A method for the synthesis of a non-natural indole amino acid

Abstract

This invention utilizes gene mining technology, random mutation, and other methods to... Schistosoma mansoni The indole monooxygenase source was modified to obtain a mutant with a high yield of DL-2-amino-3-(5-hydroxyindole)propionic acid. The recombinant vector constructed from the mutant and the recombinant genetically engineered bacteria were then used to produce DL-2-amino-3-(5-hydroxyindole)propionic acid via fermentation, resulting in a significantly increased yield, 1.5-5.2 times that of the starting strain. Therefore, this invention provides a method for producing DL-2-amino-3-(5-hydroxyindole)propionic acid that offers advantages such as mild reaction conditions, simple operation, short production cycle, environmental friendliness, and reduced production costs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an indole monooxygenase mutant and its application in the catalytic synthesis of DL-2-amino-3-(5-hydroxyindole)propionic acid. Background Technology

[0002] DL-2-Amino-3-(5-hydroxyindolyl)propionic acid is a tryptophan derivative produced by the substitution of a hydroxyl group at the 5-position of the tryptophan benzene ring by indole monooxygenase. It is referred to below as a non-natural indole amino acid. Non-natural indole amino acids can effectively treat various conditions, including depression, fibromyalgia, obesity caused by overeating, chronic headaches, and insomnia. In recent years, research and production of DL-2-amino-3-(5-hydroxyindolyl)propionic acid have developed rapidly, and its application in the pharmaceutical and related industries both domestically and internationally is expanding.

[0003] Currently, the production methods for non-natural indole amino acids include natural product extraction, chemical synthesis, and microbial fermentation. Natural product extraction remains the primary method for the commercial production of non-natural indole amino acids, mainly derived from the seeds of Ghanaian legumes. However, the long growth cycle of these plants, coupled with seasonal and geographical limitations, results in low yields, making them unsuitable for industrial applications. Chemical synthesis requires organic reagents, such as 3-methyl-4-nitrophenol, which undergo a series of addition, reduction, and hydrolysis reactions to yield non-natural indole amino acids. This method involves harsh reaction conditions, long cycles, high energy consumption, and low product yields. Synthesis also requires high-temperature and high-pressure environments, which can cause environmental pollution and is unsuitable for large-scale synthesis.

[0004] Microbial fermentation offers advantages such as being environmentally friendly, having mild reaction conditions, and enabling continuous production, making it a promising application. However, the indole monooxygenase used to catalyze the production of DL-2-amino-3-(5-hydroxyindole)propionic acid from tryptophan has low bioactivity, making it impossible to generate high yields of DL-2-amino-3-(5-hydroxyindole)propionic acid. Summary of the Invention

[0005] Currently, the industrial production of DL-2-amino-3-(5-hydroxyindole)propionic acid suffers from low yield, long production cycle, and environmental pollution. To address these shortcomings, this invention utilizes genetic engineering and computer-aided design to construct a highly active indole monooxygenase mutant, thereby significantly increasing the yield of DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0006] This invention provides a method for synthesizing DL-2-amino-3-(5-hydroxyindole)propionic acid using indole monooxygenase or its mutant.

[0007] To this end, this invention modifies indole monooxygenase (GenBank No.: AAD01923.1, hereinafter referred to as SmTPH) from Schistosoma mansoni using gene mining, directed evolution, and rational design techniques to obtain mutants with higher yields of DL-2-amino-3-(5-hydroxyindole)propionic acid, so as to utilize industrial microorganisms to produce DL-2-amino-3-(5-hydroxyindole)propionic acid more efficiently.

[0008] Therefore, the first object of the present invention is to provide a mutant obtained by mutating the indole monooxygenase SmTPH (GenBank No.: AAD01923.1) derived from Schistosoma mansoni, wherein the mutation is based on the amino acid sequence of the indole monooxygenase SmTPH and contains the following mutations selected from one or more of the following amino acid residue sites: 34, 106, 145, 175, 178, 182, 221, 223, 229, 246, 250, 275 and / or 276.

[0009] All of the above amino acid mutants possess the catalytic function of wild-type indole monooxygenases, as shown by SmTPH, in catalyzing DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0010] More specifically, the SmTPH indole monooxygenase mutant is a protein obtained by modifying SmTPH with any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all of the following thirteen types:

[0011] X1. Mutate the methionine at position 34 of SmTPH to aspartic acid, glycine, or lysine.

[0012] X2. Mutate the aspartic acid at position 106 of SmTPH to isoleucine, methionine, or serine.

[0013] X3. Mutate the tyrosine at position 145 of SmTPH to threonine, isoleucine, or serine.

[0014] X4. Mutate the threonine at position 175 of SmTPH to arginine and lysine;

[0015] X5. Mutate the proline at position 178 of SmTPH to arginine, leucine, or glycine;

[0016] X6. Mutate the histidine at position 182 of SmTPH to serine, tyrosine, valine, or threonine.

[0017] X7. Mutate the cysteine ​​at position 221 of SmTPH to asparagine and isoleucine;

[0018] X8. Mutate the phenylalanine at position 223 of SmTPH to aspartic acid, tyrosine, tryptophan, or valine.

[0019] X9. Mutate the glycine at position 229 of SmTPH to glycine, aspartic acid, phenylalanine, or serine.

[0020] X10. Mutate the serine at position 246 of SmTPH to proline, leucine, or phenylalanine.

[0021] X11. Mutate the glutamic acid at position 250 of SmTPH to aspartic acid and histidine;

[0022] X12. Mutate the leucine at position 275 of SmTPH to serine, methionine, glutamic acid, or threonine.

[0023] X13. Mutate the valine at position 276 of SmTPH to glutamine and proline;

[0024] In one embodiment of the present invention, the SmTPH indole monooxygenase mutant is:

[0025] The protein obtained by mutating the 106th position of SmTPH to isoleucine;

[0026] The protein obtained by mutating the 178th position of SmTPH to leucine;

[0027] The protein obtained by mutating the 178th position of SmTPH to glycine;

[0028] The protein obtained by mutating the 182nd site of SmTPH to valine;

[0029] The protein obtained by mutating the 182nd site of SmTPH to threonine;

[0030] The protein obtained by mutating the 221st position of SmTPH to isoleucine;

[0031] The protein obtained by mutating the 223rd site of SmTPH to aspartic acid;

[0032] The protein obtained by mutating the 223rd site of SmTPH to tyrosine;

[0033] The protein obtained by mutating the 223rd position of SmTPH to valine;

[0034] The protein obtained by mutating the 275th position of SmTPH to serine;

[0035] The protein obtained by mutating the 275th position of SmTPH to methionine.

[0036] In another embodiment of the present invention, the SmTPH indole monooxygenase mutant is:

[0037] The protein was obtained by mutating positions 106 and 178 of SmTPH to isoleucine and leucine, respectively.

[0038] The protein was obtained by mutating positions 178 and 182 of SmTPH to glycine and valine, respectively.

[0039] The protein was obtained by mutating positions 106 and 221 of SmTPH to isoleucine and isoleucine, respectively.

[0040] The protein was obtained by mutating positions 106, 182, and 221 of SmTPH to isoleucine, isoleucine, and threonine, respectively.

[0041] The protein was obtained by mutating positions 182, 221, and 223 of SmTPH to threonine, isoleucine, and tyrosine, respectively.

[0042] The protein was obtained by mutating positions 178, 223, and 275 of SmTPH to glycine, aspartic acid, and methionine, respectively.

[0043] The protein was obtained by mutating positions 106, 178, 182, and 223 of SmTPH to isoleucine, leucine, threonine, and valine, respectively.

[0044] The protein was obtained by mutating positions 106, 178, 182, 223, and 275 of SmTPH to isoleucine, glycine, threonine, valine, and serine, respectively.

[0045] The protein was obtained by mutating positions 106, 182, 221, 223, and 275 of SmTPH to isoleucine, threonine, isoleucine, aspartic acid, and methionine, respectively.

[0046] This invention also provides a recombinant strain, which is obtained by transferring the recombinant vector into a host cell. The host cell contains the recombinant vector or a gene containing the indole monooxygenase or a mutant integrated into its genome. The recombinant vector or the genetically engineered strain is used in the production of DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0047] This invention also provides a method for producing DL-2-amino-3-(5-hydroxyindole)propionic acid, which has the advantages of mild reaction conditions, simple operation, and environmental friendliness. Specifically, it includes the following steps:

[0048] 1) Production of DL-2-amino-3-(5-hydroxyindole)propionic acid using the indole monooxygenase and mutant or the recombinant genetically engineered strain described above;

[0049] 2) DL-2-amino-3-(5-hydroxyindole)propionic acid was isolated from the system in 1).

[0050] This invention discloses the following technical achievements:

[0051] This invention discloses a novel modified indole monooxygenase mutant, which obtains multiple mutants by mutating the amino acid sequence of indole monooxygenase, such as SmTPH, at specific sites; and all the mutants obtained have the function of catalyzing the production of DL-2-amino-3-(5-hydroxyindole)propionic acid from L-tryptophan, similar to the wild type of SmTPH.

[0052] This invention also discloses a recombinant vector containing the indole monooxygenase gene and its mutant gene, as well as a recombinant bacterial strain. The gene encoding the indole monooxygenase in the recombinant vector is expressed via shake-flask fermentation to obtain the corresponding enzyme. This invention, by mutating specific sites of the wild-type enzyme, yields a mutant with significantly increased yield, 1.5-5.2 times that of the starting strain, and can significantly increase the yield of DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0053] The application of the indole monooxygenase or its mutant in the synthesis of DL-2-amino-3-(5-hydroxyindole)propionic acid and its derivatives falls within the scope of protection of this invention. Attached Figure Description

[0054] Figure 1 The plasmid map of the indole monooxygenase recombinant vector.

[0055] Figure 2 The standard curve for HPLC detection of DL-2-amino-3-(5-hydroxyindole)propionic acid standard.

[0056] Figure 3 The yield ratio of DL-2-amino-3-(5-hydroxyindole)propionic acid in the dominant mutant recombinant engineered strain. Detailed Implementation

[0057] 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.

[0058] 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.

[0059] Example 1: Discovery of indole monooxygenase

[0060] The indole monooxygenase TPH from Schistosoma mansoni (GenBank ID: AAD01923.1) was obtained by searching the NCBI database. The whole gene SmTPH was synthesized and ligated into the pET24a expression vector that had been double-digested with NdeI and XhoI to obtain the recombinant expression vector pET24a-SmTPH. Figure 1 ).

[0061] Indole monooxygenase amino acid sequence (SEQ ID NO: 1):

[0062] MHHHHAKNPTLDDKVPWFPRHISDLDKVSNSVLMYGKELDADHPGFKDKEYRKRRMMF

[0063] ADIALNYKWGQQIPIVEYTEIEKTTWGRIYRELTRLYKTSACHEFQKNLGLLQDKAGYNEF

[0064] DLPQLQVVSDFLKARTGFCLRPVAGYLSARDFLSGLAFRVFYCTQYIRHQADPFYTPEPDCC

[0065] HELLGHVPMLADPKFARFSQEIGLASLGTSDEEIKKLATCYFFTIEFGLCRQDNQLKAYGAG

[0066] LLSSVAELQHALSDKAVIKPFIPMKVINEECLVTTFQNGYFETSSFEDATRQMREFVRTIKRPFDVHYNPYTQSIEIIKTPKSVAKLVQDLQFE.

[0067] The recombinant expression vector was transformed into a suitable microbial host. The host microorganism can be any conventional microorganism in the art, as long as it can stably replicate on its own and effectively express the indole monooxygenase gene. In this embodiment, the recombinant expression plasmid was introduced into E. coli BL21(DE3) competent cells via electroporation and cultured upside down on LB agar plates containing kanamycin resistance for 12-16 hours. Positive transformants were selected for DNA sequencing verification; the correctly verified transformants were the indole monooxygenase genetically engineered strains.

[0068] Example 2: Obtaining indole monooxygenase or its mutant

[0069] 2.1 Construction of SmTPH single-point saturation mutant library

[0070] Based on gene mining, molecular docking, and rational analysis of wild-type indole monooxygenase, 13 sites were rationally designed according to the substrate binding pocket and substrate molecule interaction. Therefore, this invention selected 13 sites in the amino acid sequence of wild-type indole monooxygenase from Schistosoma mansoni (GenBank ID: AAD01923.1) for modification, namely positions 34, 106, 145, 175, 178, 182, 221, 223, 229, 246, 250, 275, and 276. Single-site saturation mutant libraries were constructed for each site, as shown in Table 1.

[0071] Table 1. Single-point saturation mutant library constructed based on indole monooxygenase SmTPH

[0072]

[0073]

[0074] Note: Table 1 contains primers for multiple single-stranded DNAs: M34-F, N106-F, Y145-F, T175-F, P178-F, H182-F, C221-F, F223-F, G229-F, S246-F, E250-F, L275-F, and V276-F. The single-stranded DNAs are mixed in a molar ratio of 1:1:6:12 according to the order TGG:ATG:VMA:NDT in the table. V represents G, A, or C; M represents A or C; N represents A, T, G, or C; and D represents G, A, or T.

[0075] To obtain the indole monooxygenase SmTPH mutant, the following experiments were conducted:

[0076] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SmTPH 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 1.

[0077] PCR amplification reaction conditions: 98℃: 2min, (98℃: 10s, 55℃: 15s, 72℃: 4min) 30 cycles, 72℃: 4min.

[0078] 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 indole monooxygenase SmTPH gene mutant. Recombinant plasmids containing the target nucleotides of this strain with non-directional or directional mutations became the expression vectors for expressing the indole monooxygenase SmTPH gene mutant.

[0079] 2.2 Screening of SmTPH single-point saturation mutant libraries

[0080] After successful sequencing of the single-point saturated mutant library, 5 μL of the digested PCR product was electroporated into E. coli BL21(DE3) competent cells and plated onto Kan / Cm antibiotic plates. Using SmTPH wild-type as a control, single clones of positive and negative control were aseptically picked and transferred to 96-well deep-well plates containing 400 μL of LB medium (400 μL LB medium containing 5 μg / mL chloramphenicol and 50 μg / mL kanamycin (glycerol 8 mL / L)). After sealing, the plates were incubated at 37°C with shaking at 800 rpm for 12 h.

[0081] 700 μL of TB medium (containing tryptophan at a final concentration of 5 g / L) was added to a 96-well deep-well culture plate, along with IPTG, chloramphenicol, kanamycin, and FeSO4, to make final concentrations of 0.1 mM, 5 μg / mL, 50 μg / mL, and 50 μg / mL, respectively. The plate was incubated at 30 °C with shaking at 800 rpm for 24 h to express the protein and catalyze the conversion of tryptophan to DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0082] After culturing for 24 hours, the 96-well deep-well culture plate was centrifuged at 4000 rpm for 30 min. 20 μL of the supernatant from the 48-well plate was transferred to a 96-well glass plate, and 180 μL of ddH2O was added, resulting in a 10-fold dilution. OD 310 Absorbance values ​​are measured to screen for mutants with higher absorbance than the wild type. OD values ​​are then measured. 310 The absorbance values ​​measured were higher than those of wild-type monoclonal colonies, and single colonies were selected for sequencing identification.

[0083] Preliminary screening yielded 11 mutants with relatively high activity: SmTPH-N106I, SmTPH-P178L, SmTPH-P178G, SmTPH-H182V, SmTPH-H182T, SmTPH-C221I, SmTPH-F223D, SmTPH-F223Y, SmTPH-F223V, SmTPH-L275S, and SmTPH-L275M. These mutants all showed improved L-tryptophan substrate conversion rates compared to the wild type. The results are shown in Table 2.

[0084] Table 2. Yield ratio of recombinant engineered strain DL-2-amino-3-(5-hydroxyindole)propionic acid

[0085] strain Conversion rate (%) Relative output (times) SmTPH 15.41 1 SmTPH-N106I 34.17 2.3 SmTPH-P178L 26.73 1.7 SmTPH-P178G 36.90 2.4 SmTPH-H182V 25.45 1.7 SmTPH-H182T 23.83 1.5 SmTPH-C221I 31.04 2.1 SmTPH-F223D 35.63 2.3 SmTPH-F223Y 29.84 1.9 SmTPH-F223V 24.78 1.6 SmTPH-L275S 40.82 2.7 SmTPH-L275M 27.78 1.8

[0086] In summary, this invention, based on mutation of a specific site of wild-type indole monooxygenase SmTPH, yields a mutant with significantly increased yield, which is 1.5-2.7 times that of the starting strain, and can significantly increase the yield of DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0087] Example 3: Construction of the SmTPH combinatorial saturation mutant of indole monooxygenase or obtaining the mutant thereof

[0088] 3.1 Construction of SmTPH combinatorial mutant library

[0089] Eleven mutants (N106I, P178L, P178G, H182V, H182T, C221I, F223D, F223Y, F223V, L275S, and L275M) selected from 10 single-site saturated mutant libraries were combined with mutated at six sites. The primer designs are shown in Table 3 below.

[0090] Table 3. Combinatorial saturated mutant libraries constructed based on indole monooxygenase SmTPH

[0091] Primer name Primer sequence (5'→3') F1 CTGTCATGAATTTCAGAAAAWCCTGGGCCTGCTGCAGGATAAAG R1-1 CATCGGCACATGGCCCAGCAGTTCATGACAGCAATCTRGTTCCGG R1-2 CATCGGCACATGGCCCAGCAGTTCCACACAGCAATCTRGTTCCGG R1-3 CATCGGCACATGGCCCAGCAGTTCGGTACAGCAATCTRGTTCCGG R1-4 CATCGGCACATGGCCCAGCAGTTCATGACAGCAATCGCCTTCCGG R1-5 CATCGGCACATGGCCCAGCAGTTCCACAGCAATCGCCTTCCGG R1-6 CATCGGCACATGGCCCAGCAGTTCGGTACAGCAATCGCCTTCCGG F2 CTGCTGGGCCATGTGCCGATGCTGGCAGATCCGAAAT R2-1 GGCCAAATTCAATGGTAAAGWMATAACAGGTGCCAGTTTC R2-2 GGCCAAATTCAATGGTAAAGWMATAAATGGTGGCCAGTTTC F3 TTTACCATTGAATTTGGCCTGTGCCGTCAGGATA R3-1 GGTCACTRAGCACTCTTCATTAATCACTTTC R3-2 GGTCACCATGCACTCTTCATTAATCACTTTC

[0092] Note: Table 3 contains primers for multiple single-stranded DNAs. M represents A or C, Y represents C or T, S represents C or G, H represents A, C or T, V represents G, A or C, R represents A or G, K represents G or T, W represents A or T, D represents G, A or T, and B represents C, G or T.

[0093] To obtain the indole monooxygenase SmTPH mutant, the following experiments were conducted:

[0094] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SmTPH 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 1.

[0095] PCR amplification reaction conditions: 98℃: 2min, (98℃: 10s, 55℃: 15s, 72℃: 4min) 30 cycles, 72℃: 4min.

[0096] Overlap extension PCR procedure:

[0097] Round 1: R1-1, R1-2, R1-3, R1-4, R1-5, and R1-6 are mixed in a 1:1:1:1:1:1 ratio to form downstream primer R1. F1 and R1 amplify fragment 1 (approximately 274 bp); R2-1 and R2-2 are mixed in a 1:1 ratio to form downstream primer R2. F2 and R2 amplify fragment 2 (approximately 139 bp); R3-1 and R3-2 are mixed in a 2:1 ratio to form downstream primer R3. F3 and R3 amplify fragment 3 (approximately 162 bp).

[0098] Second round: Fragment 1, fragment 2, and fragment 3 were cut and recovered from the gel as templates. Using upstream primer F1 and mixed downstream primer R3 as upstream and downstream primers respectively, the overlapping extension PCR was used to amplify fragment 4.

[0099] Third round: Fragment 4 is used as a large primer to amplify the entire plasmid using the plasmid as a template.

[0100] 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 indole monooxygenase SmTPH gene mutant. Recombinant plasmids containing the target nucleotides of this strain with non-directional or directional mutations became the expression vectors for expressing the indole monooxygenase SmTPH gene mutant.

[0101] 3.2 Screening of SmTPH combinatorial mutant libraries

[0102] After successful sequencing of the combined mutant library, 5 μL of the digested PCR product was electroporated into E. coli BL21(DE3) competent cells and plated onto Kan / Cm antibiotic plates. Using SmTPH wild-type as a control, single clones of positive and negative control were aseptically picked and transferred to 96-well deep-well plates containing 400 μL of LB medium (400 μL LB medium containing 5 μg / mL chloramphenicol and 50 μg / mL kanamycin (glycerol 8 mL / L)). After sealing, the plates were incubated at 37°C with shaking at 800 rpm for 12 h.

[0103] 700 μL of TB medium (containing tryptophan at a final concentration of 5 g / L) was added to a 96-well deep-well culture plate, along with IPTG, chloramphenicol, kanamycin, and FeSO4, to make final concentrations of 0.1 mM, 5 μg / mL, 50 μg / mL, and 50 μg / mL, respectively. The plate was cultured at 30 °C and 800 rpm for 24 h with shaking. This was used to express the protein and catalyze the conversion of tryptophan to DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0104] After culturing for 24 hours, the 96-well deep-well culture plate was centrifuged at 4000 rpm for 30 min. 20 μL of the supernatant from the 48-well plate was transferred to a 96-well glass plate, and 180 μL of ddH2O was added, resulting in a 10-fold dilution. OD 310 Absorbance values ​​are measured to screen for mutants with higher absorbance than the wild type. OD values ​​are then measured. 310 The absorbance values ​​measured were higher than those of wild-type monoclonal colonies, and single colonies were selected for sequencing identification.

[0105] The sequenced strain, superior to the wild type, was activated by streak plating. Single colonies were picked and inoculated into 5 mL LB broth (containing 50 μg / mL kanamycin and 5 μg / mL chloramphenicol) and incubated overnight at 37°C and 220 rpm. Then, a 5% inoculum was added to 50 mL TB broth (containing 12 g / L peptone, 24 g / L yeast extract, 8 mL / L glycerol, 2.31 g KH₂PO₄, 16.43 g K₂HPO₄, and 10 g / L tryptophan). A final concentration of 50 μg / mL kanamycin and 5 μg / mL chloramphenicol were added. The mixture was incubated at 37°C and 220 rpm until the OD₀ reached approximately 0.8. Finally, 0.1 mM IPTG and 50 μg / mL FeSO₄ were added, and the mixture was incubated and fermented simultaneously at 30°C for 24 hours.

[0106] High-performance liquid chromatography (HPLC) detection: 100 μL of fermentation broth was added to 900 μL of ddH2O, centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane. The content of DL-2-amino-3-(5-hydroxyindole)propionic acid was detected using a Shimadzu LC-2030 microscope.

[0107] Liquid chromatography detection conditions: Agilent SB-Aq column (4.6 mm × 250 mm × 5 μM); detection wavelength: 275 nm; column temperature: 35 ℃; flow rate: 1 mL / min; sample loading volume: 10 μL; detection time: 15 min; mobile phase A: salt solution (0.68 g KH2PO4 dissolved in 1 L ultrapure water, 1.8 mL of 1 M trichloroacetic acid added to 1 L salt solution, pH adjusted to 3.0); mobile phase B: pure methanol; mobile phase A: mobile phase B = 92.5:7.5.

[0108] Establishment of a standard curve for DL-2-amino-3-(5-hydroxyindole)propionic acid standards: DL-2-amino-3-(5-hydroxyindole)propionic acid standards at concentrations of 0.3125 g / L, 0.625 g / L, 1.25 g / L, 2.5 g / L, and 5 g / L were prepared, and their absorption peaks were measured at 275 nm to establish a standard curve. Figure 2 ).

[0109] High-performance liquid chromatography (HPLC) analysis showed that the elution times of DL-2-amino-3-(5-hydroxyindole)propionic acid were all between 7 and 7.5 min. The results indicated that the modified recombinant strains exhibited improved fermentation efficiency for producing DL-2-amino-3-(5-hydroxyindole)propionic acid compared to the wild type. The yield of DL-2-amino-3-(5-hydroxyindole)propionic acid was calculated based on the standard curve. Compared to the wild type, the mutant strain produced a higher yield than a single wild-type colony. Single colonies were selected and sequenced for identification.

[0110] Preliminary screening yielded the following products: SmTPH-N106I / P178L, SmTPH-P178G / H182V, SmTPH-N106I / C221I, SmTPH-N106I / C221I / H182T, SmTPH-H182T / C221I / F223Y, Sm8TPH-P178G / F223D / L275M, and SmTP. Nine mutants with relatively high activity, namely H-N106I / P178L / H182T / F223V, SmTPH-N106I / P178G / H182T / F223V / L275S, and SmTPH-N106I / H182T / C221I / F223D / L275M, showed improved L-tryptophan substrate conversion rates compared to SmTPH. The results are shown in Table 4.

[0111] Table 4. Yield ratio of recombinant engineered strain DL-2-amino-3-(5-hydroxyindole)propionic acid

[0112] strain Conversion rate (%) Relative output (times) WT 15.22 1 WT-N106I / P178L 40.67 2.7 WT-P178G / H182V 55.38 3.6 WT-N106I / C221I 62.92 4.1 WT-N106I / C221I / H182T 42.05 2.8 WT-H182T / C221I / F223Y 60.60 4 WT-P178G / F223D / L275M 66.18 4.3 WT-N106I / P178L / H182T / F223V 75.15 5 WT-N106I / P178G / H182T / F223V / L275S 50.92 3.3 WT-N106I / H182T / C221I / F223D / L275M 79.58 5.2

[0113] In summary, this invention, based on the combined saturation mutation of wild-type indole monooxygenase SmTPH, yielded nine multi-site combined mutants. The yield of the mutants was significantly increased, being 2.7-5.2 times that of the starting strain, and could significantly increase the yield of DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0114] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An indole monooxygenase mutant, characterized in that, It is a protein obtained by mutating the indole monooxygenase SmTPH derived from Schistosoma mansoni, the amino acid sequence of which is shown in SEQ ID NO:

1. The mutation is based on the amino acid sequence of the indole monooxygenase SmTPH and involves the following mutations: The protein obtained by mutating the 106th position of indole monooxygenase SmTPH to isoleucine; The protein obtained by mutating positions 106 and 178 of indole monooxygenase SmTPH to isoleucine and leucine, respectively. The protein obtained by mutating positions 106 and 221 of indole monooxygenase SmTPH to isoleucine and isoleucine, respectively. The protein obtained by mutating positions 106, 182, and 221 of indole monooxygenase SmTPH to isoleucine, threonine, and isoleucine, respectively. The protein obtained by mutating positions 106, 178, 182, and 223 of indole monooxygenase SmTPH to isoleucine, leucine, threonine, and valine, respectively. The protein obtained by mutating positions 106, 178, 182, 223, and 275 of indole monooxygenase SmTPH to isoleucine, leucine, threonine, valine, and serine, respectively. The protein was obtained by mutating positions 106, 182, 221, 223, and 275 of indole monooxygenase SmTPH to isoleucine, threonine, isoleucine, aspartic acid, and methionine, respectively.

2. A gene encoding the indole monooxygenase mutant of claim 1.

3. A recombinant vector containing the encoding gene as described in claim 2.

4. A recombinant strain containing the recombinant vector as described in claim 3.

5. The recombinant strain according to claim 4, characterized in that, Its originating bacteria is Escherichia coli.

6. The recombinant strain according to claim 5, characterized in that, Its originating bacterium is E. coli BL21(DE3).

7. The use of the indole monooxygenase mutant of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the recombinant strain of claim 4, 5, or 6 in the production of DL-2-amino-3-(5-hydroxyindole)propionic acid.

8. A method for producing DL-2-amino-3-(5-hydroxyindole)propionic acid, characterized in that, The method includes the steps of culturing the recombinant strain according to claim 4, 5 or 6 to produce DL-2-amino-3-(5-hydroxyindole)propionic acid; and isolating DL-2-amino-3-(5-hydroxyindole)propionic acid.

9. The method as described in claim 8, characterized in that, It also includes a step of purifying DL-2-amino-3-(5-hydroxyindole)propionic acid.

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