Recombinant escherichia coli for producing melatonin and application thereof

Abstract

The invention relates to recombinant escherichia coli for producing melatonin and application of the recombinant escherichia coli, and belongs to the field of fermentation engineering. According to the invention, escherichia coli BL21 (DE3) is taken as a starting strain, exogenous genes luz15, ddc, aanat and asmt are introduced, in order to improve synthesis of cofactor heme of 5-tryptophan hydroxylase Luz15, an exogenous gene hemA is introduced, endogenous heme synthetic pathway genes are respectively overexpressed, and finally the melatonin high-yield strain MT2 is obtained. The invention discloses a new synthesis approach of melatonin, and the constructed melatonin production engineering strain is clear in genetic background and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation engineering, in particular to a recombinant Escherichia coli for producing melatonin and application thereof. Background Art

[0002] Melatonin (MT), also known as N-acetyl-5-methoxytryptamine, is an indole heterocyclic compound with a molecular formula of C 13 H 16 N2O2, with a relative molecular weight of 232.278. At room temperature, melatonin is a white crystal that is insoluble in water but readily soluble in organic reagents such as methanol. Melatonin is a hormone widely found in plants and animals. In plants, melatonin is found in various organs, including roots, flowers, fruits, and seeds, regulating plant growth and improving stress resistance. In animals, melatonin is secreted by the pineal gland, regulating the body's circadian rhythm and exhibiting anti-inflammatory, antioxidant, free radical scavenging, neuroendocrine immune activity, and immunity-enhancing properties. Currently, melatonin is produced and used as a health food to aid sleep and treat symptoms such as sleep disorders and jet lag.

[0003] Currently, melatonin production methods primarily include biological extraction and chemical synthesis. The earliest commercialized melatonin was produced by extracting from animal pineal glands, but this carries the risk of animal tissue contamination. Plant-based extraction, on the other hand, has limited production capacity and is unable to meet market demand. Eleven chemical synthesis methods exist across seven categories, involving numerous complex reactions such as condensation and acetylation. These methods are complex, require high substrate costs, and are prone to environmental pollution. Furthermore, they face challenges such as specificity for the tryptophan hydroxyl group, cost savings, and environmental protection, making them unsuitable for large-scale production. Microbial synthesis, however, offers advantages such as simplicity, low cost, and ease of industrialization, making it a highly promising method for producing melatonin.

[0004] Zhang et al. optimized the synthesis module of the melatonin precursor 5-HTP by modifying 4-phenylalanine hydroxylase and introducing the physostigmine synthesis gene cluster and the caffeic acid-O-methyltransferase gene comt, achieving melatonin synthesis of 0.65 g / L. Luo et al. identified a highly active phenylalanine hydroxylase through directed evolution and introduced the tBPt recycling pathway required for its catalytic cofactor, thereby constructing a strain that efficiently synthesizes 5-hydroxytryptophan. Subsequently, they introduced ddc from Canadidatus Koribacter versatilis Ellin 345, aanat from Streptomyces griseofuscus, and asmt from human sources, enabling de novo melatonin synthesis in E. coli. Melatonin production reached 2 g / L when tryptophan was supplemented into the culture medium, and 1 g / L when synthesized de novo from glucose. However, the tryptophan hydroxylase catalytic reaction involved in the above-mentioned synthesis pathway depends on cofactors. To achieve the synthesis of melatonin in E. coli, multiple exogenous genes need to be introduced to construct the cofactor synthesis and regeneration pathways, which increases the difficulty of strain construction and metabolic pressure. Summary of the Invention

[0005] To address the above technical issues, the present invention provides a recombinant Escherichia coli for melatonin production and its applications. Using E. coli BL21(DE3) as a starting strain, the present invention episomally expresses the melatonin biosynthesis pathway-related genes luz15, ddc, aanat, asmt, and hemA, successfully constructing a melatonin-producing strain.

[0006] Luz15 is a 5-tryptophan hydroxylase derived from Actinomadura luzonensis that catalyzes the hydroxylation reaction at the C5 position of the indole ring of tryptophan to produce 5-hydroxytryptophan. Unlike animal-derived tryptophan hydroxylases, Luz15 relies on the central cofactor heme to catalyze tryptophan hydroxylation. Escherichia coli can endogenously synthesize heme, which avoids the introduction of excessive exogenous genes for cofactor synthesis and regeneration. This is the first report of the introduction of exogenous genes, including luz15 from A. luzonensis, ddc from C. Koribacter versatilis Ellin, aanat from S. griseofuscus, asmt from humans, and hemA from R. palustris, to transform E. coli to achieve de novo melatonin synthesis.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a recombinant Escherichia coli for producing melatonin, wherein the recombinant Escherichia coli expresses the gene hemA encoding 5-ALA synthase HemA, the gene luz15 encoding 5-tryptophan hydroxylase Luz15, the gene ddc encoding aromatic amino acid decarboxylase Ddc, the gene aanat encoding aromatic amine N-acetyltransferase, and the gene asmt encoding acetyl serotonin O-methyltransferase ASMT.

[0009] In one embodiment of the present invention, the recombinant Escherichia coli contains a first expression system and a second expression system; the first expression system expresses the gene hemA encoding 5-ALA synthase HemA and the gene luz15 encoding 5-tryptophan hydroxylase Luz15; the gene hemA encoding 5-ALA synthase HemA is expressed upstream of the gene luz15 encoding 5-tryptophan hydroxylase Luz15;

[0010] The second expression system contains the gene ddc encoding aromatic amino acid decarboxylase Ddc, the gene aanat encoding aromatic amine N-acetyltransferase, and the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT;

[0011] The gene ddc encoding aromatic amino acid decarboxylase Ddc and the gene aanat encoding aromatic amine N-acetyltransferase are expressed upstream of the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT.

[0012] In one embodiment of the present invention, the first expression system uses the pCDFDuet-1 vector as a backbone; the second expression system uses the pET28a(+) vector as a backbone.

[0013] The free expression vector of the present invention includes pET28a-P plamdalac -ddc-aanat-P T7 -asmt and pCDF-P trc -hemA-P T7 -luz15.

[0014] In one embodiment of the present invention, the nucleotide sequence of the gene luz15 encoding 5-tryptophan hydroxylase Luz15 is shown as SEQ ID NO1; the nucleotide sequence of the gene ddc encoding aromatic amino acid decarboxylase Ddc is shown as SEQ ID NO2, the nucleotide sequence of the gene aanat encoding aromatic amine N-acetyltransferase is shown as SEQ ID NO3, the nucleotide sequence of the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT is shown as SEQ ID NO4, and the nucleotide sequence of the gene hemA encoding 5-ALA synthase HemA is shown as SEQ ID NO5.

[0015] In one embodiment of the present invention, the gene luz15 encoding 5-tryptophan hydroxylase Luz15 is derived from Actinomadura luzonensis; the gene ddc encoding aromatic amino acid decarboxylase Ddc is derived from Canadidatus Koribacter versatilis Ellin 345; the gene aanat encoding aromatic amine N-acetyltransferase is derived from Streptomyces griseofuscus; the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT is derived from humans; and the gene hemA encoding 5-ALA synthase HemA is derived from Rhodopseudomonas palustris.

[0016] In one embodiment of the present invention, the gene hemA encoding 5-ALA synthase HemA is regulated by the promoter P trc Regulate expression;

[0017] The gene encoding 5-tryptophan hydroxylase Luz15 is regulated by the promoter P T7 Regulate expression; the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT is regulated by promoter P T7 Regulate expression;

[0018] The gene ddc encoding aromatic amino acid decarboxylase Ddc and the gene aanat encoding aromatic amine N-acetyltransferase are regulated by promoter P plamdalac Regulate expression.

[0019] In one embodiment of the present invention, the promoter P T7 The nucleotide sequence of the promoter is shown in SEQ ID NO.6; plamdalac The nucleotide sequence of the promoter is shown in SEQ ID NO.7; trcThe nucleotide sequence is shown in SEQ ID NO.8.

[0020] In one embodiment of the present invention, the recombinant Escherichia coli is based on Escherichia coli BL21 (DE3) as the base strain.

[0021] The second object of the present invention is to provide the use of the recombinant Escherichia coli in the preparation of melatonin.

[0022] The third object of the present invention is to provide a method for preparing melatonin, which uses the recombinant Escherichia coli for fermentation production.

[0023] In one embodiment of the present invention, the seed solution of the recombinant Escherichia coli was 600 =0.2 was inoculated into the fermentation medium, cultured at 25℃-37℃ for 2h, then the inducer was added and the fermentation was continued for 24h-36h.

[0024] In one embodiment of the present invention, one or more of the following conditions are met:

[0025] (1) The system also includes an inducer IPTG at a concentration of 0.1 mM-1 mM;

[0026] (2) The fermentation conditions are 25°C-37°C for 24h-36h;

[0027] (3) The fermentation medium: yeast powder 2 g / L, (NH4)2SO4 16 g / L, KH2PO4 3 g / L, Na2HPO4·12H2O 16 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 0.01 g / L, tryptophan 1 g / L-5 g / L, glycine 1 g / L-5 g / L, methionine 1 g / L-5 g / L;

[0028] (4) The seed culture medium includes 5 g / L yeast powder, 10 g / L tryptone, and 10 g / L NaCl.

[0029] The above technical solution of the present invention has the following advantages over the prior art:

[0030] The present invention provides a recombinant Escherichia coli for producing melatonin and its applications. Using Escherichia coli BL21(DE3), a starting strain with a short growth cycle, clear genetic background and metabolic pathways, and simple molecular manipulation, a de novo synthesis pathway for melatonin was constructed. The effect of the cofactor heme on melatonin production was further explored. Ultimately, the resulting strain, MT2, possessed a clear genetic background and was capable of stable melatonin production. The yield of melatonin produced by shake flask fermentation reached 105.5 mg / L, and that produced by a 3-L bioreactor reached 1168 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0032] Figure 1 A roadmap for the melatonin synthesis pathway;

[0033] Figure 2 To construct the recombinant plasmid map, (A): pCDF-P trc -hemA-P T7 -luz15, (B): pET28a-P plamdalac -ddc-aanat-P T7 -asmt;

[0034] Figure 3 This is the HPLC chart of melatonin standard;

[0035] Figure 4 HPLC profile of melatonin production by strain MT1;

[0036] Figure 5 Melatonin production and OD for production strain MT1 600 Time-varying graphs;

[0037] Figure 6 These are the fermentation diagrams of the production strains MT2, MT3, MT4, MT5, MT6, and MT7, where A is the yield versus time, and B is the OD 600 Time-varying graphs;

[0038] Figure 7 Figure 2 shows the fermentation process of the production strain MT2 in a 3-L bioreactor. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0040] The present invention provides a recombinant Escherichia coli for producing melatonin, which expresses the gene luz15 encoding 5-tryptophan hydroxylase Luz15, the gene ddc encoding aromatic amino acid decarboxylase Ddc, the gene aanat encoding aromatic amine N-acetyltransferase, the gene asmt encoding acetyl serotonin O-methyltransferase ASMT, and the gene hemA encoding 5-ALA synthase HemA, and is used for synthesizing melatonin.

[0041] Furthermore, the gene luz15 is derived from Actinomadura luzonensis, the gene ddc is derived from Canadidatus Koribacter versatilis Ellin 345, the gene aanat is derived from Streptomyces griseofuscus, the gene asmt is derived from humans, and the gene hemA is derived from Rhodopseudomonas palustris.

[0042] Furthermore, the gene luz15 was expressed using pCDFDuet-1 as the expression vector and P T7 As the promoter, an inducible expression cassette was formed, and the expression was induced by IPTG in the engineered E. coli; the gene hemA was expressed in pCDFDuet-1 vector and P trc As the promoter, an inducible expression cassette was formed, and the expression was induced by IPTG in the engineered E. coli; the gene ddc was expressed in pET-28a(+) with P plamdalac As the promoter, an inducible expression cassette was formed, and the expression was induced by IPTG in the engineered E. coli; the gene aanat was expressed in pET-28a(+) vector and P plamdalac As the promoter, an inducible expression cassette was formed, and the expression was induced by IPTG in the engineered E. coli; the gene asmt was expressed in pET-28a(+) vector and P T7 The promoter is used to form an inducible expression cassette, which is expressed in engineered E. coli using IPTG induction.

[0043] Furthermore, the nucleotide sequences of the gene luz15, gene ddc, gene aanat, gene asmt and gene hemA are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0044] Furthermore, the inducible promoter P T7 、P plamdalac and P trcThe amino acid sequences are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.

[0045] The present invention also provides a method for producing melatonin by fermentation with the recombinant Escherichia coli (synthetic pathway diagram as shown in Figure 1 as shown); comprising the following steps:

[0046] The first step is to prepare the seed solution: a single colony of the production strain is inoculated into the seed culture medium and cultured at 37°C overnight;

[0047] The second step is shake flask fermentation: the seed liquid is fermented at an initial OD 600 =0.2 was inoculated into the fermentation medium, cultured at 25°C-37°C for 2h, then the inducer IPTG was added with a final concentration of 0.1mM-1mM, and fermented at 25°C-37°C for 24h-36h to produce melatonin.

[0048] Furthermore, the seed culture medium includes 5 g / L yeast powder, 10 g / L tryptone, and 10 g / L NaCl.

[0049] Furthermore, the fermentation medium formula is yeast powder 2g / L, (NH4)2SO4 16 g / L, KH2PO4 3 g / L, Na2HPO4·12H2O 16g / L, MgSO4·7H2O 1g / L, MnSO4·H2O 0.01g / L, tryptophan 1g / L-5g / L, glycine 1g / L-5g / L, and methionine 1g / L-5g / L.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0051] The nucleotide sequence information involved in the embodiments of the present invention is as follows:

[0052] The nucleotide sequence of luz15 is shown in SEQ ID NO. 1;

[0053] The nucleotide sequence of ddc is shown in SEQ ID NO. 2;

[0054] The nucleotide sequence of aanat is shown in SEQ ID NO.3;

[0055] The nucleotide sequence of asmt is shown in SEQ ID NO. 4;

[0056] The nucleotide sequence of hemA is shown in SEQ ID NO. 5;

[0057] Inducible promoter P T7The nucleotide sequence is shown in SEQ ID NO.6;

[0058] Inducible promoter P plamdalac The nucleotide sequence is shown in SEQ ID NO.7;

[0059] Inducible promoter P trc The nucleotide sequence is shown in SEQ ID NO.8.

[0060] (1) Strains

[0061] The plasmid was constructed in E. coli JM109 and then transformed into E. coli BL21 (DE3) for expression and production.

[0062] (2) Culture medium

[0063] LB medium was used as the seed culture medium for E. coli, and fermentation medium was used for shake flask fermentation;

[0064] LB medium: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; solid medium requires the addition of 2% (w / v) agar powder.

[0065] Shake flask fermentation medium: Yeast extract 2 g / L, (NH₄)₂SO₄ 16 g / L, KH₂PO₄ 3 g / L, Na₂HPO₄·12H₂O 16 g / L, MgSO₄·7H₂O 1 g / L, MnSO₄·H₂O 0.01 g / L, tryptophan 1 g / L, glycine 1 g / L, methionine 1 g / L. Glucose, MgSO₄·7H₂O, MnSO₄·H₂O, tryptophan, glycine, and methionine must be sterilized separately from the other ingredients and added prior to fermentation.

[0066] The final concentrations of antibiotics used were: Kanamycin 50 μg / mL, Chloramphenicol 15 μg / mL, and Streptomycin 100 μg / mL.

[0067] (3) Melatonin determination

[0068] An appropriate amount of fermentation broth was centrifuged at 12,000 rpm for 5 minutes to allow sedimentation. The supernatant was mixed with acetonitrile at a ratio of 60:40 (v / v) and centrifuged at 12,000 rpm for 10 minutes to allow sedimentation. The fermentation broth was filtered using a disposable syringe filter and a 0.22 μm membrane filter to remove bacterial cells and other impurities. The broth was then transferred to a liquid phase injection vial. Melatonin content was measured using an Agilent 1260 high-performance liquid chromatograph at a UV wavelength of 290 nm. The chromatographic column was an Agilent ZORBAX Eclipse XDB-C18 analytical column (3.0 mm × 250 mm, 5 μm), the injection volume was 10 μL, the column temperature was 30°C, the flow rate was 0.8 mL / min, the mobile phases were water (phase A) and acetonitrile (phase B) (each with 0.1% (v / v) formic acid added), and the gradient elution was: 0 min, 95% A; 12 min, 41% A; 13 min, 5% A; 17 min, 5% A; 25 min, 95% A.

[0069] The retention time of melatonin is 10.08 min.

[0070] Molecular biology experimental methods not specifically described in the following examples were all performed with reference to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0071] Table 1 Primer sequences involved in the examples

[0072]

[0073]

[0074]

[0075] Example 1: Melatonin synthesis pathway expression vector pCDF-P T7 -luz15 and pET28a-P plamdalac -ddc-aanat-P T7 -asmt construction

[0076] Use primers pCDF-vector-F / R to amplify the pCDFDuet-1 fragment and add homology arms; use primers luz15-fg-F / R to amplify the luz15 gene expression cassette. The above fragments were ligated using a one-step cloning kit to obtain pCDF-P T7 -luz15 and the sequence accuracy was confirmed by Sanger sequencing.

[0077] Use primers pET-vector-1F / 1R and pET-vector-2F / 2R to amplify pET28a(+), use amplification primers or vector fragments and add homology arms; use primers ddc-fg-F / R, aanat-fg-F / R, asmt-fg-F / R to amplify ddc, aanat, and asmt gene expression cassettes. The above fragments were connected using a one-step cloning kit to obtain pET28a-P plamdalac -ddc-aanat-P T7 -asmt and confirmed its correctness by Sanger sequencing, as Figure 2 B in.

[0078] Example 2: Construction of heme synthesis pathway overexpression vector

[0079] In E. coli, heme synthesis is divided into two modules: the synthesis of the heme precursor 5-ALA and the synthesis of heme from 5-ALA. First, there are two 5-ALA synthesis pathways in nature: the C4 pathway and the C5 pathway. The C4 pathway is primarily found in animals, yeast, a small number of chemotrophic bacteria, and purple non-sulfur photosynthetic bacteria. The 5-ALA synthase, encoded by hemA or hemT, catalyzes the condensation of succinyl-CoA and glycine to synthesize 5-ALA. The C5 pathway, primarily found in higher plants, algae, and bacteria, involves three genes: gltX, hemA, and heml. In this example, the 5-ALA synthase gene hemA from R. palustris was selected to promote the synthesis of 5-ALA in E. coli. In addition, the synthesis of heme from 5-ALA in E. coli involves seven genes: hemB, hemC, hemD, hemE, hemF, hemG, and hemH.

[0080] Use primers pCDF-luz15-vector-F / R to pCDF-P T7 -luz15, using amplification primers or vector fragments and adding homology arms; using primers hemA-fg-F / R to amplify the hemA gene expression cassette. The above fragments were ligated using a one-step cloning kit to obtain pCDF-P trc -hemA-P T7 -luz15 and confirm the sequence correctness by Sanger sequencing, as Figure 2 A in.

[0081] Primers pACYC-vector-1F / 1R and pACYC-vector-2F / 1R were used to amplify the primers or vector fragments of pACYCDuet-1 and add homology arms; primers hemE-fg-1F / 1R, hemE-fg-1F / 2R, and hemF-fg-F / R were used to amplify the hemE and hemF gene expression cassettes. The above fragments were connected using a one-step cloning kit to obtain pACYC-P trc -hemE、pACYC-P trc -hemE-hemF and sequence accuracy was confirmed by Sanger sequencing.

[0082] Use primers pACYC-hemE-hemF-vector-F / R to pACYC-P trc -hemE-hemF, using amplification primers or vector fragments and adding homology arms; using primers hemG-fg-F / R and hemH-fg-1F / 1R to amplify the hemG and hemH gene expression cassettes respectively. The above fragments were connected using a one-step cloning kit to obtain pACYC-P trc -hemE-hemF-P plamdalac -hemG, pACYC-P trc -hemE-hemF-P plamdalac -hemH and sequence accuracy was confirmed by Sanger sequencing.

[0083] Use primers pACYC-hemE-hemF-hemG-vector-F / R to pACYC-P trc -hemE-hemF-P plamdalac -hemG, using amplification primers or vector fragments and adding homology arms; using primers hemH-fg-2F / 2R to amplify the hemH gene expression cassette. The above fragments were ligated using a one-step cloning kit to obtain pACYC-P trc -hemE-hemF-P plamdalac -hemG-hemH and sequence accuracy was confirmed by Sanger sequencing.

[0084] Example 3: Construction and production of melatonin-producing strain MT1

[0085] pCDF-P T7 -luz15 and pET28a-P plamdalac -ddc-aanat-P T7 The -asmt target plasmid was transformed into Escherichia coli BL21 (DE3), spread on the corresponding resistance LB solid culture medium, and then placed in a constant temperature culture medium at 37°C for inverted culture to obtain the melatonin-producing strain MT1.

[0086] Pick a single colony from the corresponding resistance LB solid medium and inoculate it into a 50 mL shaking tube containing 5 mL of the corresponding resistance LB liquid medium, and culture it in a shaking incubator at 37°C overnight as the seed liquid. 600 =0.2 was inoculated into a 250 mL flat-bottom shake flask containing 25 mL of the corresponding resistance fermentation medium and cultured in a shaker at 30°C and 220 rpm. After culturing for 2 h, IPTG was added to a final concentration of 0.1 mM to induce the expression of melatonin-related enzymes, and samples were taken at intervals.

[0087] After 36 h of fermentation, the production of melatonin was successfully detected by HPLC. Figure 4 As shown ( Figure 3 The HPLC chart of melatonin standard product was successfully constructed, and the melatonin production strain was 56.9 mg / L. Figure 5 shown.

[0088] Example 4: Construction and production of melatonin-producing strains MT2, MT3, MT4, MT5, MT6, and MT7

[0089] pCDF-P trc -hemA-P T7 -luz15 and pET28a-P plamdalac -ddc-aanat-P T7 The -asmt target plasmid was transformed into Escherichia coli BL21 (DE3), spread on the corresponding resistance LB solid medium, and then placed in a constant temperature medium at 37°C for inverted culture to obtain the melatonin-producing strain MT2.

[0090] pACYC-P trc -hemE、pACYC-P trc -hemE-hemF, pACYC-P trc -hemE-hemF-P plamdalac -hemG,

[0091] pACYC-P trc -hemE-hemF-P plamdalac -hemH,

[0092] pACYC-P trc -hemE-hemF-P plamdalac -hemG-hemH were transformed into the production strain MT2 respectively, spread on the corresponding resistance LB solid culture medium and then placed in a constant temperature culture medium at 37°C for inverted culture to obtain melatonin-producing strains MT3, MT4, MT5, MT6 and MT7.

[0093] Pick a single colony from the corresponding resistance LB solid medium and inoculate it into a 50 mL shaking tube containing 5 mL of the corresponding resistance LB liquid medium, and culture it in a shaking incubator at 37°C overnight as the seed liquid. 600 =0.2 was inoculated into a 250 mL flat-bottom shake flask containing 25 mL of the corresponding resistance fermentation medium and cultured in a shaker at 30°C and 220 rpm. After culturing for 2 h, IPTG was added to a final concentration of 0.1 mM to induce the expression of melatonin-related enzymes, and samples were taken at intervals.

[0094] After 36h of fermentation, HPLC detection showed that MT2 had the highest melatonin production, reaching 105.5mg / L. Figure 6 shown.

[0095] Example 5: Expanded culture and fermentation of melatonin-producing strain MT2 in a 3-L bioreactor

[0096] After streaking MT2 onto the corresponding resistance LB solid medium and culturing overnight in a 37°C incubator, pick a single colony and inoculate it into a 250mL flat-bottom shake flask containing 25mL of the corresponding resistance LB liquid medium. Incubate it in a shaker at 37°C, 220rpm, and use it as the primary seed liquid. Inoculate 2% of the primary seed liquid into a 250mL flat-bottom shake flask containing 25mL of the corresponding resistance fermentation medium. Incubate it in a shaker at 37°C, 220rpm, and use it as the secondary seed liquid. Centrifuge the secondary seed liquid at 4000rpm for 5 minutes, discard the supernatant, retain the bacteria, resuspend them in fermentation medium, and inoculate them into a 3-L bioreactor.

[0097] During the expansion culture, samples were taken every 4 hours to measure the residual glucose content in the culture medium, and batch feeding was performed based on the glucose concentration to maintain the glucose concentration in the fermentation broth at 0-5 g / L. After 8 hours of expansion culture, IPTG was added at a final concentration of 0.3 mM to induce the expression of melatonin-related enzymes, and samples were taken at intervals to measure melatonin production and OD 600 After 36 hours of fermentation, the melatonin production reached 1168 mg / L. Figure 7 shown.

[0098] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A recombinant Escherichia coli for producing melatonin, characterized in that: The recombinant Escherichia coli expresses the gene hemA encoding 5-ALA synthase HemA, the gene luz15 encoding 5-tryptophan hydroxylase Luz15, the gene ddc encoding aromatic amino acid decarboxylase Ddc, the gene aanat encoding aromatic amine N-acetyltransferase, and the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT.

2. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli contains a first expression system and a second expression system; the first expression system expresses the gene hemA encoding 5-ALA synthase HemA and the gene luz15 encoding 5-tryptophan hydroxylase Luz15; the gene hemA encoding 5-ALA synthase HemA is expressed upstream of the gene luz15 encoding 5-tryptophan hydroxylase Luz15; The second expression system contains the gene ddc encoding aromatic amino acid decarboxylase Ddc, the gene aanat encoding aromatic amine N-acetyltransferase, and the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT; The gene ddc encoding aromatic amino acid decarboxylase Ddc and the gene aanat encoding aromatic amine N-acetyltransferase are expressed upstream of the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT.

3. The recombinant Escherichia coli according to claim 1, characterized in that The first expression system uses the pCDFDuet-1 vector as the backbone; the second expression system uses the pET28a(+) vector as the backbone.

4. The recombinant Escherichia coli according to claim 1, characterized in that The gene luz15 encoding 5-tryptophan hydroxylase Luz15 is derived from Actinomadura luzonensis; the gene ddc encoding aromatic amino acid decarboxylase Ddc is derived from Canadidatus Koribacterversatilis Ellin 345; the gene aanat encoding aromatic amine N-acetyltransferase is derived from Streptomyces griseofuscus; the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT is derived from humans; and the gene hemA encoding 5-ALA synthase HemA is derived from Rhodopseudomonas palustris.

5. The recombinant Escherichia coli according to claim 1, characterized in that The gene hemA encoding 5-ALA synthase HemA is regulated by promoter P trc Regulate expression; The gene encoding 5-tryptophan hydroxylase Luz15 is regulated by the promoter P T7 Regulate expression; the gene asmt encoding acetyl 5-hydroxytryptamine O-methyltransferase ASMT is regulated by promoter P T7 Regulate expression; The gene ddc encoding aromatic amino acid decarboxylase Ddc and the gene aanat encoding aromatic amine N-acetyltransferase are regulated by promoter P plamdalac Regulate expression.

6. The recombinant Escherichia coli according to claim 5, characterized in that The promoter P T7 The nucleotide sequence of the promoter is shown in SEQ ID NO.6; plamdalac The nucleotide sequence of the promoter is shown in SEQ ID NO.7; trc The nucleotide sequence is shown in SEQ ID NO.

8.

7. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as the base bacteria.

8. Use of the recombinant Escherichia coli according to any one of claims 1 to 7 in the preparation of melatonin.

9. A method for preparing melatonin, characterized in that: The recombinant Escherichia coli according to any one of claims 1 to 7 is used for fermentation production.

10. The method according to claim 9, characterized in that One or more of the following conditions are met: (1) The system also includes an inducer IPTG at a concentration of 0.1 mM-1 mM; (2) The fermentation conditions are 25°C-37°C for 24h-36h.

Images