanced activity was obtained, and its activity was 7.2 times that of the parent.

[0107] Example 4: UstD mutant transformation rate test

[0108] By performing single-point saturation mutations and iterative saturation mutations on key sites, several single-point, two-point, and three-point mutants exhibiting enhanced activity were screened and obtained. To further evaluate their catalytic performance, nine mutants with significant activity enhancements were selected.

[0109] The reaction system contained 40 μL L-aspartic acid (50 mM), 10 μL benzaldehyde (25 mM), 2 μL PLP (0.1 mM), 20 μL purified protein (125 μg / mL) of single-point, double-point, and triple-point mutants obtained in step 2.1, and 128 μL phosphate buffer solution (50 mM, pH 7.4). The reaction was carried out at 37°C for 7 hours, and the conversion rate was calculated. The conversion rate was determined according to the high-performance liquid chromatography method of Example 2 and was defined as the percentage of substrate consumed relative to the initial feed amount.

[0110] As shown in Table 6, the mutant transformation rate obtained by this invention is relative to UstD. v2.0 All showed some improvement, with the three-point mutation G128V / L392M / M393K showing the highest activity improvement, achieving a conversion rate of 87%.

[0111] Table 6. Transformation rate test of activity-enhancing mutants

[0112]

[0113] As shown in Table 6, the relationship with the parent UstD v2.0Compared to the previous mutants, the G128T mutant showed a 1.82-fold increase in activity and a 26.1% increase in transformation efficiency; the G128I mutant showed a 1.75-fold increase in activity and a 10.9% increase in transformation efficiency; the G128V mutant showed a 1.94-fold increase in activity and a 26.1% increase in transformation efficiency; the L392M mutant showed a 1.91-fold increase in activity and a 19.6% increase in transformation efficiency; and the G128V / L392C mutant showed a 2.88-fold increase in activity and a 26.1% increase in transformation efficiency. The activity of the mutant containing G128V / L392M increased by 2.84 times, and the transformation rate increased by 50%; the activity of the mutant containing G128V / M393Y increased by 2.4 times, and the transformation rate increased by 13.0%; the activity of the mutant containing G128V / L392M / M393H increased by 6.4 times, and the transformation rate increased by 30.43%; the activity of the mutant containing G128V / L392M / M393K increased by 7.27 times, and the transformation rate increased by 89.1%.

[0114] Example 5: Synthesis and Application of γ-hydroxy non-natural amino acids based on UstD mutant

[0115] UstD enzymes can catalyze the decarboxylation and aldol condensation of L-aspartic acid and aldehydes as substrates, thereby generating non-natural amino acids containing γ-hydroxyl groups. The reaction formula is as follows: Figure 1 As shown.

[0116] The non-natural amino acids containing γ-hydroxyl groups, and their pharmaceutically acceptable salts or esters, synthesized by the method of this invention, have broad industrial application value. Specifically, the non-natural amino acids containing γ-hydroxyl groups can be used in any of the following fields:

[0117] In the pharmaceutical field: it serves as a key chiral building block for the synthesis of chiral drugs, prodrugs, or bioactive molecules. In particular, it can serve as a synthetic intermediate for neurotransmitter analogs, enzyme inhibitors, or receptor modulators.

[0118] In the food and nutrition field: as an ingredient in non-natural amino acid nutrients or dietary supplements.

[0119] In the field of materials chemistry: monomers used to synthesize novel biodegradable polymer materials, chiral ligands, or ion chelating agents.

[0120] In the field of chemical synthesis: as a chiral auxiliary or building block in organic synthesis, it is used to construct more complex molecular structures.

[0121] In the field of agricultural chemistry: as an intermediate in the synthesis of agricultural chemicals (such as herbicides and pesticides).

[0122] The UstD three-point mutant G128V / L392M / M393K provided by this invention exhibits higher reactivity than UstD in the catalytic synthesis of 4-hydroxy-4-phenyl-2-aminobutyric acid. v2.0 It achieved an improvement of approximately 7.27 times.

[0123] Existing technologies (such as WO2025230736) indicate that UstD v2.0 The enzyme has exhibited catalytic activity against a variety of macromolecular aldehyde substrates, such as benzaldehyde mono- or poly-substituted with alkyl, alkoxy, halogen, or nitro groups, as well as aldehydes containing aromatic or heteroaromatic structures such as naphthalene, pyridine, furan, and thiophene. Since the natural substrates of this enzyme are macromolecular structures, its catalytic activity against large-sized substrates has been optimized.

[0124] This invention modifies the enzyme substrate binding pocket to obtain a three-point mutant with improved performance. Based on the analysis of the mutant structure, those skilled in the art believe that the mutation direction may have reduced the substrate cavity while maintaining certain hydrophobic interactions, thus enabling better immobilization and activation of small molecule aldehyde substrates. Therefore, it is reasonable to expect that the catalytic range of the mutant for small molecule aldehyde substrates will be effectively expanded. These substances include formaldehyde, acetaldehyde, propionaldehyde, isobutyraldehyde, and small molecule aldehydes containing hydroxyl, halogen, amino, and other functional groups. This will, to some extent, complement existing technologies in terms of function, providing a more powerful tool enzyme for the construction of libraries of γ-hydroxy non-natural amino acids.

Claims

1. A PLP-dependent decarboxylallocondensate mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the mutant is any one of the following: 1) Replacement of G128V; 2) Replacement of G128V and L392M; 3) Replacement of G128V, L392M and M393K; 4) Replacement of G128V, L392M and M393H.

2. An isolated polynucleotide, characterized in that, The mutant as described in claim 1 is encoded.

3. A nucleic acid construct, characterized in that, It contains the polynucleotide as described in claim 2.

4. A genetically engineered bacterium, characterized in that, It includes the nucleic acid construct as described in claim 3 or the genome in which the polynucleotide as described in claim 2 is integrated.

5. The use of the mutant of claim 1, the polynucleotide of claim 2, the nucleic acid construct of claim 3, or the genetically engineered bacteria of claim 4 in the preparation of non-natural amino acids.

6. A method for preparing non-natural amino acids, characterized in that, The mutant as described in claim 1, the polynucleotide as described in claim 2, the nucleic acid construct as described in claim 3, or the genetically engineered bacterial catalytic substrate as described in claim 4, wherein the substrate comprises aldehydes and aspartic acid.

7. The method as described in claim 6, characterized in that, The aldehydes are selected from benzaldehyde, naphthaldehyde, pyridine formaldehyde, furanaldehyde, and thiophene formaldehyde.

8. The method as described in claim 6, characterized in that, The non-natural amino acids mentioned are non-natural amino acids containing γ-hydroxyl groups.

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