An aralkylamine n-acetyltransferase mutant and its use in catalyzing synthesis of n-acetyl-5-hydroxytryptamine

Abstract

The present application provides an aralkylamine N - Acetyltransferase mutants and their use in the synthesis N - Acetyl-5-hydroxytryptamine. The present application obtains a series of mutants with N - acetylation activity through gene mining, directed evolution, etc. These mutants can directly acetylate the substrate 5-hydroxytryptamine to generate N - acetyl-5-hydroxytryptamine. Therefore, these aralkylamines N - acetyltransferase mutants have important application value in industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an aralkylamine N-acetyltransferase mutant and its application in catalyzing synthesis of N-acetyl-5-hydroxytryptamine. BACKGROUND

[0002] Melatonin (N-acetyl-5-methoxytryptamine, MT) is widely present in animals, plants and microorganisms. As an important indole neurohormone, it is involved in the regulation of body reproduction and immunity. The main pharmacological activities of melatonin are manifested in the rhythm balance of biological clock sleep-wake, sleep improvement, immune enhancement, memory improvement, anti-anxiety and other multiple effects. So far, the synthesis of melatonin mainly uses chemical method, but the method has long synthesis route, harsh reaction conditions, and is not feasible for large-scale production due to high energy consumption, low yield and serious environmental pollution. In contrast, the biological enzyme method has the advantages of green environmental protection, short cycle, continuous production and mild reaction conditions. The biological enzyme method for synthesizing melatonin is to synthesize L-tryptophan as a substrate through four enzyme cascade continuous catalysis: 1) L-tryptophan hydroxylation to form 5-hydroxytryptamine, 2) decarboxylation to produce 5-hydroxytryptamine, 3) acetylation to form N-acetyl-5-hydroxytryptamine, and 4) methylation to form melatonin. In 2020, Bernhard Palsson's team constructed a melatonin synthesis pathway in E. coli (L Hao et al. Microbial Synthesis of Human-Hormone Melatonin at Gram Scales. 2020. DOI: 10.1021 / acssynbio.0c00065), which achieved a yield of 1 g / L melatonin from glucose and 2 g / L melatonin from tryptophan, respectively. Palsson team in E. coli (L Hao et al. Microbial Synthesis of Human-Hormone Melatonin at Gram Scales. 2020. DOI: 10.1021 / acssynbio.0c00065), which achieved a yield of 1 g / L melatonin from glucose and 2 g / L melatonin from tryptophan, respectively.

[0003] At present, due to the unsolubility or low enzyme activity of the genes involved in the melatonin synthesis pathway, it is difficult to produce high titer of melatonin in heterologous hosts. The aralkylamine N-acetyltransferase (AANAT) involved in the synthesis of melatonin is a key rate-limiting enzyme for the biosynthesis of melatonin. The aralkylamine N-acetyltransferase (AANAT) is an acetyl-CoA-dependent enzyme that catalyzes the transfer of acetyl group from acetyl-CoA to 5-hydroxytryptamine to form N-acetyl-5-hydroxytryptamine. AANAT is an important rate-limiting enzyme for regulating the synthesis of melatonin, but the wild-type AANAT enzyme has low catalytic efficiency for the substrate 5-hydroxytryptamine. Therefore, it is necessary to develop an aralkylamine N-acetyltransferase (AANAT) mutant that can effectively convert 5-hydroxytryptamine to N-acetyl-5-hydroxytryptamine. SUMMARY

[0004] In view of the above-mentioned deficiencies of the prior art, the present application provides a mutant of aralkylamine N-acetyltransferase (AANAT) for preparing N-acetyl-5-hydroxytryptamine. The present application utilizes genetic engineering technology and computer-aided design to construct a mutant of aralkylamine N-acetyltransferase with higher activity, thereby greatly improving the yield of N-acetyl-5-hydroxytryptamine.

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

[0006] To this end, the present application reengineers aralkylamine N-acetyltransferase derived from Sus scrofa (GenBank No. XP_005656968.1, hereinafter referred to as SsAANAT) by gene mining, directed evolution and rational design, etc., to obtain a mutant with higher yield of N-acetyl-5-hydroxytryptamine, so as to more efficiently produce N-acetyl-5-hydroxytryptamine by using industrial microorganisms.

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

[0008] All of the above-mentioned amino acid mutants have the function of catalyzing N-acetyl-5-hydroxytryptamine as the wild-type aralkylamine N-acetyltransferase shown in SsAANAT.

[0009] More specifically, the SsAANAT aralkylamine N-acetyltransferase mutant is a protein obtained by reengineering SsAANAT as follows:

[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, mutating the proline at position 58 of SsAANAT to serine;

[0013] X4, mutating the threonine at position 62 of SsAANAT to glutamic acid;

[0014] X5, mutating the asparagine at position 66 of SsAANAT to histidine;

[0015] X6, mutating the methionine at position 82 of SsAANAT to leucine;

[0016] X7, mutating the leucine at position 91 of SsAANAT to valine;

[0017] X8, mutating the isoleucine at position 104 of SsAANAT to leucine;

[0018] X9, mutating the valine at position 138 of SsAANAT to isoleucine;

[0019] X10, mutating the arginine at position 163 of SsAANAT to phenylalanine;

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

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

[0022] a protein obtained by mutating the 5th site of SsAANAT to serine;

[0023] a protein obtained by mutating the 8th site of SsAANAT to proline;

[0024] a protein obtained by mutating the 58th site of SsAANAT to serine;

[0025] a protein obtained by mutating the 104th site of SsAANAT to leucine;

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

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

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

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

[0030] a protein obtained by mutating the 104th and 91st sites of SsAANAT to leucine and valine, respectively;

[0031] The protein obtained by mutating the 104th and 182nd sites 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 the 104th, 91st and 66th sites of SsAANAT to leucine, valine and histidine respectively.

[0036] The present application provides a gene encoding the mutant of aralkylamine N-acetyltransferase, and a recombinant vector containing the gene.

[0037] The present application further provides a recombinant strain obtained by transforming the recombinant vector into a host cell. The host cell contains the recombinant vector or a gene containing the aralkylamine N-acetyltransferase or mutant thereof integrated in the genome. Preferably, the starting bacterium is Escherichia coli, and more preferably, it is E. coli BL21 (DE3).

[0038] The present application thus provides the use of the aralkylamine N-acetyltransferase mutant, the gene encoding the mutant, the recombinant vector or the recombinant strain in the production of N-acetyl-5-hydroxytryptamine.

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

[0040] The aralkylamine N-acetyltransferase mutant or the recombinant genetically engineered strain is used to produce N-acetyl-5-hydroxytryptamine by whole-cell catalysis with 5-hydroxytryptamine as the substrate. Optionally, the method further comprises the step of isolating N-acetyl-5-hydroxytryptamine.

[0041] The present application discloses the following technical achievements: the newly modified aralkylamine N-acetyltransferase mutant of the present application is used to produce N-acetyl-5-hydroxytryptamine by whole-cell catalysis with 5-hydroxytryptamine as the substrate, and the yield is significantly improved. Therefore, these aralkylamine N-acetyltransferases have important application value in industry. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Plasmid map of recombinant vector for aralkylamine N-acetyltransferase.

[0043] Figure 2 Standard curve map for HPLC liquid chromatography detection of N-acetyl-5-hydroxytryptamine standard.

[0044] Figure 3 Relative activity ratio of N-acetyl-5-hydroxytryptamine of dominant mutant recombinant engineering strain. DETAILED DESCRIPTION

[0045] The present application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0046] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0047] Example 1, Mining of aralkylamine N-acetyltransferase

[0048] The Sus scrofa-derived aralkylamine N-acetyltransferase AANAT (GenBank No. XP_005656968.1) was obtained by searching the NCBI database, and the SsAANAT was synthesized and connected to the pET24a expression vector digested by NdeI and XhoI to obtain the recombinant expression vector pET24a-SsAANAT. Figure 1

[0049] Aralkylamine 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 various host microorganisms that are conventional in the art, as long as the above-mentioned recombinant expression vector can stably self-replicate and the aralkylamine N-acetyltransferase gene can be effectively expressed. In the present embodiment, the aforementioned recombinant expression plasmid is introduced into E. coli BL21 (DE3) competent cells by electroporation, and inverted culture is performed on a LB solid plate containing kanamycin resistance for 12-16 h, and positive transformants are selected for DNA sequencing verification, and the correct transformants are verified as aralkylamine N-acetyltransferase gene engineering strains.

[0051] Example 2, Construction of aralkylamine N-acetyltransferase single-point mutant or obtaining of a mutant 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 No. XP_005656968.1), 10 sites were designed for rational design and single-point mutation. Therefore, 10 sites were selected for the first round of modification, namely 3, 5, 6, 8, 58, 67, 91, 104, 123, and 163, and site-directed mutagenesis was performed for each site, as shown in Table 1.

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

[0055]

[0056] In order to obtain the aralkylamine N-acetyltransferase SsAANAT single-point mutant, the following experiments were performed:

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

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

[0059] The obtained PCR product was subjected to the following operation: 1 μL of Dpn I enzyme was added to 20 μL of the PCR product for plasmid template digestion, and the reaction 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, and the electroporated E. coli BL21(DE3) bacterial solution was uniformly spread on a kanamycin-resistant (concentration of 50 μg / mL) LB plate. After being cultured at 37°C for 14 h, single colonies were grown, which were the aralkylamine N-acetyltransferase SsAANAT gene mutant engineering strain. The recombinant plasmid containing the nucleotide of interest which was subjected to non-directional mutation or directional mutation was the expression vector for expressing the aralkylamine N-acetyltransferase SsAANAT gene mutant.

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

[0061] After each single-point mutant was successfully sequenced, the plasmid was electroporated into E. coli BL21(DE3) competent cells and spread on a Kan-resistant plate. A single colony was picked and inoculated into 5 mL of LB broth containing 5 μL of kanamycin (final concentration of 50 μg / mL) and cultured at 37°C and 220 rpm for 12 h. 1 mL of the bacterial solution was aseptically inoculated (1% inoculation amount) into 100 mL of TB medium, 100 μL of kanamycin (final concentration of 50 μg / mL) was added, and the culture was incubated at 37°C and 220 rpm for about 3 h until the OD600 was 0.6-0.8. Then, 100 μL of isopropyl-β-D-thiogalactoside (IPTG) (final concentration of 0.1 μmol / L) was added to induce protein expression. The culture was incubated at 18°C for 15 h. Subsequently, the protein was centrifuged at 4°C and 4000 rpm for 15 min, washed with PBK buffer (50 mM, pH 7.4), and resuspended. 600 0.6-0.8, 100 μL of isopropyl-β-D-thiogalactoside (IPTG) (final concentration of 0.1 μmol / L) was added to induce protein expression. The culture was incubated at 18°C for 15 h. Subsequently, the protein was centrifuged at 4°C and 4000 rpm for 15 min, washed with PBK buffer (50 mM, pH 7.4), and resuspended.

[0062] A 500 μL reaction system was prepared: 50 mM KH2PO4 / K2HPO4 buffer (pH 7.4), 0.1 g / mL whole cells, 5 mM 5-hydroxytryptamine, and the reaction was performed at 30°C and 1000 rpm for 4 h. Then, 500 μL of the reaction mixture was mixed with 500 μL of methanol, and the mixture was uniformly shaken and centrifuged at 12000 rpm for 5 min. The supernatant was subjected to HPLC analysis. The detection conditions were as follows: liquid chromatography column Zorbax SB-C18 (4.6 mm x 150 mm, 5 μm), 275 nm, 35°C, and a flow rate of 1 mL / min. The injection volume was 10 μL, the A pump was pure water (containing 0.1% trifluoroacetic acid), the B pump was methanol, and the elution was performed at a constant ratio for 10 min.

[0063] SsAANAT-T5S, SsAANAT-Y8P, SsAANAT-P58S, SsAANAT-I104L, a total of 4 mutants with relatively high activity were obtained by preliminary screening, and the conversion rate of 5-hydroxytryptamine substrate of the mutants was improved compared with the wild type, and the results are shown in Table 2.

[0064] Table 2, the results of the first round of single point mutation of SsAANAT

[0065] Strain Conversion rate (%) Relative yield (fold) 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 application is based on the mutation of specific sites of wild-type aralkylamine N-acetyltransferase SsAANAT, and the conversion rate of the obtained mutant to the substrate 5-hydroxytryptamine is increased by 1.16-1.31 times of the starting strain, and the yield of N-acetyl-5-hydroxytryptamine is increased.

[0067] Example 3, construction of aralkylamine N-acetyltransferase double site mutant or obtaining of the mutant thereof

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

[0069] Based on the first round of advantage mutants SsAANAT-T5S, SsAANAT-Y8P, SsAANAT-P58S, SsAANAT-I104L, the second round of double site mutant modification was continued. According to the substrate binding pocket and the interaction of the substrate molecule, a total of 9 sites were designed for rational design, and double site mutation was carried out. Therefore, the four advantage mutants SsAANAT-T5S, SsAANAT-Y8P, SsAANAT-P58S, SsAANAT-I104L of the first round were used as templates for the second round of modification, and the positions of 62, 82, 187, 104, 58, 91, 67, 123 and 182 were selected for site-directed mutation, as shown in Table 3.

[0070] Table 3, the second round of double site mutation based on SsAANAT

[0071]

[0072] In order to obtain aralkylamine N-acetyltransferase SsAANAT mutant, the following experiments were carried out:

[0073] PCR amplification reaction system (50 μL): PrimeSTAR (2x) 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 subjected to the following operation: 1 μL of Dpn I enzyme was added to 20 μL of the PCR product for plasmid template digestion, and the reaction was carried out at 37℃ for 2h. 5 μL of the digested PCR product was electroporated into 100 μL of E. coli BL21 (DE3) competent cells, and the electroporated E. coli BL21 (DE3) bacterial solution was evenly spread on a kanamycin-resistant (concentration of 50 μg / mL) LB plate. After incubation at 37℃ for 14h, single colonies grew, which were the aralkylamine N-acetyltransferase SsAANAT gene mutant engineering strains. The recombinant plasmid containing the target nucleotide subjected to non-directional mutation or directional mutation in the strain was the expression vector for expressing the aralkylamine N-acetyltransferase SsAANAT gene mutant.

[0076] 3.2 Screening of the second round of double-site mutants of SsAANAT

[0077] After each double-site mutant was successfully sequenced, the plasmid was electroporated into E. coli BL21 (DE3) competent cells and spread on a Kan-resistant plate. A single colony was inoculated into 5 mL of LB broth containing 5 μL of kanamycin (final concentration of 50 μg / mL) and incubated at 37℃ with shaking at 220 rpm for 12h. 1 mL of the bacterial solution was used to inoculate (1% inoculation amount) 100 mL of TB medium, and 100 μL of kanamycin (final concentration of 50 μg / mL) was added. After incubation at 37℃ with shaking at 220 rpm for about 3h until the OD600 value reached 0.6-0.8, 100 μL of isopropyl-β-D-thiogalactoside (IPTG) (final concentration of 0.1 μmol / L) was added to induce protein expression. The culture was then incubated at 18℃ for 15h. The protein was then centrifuged at 4℃ and 4000 rpm for 15min, washed with PBK buffer (50 mM, pH 7.4), and resuspended. 600 0.6-0.8, and 100 μL of isopropyl-β-D-thiogalactoside (IPTG) (final concentration of 0.1 μmol / L) was added to induce protein expression. The culture was then incubated at 18℃ for 15h. The protein was then centrifuged at 4℃ and 4000 rpm for 15min, washed with PBK buffer (50 mM, pH 7.4), and resuspended.

[0078] Prepare 500 μL reaction system: 50 mM KH2PO4 / K2HPO4 buffer (pH 7.4), 0.1 g / mL whole cell, 5 mM 5-hydroxytryptamine, react at 30°C, 1000 rpm for 4 h. Then mix 500 μL reaction mixture with 500 μL methanol, shake well, centrifuge at 12000 rpm for 5 min, take the supernatant for HPLC analysis.

[0079] As a control of SsAANAT wild type, by screening, obtain SsAANAT-T5S-M82L, SsAANAT-T5S-L91V, SsAANAT-P58S-T62E, SsAANAT-I104L-S182T, SsAANAT-I104L-L91V, a total of 5 mutants with relatively high activity, which have improved conversion rate of 5-hydroxytryptamine substrate compared with the wild type, and the results are shown in Table 4.

[0080] Table 4, results of the second round of double-site mutation of SsAANAT

[0081] Strain Conversion rate (%) Relative yield (fold) 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, based on the double-site combination mutation of the single-site advantageous mutant of SsAANAT, the conversion rate of the obtained mutant to the substrate 5-hydroxytryptamine is increased by 1.37-1.66 times of the starting strain, and the yield of N-acetyl-5-hydroxytryptamine is increased.

[0083] Example 4, construction of aralkylamine N-acetyltransferase three-site mutant or obtaining of the mutant thereof

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

[0085] Based on the second round of advantageous mutants SsAANAT-T5S-M82L, SsAANAT-T5S-L91V, SsAANAT-P58S-T62E, SsAANAT-I104L-S182T, SsAANAT-I104L-L91V, continue to perform the third round of three-site mutant modification. According to the substrate binding pocket and substrate molecule interaction, a total of 11 sites are rationally designed for three-site mutation. Therefore, taking the five advantageous mutants SsAANAT-T5S-M82L, SsAANAT-T5S-L91V, SsAANAT-P58S-T62E, SsAANAT-I104L-S182T, SsAANAT-I104L-L91V of the second round as templates for the third round of modification, respectively, 163, 123, 138, 82, 104, 91, 67, 62, 66, 58, 86, site-directed mutation is performed for each site, as shown in Table 5.

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

[0087]

[0088] To obtain the aralkylamine N-acetyltransferase SsAANAT mutants, the following experiments were performed:

[0089] PCR amplification reaction system (50 μL): PrimeSTAR (2x) 25 μL, template SsAANAT-T5S-M82L, SsAANAT-T5S-L91V, SsAANAT-P58S-T62E, SsAANAT-I104L-S182T, SsAANAT-I104L-L91V 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 6.

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

[0091] The obtained PCR product was subjected to the following operation: 1 μL of Dpn I enzyme was added to 20 μL of the PCR product for plasmid template digestion, and the digestion was performed 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, and the electroporated E. coli BL21 (DE3) bacterial solution was evenly spread on a kanamycin-resistant (concentration of 50 μg / mL) LB plate. After incubation at 37°C for 14 h, single colonies grew, which were the aralkylamine N-acetyltransferase SsAANAT gene mutant engineering strains. The recombinant plasmid containing the target nucleotide with non-directional mutation or directional mutation in the strain was the expression vector for expressing the aralkylamine N-acetyltransferase SsAANAT gene mutant.

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

[0093] After each three-site mutant sequencing success, the plasmid was electroporated into E. coli BL21 (DE3) competent, coated on Kan resistance plate. A single colony was inoculated into 5 mL of LB broth containing 5 μL of kanamycin (final concentration of 50 μg / mL) and cultured at 37°C, 220 rpm for 12 h. 1 mL of bacterial solution was taken for expansion culture (1% inoculation) into 100 mL of TB medium, 100 μL of kanamycin (final concentration of 50 μg / mL) was added, and the culture was incubated at 37°C, 220 rpm for about 3 h until the OD600 reached 0.6-0.8. 600 0.6-0.8, 100 μL of isopropyl-β-D-thiogalactoside (IPTG) (final concentration of 0.1 μmol / L) was added, and the culture was incubated at 18°C for 15 h after induction, and the protein expression was induced. Then the protein was centrifuged at 4°C, 4000 rpm for 15 min, washed with PBK buffer (50 mM, pH 7.4) and resuspended.

[0094] A 500 μL reaction system was prepared: 50 mM KH2PO4 / K2HPO4 buffer (pH 7.4), 0.1 g / mL whole cell, 5 mM 5-hydroxytryptamine, and the reaction was carried out at 30°C, 1000 rpm for 4 h. Then 500 μL of the reaction mixture was mixed with 500 μL of methanol, and the mixture was mixed uniformly by shaking, centrifuged at 12000 rpm for 5 min, and the supernatant was analyzed by HPLC.

[0095] As a control, SsAANAT wild type, four mutants SsAANAT-P58S-T62E-R163F, SsAANAT-P58S-T62E-V138I, SsAANAT-P58S-T62E-L91V and SsAANAT-I104L-L91V-N66H were obtained by screening, and the conversion rate of 5-hydroxytryptamine substrate of the four mutants was higher than that of the wild type, and the results are shown in Table 6.

[0096] Table 6, results of the third round of three-site mutation of SsAANAT

[0097] Strain Conversion rate (%) Relative yield (fold) 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, the present application is based on the three-site combination mutation of the SsAANAT double-site advantage mutant, and the conversion rate of the mutant to the substrate 5-hydroxytryptamine is increased by 1.70-2.08 times of the starting strain, and the yield of N-acetyl-5-hydroxytryptamine is increased.

[0099] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a specific example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. An aralkylamine N-acetyltransferase mutant, characterized in that, (1) the protein obtained by mutating the 58th site of the aralkylamine N-acetyltransferase to serine; (2) the protein obtained by mutating the 58th site of the aralkylamine N-acetyltransferase to serine and the 62nd site to glutamic acid; (3) the protein obtained by mutating the 58th site of the aralkylamine N-acetyltransferase to serine, the 62nd site to glutamic acid, and the 163rd site to phenylalanine; (4) the protein obtained by mutating the 58th site of the aralkylamine N-acetyltransferase to serine, the 62nd site to glutamic acid, and the 138th site to isoleucine; (5) the protein obtained by mutating the 58th site of the aralkylamine N-acetyltransferase to serine, the 62nd site to glutamic acid, and the 91st site to valine.

2. A coding gene comprising the aralkylamine N-acetyltransferase mutant of claim 1.

3. A recombinant vector comprising the coding gene of claim 2.

4. A recombinant strain comprising the recombinant vector of claim 3. The starting strain is E. coli.

5. The recombinant bacterial strain of claim 4, wherein The starting strain is E. coli BL21 (DE3).

6. The recombinant bacterial strain of claim 5, wherein 7. Use of the aralkylamine N-acetyltransferase mutant of claim 1, the coding gene of claim 2, the recombinant vector of claim 3, or the recombinant strain of any one of claims 4 to 6 in the production of N-acetyl-5-hydroxytryptamine. It comprises culturing the recombinant strain of claim 6 to obtain whole cells, using 5-hydroxytryptamine as a substrate, and catalyzing a reaction to obtain N-acetyl-5-hydroxytryptamine.

8. A method of producing N-acetyl-5-hydroxytryptamine, characterized by, It also comprises the step of isolating N-acetyl-5-hydroxytryptamine.

9. The method of claim 8, wherein, ​

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