Arylalkylamine N-acetyltransferase mutant and application thereof in catalytic synthesis of N-acetyl-5-hydroxytryptamine

Abstract

The invention provides an arylalkylamine N-acetyltransferase mutant and an application of the arylalkylamine N-acetyltransferase mutant in synthesis of N-acetyl-5-hydroxytryptamine. According to the invention, a series of mutants with N-acetylation activity are obtained through gene mining, directed evolution and the like, and the mutants can directly acetylate a substrate 5-hydroxytryptamine to generate N-acetyl-5-hydroxytryptamine. Therefore, the arylalkylamine N-acetyltransferase mutants have important application value in the industry.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an arylalkylamine N-acetyltransferase mutant and its application in the catalytic synthesis of N-acetyl-5-hydroxytryptamine. Background Art

[0002] Melatonin (N-acetyl-5-methoxytryptamine, MT) is widely present in animals, plants, and microorganisms. As an important indole neurohormone, it participates in the body's reproductive and immune regulation. The main pharmacological activities of melatonin are manifested in multiple effects such as balancing the rhythm of biological clock sleep and wakefulness, improving sleep, enhancing immunity, improving memory, and anti-anxiety. So far, the synthesis of melatonin mainly uses chemical methods, but the method has a long synthesis route, harsh reaction conditions, high energy consumption, low yield, and serious environmental pollution. Large-scale production is not feasible. In comparison, the bioenzymatic method has the advantages of being green and environmentally friendly, short cycle, continuous production, and mild reaction conditions. The bioenzymatic synthesis of melatonin is a continuous catalytic synthesis of four enzyme cascades using L-tryptophan as a substrate: 1) L-tryptophan is hydroxylated to form 5-hydroxytryptophan, 2) decarboxylation to produce 5-hydroxytryptamine, 3) acetylation to form N-acetyl-5-hydroxytryptamine, and 4) methylation to produce melatonin. In 2020, Bernhard Palsson's team constructed a melatonin synthesis pathway in E. coli (L Hao et al. Microbial Synthesis of Human-Hormone Melatoninat Gram Scales.2020.DOI:10.1021 / acssynbio.0c00065), achieving melatonin production of 1 g / L from glucose and 2 g / L from tryptophan, respectively.

[0003] Currently, high titers of melatonin cannot be produced in heterologous hosts because genes involved in the melatonin biosynthesis pathway exhibit insolubility or low enzyme activity. Arylalkylamine N-acetyltransferase (AANAT), involved in melatonin biosynthesis, is a key rate-limiting enzyme in melatonin biosynthesis. Arylalkylamine N-acetyltransferase (AANAT) is an acetyl-CoA-dependent enzyme that catalyzes the transfer of the acetyl group in acetyl-CoA to 5-hydroxytryptamine to produce N-acetyl-5-hydroxytryptamine. AANAT is an important rate-limiting enzyme regulating melatonin synthesis, but the wild-type AANAT enzyme has low catalytic efficiency for the substrate 5-hydroxytryptamine. Therefore, it is very necessary to develop arylalkylamine N-acetyltransferase (AANAT) mutants that can effectively convert 5-hydroxytryptamine to N-acetyl-5-hydroxytryptamine. Summary of the Invention

[0004] To address the shortcomings of the prior art, the present invention provides an arylalkylamine N-acetyltransferase (AANAT) mutant for use in preparing N-acetyl-5-hydroxytryptamine. The present invention utilizes genetic engineering techniques and computer-aided design to construct a highly active arylalkylamine N-acetyltransferase mutant, thereby significantly increasing the production of N-acetyl-5-hydroxytryptamine.

[0005] The present invention provides a method for synthesizing N-acetyl-5-hydroxytryptamine by using arylalkylamine N-acetyltransferase or a mutant thereof.

[0006] To this end, the present invention modified the arylalkylamine N-acetyltransferase (SsAANAT) from Sus scrofa (GenBank No. XP_005656968.1) through gene mining, directed evolution, and rational design techniques to obtain mutants with higher N-acetyl-5-hydroxytryptamine production, thereby enabling more efficient production of N-acetyl-5-hydroxytryptamine using industrial microorganisms.

[0007] Therefore, the first object of the present invention is to provide a protein obtained by mutating an aralkylamine N-acetyltransferase AANAT (SsAANAT, GenBank No.: XP_005656968.1) derived from the wild boar species (Sus scrofa), wherein the mutation is based on the amino acid sequence of the aralkylamine N-acetyltransferase AANAT and contains the following mutations at one or more of the following amino acid residue sites: position 5, position 8, position 58, position 62, position 66, position 82, position 91, position 104, position 138, position 163 and / or position 182.

[0008] All of the above amino acid mutants possess the catalytic function of wild-type aralkylamine N-acetyltransferases, as shown by SsAANAT, in catalyzing N-acetyl-5-hydroxytryptamine.

[0009] More specifically, the SsAANAT arylalkylamine N-acetyltransferase mutant is a protein obtained by subjecting SsAANAT to any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten or all of the following eleven modifications:

[0010] X1, mutating the threonine at position 5 of SsAANAT to serine;

[0011] X2, mutating the tyrosine at position 8 of SsAANAT to proline;

[0012] X3. Mutate the proline at position 58 of SsAANAT to serine;

[0013] X4. Mutate the threonine at position 62 of SsAANAT to glutamic acid;

[0014] X5. Mutate the asparagine at position 66 of SsAANAT to histidine;

[0015] X6. Mutate the methionine at position 82 of SsAANAT to leucine;

[0016] X7. Mutate the leucine at position 91 of SsAANAT to valine;

[0017] X8. Mutate the isoleucine at position 104 of SsAANAT to leucine;

[0018] X9. Mutate the valine at position 138 of SsAANAT to isoleucine;

[0019] X10. Mutate the arginine at position 163 of SsAANAT to phenylalanine;

[0020] X11. Mutate the serine at position 182 of SsAANAT to threonine.

[0021] In one embodiment of the present invention, the SsAANAT arylalkylamine N-acetyltransferase mutant is:

[0022] The protein obtained by mutating the 5th position of SsAANAT to serine;

[0023] The protein obtained by mutating the 8th position of SsAANAT to proline;

[0024] The protein obtained by mutating the 58th position of SsAANAT to serine;

[0025] The protein obtained by mutating the 104th position of SsAANAT to leucine;

[0026] In another embodiment of the present invention, the SsAANAT arylalkylamine N-acetyltransferase mutant is:

[0027] The protein obtained by mutating the 5th and 82nd sites of SsAANAT to serine and leucine, respectively;

[0028] The protein obtained by mutating the 5th and 91st sites of SsAANAT to serine and valine, respectively;

[0029] The protein obtained by mutating the 58th and 62nd sites of SsAANAT to serine and glutamic acid, respectively;

[0030] The protein obtained by mutating SsAANAT at positions 104 and 91 to leucine and valine, respectively.

[0031] The protein was obtained by mutating the 104th and 182th positions of SsAANAT to leucine and threonine, respectively;

[0032] The protein obtained by mutating the 58th, 62nd, and 163rd sites of SsAANAT to serine, glutamic acid, and phenylalanine, respectively.

[0033] The protein obtained by mutating the 58th, 62nd, and 138th sites of SsAANAT to serine, glutamic acid, and isoleucine, respectively;

[0034] The protein obtained by mutating the 58th, 62nd, and 91st sites of SsAANAT to serine, glutamic acid, and valine, respectively;

[0035] The protein obtained by mutating positions 104, 91, and 66 of SsAANAT to leucine, valine, and histidine, respectively.

[0036] The present invention provides a coding gene for the arylalkylamine N-acetyltransferase mutant, and a recombinant vector containing the coding gene.

[0037] The present invention further provides a recombinant bacterial strain, which is obtained by transferring the recombinant vector into a host cell. The host cell contains the recombinant vector or its genome integrated with a gene containing the arylalkylamine N-acetyltransferase or a mutant. Preferably, the originating bacterium is *Escherichia coli*, more preferably *E. coli* BL21(DE3).

[0038] The present invention therefore provides the use of the arylalkylamine N-acetyltransferase mutant, the encoding gene, the recombinant vector or the recombinant strain in producing N-acetyl-5-hydroxytryptamine.

[0039] The present invention also provides a method for producing N-acetyl-5-hydroxytryptamine, which has the advantages of mild reaction conditions, simple operation, and environmental friendliness. Specifically, the method comprises the following steps:

[0040] Using the aforementioned arylalkylamine N-acetyltransferase mutant or the aforementioned recombinant genetically engineered strain, N-acetyl-5-hydroxytryptamine is produced in a whole-cell catalytic manner using 5-hydroxytryptamine as a substrate; optionally, the method further includes a step of isolating N-acetyl-5-hydroxytryptamine.

[0041] This invention discloses the following technical achievements: The newly modified arylalkylamine N-acetyltransferase mutant of this invention produces N-acetyl-5-hydroxytryptamine using 5-hydroxytryptamine as a substrate through whole-cell catalysis, resulting in a significantly improved yield. Therefore, these arylalkylamine N-acetyltransferases have important industrial applications. Attached Figure Description

[0042] Figure 1 Plasmid map for the construction of the recombinant vector encoding arylalkylamine N-acetyltransferase.

[0043] Figure 2 The standard curve for the HPLC detection of N-acetyl-5-hydroxytryptamine standard.

[0044] Figure 3 The relative activity ratio of N-acetyl-5-hydroxytryptamine of the dominant mutant recombinant engineering strain is shown in FIG. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0047] Example 1: Discovery of Arylalkylamine N-acetyltransferases

[0048] The arylalkylamine N-acetyltransferase AANAT (GenBank ID: XP_005656968.1) from the Sus scrofa gene was obtained from the NCBI database. The full-gene SsAANAT was synthesized and ligated into the pET24a expression vector, which had been double-digested with NdeI and XhoI, to obtain the recombinant expression vector pET24a-SsAANAT. Figure 1 ).

[0049] Arylalkylamine N-acetyltransferase amino acid sequence (SEQ ID NO: 1): MSAQTSHYLKPRPLSLPPTIPE SPSRQRRHTLPASEFRCLTPEDAAGVFELEREAFIPVSGTCPLNVDEVRHFLTLCPELSMGWF LEGRLLAFIIGSLWDKERITQESLTLHRPGGRTAHLHLLAVHRAFRQQGKGSVLLWRYLHHL GSQPAVRRAVLMCEARLVPFYQRFGFRPVGPCAVSVGSLVFTEMQCSMRDLVSQRRNSDC.

[0050] The above-mentioned recombinant expression vector is transformed into a suitable microbial host. The host microorganism is any of the conventional host microorganisms in the art, as long as the above-mentioned recombinant expression vector can stably replicate and the arylalkylamine N-acetyltransferase gene can be effectively expressed. In the present embodiment, the above-mentioned recombinant expression plasmid is introduced into E.coli BL21 (DE3) competent cells by electroporation, and cultured in an inverted manner on a LB solid plate containing kanamycin resistance for 12-16 hours. Positive transformants are selected for DNA sequencing verification to verify that the correct transformants are arylalkylamine N-acetyltransferase genetically engineered strains.

[0051] Example 2: Construction of single-point mutants of arylalkylamine N-acetyltransferase or acquisition of mutants thereof

[0052] 2.1 Construction of the first round of single-point mutants of SsAANAT

[0053] Based on the substrate binding pocket and substrate molecule interaction of wild-type aralkylamine N-acetyltransferase AANAT (GenBank ID: XP_005656968.1), a rational design was implemented using 10 sites for single-point mutagenesis. Therefore, the first round of modification selected 10 sites: positions 3, 5, 6, 8, 58, 67, 91, 104, 123, and 163. Site-directed mutagenesis was performed at each site, as shown in Table 1.

[0054] Table 1. First round of single-point mutations based on SsAANAT

[0055]

[0056] To obtain a single-point mutant of arylalkylamine N-acetyltransferase SsAANAT, the following experiment was performed:

[0057] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SsAANAT plasmid 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of forward and reverse primers are shown in Table 1.

[0058] PCR amplification reaction conditions: 98°C: 2 min, (98°C: 10 s, 55°C: 15 s, 72°C: 4 min) 30 cycles, 72°C: 4 min.

[0059] The obtained PCR product was processed as follows: 1 μL of Dpn I enzyme was added to 20 μL of the PCR product to digest the plasmid template, and the mixture was treated at 37°C for 2 h. 5 μL of the digested PCR product was electroporated into 100 μL of E. coli BL21(DE3) competent cells. The electroporated E. coli BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 μg / mL) LB agar plates and cultured at 37°C for 14 h. Single colonies grew, which were the engineered strain of the arylalkylamine N-acetyltransferase SsAANAT gene mutant. Recombinant plasmids containing the target nucleotides of this strain with non-directional or directional mutations were used as expression vectors for expressing the arylalkylamine N-acetyltransferase SsAANAT gene mutant.

[0060] 2.2 Screening of single-point mutants in the first round of SsAANAT

[0061] After successful sequencing of each single-point mutant, the plasmid was electroporated into E. coli BL21(DE3) competent cells and plated onto Kansas plates. Single colonies were picked and inoculated into 5 mL of LB medium containing 5 μL kanamycin (final concentration 50 μg / mL), and cultured at 37°C and 220 rpm for 12 h. Aseptically, 1 mL of the bacterial culture was inoculated into 100 mL of TB medium (1% inoculum), and 100 μL of kanamycin (final concentration 50 μg / mL) was added. The culture was then incubated at 37°C and 220 rpm for approximately 3 h until OD (outcome limit) was reached. 600 Add 100 μL of isopropyl-β-D-thiogalactoside (IPTG) (final concentration 0.1 μmol / L) to a solution of 0.6-0.8 μL for induction, then incubate at 18℃ for 15 h to induce protein expression. Subsequently, centrifuge the protein at 4℃, 4000 rpm for 15 min, wash with PBK buffer (50 mM, pH 7.4), and resuspend.

[0062] Prepare a 500 μL reaction mixture: 50 mM KH₂PO₄ / K₂HPO₄ buffer (pH 7.4), 0.1 g / mL whole cells, and 5 mM 5-hydroxytryptamine. React at 30 °C and 1000 rpm for 4 h. Then, mix the 500 μL reaction mixture with 500 μL of methanol by shaking, centrifuge at 12000 rpm for 5 min, and collect the supernatant for HPLC analysis. Detection conditions: Zorbax SB-C18 column (4.6 mm × 150 mm, 5 μm), 275 nm, flow rate 1 mL / min at 35 °C. Injection volume: 10 μL; pump A: pure water (containing 0.1% trifluoroacetic acid); pump B: methanol; elution at equal ratios for 10 min.

[0063] Using the wild type of SsAANAT as a control, four mutants with relatively high activity, namely SsAANAT-T5S, SsAANAT-Y8P, SsAANAT-P58S, and SsAANAT-I104L, were obtained through preliminary screening. Their conversion rates of 5-hydroxytryptamine substrate were improved compared with the wild type. The results are shown in Table 2.

[0064] Table 2. Results of the first round of single-point mutations constructed by SsAANAT

[0065] strain Conversion rate (%) Relative output (times) SsAANAT 42.53 1 SsAANAT-T5S 55.56 1.31 SsAANAT-Y8P 54.38 1.28 SsAANAT-P58S 54.04 1.27 SsAANAT-I104L 49.43 1.16

[0066] In summary, the present invention is based on mutating a specific site of the wild-type arylalkylamine N-acetyltransferase SsAANAT. The resulting mutant has an improved conversion rate of the substrate 5-hydroxytryptamine, which is 1.16-1.31 times that of the starting strain, thereby increasing the production of N-acetyl-5-hydroxytryptamine.

[0067] Example 3: Construction of a two-site mutant of arylalkylamine N-acetyltransferase or obtaining a mutant thereof

[0068] 3.1 Construction of the second round of two-site mutants of SsAANAT

[0069] Based on the first round of dominant mutants SsAANAT-T5S, SsAANAT-Y8P, SsAANAT-P58S, and SsAANAT-I104L, a second round of two-site mutant modification was carried out. Based on the substrate binding pocket and substrate molecule interaction, a total of nine sites were rationally designed for two-site mutation. Therefore, using the four dominant mutants from the first round (SsAANAT-T5S, SsAANAT-Y8P, SsAANAT-P58S, and SsAANAT-I104L) as templates, the second round of modification was performed at positions 62, 82, 187, 104, 58, 91, 67, 123, and 182, respectively. Site-directed mutations were performed at each site, as shown in Table 3.

[0070] Table 3. Second round of two-site mutations constructed based on SsAANAT

[0071]

[0072] To obtain the arylalkylamine N-acetyltransferase SsAANAT mutant, the following experiment was performed:

[0073] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SsAANAT-T5S, SsAANAT-Y8P, SsAANAT-P58S, SsAANAT-I104L plasmid 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of the forward and reverse primers are shown in Table 2.

[0074] PCR amplification reaction conditions: 98℃: 2min, (98℃: 10s, 55℃: 15s, 72℃: 4min) 30 cycles, 72℃: 4min.

[0075] The obtained PCR product was processed as follows: 1 μL of Dpn I enzyme was added to 20 μL of the PCR product to digest the plasmid template, and the mixture was treated at 37°C for 2 h. 5 μL of the digested PCR product was electroporated into 100 μL of E. coli BL21(DE3) competent cells. The electroporated E. coli BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 μg / mL) LB agar plates and cultured at 37°C for 14 h. Single colonies grew, which were the engineered strain of the arylalkylamine N-acetyltransferase SsAANAT gene mutant. Recombinant plasmids containing the target nucleotides of this strain with non-directional or directional mutations were used as expression vectors for expressing the arylalkylamine N-acetyltransferase SsAANAT gene mutant.

[0076] 3.2 Second round of screening for SsAANAT two-site mutants

[0077] After successful sequencing of each dual-site mutant, the plasmid was electroporated into E. coli BL21(DE3) competent cells and plated onto Kansas plates. Single colonies were picked and inoculated into 5 mL of LB medium containing 5 μL kanamycin (final concentration 50 μg / mL), and cultured at 37°C and 220 rpm for 12 h. Aseptically, 1 mL of the bacterial culture was inoculated into 100 mL of TB medium (1% inoculum), and 100 μL of kanamycin (final concentration 50 μg / mL) was added. The culture was then incubated at 37°C and 220 rpm for approximately 3 h until OD (occurrence limit) was reached. 600 Add 100 μL of isopropyl-β-D-thiogalactoside (IPTG) (final concentration 0.1 μmol / L) to a solution of 0.6-0.8 μL for induction, then incubate at 18℃ for 15 h to induce protein expression. Subsequently, centrifuge the protein at 4℃, 4000 rpm for 15 min, wash with PBK buffer (50 mM, pH 7.4), and resuspend.

[0078] Prepare a 500 μL reaction mixture: 50 mM KH₂PO₄ / K₂HPO₄ buffer (pH 7.4), 0.1 g / mL whole cells, and 5 mM 5-hydroxytryptamine. React at 30 °C and 1000 rpm for 4 h. Then, mix the 500 μL reaction mixture with 500 μL of methanol by shaking, centrifuge at 12000 rpm for 5 min, and collect the supernatant for HPLC analysis.

[0079] Using the wild-type SsAANAT as a control, five mutants with relatively high activity were obtained through screening: SsAANAT-T5S-M82L, SsAANAT-T5S-L91V, SsAANAT-P58S-T62E, SsAANAT-I104L-S182T, and SsAANAT-I104L-L91V. All of them showed improved conversion rates of 5-hydroxytryptamine substrates compared to the wild-type. The results are shown in Table 4.

[0080] Table 4. Results of the second round of two-site mutations constructed using SsAANAT

[0081] strain Conversion rate (%) Relative output (times) SsAANAT 42.53 1 SsAANAT-T5S-M82L 58.26 1.37 SsAANAT-T5S-L91V 61.20 1.44 SsAANAT-P58S-T62E 70.77 1.66 SsAANAT-I104L-S182T 59.05 1.39 SsAANAT-I104L-L91V 64.59 1.52

[0082] In summary, this invention is based on a two-site combination mutation of the SsAANAT single-site dominant mutant. The resulting mutant has an increased conversion rate of the substrate 5-hydroxytryptamine, which is 1.37-1.66 times that of the starting strain, thus increasing the yield of N-acetyl-5-hydroxytryptamine.

[0083] Example 4: Construction of a three-point mutant of arylalkylamine N-acetyltransferase or obtaining a mutant thereof

[0084] 4.1 Construction of the third round of SsAANAT three-point mutants

[0085] Based on the second round of advantageous mutants SsAANAT-T5S-M82L, SsAANAT-T5S-L91V, SsAANAT-P58S-T62E, SsAANAT-I104L-S182T, and SsAANAT-I104L-L91V, a third round of three-point mutant modification was carried out. Based on the substrate binding pocket and substrate molecule interactions, a total of 11 sites were rationally designed for three-point mutation. Therefore, the five dominant mutants from the second round, SsAANAT-T5S-M82L, SsAANAT-T5S-L91V, SsAANAT-P58S-T62E, SsAANAT-I104L-S182T, and SsAANAT-I104L-L91V, were used as templates for the third round of modification. Site-directed mutations were performed at positions 163, 123, 138, 82, 104, 91, 67, 62, 66, 58, and 86, respectively, as shown in Table 5.

[0086] Table 5. Third-round three-point mutations constructed based on SsAANAT

[0087]

[0088] To obtain the arylalkylamine N-acetyltransferase SsAANAT mutant, the following experiment was performed:

[0089] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, templates SsAANAT-T5S-M82L, SsAANAT-T5S-L91V, SsAANAT-P58S-T62E, SsAANAT-I104L-S182T, SsAANAT-I104L-L91V plasmids 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of the forward and reverse primers are shown in Table 6.

[0090] PCR amplification reaction conditions: 98℃: 2min, (98℃: 10s, 55℃: 15s, 72℃: 4min) 30 cycles, 72℃: 4min.

[0091] The obtained PCR product was processed as follows: 1 μL of Dpn I enzyme was added to 20 μL of the PCR product to digest the plasmid template, and the mixture was treated at 37°C for 2 h. 5 μL of the digested PCR product was electroporated into 100 μL of E. coli BL21(DE3) competent cells. The electroporated E. coli BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 μg / mL) LB agar plates and cultured at 37°C for 14 h. Single colonies grew, which were the engineered strain of the arylalkylamine N-acetyltransferase SsAANAT gene mutant. Recombinant plasmids containing the target nucleotides of this strain with non-directional or directional mutations were used as expression vectors for expressing the arylalkylamine N-acetyltransferase SsAANAT gene mutant.

[0092] 4.2 Screening of SsAANAT third-round three-point mutants

[0093] After successful sequencing of each three-point mutant, the plasmid was electroporated into E. coli BL21(DE3) competent cells and plated onto Kansas plates. Single colonies were picked and inoculated into 5 mL of LB medium containing 5 μL kanamycin (final concentration 50 μg / mL), and cultured at 37°C and 220 rpm for 12 h. Aseptically, 1 mL of the bacterial culture was inoculated into 100 mL of TB medium (1% inoculum), and 100 μL of kanamycin (final concentration 50 μg / mL) was added. The culture was then incubated at 37°C and 220 rpm for approximately 3 h until OD (occurrence limit) was reached. 600 Add 100 μL of isopropyl-β-D-thiogalactoside (IPTG) (final concentration 0.1 μmol / L) to a solution of 0.6-0.8 μL for induction, then incubate at 18℃ for 15 h to induce protein expression. Subsequently, centrifuge the protein at 4℃, 4000 rpm for 15 min, wash with PBK buffer (50 mM, pH 7.4), and resuspend.

[0094] Prepare a 500 μL reaction mixture: 50 mM KH₂PO₄ / K₂HPO₄ buffer (pH 7.4), 0.1 g / mL whole cells, and 5 mM 5-hydroxytryptamine. React at 30 °C and 1000 rpm for 4 h. Then, mix the 500 μL reaction mixture with 500 μL of methanol by shaking, centrifuge at 12000 rpm for 5 min, and collect the supernatant for HPLC analysis.

[0095] Using the wild-type SsAANAT as a control, four mutants with relatively high activity were obtained through screening: SsAANAT-P58S-T62E-R163F, SsAANAT-P58S-T62E-V138I, SsAANAT-P58S-T62E-L91V, and SsAANAT-I104L-L91V-N66H. All of them showed improved conversion rates of 5-hydroxytryptamine substrates compared to the wild-type. The results are shown in Table 6.

[0096] Table 6. Results of the third round of three-point mutations constructed using SsAANAT

[0097] strain Conversion rate (%) Relative yield (times) SsAANAT 42.53 1 SsAANAT-P58S-T62E-R163F 73.84 1.74 SsAANAT-P58S-T62E-V138I 88.54 2.08 SsAANAT-P58S-T62E-L91V 72.36 1.70 SsAANAT-I104L-L91V-N66H 75.15 1.77

[0098] In summary, this invention is based on three-site combination mutation of the SsAANAT two-site dominant mutant. The resulting mutant has a higher conversion rate of the substrate 5-hydroxytryptamine, which is 1.70-2.08 times that of the starting strain, and increases the yield of N-acetyl-5-hydroxytryptamine.

[0099] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. An arylalkylamine N -acetyltransferase mutant, characterized in that It is a protein obtained by mutating an aralkylamine Sus scrofa -acetyltransferase derived from the wild boar species N -acetyltransferase, and the mutation is as follows based on the amino acid sequence of the aralkylamine N -acetyltransferase AANAT: selected from one or several of the following amino acid residue sites: position 5, position 8, position 58, position 62, position 66, position 82, position 91, position 104, position 138, position 163 and / or position 182.

2. The arylalkylamine according to claim 1 N -acetyltransferase mutant, characterized in that There are proteins obtained by any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten or all of the following eleven types of transformation mutations: X1. Mutate the threonine at position 5 of the aralkylamine N -acetyltransferase to serine; X2. Mutate the tyrosine at position 8 of the aralkylamine N -acetyltransferase to proline; X3. Mutate the proline at position 58 of the aralkylamine N -acetyltransferase to serine; X4. Mutate the threonine at position 62 of the aralkylamine N -N-acetyltransferase to glutamate; X5. Mutate the asparagine at position 66 of the aralkylamine N -acetyltransferase to histidine; X6. Mutate the methionine at position 82 of the aralkylamine N -N-acetyltransferase to leucine; X7. Mutate the leucine at position 91 of the aralkylamine N -acetyltransferase to valine; X8. Mutate the isoleucine at position 104 of the aralkylamine N -N-acetyltransferase to leucine; X9. Mutate the valine at position 138 of the aralkylamine N -N-acetyltransferase to isoleucine; X10. Mutate the arginine at position 163 of the aralkylamine N -N-acetyltransferase to phenylalanine; X11. Mutate the serine at position 182 of the aralkylamine N -acetyltransferase to threonine.

3. The arylalkylamine according to claim 2 N -acetyltransferase mutant, characterized in that It is one of the following mutants: The protein obtained by mutating the 5th and 82nd sites of aralkylamine N -acetyltransferase to serine and leucine respectively; The protein obtained by mutating the 5th and 91st sites of the aralkylamine N -acetyltransferase to serine and valine respectively; The protein obtained by mutating the 58th and 62nd positions of aralkylamine N -N-acetyltransferase to serine and glutamate respectively; The protein obtained by mutating the 104th and 91st sites of the aralkylamine N -acetyltransferase into leucine and valine respectively; The protein obtained by mutating the 104th and 182nd sites of aralkylamine N -N-acetyltransferase into leucine and threonine respectively; The protein obtained by mutating the 58th, 62nd, and 163rd sites of the aralkylamine N -acetyltransferase to serine, glutamate, and phenylalanine respectively; The protein obtained by mutating the 58th, 62nd, and 138th sites of aralkylamine N -acetyltransferase into serine, glutamic acid, and isoleucine respectively; The protein obtained by mutating the 58th, 62nd, and 91st sites of aralkylamine N -acetyltransferase to serine, glutamate, and valine respectively; The protein obtained by mutating the 104th, 91st, and 66th sites of the aralkylamine N -acetyltransferase to leucine, valine, and histidine respectively.

4. An encoding gene of an arylalkylamine N -acetyltransferase mutant according to any one of claims 1 to 3 N -acetyltransferase mutant.

5. A recombinant vector containing the coding gene as described in claim 4.

6. A recombinant strain containing the recombinant vector as described in claim 4.

7. The recombinant strain according to claim 6, characterized in that, The starting bacterium is Escherichia coli, more preferably E.coli BL21(DE3).

8. An aralkylamine according to any one of claims 1-3 N -acetyltransferase mutant, the coding gene according to claim 4, the recombinant vector according to claim 5, or the recombinant strain according to claim 6 or 7 in the production of N -acetyl-5-hydroxytryptamine.

9. A method for producing N -acetyl-5-hydroxytryptamine, characterized in that Including obtaining whole cells by culturing the recombinant strain described in claim 6, using serotonin as a substrate, and obtaining N N-acetylserotonin through a catalytic reaction.

10. The method according to claim 9, characterized in that, It also includes the step of separating N -acetyl-5-hydroxytryptamine.

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