Synthesis method of non-natural indole amino acid

Abstract

According to the invention, indole monooxygenase from Schistosoma mansoni is modified by methods such as a gene mining technology, random mutation and the like, so as to obtain a mutant with relatively high yield of DL-2-amino-3-(5-hydroxyindole) propionic acid. The DL-2-amino-3-(5-hydroxyindole) propionic acid is produced from a recombinant vector and a recombinant genetically engineered bacterium constructed by the mutant in a fermentation culture manner, and the yield of the DL-2-amino-3-(5-hydroxyindole) propionic acid is remarkably improved and is 1.5-5.2 times that of an original strain. Therefore, the method for producing the DL-2-amino-3-(5-hydroxyindole) propionic acid, provided by the invention, has the advantages of mild reaction conditions, simplicity in operation, short production period, environmental friendliness, reduction of production cost and the like.

Description

Technical Field

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

[0002] DL-2-Amino-3-(5-hydroxyindolyl)propionic acid is a tryptophan derivative. It is catalyzed by indole monooxygenase to replace the 5-position hydrogen atom on the benzene ring of tryptophan with a hydroxyl group. It is hereinafter referred to as non-natural indole amino acids. Non-natural indole amino acids can effectively treat various diseases, including depression, fibromyalgia, obesity caused by overeating, chronic headaches, and insomnia. In recent years, the production research of DL-2-Amino-3-(5-hydroxyindolyl)propionic acid has developed rapidly, and its application in domestic and foreign pharmaceutical and related industries is also expanding.

[0003] At present, the production methods of non-natural indole amino acids include natural product extraction, chemical synthesis and microbial fermentation. Among them, natural product extraction is still the main way to commercialize non-natural indole amino acids. The main source is extracted from the seeds of the African legume plant Ghana, but the plant has a long growth cycle and is restricted by seasons and regions, with low yields, which is not suitable for industrial applications. The chemical synthesis method requires organic reagent raw materials, such as 3-methyl-4-nitrophenol, and then undergoes a series of addition reactions, reduction reactions, hydrolysis reactions, etc. to obtain non-natural indole amino acids. The reaction conditions used are harsh, the cycle is long, the energy consumption is large, the product yield is low, and it needs to be synthesized under high temperature and high pressure, which is easy to cause environmental pollution and is not suitable for large-scale synthesis.

[0004] The microbial fermentation method has the advantages of being green, environmentally friendly, mild reaction conditions, and continuous production, and has great application prospects. However, the indole monooxygenase used to catalyze the production of DL-2-amino-3-(5-hydroxyindole) propionic acid from tryptophan has low biological activity and cannot produce high-yield DL-2-amino-3-(5-hydroxyindole) propionic acid. Summary of the invention

[0005] At present, the industrial production of DL-2-amino-3-(5-hydroxyindole) propionic acid has the problems of low yield, long cycle, easy environmental pollution, etc. Aiming at the shortcomings of the prior art, the present invention utilizes genetic engineering technology and computer-aided design to construct an indole monooxygenase mutant with higher activity, thereby greatly improving the yield of DL-2-amino-3-(5-hydroxyindole) propionic acid.

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

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

[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 exists in one or more of the following amino acid residue positions: 34, 106, 145, 175, 178, 182, 221, 223, 229, 246, 250, 275 and / or 276.

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

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

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

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

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

[0014] X4, mutating the threonine at position 175 of SmTPH to arginine or lysine;

[0015] X5, mutating 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, mutating the cysteine ​​at position 221 of SmTPH to asparagine and isoleucine;

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

[0019] X9, mutating the glycine at position 229 of SmTPH to glycine, aspartic acid, phenylalanine, and serine;

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

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

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

[0023] X13, mutating 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 position 178 of SmTPH to leucine;

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

[0028] The protein obtained by mutating position 182 of SmTPH to valine;

[0029] The protein obtained by mutating position 182 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 position of SmTPH to aspartic acid;

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

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

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

[0035] Protein obtained by mutating position 275 of SmTPH to methionine.

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

[0037] The proteins were obtained by mutating the 106th and 178th positions of SmTPH to isoleucine and leucine, respectively.

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

[0039] Proteins obtained by mutating the 106th and 221st positions of SmTPH to isoleucine and isoleucine, respectively.

[0040] Proteins were obtained by mutating the 106th, 182th, and 221st positions of SmTPH to isoleucine, isoleucine, and threonine, respectively.

[0041] The proteins were obtained by mutating the 182nd, 221st, and 223rd positions of SmTPH to threonine, isoleucine, and tyrosine, respectively.

[0042] The proteins were obtained by mutating the 178th, 223rd, and 275th positions of SmTPH to glycine, aspartic acid, and methionine, respectively.

[0043] The proteins were obtained by mutating the 106th, 178th, 182th and 223rd positions of SmTPH to isoleucine, leucine, threonine and valine, respectively.

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

[0045] The proteins were obtained by mutating the 106th, 182th, 221st, 223th and 275th positions of SmTPH to isoleucine, threonine, isoleucine, aspartic acid and methionine, respectively.

[0046] The present 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 mutant integrated into its genome, and the recombinant vector or the genetically engineered strain is used in the production of DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0047] The present 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, the method comprises the following steps:

[0048] 1) Producing DL-2-amino-3-(5-hydroxyindole)propionic acid by using the indole monooxygenase and mutant or the recombinant genetic engineering strain;

[0049] 2) Separating and obtaining DL-2-amino-3-(5-hydroxyindole)propionic acid from the system of 1).

[0050] The present invention discloses the following technical achievements:

[0051] The invention discloses a newly modified indole monooxygenase mutant, wherein a plurality of mutants are obtained by performing specific site mutation on the amino acid sequence of the indole monooxygenase as shown in SmTPH; and the obtained mutants all have the function of catalyzing L-tryptophan to generate DL-2-amino-3-(5-hydroxyindole)propionic acid as the wild type SmTPH.

[0052] The present invention also discloses a recombinant vector and a recombinant strain comprising an indole monooxygenase gene and a mutant gene thereof. The gene encoding the indole monooxygenase in the recombinant vector is expressed by a shake flask fermentation culture method to obtain the corresponding enzyme. The present invention significantly improves the yield of the mutant obtained by mutating a specific site of the wild-type enzyme, which is 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 indole monooxygenase mutant in the synthesis of DL-2-amino-3-(5-hydroxyindole)propionic acid and its derivatives belongs to the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Plasmid map for the construction of indole monooxygenase recombinant vector.

[0055] Figure 2 This is a standard curve diagram of HPLC liquid chromatography 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 of the dominant mutant recombinant engineering strain is shown in Table 1. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] Example 1: Discovery of indole monooxygenase

[0060] The indole monooxygenase TPH from Schistosoma mansoni (GenBank No.: AAD01923.1) was retrieved from the NCBI database, and the whole gene synthesized SmTPH was connected to the pET24a expression vector 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] MHHHHAKNPTLDDKVPWFPRHISDLDKVSNSVLMYGKELDADHPGFKDKEYRKRRMMFADIALNYKWGQQIPIVEYTEIEKTTWGRIYRELTRLYKTSACHEFQKNLGLLQDKAGYNEFDLPQLQVVSDFLKARTGFCLRPVAGYLSARDFLSGLAFRVFYCTQYIRHQ ADPFYTPEPDCCHELLGHVPMLADPKFARFSQEIGLASLGTSDEEIKKLATCYFFTIEFGLCRQDNQLKAYGAGLLSSVAELQHALSDKAVIKPFIPMKVINEECLVTTFQNGYFETSSSFEDATRQMREFVRTIKRPFDVHYNPYTQSIEIIKTPKSVAKLVQDLQFE.

[0063] The above-mentioned recombinant expression vector is transformed into a suitable microbial host. The host microorganism is any conventional host microorganism in the art, as long as the above-mentioned recombinant expression vector can stably replicate itself and the indole monooxygenase gene can be effectively expressed. In this embodiment, the above-mentioned recombinant expression plasmid is introduced into the competent state of E. coli BL21 (DE3) by electrotransformation, and inverted culture is carried out on a LB solid plate containing kanamycin resistance for 12-16 hours, and positive transformants are selected for DNA sequencing verification, and the correct transformant is the indole monooxygenase gene engineering strain.

[0064] Example 2. Obtaining indole monooxygenase or its mutant

[0065] 2.1 Construction of SmTPH single-site saturation mutant library

[0066] Based on gene mining, molecular docking, rational analysis and other methods of wild-type indole monooxygenase, a total of 13 sites were designed for rational design according to the substrate binding pocket and the interaction between substrate molecules. Therefore, the present invention selects 13 sites in the amino acid sequence of wild-type indole monooxygenase from Schistosoma mansoni (GenBank No.: AAD01923.1) for transformation, which are 34, 106, 145, 175, 178, 182, 221, 223, 229, 246, 250, 275, and 276, respectively, and constructs a single-site saturation mutant library for each site, as shown in Table 1.

[0067] Table 1. Single-site saturation mutant library constructed based on indole monooxygenase SmTPH

[0068]

[0069]

[0070] Note: In Table 1, there are multiple single-stranded DNA primers, primers M34-F, N106-F, Y145-F, T175-F, P178-F, H182-F, C221-F, F223-F, G229-F, S246-F, E250-F, L275-F, V276-F, and the single-stranded DNAs are mixed in the order of TGG:ATG:VMA:NDT in the table at a molar ratio of 1:1:6:12; V represents G or A or C, M represents A or C, N represents A or T or G or C, and D represents G or A or T.

[0071] To obtain the indole monooxygenase SmTPH mutant, the following experiment was performed:

[0072] 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 forward and reverse primers are shown in Table 1.

[0073] 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.

[0074] The obtained PCR product was operated as follows: 1 μL of Dpn I enzyme was added to 20 μL of PCR product to digest the plasmid template, and the product was treated at 37°C for 2 hours. 5 μL of the digested PCR product was electrotransformed into 100 μL of E. coli BL21 (DE3) competent cells, and the electrotransformed E. coli BL21 (DE3) bacterial solution was evenly coated on a kanamycin-resistant (concentration of 50 μg / mL) LB plate, and a single colony was grown after culturing at 37°C for 14 hours, which was the engineering strain of the indole monooxygenase SmTPH gene mutant. The recombinant plasmid containing the target nucleotide in the strain that underwent undirected mutation or directed mutation was the expression vector for expressing the indole monooxygenase SmTPH gene mutant.

[0075] 2.2 Screening of the SmTPH single-site saturation mutant library

[0076] After sequencing of the single-point saturation mutant library, 5 μL of the digested PCR product was electrotransferred to the competent medium of E. coli BL21 (DE3) and spread on the Kan / Cm resistance plate. Using the wild type of SmTPH as a control, the positive and negative control monoclonal clones were aseptically picked and transferred to a 96-well deep-well culture plate containing 400 μL LB medium, 400 μL LB containing 5 μg / mL chloramphenicol and 50 μg / mL kanamycin (glycerol 8 mL / L), and the film was sealed and cultured at 37°C with shaking at 800 rpm for 12 h.

[0077] 700 μL of TB medium (TB medium containing tryptophan at a final concentration of 5 g / L) was added to a 96-well deep-well culture plate, and IPTG, chloramphenicol, kanamycin and FeSO4 were added to make the final concentrations of 0.1 mM, 5 μg / mL, 50 μg / mL and 50 μg / mL, respectively. The culture was shaken at 30°C and 800 rpm for 24 h to express the protein and catalyze the conversion of tryptophan into DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0078] After 24 hours of culture, the 96-well deep-well culture plate was centrifuged at 4000 rpm for 30 minutes. 20 μL of the supernatant from the 48-well plate was taken into a glass 96-well plate and 180 μL of ddH2O was added to dilute it 10 times. The OD 310 Determine the absorbance value to screen mutants with higher absorbance than the wild type. 310 The single clones whose absorbance values ​​were higher than those of the wild type were picked and sequenced for identification.

[0079] Through preliminary screening, 11 mutants with relatively high activity were obtained, including SmTPH-N106I, SmTPH-P178L, SmTPH-P178G, SmTPH-H182V, SmTPH-H182T, SmTPH-C221I, SmTPH-F223D, SmTPH-F223Y, SmTPH-F223V, SmTPH-L275S, and SmTPH-L275M. Their conversion rates of L-tryptophan substrate were improved compared with the wild type. The results are shown in Table 2.

[0080] Table 2. Recombinant engineering strain DL-2-amino-3-(5-hydroxyindole) propionic acid production ratio

[0081] strains Conversion rate (%) Relative yield (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

[0082] In summary, the present invention is based on mutating specific sites of wild-type indole monooxygenase SmTPH, and the yield of the obtained mutant is significantly improved, 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.

[0083] Example 3: Construction of a combined saturation mutant of indole monooxygenase SmTPH or acquisition of its mutant

[0084] 3.1 Construction of SmTPH combinatorial mutant library

[0085] Eleven mutants N106I, P178L, P178G, H182V, H182T, C221I, F223D, F223Y, F223V, L275S, and L275M screened from 10 single-site saturation mutant libraries were subjected to combined mutations, for a total of 6 sites. The primer designs are shown in Table 3.

[0086] Table 3. Combinatorial saturation mutant library constructed based on indole monooxygenase SmTPH

[0087] 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

[0088] Note: In Table 3, there are multiple single-stranded DNA primers, M represents A or C, Y represents C or T, S represents C or G, H represents A or C or T, V represents G or A or C, R represents A or G, K represents G or T, W represents A or T, D represents G or A or T, and B represents C or G or T.

[0089] To obtain the indole monooxygenase SmTPH mutant, the following experiment was performed:

[0090] 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 forward and reverse primers are shown in Table 1.

[0091] 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.

[0092] Overlap extension PCR process:

[0093] First round: R1-1, R1-2, R1-3, R1-4, R1-5, and R1-6 were mixed in a ratio of 1:1:1:1:1:1 to form downstream primer R1, and F1 and R1 amplified fragment 1 (about 274 bp); R2-1 and R2-2 were mixed in a ratio of 1:1 to form downstream primer R2, and F2 and R2 amplified fragment 2 (about 139 bp); R3-1 and R3-2 were mixed in a ratio of 2:1 to form downstream primer R3, and F3 and R3 amplified fragment 3 (about 162 bp);

[0094] Second round: Fragment 1, fragment 2, and fragment 3 were excised and recovered as templates, and upstream primer F1 and mixed downstream primer R3 were used as upstream and downstream primers respectively, and fragment 4 was amplified by overlapping extension PCR;

[0095] Round 3: Fragment 4 was used as a large primer to amplify the entire plasmid using the plasmid as a template.

[0096] The obtained PCR product was operated as follows: 1 μL of Dpn I enzyme was added to 20 μL of PCR product to digest the plasmid template, and the product was treated at 37°C for 2 hours. 5 μL of the digested PCR product was electrotransformed into 100 μL E.coli BL21 (DE3) competent cells, and the electrotransformed E.coli BL21 (DE3) bacterial solution was evenly coated on a kanamycin-resistant (concentration of 50 μg / mL) LB plate, and a single colony was grown after culturing at 37°C for 14 hours, which was the engineering strain of the indole monooxygenase SmTPH gene mutant. The recombinant plasmid containing the target nucleotide in the strain that underwent undirected mutation or directed mutation was the expression vector for expressing the indole monooxygenase SmTPH gene mutant.

[0097] 3.2 Screening of SmTPH combinatorial mutant library

[0098] After successful sequencing of the combined mutant library, 5 μL of the digested PCR product was electrotransferred to E. coli BL21 (DE3) competent medium and spread on Kan / Cm resistance plates. Using the wild type of SmTPH as a control, the positive and negative control monoclonal clones were aseptically picked and transferred to a 96-well deep-well culture plate containing 400 μL LB medium, 400 μL LB containing 5 μg / mL chloramphenicol and 50 μg / mL kanamycin (glycerol 8 mL / L), and the film was sealed and cultured at 37°C 800 rpm for 12 h.

[0099] 700 μL of TB medium (TB medium containing tryptophan at a final concentration of 5 g / L) was added to a 96-well deep-well culture plate, and IPTG, chloramphenicol, kanamycin and FeSO4 were added to make the final concentrations of 0.1 mM, 5 μg / mL, 50 μg / mL and 50 μg / mL, respectively. The culture was shaken at 30°C and 800 rpm for 24 h to express the protein and catalyze the conversion of tryptophan into DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0100] After 24 hours of culture, the 96-well deep-well culture plate was centrifuged at 4000 rpm for 30 minutes. 20 μL of the supernatant from the 48-well plate was taken into a glass 96-well plate and 180 μL of ddH2O was added to dilute it 10 times. The OD 310 Determine the absorbance value to screen mutants with higher absorbance than the wild type. 310 The single clones whose absorbance values ​​were higher than those of the wild type were picked and sequenced for identification.

[0101] The strains that are higher than the wild type and have been sequenced were activated by streaking on a plate, and a single colony was picked and inoculated into 5 mL of LB test tube medium (containing a final concentration of 50 μg / mL kanamycin and 5 μg / mL chloramphenicol), cultured at 37°C and 220 rpm overnight, and inoculated into 50 mL of TB medium (12 g / L peptone, 24 g / L yeast powder, 8 mL / L glycerol, 2.31 g KH2PO4, 16.43 g K2HPO4, 10 g / L tryptophan) at a 5% inoculum, and kanamycin and chloramphenicol were added at a final concentration of 50 μg / mL. The OD600 was cultured at 37°C and 220 rpm until it reached about 0.8, and IPTG and 50 μg / mL FeSO4 were added at a final concentration of 0.1 mM, and the culture and fermentation were carried out at 30°C for 24 hours.

[0102] 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 DL-2-amino-3-(5-hydroxyindole) propionic acid content was detected using Shimadzu LC-2030.

[0103] Liquid phase detection conditions: chromatographic column Agilent SB-Aq (4.6mm×250mm×5μM); detection wavelength: 275nm; column temperature: 35℃; flow rate: 1mL / min; sample volume: 10μL; detection time: 15min; mobile phase A: saline solution (0.68g KH2PO4 dissolved in 1L ultrapure water, 1.8mL of 1M trichloroacetic acid added to 1L of saline solution, pH adjusted to 3.0); mobile phase B: pure methanol; mobile phase A: mobile phase B = 92.5:7.5.

[0104] DL-2-amino-3-(5-hydroxyindole) propionic acid standard to establish a standard curve: prepare 0.3125g / L, 0.625g / L, 1.25g / L, 2.5g / L, and 5g / L of DL-2-amino-3-(5-hydroxyindole) propionic acid standard, measure its absorption peak at 275nm, and establish a standard curve ( Figure 2 ).

[0105] According to the HPLC detection, the peak time of DL-2-amino-3-(5-hydroxyindole) propionic acid was 7min-7.5min. The results showed that the ability of the recombinant engineering strain to produce DL-2-amino-3-(5-hydroxyindole) propionic acid by fermentation was improved compared with the wild type. The production of DL-2-amino-3-(5-hydroxyindole) propionic acid was calculated based on the standard curve of the standard product. Compared with the wild type, the mutant had a higher production than the wild type strain. The single clone was selected for sequencing and identification.

[0106] Through preliminary screening, SmTPH-N106I / P178L, SmTPH-P178G / H182V, SmTPH-N106I / C221I, SmTPH-N106I / C221I / H182T, SmTPH-H182T / C221I / F223Y, Sm8TPH-P178G / F223D / L275M, SmTPH- There are nine relatively active mutants, including H-N106I / P178L / H182T / F223V, SmTPH-N106I / P178G / H182T / F223V / L275S, and SmTPH-N106I / H182T / C221I / F223D / L275M, which have improved L-tryptophan substrate conversion rates compared with SmTPH. The results are shown in Table 4.

[0107] Table 4 Recombinant engineering strain DL-2-amino-3-(5-hydroxyindole) propionic acid production ratio

[0108] strains Conversion rate (%) Relative yield (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

[0109] In summary, the present invention is based on the combined saturation mutagenesis of the wild-type indole monooxygenase SmTPH, and 9 multi-site combined mutants are obtained respectively. The yield of the obtained mutants is significantly improved, which is 2.7-5.2 times that of the starting strain, and can significantly increase the yield of DL-2-amino-3-(5-hydroxyindole)propionic acid.

[0110] 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. An indole monooxygenase mutant, characterized in that It is derived from Schistosoma mansoni ( Schistosoma mansoni ) Sm TPH (GenBank No.: AAD01923.1) was mutated to obtain a protein, wherein the mutation was in the indole monooxygenase Sm The following mutations exist based on the amino acid sequence of TPH: selected from one or more of the following amino acid residue positions: 34, 106, 145, 175, 178, 182, 221, 223, 229, 246, 250, 275 and / or 276.

2. The indole monooxygenase mutant according to claim 1, characterized in that There is any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve or all of the following thirteen proteins obtained by modification and mutation: X1, indole monooxygenase Sm The methionine at position 34 of TPH was mutated to aspartic acid, glycine, and lysine; X2, indole monooxygenase Sm The aspartic acid at position 106 of TPH was mutated to isoleucine, methionine, and serine; X3, indole monooxygenase Sm The tyrosine at position 145 of TPH was mutated to threonine, isoleucine, and serine; X4, indole monooxygenase Sm The threonine at position 175 of TPH was mutated to arginine and lysine; X5, indole monooxygenase Sm The proline at position 178 of TPH was mutated to arginine, leucine, and glycine; X6, indole monooxygenase Sm The histidine at position 182 of TPH was mutated to serine, tyrosine, valine, or threonine; X7, indole monooxygenase Sm The cysteine ​​at position 221 of TPH was mutated to asparagine and isoleucine; X8, indole monooxygenase Sm The phenylalanine at position 223 of TPH was mutated to aspartic acid, tyrosine, tryptophan, and valine; X9, indole monooxygenase Sm The glycine at position 229 of TPH was mutated to glycine, aspartic acid, phenylalanine, and serine; X10, indole monooxygenase Sm The serine at position 246 of TPH was mutated to proline, leucine, and phenylalanine; X11, indole monooxygenase Sm The glutamic acid at position 250 of TPH was mutated to aspartic acid and histidine; X12, indole monooxygenase Sm The leucine at position 275 of TPH was mutated to serine, methionine, glutamic acid, or threonine; X13, indole monooxygenase Sm The valine at position 276 of TPH was mutated to glutamine and proline.

3. The indole monooxygenase mutant according to claim 2, characterized in that It is one of the following mutants: indole monooxygenase Sm The protein obtained by mutation of position 106 of TPH to isoleucine; indole monooxygenase Sm The protein obtained by mutation of position 178 of TPH to leucine; indole monooxygenase Sm The protein obtained by mutation of position 178 of TPH to glycine; indole monooxygenase Sm The protein obtained by mutation of position 182 of TPH to valine; indole monooxygenase Sm The protein obtained by mutation of position 182 of TPH to threonine; indole monooxygenase Sm The protein obtained by mutation of position 221 of TPH to isoleucine; indole monooxygenase Sm The protein obtained by mutation of position 223 of TPH to aspartic acid; indole monooxygenase Sm The protein obtained by mutation of position 223 of TPH to tyrosine; indole monooxygenase Sm The protein obtained by mutation of position 223 of TPH to valine; indole monooxygenase Sm The protein obtained by mutation of position 275 of TPH to serine; indole monooxygenase Sm The protein obtained by mutation of position 275 of TPH to methionine; indole monooxygenase Sm The proteins obtained by mutating positions 106 and 178 of TPH to isoleucine and leucine, respectively; indole monooxygenase Sm The proteins obtained by mutating positions 178 and 182 of TPH to glycine and valine, respectively; indole monooxygenase Sm The proteins obtained by mutating positions 106 and 221 of TPH to isoleucine and isoleucine, respectively; indole monooxygenase Sm The proteins obtained by mutating the 106th, 182th, and 221st positions of TPH to isoleucine, isoleucine, and threonine, respectively; indole monooxygenase Sm The proteins obtained by mutating the 182nd, 221st, and 223rd positions of TPH to threonine, isoleucine, and tyrosine, respectively; indole monooxygenase Sm The proteins obtained by mutating the 178th, 223rd, and 275th positions of TPH to glycine, aspartic acid, and methionine, respectively; indole monooxygenase Sm The proteins obtained by mutating the 106th, 178th, 182th, and 223rd positions of TPH to isoleucine, leucine, threonine, and valine, respectively; indole monooxygenase Sm The proteins obtained by mutating the 106th, 178th, 182th, 223th, and 275th positions of TPH to isoleucine, glycine, threonine, valine, and serine, respectively; indole monooxygenase Sm The proteins obtained were obtained by mutating the 106th, 182th, 221st, 223th, and 275th positions of TPH to isoleucine, threonine, isoleucine, aspartic acid, and methionine, respectively.

4. A gene encoding the indole monooxygenase mutant according to any one of claims 1 to 3.

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

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

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

8. Use of the indole monooxygenase mutant according to any one of claims 1 to 3, the encoding 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 DL-2-amino-3-(5-hydroxyindole)propionic acid.

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

10. The method according to claim 9, characterized in that The method also includes a step of purifying DL-2-amino-3-(5-hydroxyindole)propionic acid.

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