Mutant of pita-r6 transaminase and construction method and application thereof

Abstract

This invention discloses a mutant of PjTA-R6 transaminase, its construction method, and its applications. Using schizosphingine, a key precursor of large-volume monoterpene indole alkaloids, as the target for reductive amination, this invention rationally designs the PjTA-R6 transaminase through a global free energy optimization strategy, obtaining 10 mutants with significantly higher catalytic efficiencies than the original PjTA-R6 transaminase. Recombinant *E. coli* strains based on these mutants are then constructed, resulting in a substantial improvement in reductive amination catalytic efficiency. The PjTA-R6 L164E / L57F mutant provided by this invention achieves a 99% conversion rate of schizosphingine to bacamargine via whole-cell reductive amination, a 46% increase compared to the original strain. The PjTA-R6 transaminase mutant provided by this invention exhibits significant catalytic efficiency, offering a powerful tool enzyme for overcoming the industrial bottleneck of transaminase catalysis of large-volume substrates, and possesses broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a mutant of PjTA-R6 transaminase, its construction method and application. It is a PjTA-R6 transaminase mutant with enhanced catalytic activity against secologanin, its encoding gene, preparation method and its application in biocatalytic synthesis. Background Technology

[0002] Transaminases (TAs; EC 2.6.1) are a class of pyridoxal phosphate-dependent biocatalysts that efficiently catalyze aminotransfer reactions, exhibiting unique advantages in the synthesis of chiral amine drug intermediates and natural products. Compared with traditional chemical synthesis methods, transaminase catalytic pathways offer significant advantages such as milder conditions, high stereoselectivity, and environmental friendliness. A typical industrial application example is Merck's use of ω-transaminases to achieve the efficient synthesis of chiral amine intermediates for the antidiabetic drug sitagliptin, fully demonstrating its industrial application value.

[0003] However, the catalytic efficiency of natural transaminases for sterically hindered substrates is generally low, a bottleneck that severely restricts their application in the synthesis of complex natural products. Stericly hindered substrates include important reactive molecules such as terpenes, steroids, and alkaloids. The main reasons for their limited catalytic activity are: steric hindrance of the active pocket hindering substrate entry; rigid conformational constraints on substrate channels; insufficient enzyme-substrate binding affinity; and poor stability of PLP reaction intermediates.

[0004] Secologanin, as a common precursor to over 2000 monoterpenoid indole alkaloids (MIAs), plays a crucial role in drug development. Drugs derived from it, such as vinblastine, quinine, and reserpine, are indispensable in clinical treatment. Currently, the acquisition of these high-value compounds mainly relies on plant extraction and semi-synthesis, which suffers from low yields, long cycles, and high costs. Therefore, developing efficient enzyme-catalyzed synthetic routes, particularly for the precise amination modification of secologanin, has become a research hotspot in this field. However, the complex molecular structure of secologanin (molecular weight 388 Da, containing aldehyde, ester, double bonds, and multiple chiral centers), its large size, and multifunctional characteristics make it difficult for natural transaminases to effectively recognize and catalyze it.

[0005] Protein engineering strategies such as directed evolution and rational design have been used to expand the substrate profile of transaminases. The transaminase substrate used in this study was derived from *Pseudomonas japonicus* (…). Pseudomonas jesseniiTaking the transaminase PjTA-R6 (PDB: 6BT1) as an example, this enzyme exhibits good thermostability and organic solvent tolerance, showing potential for industrial applications. However, traditional strategies still have significant shortcomings when dealing with complex substrates such as schizokinin: the rigid structure of the active pocket restricts substrate conformational adaptation; local mutations easily disrupt the global stability of the enzyme; and existing designs have limited efficiency in modifying substrates with complex stereoconfigurations.

[0006] In summary, there is an urgent need in this field to develop a new strategy that can systematically optimize the interaction between transaminases and large-volume substrates, and based on this, to create mutants of novel transaminases with high catalytic activity against schizosphingine, so as to promote the biosynthesis of related high-value natural products. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a mutant of PjTA-R6 transaminase, which is a transaminase PjTA-R6 mutant with significantly improved catalytic efficiency, especially its amination activity against the large substrate secologanin. This invention provides a transaminase PjTA-R6 mutant with significantly enhanced catalytic activity against secologanin, its encoding gene, expression vector, and engineered bacteria.

[0008] The present invention provides a mutant of PjTA-R6 transaminase, wherein the mutant is based on the PjTA-R6 transaminase shown in SEQ ID NO.2 and has at least one of the following mutations: N167Y, L57F, L64D, L164E, L164Y, P19E, P19F, P19Y, Y20R, Y20K.

[0009] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating asparagine at position 167 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine, and the mutant is named N167Y.

[0010] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating leucine at position 57 of the amino acid sequence shown in SEQ ID NO.2 to phenylalanine, and the mutant is named L57F.

[0011] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to aspartic acid, and the mutant is named L164D.

[0012] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to glutamic acid, and the mutant is named L164E.

[0013] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine, and the mutant is named L164Y.

[0014] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating proline at position 19 of the amino acid sequence shown in SEQ ID NO.2 to glutamic acid, and the mutant is named P19E.

[0015] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating proline at position 19 of the amino acid sequence shown in SEQ ID NO.2 to phenylalanine, and the mutant obtained is named P19F.

[0016] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating proline at position 19 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine, and the mutant is named P19Y.

[0017] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating tyrosine at position 20 of the amino acid sequence shown in SEQ ID NO.2 to arginine, and the resulting mutant is named Y20R.

[0018] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating the tyrosine at position 20 of the amino acid sequence shown in SEQ ID NO.2 to lysine, and the mutant obtained is named Y20K.

[0019] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to glutamic acid, and proline at position 19 of the amino acid sequence to tyrosine. The mutant obtained is named L164E / P19Y.

[0020] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO:2 to glutamic acid, and simultaneously mutating leucine at position 57 of the amino acid sequence to phenylalanine. The mutant obtained is named L164E / L57F, and its amino acid sequence is shown in SEQ ID NO:4.

[0021] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to glutamic acid, and simultaneously mutating asparagine at position 167 of the amino acid sequence to tyrosine. The mutant obtained is named L164E / N167Y.

[0022] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to glutamic acid, and simultaneously mutating tyrosine at position 20 of the amino acid sequence to lysine. The mutant obtained is named L164E / Y20K.

[0023] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine, and simultaneously mutating proline at position 19 of the amino acid sequence to tyrosine. The mutant obtained is named L164Y / P19Y.

[0024] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine, and simultaneously mutating leucine at position 57 of the amino acid sequence to phenylalanine. The mutant obtained is named L164Y / L57F.

[0025] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine, and simultaneously mutating asparagine at position 167 of the amino acid sequence to tyrosine, and the resulting mutant is named L164Y / N167Y.

[0026] In one embodiment, the mutant of the PjTA-R6 transaminase is constructed by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine, and simultaneously mutating tyrosine at position 20 of the amino acid sequence to lysine. The mutant obtained is named L164Y / Y20K.

[0027] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to glutamic acid, and tyrosine at position 20 of the amino acid sequence to arginine. The mutant obtained is named L164E / Y20R.

[0028] In one embodiment, the mutant of the PjTA-R6 transaminase is obtained by mutating leucine at position 164 of the amino acid sequence shown in SEQ ID NO.2 to tyrosine, and tyrosine at position 20 of the amino acid sequence to arginine. The mutant obtained is named L164Y / Y20R.

[0029] The present invention also provides an enzyme preparation containing a mutant of the PjTA-R6 transaminase.

[0030] In one embodiment, the enzyme preparation contains a protein of the mutant PjTA-R6 transaminase, the sequence of which is shown in SEQ ID NO.4.

[0031] In one embodiment, the enzyme preparation is an immobilized enzyme of a mutant of the PjTA-R6 transaminase.

[0032] The present invention also provides a gene encoding the mutant of the PjTA-R6 transaminase.

[0033] In one embodiment, the nucleotide sequence of the gene encoding the mutant L164E is shown in SEQ ID NO.4.

[0034] The present invention also provides a recombinant plasmid containing the said gene.

[0035] The present invention also provides a recombinant microbial cell expressing a mutant of PjTA-R6 transaminase.

[0036] In one embodiment, the method for constructing the recombinant microorganism includes: based on the reported wild-type gene sequence of PjTA-R6 transaminase (reference: [Dick B. Janssen, 2020, ACS Catalysis, 10(5): 2915-2928]), introducing the desired mutation (such as L164E) through gene synthesis or PCR site-directed mutagenesis, and then ligating the mutated gene into the pET-28a vector and transforming it into Escherichia coli BL21(DE3) to achieve the expression of the PjTA-R6 transaminase mutant.

[0037] Another object of the present invention is to provide a method for preparing the mutant of the PjTA-R6 transaminase, the method being as follows: (1) Construct a recombinant plasmid expressing a mutant of PjTA-R6 transaminase and transform it into competent Escherichia coli cells to obtain recombinant engineered Escherichia coli; (2) The recombinant Escherichia coli was fermented in a culture medium and induced with isopropyl-β-D-thiogalactoside (IPTG) during the fermentation process; (3) The Escherichia coli cell suspension harvested after fermentation was ultrasonically broken, and then subjected to a one-step Ni-NTA affinity chromatography treatment to obtain the mutant protein of PjTA-R6 transaminase with a purity of >95%.

[0038] In one embodiment, the method involves culturing the recombinant microorganisms to OD200. 600 When the concentration reaches 0.8-1, add IPTG to a final concentration of 1 mM as an inducer and induce culture at 16℃ for 20 h.

[0039] In one embodiment, the fermentation is carried out in TB medium and / or LB medium.

[0040] In one embodiment, the method involves culturing the recombinant Escherichia coli in LB medium at 37°C until OD reaches [a certain value]. 600 When the concentration reaches 0.8-1, induce with IPTG at a final concentration of 1 mM for 20 h at 16°C. Then collect the cells and transfer them to TB / LB medium, and continue to culture at 36°C for at least 20 h.

[0041] Another object of the present invention is to provide the mutant protein of the PjTA-R6 transaminase as a catalyst for the catalytic production of bacamaroline (CAS No.: 1398-17-0).

[0042] The application is achieved through the following steps: using the obtained PjTA-R6 transaminase (PjTA-R6) or a mutant of PjTA-R6 transaminase (PjTA-R6 mutant) as a catalyst, pyridoxal phosphate (PLP) as a coenzyme, and schizosporine (1a) and para-aminonaphthylamine (1b) as raw materials to catalyze the generation of baccacosporine (1c). The beneficial effects of this invention are as follows: This invention innovatively employs a comprehensive and rational design and screening strategy for transaminase modification. Based on the concept of global free energy optimization, it uses the Rosetta energy scanning system to evaluate the binding free energy distribution of the enzyme-substrate complex, accurately identifies key residues affecting substrate binding and conversion, and constructs single-site and combinatorial mutant libraries to systematically reduce the overall free energy of the enzyme-substrate complex. The obtained preferred mutant L164E / L57F achieves a catalytic efficiency (Kcat / Km) of 0.660 mM for schizocyclic strychnine. -1 ·s -1 It is wild type (0.0269 mM) -1 ·s -1 It is 24.5 times that of the original, and it synergistically optimizes substrate binding affinity and catalytic conversion rate, achieving a leap in catalytic performance.

[0043] 2. The optimal mutant PjTA-R6 L164E / L57F increased the conversion rate from 53% of PjTA-R6 to 99%. The high-performance mutant provided by this invention provides a powerful tool enzyme to solve the industrial bottleneck of transaminase catalysis of large-volume substrates, and lays a solid foundation for the green biomanufacturing of schizosporine-derived alkaloids, with broad application prospects. Attached Figure Description

[0044] Figure 1 This is a gene map of PjTA-R6 transaminase provided in a specific embodiment of the present invention.

[0045] Figure 2 The diagram shows the docking conformation of PjTA-R6 WT with the substrate sclerophyllin and a schematic diagram of the mutant protein simulated crystal structure of the optimal mutant PjTA-R6 L164E / L57F transaminase, as provided in a specific embodiment of the present invention.

[0046] Figure 3 The images show the sequence of different amino acid binding energies, single-point mutations, double-point mutations, and the kinetic curves of the optimal mutant and PjTA-R6 WT enzyme obtained based on the Rosetta global free energy optimization strategy.

[0047] Figure 4 This is a standard curve of fluorescence intensity versus concentration of the enzyme-catalyzed product p-aminonaphthylone, measured using p-aminonaphthylamine J2 as the amine donor, based on a fluorescence coupling strategy.

[0048] Figure 5 This is a high-performance liquid chromatography (HPLC) chromatogram of the whole-cell catalytic products.

[0049] Figure 6 This is a protein gel electrophoresis image of the mutant PjTA-R6 transaminase with enhanced activity prepared in this invention. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0051] (a) Materials The substrate secologanin and the amine donor p-aminonaphthylamine J2 for transaminase-catalyzed reactions were composed of the following and purified.

[0052] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (ThermoScientific™ Oxoid).

[0053] TB medium: Tryptone 12 g / L, Yeast Extract 24 g / L, Glycerol 4 mL / L, and a phosphate buffer system (KH2PO4 2.31 g / L and K2HPO4 12.54 g / L). Oxoid (a brand under Thermo Fisher Scientific) was commonly used for tryptone and yeast extract. Glycerol and phosphate were purchased from Sigma-Aldrich. The inorganic salts (Na2HPO4, KH2PO4, NH4Cl, NaCl, MgSO4 and CaCl2) involved in this application were purchased from Sigma-Aldrich, and the glucose was purchased from Sinopharm.

[0054] Escherichia coli BL21(DE3) was purchased from Beijing TransGen Biotech Co., Ltd.

[0055] (II) Activity Assay Method (1) Microplate detection method: To rapidly screen enzyme mutants with higher activity, the growth of the product fluorescence signal was detected using p-aminonaphthylamine J2 as an amine donor to reflect enzyme activity; that is, the activity was measured by microplate testing with OD... 600 The catalytic activity of the supernatant from the bacterial lysate was compared to that of the original enzyme PjTA-R6. The steps were as follows: A mutant with higher enzyme activity was rapidly screened. The growth of the product fluorescence signal was detected using p-aminonaphthylamine as an amine donor to reflect enzyme activity, i.e., by measuring the OD value in a microplate. 600The catalytic activity of the supernatant from the bacterial lysate was compared to the activity of the mutant relative to the original enzyme PjTA-R6. The steps were as follows: After the target plasmid was transformed into Escherichia coli BL21(DE3) strain, it was cultured on Kana LB agar plates. A single colony was picked and inoculated into 3 mL of liquid LB medium to prepare a primary seed culture, which was then incubated overnight (12 hours) at 37°C. The next day, 2% of the culture was transferred to 3 mL of LB liquid medium containing 100 μg / mL and incubated at 37℃ and 220 rpm for about 2.5 h until the OD600 reached 0.6. Then, 3 μL of 1M IPTG was added, and the culture was incubated at 16℃ with shaking at 220 rpm for 16 h. After induction, 1 mL of each bacterial culture was taken, centrifuged at 12000 rpm for 1 min, the supernatant was discarded, and the culture was resuspended in 1 mL of 10 mM PBS. The OD600 was measured using a spectrophotometer. The remaining bacterial culture was appropriately diluted to make its OD600 the same, centrifuged at 12000 rpm for 1 min, the supernatant was discarded, and the bacterial cells were resuspended in 100 μL of 10 mM PBS containing 30% BugBuster protein extraction reagent. The cells were lysed in an ice bath for 15 min to obtain the crude enzyme solution. Simultaneously, the reaction system was prepared as follows: 100 μL of 10 mM PBS containing 10 μL of crude enzyme solution, 0.2 mM J2, 0.1 mM PLP, and 1 mM schizophylline. After lysis, the reaction system was placed in a Synergy H1 microplate reader (Bio-Tek Instruments, Inc.) and detected at 37 ℃ for 30 min; the detection parameters were set to 357 nm excitation and 525 nm emission.

[0056] (2) Liquid phase detection of activity: A reversed-phase column (Thermo Fisher Science, Hypersil™ ODS C18, 4.6 × 250 mm, 5 µm particle size) with a 254 nm UV detector was used. The analytical method involved 30 minutes of isocratic elution of each sample using an 80:20 mobile phase of acetonitrile (0.1% TFA):water (0.1% TFA). The flow rate was 0.5 mL / min, the detection volume was 10 µL, and the column temperature was maintained at 25 °C. After terminating the enzyme reaction by adding an equal volume of acetonitrile, the samples were centrifuged at 12,000 rpm for 5 minutes, filtered through a 0.22 μm microporous membrane, and then analyzed by high-performance liquid chromatography (HPLC).

[0057] Example 1: Construction of recombinant bacteria expressing PjTA-R6 transaminase.

[0058] The source of Pseudomonas jejuni ( Pseudomonas jesseniiThe PjTA-R6 transaminase gene was codon optimized using Escherichia coli as the host cell. The gene fragment with the nucleotide sequence shown in SEQ ID NO.1 was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and amplified using upstream and downstream amplification primers. The nucleic acid sequences of the upstream and downstream primers are shown in Table 1.

[0059] The amplification conditions were as follows: amplification at 98℃ for 30 seconds, then amplification at 98℃ for 30 seconds, amplification at 60℃ for 10 seconds, amplification at 72℃ for 70 seconds, for a total of 35 cycles, and finally amplification at 72℃ for 5 minutes.

[0060] After the reaction was complete, the PCR amplification product was detected by 1% agarose gel electrophoresis, yielding a band of approximately 1.4 kb, the length of which was in line with expectations. The PCR product was purified using a Novizan PCR product purification kit to remove primers, dNTPs, enzymes, and other impurities. The target fragment was recovered and purified according to the kit's standard procedures. The purified target gene fragment was recombined with the vector pET-28a(+) (Addgene: Plasmid #69864). The resulting ligation product was transformed into *E. coli* BL21(DE3) competent cells. The transformed cells were plated on LB plates containing 50 μg / ml Kana, and positive clone plasmids were extracted and sequenced. The results showed that the cloned PjTA-R6 transaminase gene sequence was correct and correctly inserted into the pET-28a(+) plasmid vector, yielding the recombinant plasmid pET-28a(+)-PjTA-R6. The full plasmid map is shown below. Figure 1 .

[0061] Example 2 Expression and purification of PjTA-R6 transaminase.

[0062] The engineered bacteria from the glycerol tube were inoculated at a volume ratio of 1% into 3 mL LB medium tubes containing 50 μg / mL kanamycin and cultured at 37℃ and 220 rpm for 12 h. 800 μL of the bacterial suspension was then transferred to a 50 mL TB medium shake flask containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 2 h until the OD600 reached approximately 1. 1% IPTG inducer was then added, and the culture was induced at 16℃ and 200 rpm for 18 h. The harvested *E. coli* bacterial suspension was ultrasonically disrupted and then subjected to a one-step Ni-NTA affinity chromatography to obtain PjTA-R6 transaminase protein with a purity >95%. The amino acid sequence is SEQ ID NO.1, and the gel electrophoresis image is shown below. Figure 6 .

[0063] Example 3: Rational design and screening of mutants of PjTA-R6 transaminase.

[0064] To achieve efficient and rational design of PjTA-R6 transaminase and enhance its catalytic activity against the bulky substrate sclerophyllin, this invention employs a comprehensive strategy that combines computational simulation with experimental verification and screening.

[0065] The specific design and screening process is as follows: (1) Construction and optimization of the computational model Derived from Pseudomonas japonicus ( Pseudomonas jessenii The crystal structure of transaminase PjTA-R6 (PDB: 6BT1) was used as the initial template for calculations. First, the original crystal structure was preprocessed using molecular modeling software PyMOL and Rosetta, including completing missing amino acid residues, adding hydrogen atoms, and optimizing the side chain conformation, to obtain a complete and energy-optimal three-dimensional enzyme structure model for subsequent calculations.

[0066] (2) Substrate docking and conformational sampling The three-dimensional structure of the target substrate, schizosporine, was constructed and its energy minimized in ChemOffice software. Subsequently, the Rosetta Ligand module was used to dock schizosporine into the active pocket of PjTA-R6. The docking process considered the substrate's flexibility, sampling around its rotatable bonds, resulting in 49 different initial conformations (Poses) of the substrate-enzyme complex. By visually inspecting these conformations and using the Rosetta Total Score, we selected the lowest-energy conformation as the representative binding mode (e.g., ...). Figure 2 As shown in Figure A), in this conformation, the aldehyde functional group of the cleaved strychnine is correctly located near the catalytic center lysine (K288), and its large iridoid skeleton is oriented and contained in a large binding pocket composed of residues such as L57, Y20, and L164, which lays the foundation for subsequent energy analysis.

[0067] (3) Global free energy scanning and identification of key residues Based on the optimal enzyme-substrate complex conformation selected above, we performed a systematic global binding free energy (BFE) scan using the Rosetta ddG_monomer application. This calculation quantifies the contribution of each amino acid residue to the overall BFE of the enzyme-substrate complex. Through analysis, we successfully located eight key residues that significantly contribute to the BFE (Delta binding energy < -1.0 kcal / mol): M419, Y151, L57, Y20, L164, R417, P19, and A230. These residues were preliminarily identified as key sites affecting substrate binding and potential mutation hotspots.

[0068] (4) Verification by alanine scanning experiment To screen for the optimal sites for functional optimization from the eight key residues predicted by calculation, we performed an alanine scan. Using site-directed mutagenesis (specific primers are shown in Table 1), we mutated each of the eight sites to alanine (Ala) to eliminate all atoms of the side chain beyond Cβ, leaving only the simplest methyl side chain, thereby evaluating the role of this side chain in catalysis.

[0069] The constructed alanine mutant plasmid was transformed into Escherichia coli BL21(DE3) for expression, and the relative activity of the crude enzyme solution of each mutant against schizosphingine was rapidly determined by microplate fluorescence detection method (see Example 5(1) for details). Experimental results ( Figure 3 A) Clearly displayed.

[0070] The mutants L57A, Y20A, L164A, and P19A showed comparable or slightly enhanced activity compared to the wild type, indicating that the wild-type residues at these positions are not strictly necessary and their side chains can be modified to optimize performance, thus identifying them as promising engineering hotspots.

[0071] The mutants M419A, R417A, and Y151A resulted in significant loss of enzyme activity or even complete inactivation, demonstrating that these residues play an irreplaceable role in maintaining the structural integrity or catalytic function of the enzyme. Therefore, they were excluded from the subsequent saturation mutation range in this strategy.

[0072] (5) Consensus-based directed saturation mutation Based on the results of alanine scanning, we focused on in-depth modification of four promising hotspot residues (L57, Y20, L164, and P19). To overcome the limitations of traditional single-type mutations, we adopted a consensus-based targeted saturation mutation strategy. The core of this strategy is that for each hotspot site, instead of introducing amino acids with similar properties, we purposefully replace them with multiple amino acids that differ significantly in physicochemical properties such as charge, hydrophobicity, and side chain size. For example, we mutated the hydrophobic leucine (L) to negatively charged glutamic acid (E), positively charged arginine (R), or larger aromatic amino acids such as tyrosine (Y) and phenylalanine (F). The single-mutant protein gel image is shown below. Figure 6 A.

[0073] Using this strategy, we constructed a library containing multiple single-point mutants. High-throughput activity screening of these single-point mutants revealed that 48.1% of the variants exhibited enhanced activity compared to wild-type PjTA-R6. Figure 3 (B) This fully demonstrates the effectiveness of the rational design strategy.

[0074] (6) Construction and screening of combined mutants After obtaining a batch of single-point mutants with positive effects (such as L57F, L164E, L164Y, Y20R, etc.), we performed combinatorial mutagenesis to further explore the synergistic effects between sites. We combined different single-point mutations to construct several double-point mutants (such as L164E / L57F, L164Y / Y20L, L164E / Y20L, etc.). The proteoglycan images of the double mutants are shown below. Figure 6 B.

[0075] Activity assays were performed on these combined mutants, and the results were ( Figure 3 C) indicates that multiple double-point mutants exhibit significantly greater catalytic activity than single-point mutants. Among them, the L164E / L57F mutant showed the most significant effect (its protein structure was predicted using AlphaFold structural modeling, as shown in Figure 1). Figure 2 B), whose specific activity was increased by 61.5 times compared to the wild type. This demonstrates that the mutant combination obtained through rational design can produce a synergistic effect of "1+1>2", significantly breaking through the catalytic bottleneck of the wild-type enzyme.

[0076] Example 4: Construction, expression, and purification of single / double point mutants of PjTA-R6 transaminase.

[0077] (1) Construction of recombinant bacteria expressing PjTA-R6 mutant Using the pET-28a(+)-PjTA-R6 plasmid (nucleotide sequence as shown in SEQ ID NO.1) as a template, the target mutant was constructed using the primers shown in Table 1 of the instruction manual and a site-directed mutagenesis kit (purchased from Novizan).

[0078] Table 1. Amplification primer sequences for alanine mutations, single-point mutations, and double-point mutations. The reaction mixture (50 μL) consisted of: 1 μL template DNA (PjTA-R6), 2 μL forward primer (F), 2 μL reverse primer (R), 25 μL 2×Phanta Max Buffer, 2 μL dNTP Mix, 1 μL Phanta Max Super-Fidelity DNA Polymerase, and ddH2O to a final volume of 50 μL. The PCR program was set as follows: 98℃ pre-denaturation for 30 s; 35 cycles of amplification (98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 70 s, adjusting the extension time by 1 kb / 10 s); and a final extension at 72℃ for 300 s. After amplification, 4 μL of PCR product was mixed with 1 μL of 5× Loading buffer and electrophoresed on a 1% agarose gel (containing 0.5 μg / mL GoldView™ dye) (120 V, 50 min), and imaged using the GelDoc XR+ system. The PCR product was recovered from the gel, and the product was digested with DpnI enzyme at 37℃ for 1 h to degrade the initial template. The digested product was then recombined to obtain the reconstructed plasmid. The double-point mutant plasmid was obtained by further processing the above steps using the single-point mutant plasmid as a vector. The reconstructed plasmid was transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin resistance, and cultured overnight at 37℃. Positive clones were screened and sequenced to verify the results, yielding recombinant bacteria containing single / double-point mutants of PjTA-R6 transaminase.

[0079] The PCR amplification described above was performed using the Vazyme high-fidelity PCR kit.

[0080] The DpnI enzyme mentioned above was provided by Fermentas.

[0081] (2) Expression and purification of PjTA-R6 mutant The recombinant bacteria constructed in step (1) were inoculated into a 3 mL LB medium tube containing 50 μg / mL kanamycin and cultured at 37℃ and 220 rpm for 12 h. 800 μL of the bacterial culture was then transferred to a 50 mL TB medium shake flask containing 50 μg / mL kanamycin and cultured at 37℃ and 220 rpm for 2.5 h to allow the OD to adjust. 600 When the concentration reaches 0.8-1, add 1 mM MIPTG inducer and induce culture at 16℃ and 200 rpm for 20 h. Collect the E. coli cell suspension, sonicate and then perform a one-step Ni-NTA affinity chromatography to obtain the mutant protein of PjTA-R6 transaminase with a purity >95%; the protein gel image is shown below. Figure 6 As shown.

[0082] Example 5: Detection of catalytic activity of PjTA-R6 transaminase mutant.

[0083] (1) Microplate detection To rapidly screen enzyme mutants with higher activity, p-aminonaphthylamine J2 was used as the amine donor. Enzyme activity was reflected by detecting the increase in fluorescence signal of its corresponding product, p-aminonaphthylone (J1), using a microplate assay with OD... 600 The catalytic activity of the supernatant from the bacterial lysate was compared to the activity of the mutant relative to the original enzyme PjTA-R6. The procedure was as follows: The target plasmid was transformed into *E. coli* BL21(DE3) cells and then cultured on LB agar plates containing kanamycin. Single colonies were picked and inoculated into 3 mL of liquid LB medium as primary seed culture and incubated overnight (12 hours). The next day, 2% was transferred to 3 mL of liquid LB medium containing 100 μg / mL and cultured at 37°C and 220 rpm for approximately 2.5 h until OD was reached. 600 Add 3 μL of 1M IPTG to the culture at 0.6, and incubate the culture at 16°C with shaking at 220 rpm for 16 hours.

[0084] After induction, take 1 mL of bacterial culture from each culture, centrifuge at 12000 rpm for 1 min, discard the supernatant, resuspend in 1 mL of 10 mM PBS, and measure the OD using a spectrophotometer. 600 Dilute the remaining bacterial culture appropriately to adjust its OD value. 600 Similarly, the cells were centrifuged at 12000 rpm for 1 min, the supernatant was discarded, and the cells were resuspended in 100 μL of 10 mM PBS containing 30% BugBuster protein extraction reagent. The cells were then lysed on ice for 15 min to obtain the crude enzyme solution. Simultaneously, a reaction system was prepared: 100 μL of 10 mM PBS containing 10 μL of the crude enzyme solution, 0.2 mM J2, 0.1 mM PLP, and 1 mM schizophylline. After lysis, the reaction system was placed in a Synergy H1 microplate reader (Bio-Tek Instruments, Inc.) and detected at 37°C for 30 min; the detection parameters were set to 357 nm excitation and 525 nm emission. The relative enzyme activities of single-point mutations and double-point combination mutations are as follows: Figure 3 .

[0085] For the highly active crude enzymes identified through rapid screening, the activity of the corresponding mutant pure enzymes can be further precisely measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0086] The reaction system consisted of 100 μL of 10 mM PBS (pH 8.0), containing 10 μL of appropriately diluted purified enzyme, 0.2 mM amine donor J2, 0.1 mM PLP, and 1 mM substrate schizophylline. The reaction was performed at 37°C using a microplate reader (Synergy H1), and the fluorescence signal was monitored over 30 minutes (excitation wavelength 357 nm, emission wavelength 525 nm). Enzyme activity was calculated based on the rate of increase in fluorescence intensity per unit time. The results are shown below. Figure 3 As shown in C, the relative enzyme activity of the double-point mutant L164E / L57F reached 61.5 times that of the wild type.

[0087] (2) High performance liquid chromatography (HPLC) detection A reversed-phase column (Thermo Fisher Science, Hypersil™ ODS C18, 4.6 × 250 mm, 5 µm particle size) with a 254 nm UV detector was used. The analytical method involved 30 minutes of isocratic elution for each sample using an 80:20 mobile phase of acetonitrile (0.1% TFA):water (0.1% TFA). The flow rate was 0.5 mL / min, the detection volume was 10 µL, and the column temperature was maintained at 25 °C. After terminating the enzyme reaction by adding an equal volume of acetonitrile, the samples were centrifuged at 12000 rpm for 5 minutes, filtered through a 0.22 μm microporous membrane, and then analyzed by high-performance liquid chromatography (HPLC). The total detection time was 30 minutes, primarily examining the reactions of several major mutants and wild-type samples. The two-point mutant combination showed varying degrees of improvement in conversion rate and reaction rate compared to the PjTA-R6 wild-type.

[0088] Example 6: Determination of enzyme kinetic parameters.

[0089] To accurately quantify the performance improvement of the PjTA-R6 mutant described in this invention compared to the wild type in the catalytic cleavage of Strychnos nux-vomica alkaline amination reaction, we conducted a systematic steady-state enzyme kinetic analysis on the wild type and representative high-activity mutants (taking L164E / L57F as an example).

[0090] (1) Enzyme sample preparation Wild-type PjTA-R6 and mutant L164E / L57F proteins used for kinetic testing were expressed and purified according to the method described in Example 2, yielding pure enzymes with a purity >95%. Protein concentration was determined using a NanoDrop microspectrophotometer and calculated based on absorbance at 280 nm and theoretical extinction coefficient to ensure concentration accuracy. The purified enzyme solution was diluted with reaction buffer (10 mM PBS, pH 8.0) to a series of suitable concentrations for subsequent kinetic analysis.

[0091] (2) Kinetic reaction system and conditions Enzyme kinetics assays were performed in a 100 μL system containing a fixed concentration of amine donor: 200 μM p-aminonaphthylamine (J2).

[0092] Gradient concentration amine receptor: schizocyclic strychnine, with concentration gradients set at 1, 2, 5, 10, 20, 40, 60, 80, 100, and 120 mM to adequately cover the kinetic range of the enzymatic reaction from zero to first order. Cofactor: 0.1 mM. Pyridoxal phosphate (PLP) enzyme amount: Optimized through preliminary experiments, the final concentration of wild-type PjTA-R6 was 0.05 mg / mL, while the final concentration of the highly active mutant L164E / L57F was 0.01 mg / mL. Buffer system: 10 mM PBS (pH 8.0).

[0093] All reactions were performed at 37°C using a microplate reader (Synergy H1, BioTek).

[0094] (3) Initial reaction rate determination The initial reaction rate was determined using continuous fluorescence monitoring (CFRM). After reaction initiation, the fluorescence intensity was continuously monitored at 37°C for 30 minutes, with readings taken every 30 seconds at the excitation wavelength of 357 nm and the emission wavelength of 525 nm. The reaction progress was characterized by monitoring the increase in the fluorescence signal of product J1. At each substrate concentration, the fluorescence intensity-time curve was selected during the initial linear phase of the reaction (typically the first 5-10 minutes), and its slope was calculated as the initial rate (v0), expressed in fluorescence intensity units per minute (RFU / min). Three parallel experiments were conducted at each concentration point, and the average value was taken.

[0095] (4) Calibration of the standard curve of fluorescence signal versus product concentration To ensure that the initial rate (v0, RFU / min) can be accurately converted to the absolute reaction rate (v, μM / s or mM / s), we pre-plotted a product concentration-fluorescence intensity standard curve (as shown in the figure). Fluorescence detection was performed on pure product J1 at known concentrations under the same reaction conditions to establish a linear relationship between fluorescence intensity and product concentration. The linear regression coefficient (R²) of this standard curve is... 2 The value is greater than 0.99, which ensures the accuracy of the dynamic data calculation.

[0096] (5) Fitting the Michaelis equation and calculating dynamic parameters The measured initial reaction rate (v) was plotted against the corresponding substrate cleavage strychnine concentration. The Michaelis-Menten equation was fitted using nonlinear regression with the professional data analysis software GraphPad Prism 10.2. v=(Vmax×[S]) / (Km+[S]) The fitting process uses the least squares method, and the software automatically calculates the following key enzyme kinetic parameters: Michaelis constant (Km): Characterizes the apparent affinity between an enzyme and its substrate; the lower the Km value, the higher the affinity. Maximum reaction rate (Vmax): The maximum reaction rate when the enzyme is saturated with substrate.

[0097] Catalytic constant (Kcat): represents the number of catalytic conversions per unit time for each enzyme active site. The calculation formula is Kcat=Vmax / [E], where [E] is the molar concentration of the enzyme in the reaction system.

[0098] Catalytic efficiency (Kcat / Km): This is the core comprehensive indicator for evaluating the catalytic performance of an enzyme. The higher the value, the higher the catalytic efficiency of the enzyme.

[0099] (6) Dynamic results and analysis The fitting results are as follows Figure 4 As shown, the enzyme kinetic curves of wild type and mutant L164E / L57F (e.g.) Figure 3 D) shows significant differences, and the specific dynamic parameters are summarized in Table 2.

[0100] Table 2. Enzyme kinetic parameters of the optimal mutant (L164E / L57F) and wild-type PjTA-R6 Example 7: Whole-cell catalysis application.

[0101] To verify the performance of the PjTA-R6 mutant described in this invention in practical biocatalytic applications, we conducted an application study on the whole-cell catalytic synthesis of bacamaroline, using the optimal mutant L164E / L57F as a representative and comparing it with the wild type (WT).

[0102] (1) Preparation of catalytic cells Recombinant *E. coli* BL21(DE3) carrying the plasmids pET-28a(+)-PjTA-R6 and pET-28a(+)-PjTA-R6(L164E / L57F) (construction and validation are described in Examples 1 and 4) were streaked onto LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 12–16 hours. Single, plump colonies were picked from each plate and inoculated into 3 mL of liquid LB medium containing 50 μg / mL kanamycin. The culture was then incubated at 37°C and 220 rpm with shaking for approximately 12 hours to obtain the primary seed culture.

[0103] Expanded culture and induced expression: At an inoculum rate of 2% (v / v), the primary seed culture was transferred to 50 mL of TB medium containing 50 μg / mL kanamycin and cultured at 37℃ and 220 rpm until OD500 was achieved. 600 The concentration was increased to 0.8–1.0. Isopropyl-β-D-thiogalactoside (IPTG) was then added to a final concentration of 1 mM, and the culture temperature was lowered to 16°C. Expression was induced for another 20 hours at 200 rpm to express soluble transaminases. After induction, 50 mL of culture medium was centrifuged at 4°C and 5000 × g for 10 minutes, and the supernatant was discarded. The bacterial pellet was washed once with 10 mM PBS buffer (pH 8.0), centrifuged again, and the wet bacterial cells were collected and weighed for later use.

[0104] (2) Whole-cell catalytic reaction Establish a catalytic reaction system in a 250 mL Erlenmeyer flask. The total volume of the system is 50 mL, containing: ① Wet bacterial cells: equivalent to 0.5 g dry weight of L164E / L57F or WT wet bacterial cells (preliminary experiments determined the wet bacterial cell to dry weight ratio to be approximately 5:1, therefore the actual wet bacterial cell addition amount is 2.5 g); ② Substrate: 1 mM schizophyllin; ③ Amine donor: 0.2 mM p-aminonaphthylamine (J2); ④ Cofactor: no need to add pyridoxal phosphate (PLP); ⑤ Reaction medium: 10 mM PBS buffer (pH 8.0), bring to 50 mL.

[0105] The conical flask was placed in a constant-temperature shaker at 37℃ and 220 rpm for the catalytic reaction. The reaction lasted for 3 days. At time points of 0h, 6h, 12h, 24h, 2d, and 3d, 1.0 mL of the reaction solution was accurately pipetted into a 1.5 mL centrifuge tube, and an equal volume (1.0 mL) of pre-cooled acetonitrile was immediately added. The mixture was then vortexed to terminate the enzyme reaction.

[0106] (3) Sample processing and analysis Sample pretreatment: After the reaction was terminated, the sample was centrifuged at 4°C and 12,000 × g for 10 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane, and the filtrate was used for high-performance liquid chromatography (HPLC) analysis.

[0107] A reversed-phase column (Thermo Fisher Science, Hypersil™ ODS C18, 4.6 × 250 mm, 5 µm particle size) with a 254 nm UV detector was used. The analytical method involved 30 minutes of isocratic elution of each sample using an 80:20 mobile phase of acetonitrile (0.1% TFA):water (0.1% TFA). The flow rate was 0.5 mL / min, the detection volume was 10 µL, and the column temperature was maintained at 25 °C. After terminating the enzyme reaction by adding an equal volume of acetonitrile, the samples were centrifuged at 12,000 rpm for 5 minutes, filtered through a 0.22 μm microporous membrane, and then analyzed by high-performance liquid chromatography (HPLC).

[0108] Quantitative analysis: Quantitative analysis was performed using the external standard method. The standard of the schizocyclic strychnine reduction-amination product was accurately weighed, and a series of standard solutions with varying concentrations were prepared. These solutions were analyzed under the same HPLC conditions as the sample, and a peak area-concentration standard curve (linear regression coefficient R0) was plotted. 2 >0.999). Based on the standard curve, convert the peak area of ​​the sample into product concentration and calculate the conversion rate.

[0109] Results and Analysis: Whole-cell catalysis results are as follows: Figure 5 As shown in Table 3.

[0110] Table 3 Enzyme kinetic parameters of the optimal mutant (L164E / L57F) and wild-type PjTA-R6 The results analysis shows that: 1. Significantly improved catalytic efficiency: Within the same reaction time, the whole-cell catalyst expressing the mutant L164E / L57F achieved a much higher final product concentration than the wild-type control group. The mutant achieved near-complete substrate conversion (conversion rate > 99%) within 24 hours, while the wild-type conversion rate was only 53%.

[0111] 2. Superior application performance: The mutant's relative wild-type activity is increased to 99%. This fully demonstrates that the highly active mutant L164E / L57F obtained through rational design in this invention not only exhibits excellent kinetic parameters at the pure enzyme level, but also demonstrates superior application performance in a simpler, more economical, and easily scaled-up whole-cell catalytic system, efficiently converting schizosphingine into the target reduced amination product.

[0112] This embodiment demonstrates that the PjTA-R6 mutant provided by the present invention has great industrial application potential and market value in whole-cell catalysis processes that simplify downstream processes and reduce production costs.

[0113] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A mutant of PjTA-R6 transaminase, characterized in that, Based on the PjTA-R6 transaminase with the amino acid sequence shown in SEQ ID NO.2, it has at least one of the following mutations: N167Y, L57F, L164D, L164E, L164Y, P19E, P19Y, Y20R, Y20K.

2. The mutant of PjTA-R6 transaminase according to claim 1, characterized in that, The amino acid sequence corresponding to the mutant of the PjTA-R6 transaminase is as shown in SEQ ID NO.

2. The parent PjTA-R6 transaminase is subjected to any of the following mutations: (1) The lysine at position 167 is mutated to tyrosine; (2) The lysine at position 57 is mutated to phenylalanine; (3) The lysine at position 164 is mutated to aspartic acid; (4) Mutate the lysine at position 164 to glutamic acid; (5) Mutate the lysine at position 164 to tyrosine; (6) The lysine at position 19 is mutated to glutamic acid; (7) The lysine at position 19 is mutated to phenylalanine; (8) The lysine at position 19 is mutated to tyrosine; (9) The lysine at position 20 is mutated to arginine; (10) The lysine at position 20 is mutated to lysine; (11) The leucine at position 164 is mutated to glutamic acid, and the proline at position 19 is mutated to tyrosine. (12) The leucine at position 164 is mutated to glutamic acid, and the leucine at position 57 is mutated to phenylalanine. (13) The leucine at position 164 is mutated to glutamic acid, and the asparagine at position 167 is mutated to tyrosine. (14) The leucine at position 164 is mutated to glutamic acid, and the tyrosine at position 20 is mutated to lysine. (15) The leucine at position 164 is mutated to tyrosine, and the proline at position 19 is mutated to tyrosine. (16) The leucine at position 164 is mutated to tyrosine, and the leucine at position 57 is mutated to phenylalanine. (17) The leucine at position 164 is mutated to tyrosine, and the asparagine at position 167 is mutated to tyrosine. (18) The leucine at position 164 is mutated to tyrosine, and the tyrosine at position 20 is mutated to lysine. (19) The leucine at position 164 is mutated to glutamic acid, and the tyrosine at position 20 is mutated to arginine. (20) The leucine at position 164 is mutated to tyrosine, and the tyrosine at position 20 is mutated to arginine.

3. An enzyme preparation containing a mutant of the PjTA-R6 transaminase according to claim 1 or 2, characterized in that, The protein contains a mutant of the PjTA-R6 transaminase, and the enzyme preparation is an immobilized enzyme of the mutant of the PjTA-R6 transaminase.

4. The method for preparing the mutant of PjTA-R6 transaminase according to claim 1 or 2, wherein the method is as follows: (1) Construct a recombinant plasmid expressing a mutant of PjTA-R6 transaminase and transform it into competent Escherichia coli cells to obtain recombinant engineered Escherichia coli. The recombinant microorganisms were cultured to OD200. 600 When the concentration reaches 0.8~1, add 1 mMIPTG as an inducer and induce culture at 16℃ for 20 h; (2) The recombinant Escherichia coli was fermented in a culture medium, and the fermentation was induced by isopropyl-β-D-thiogalactoside (IPTG). The fermentation was carried out in TB medium and / or LB medium. (3) The Escherichia coli cell suspension harvested after fermentation was ultrasonically broken, and then subjected to a one-step Ni-NTA affinity chromatography treatment to obtain the mutant protein of PjTA-R6 transaminase with a purity of >95%.

5. The preparation method according to claim 4, characterized in that, Step (3) The recombinant Escherichia coli was cultured in LB medium at 37°C until OD200 reached. 600 When the concentration reaches 0.8-1, induce with IPTG at a final concentration of 1 mM for 20 h at 16°C. Then collect the cells and transfer them to TB / LB medium, and continue to culture at 36°C for at least 20 h.

6. The mutant protein of PjTA-R6 transaminase obtained by the method of claim 4 is used as a catalyst in the catalytic production of bacamaroline.

7. The application according to claim 6, characterized in that, The application utilizes the obtained PjTA-R6 transaminase (PjTA-R6) or a mutant of PjTA-R6 transaminase (PjTA-R6 mutant) as a catalyst, and uses schizosporine (1a) and p-aminonaphthylamine (1b) as raw materials to catalyze the production of baccacomarine (1c), as shown in the following reaction formula: 。

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