5-HT producing Escherichia coli strain and its construction method and application

Abstract

The present invention provides a 5-hydroxytryptamine Escherichia coli production strain and a construction method and application thereof, wherein the strain lacks tnaA, pykA, trpR, ldhA and trpL genes, and upregulates trpE fbr 、aroG fbr ,serA fbr , tktA, prsA genes, heterologous expression of gdh and glnA fbr Gene, dynamically regulated the pykF gene, and the production bacteria carried plasmids pACYC-FYSPD and plasmids pETDuet-TM2-RBS-AnTDC; the constructed strain has the characteristics of metabolic balance, and by optimizing the expression intensity of key genes of each precursor, the expression amount of tryptophan hydroxylation-decarboxylation system and coenzyme tetrahydrobiopterin synthesis and regeneration system is regulated by a dual-plasmid system, so that 5-hydroxytryptamine can be efficiently synthesized in a culture medium without by-products.

Description

Technical Field

[0001] The invention relates to the field of biotechnology production, in particular to a 5-hydroxytryptamine Escherichia coli production strain and a construction method and application thereof. Background Art

[0002] 5-hydroxytryptamine (5-HT) is a secondary metabolite formed by the two-step reaction of hydroxylation and decarboxylation of L-tryptophan. It was first extracted from serum in 1948, so it is also called serotonin. As a monoamine neurotransmitter, it has multiple functions such as regulating emotions, treating insomnia and scavenging free radicals. In addition, 5-HT plays an important role in many fields such as agriculture, medicine and scientific research because it can improve plant stress resistance, treat depression and promote human tissue regeneration and repair.

[0003] At present, the production of 5-HT mainly relies on plant tissue extraction and chemical synthesis, but these two methods cannot be mass-produced due to cost and raw materials, and the price of 5-HT on the market is also high. In addition, the 5-HT synthesis pathway is lengthy and involves a variety of precursors. How to balance the supply of various precursors to build a microbial cell factory with balanced metabolic flow and full utilization of intracellular resources is a technical problem that needs to be solved urgently. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a 5-hydroxytryptamine producing Escherichia coli strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned 5-HT producing Escherichia coli strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned 5-hydroxytryptamine producing Escherichia coli strain.

[0007] In order to solve the above technical problems, the technical solution of the present invention is:

[0008] A 5-HT production strain of Escherichia coli, designated as strain WW14, lacks tnaA, pykA, trpR, ldhA and trpL genes and upregulates trpE fbr 、aroG fbr ,serA fbr , tktA, and prsA genes, and heterologously expressed gdh and glnA from B. subtilis 168 fbr Gene, dynamically regulated the pykF gene, and the production bacteria carried the low copy plasmid pACYC-FYSPD and the high copy plasmid pETDuet-TM2-RBS-AnTDC.

[0009] Preferably, in the above-mentioned 5-HT producing Escherichia coli strain, the nucleotide sequence of the tnaA gene is shown in the sequence listing SEQ ID NO.1; the nucleotide sequence of the trpR gene is shown in the sequence listing SEQ ID NO.4; the nucleotide sequence of the pykA gene is shown in the sequence listing SEQ ID NO.7; the nucleotide sequence of the ldhA gene is shown in the sequence listing SEQ ID NO.11; and the nucleotide sequence of the trpL gene is shown in the sequence listing SEQ ID NO.31.

[0010] Preferably, the above-mentioned 5-HT producing Escherichia coli strain, the trpE fbr The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.3; aroG fbr The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.5; serA fbr The nucleotide sequence of the gene is shown in the sequence list as SEQ ID NO.10; the nucleotide sequence of the tktA gene is shown in the sequence list as SEQ ID NO.9; the nucleotide sequence of the prsA gene is shown in the sequence list as SEQ ID NO.8; the nucleotide sequence of the gdh gene is shown in the sequence list as SEQ ID NO.12; fbr The nucleotide sequence of the gene is shown in the sequence listing as SEQ ID NO.6; the nucleotide sequence of the pykF gene is shown in the sequence listing as SEQ ID NO.30.

[0011] Preferably, in the above-mentioned 5-HT producing Escherichia coli strain, the nucleotide sequence of the plasmid pACYC-FYSPD is shown in the sequence listing SEQ ID NO.27.

[0012] Preferably, in the above-mentioned 5-HT producing Escherichia coli strain, the nucleotide sequence of the plasmid pETDuet-TM2-RBS-AnTDC is shown in the sequence listing SEQ ID NO.28.

[0013] The low copy plasmid pACYC-FYSPD expressed folE from Escherichia coli. T198I , ygcM genes and SPR, DHPR and PCD genes from humans; plasmid pACYC-FYSPD has Figure 1 The structure shown in the figure shows that the plasmid pACYC-FYSPD carries plasmid elements such as the p15A replication origin, chloramphenicol resistance, T7 promoter, and terminator, and also carries folE from Escherichia coli. fbr , ycgM genes and SPR, DHPR and PCD genes from Homo sapiens, folE fbrThe M1-12 promoter is used to enhance transcription of the ycgM and SPR genes, the M1-93 promoter is used to enhance transcription of the PCD gene, and the trc promoter is used to enhance transcription of the DHPR gene. The high-copy plasmid pETDuet-TM2-RBS-AnTDC expresses the human TM2 gene and the AnTDC gene from Aspergillus niger; the plasmid pETDuet-TM2-RBS-AnTDC has Figure 2 The structure shown in the figure is that the plasmid pETDuet-TM2-RBS-AnTDC carries plasmid elements such as ColE1 replication start site, ampicillin resistance, T7 promoter, terminator, etc., and also carries the TM2 gene derived from Homo sapiens and the AnTDC gene derived from Aspergillus niger. The TM2 gene and the AnTDC gene are connected through RBS, the nucleotide sequence of the RBS is shown in the sequence table SEQ ID NO.29, and transcription is driven by the same T7 promoter to construct an artificial mini gene cluster.

[0014] The above folE fbr The nucleotide sequence of the gene is shown in SEQ ID NO.13 in the sequence listing; the nucleotide sequence of the ycgM gene is shown in SEQ ID NO.14 in the sequence listing; the nucleotide sequence of the SPR gene is shown in SEQ ID NO.15 in the sequence listing; the nucleotide sequence of the PCD gene is shown in SEQ ID NO.16 in the sequence listing; the nucleotide sequence of the DHPR gene is shown in SEQ ID NO.17 in the sequence listing; the nucleotide sequence of the TM2 gene is shown in SEQ ID NO.18 in the sequence listing; the nucleotide sequence of the AnTDC gene is shown in SEQ ID NO.19 in the sequence listing; the nucleotide sequence of the M1-12 promoter is shown in SEQ ID NO.26 in the sequence listing; and the nucleotide sequence of the T7 promoter is shown in SEQ ID NO.21 in the sequence listing.

[0015] Preferably, the above-mentioned 5-HT producing Escherichia coli strain is E.coli W3110 ΔlacIZ::P XylF -T7RNAP (the strain E. coli HT01 obtained in Example 2 of CN116590210A) was used as the chassis strain, and the tnaA, pykA, trpR, ldhA and trpL genes on the strain genome were knocked out; the aroG fbr The gene was transcribed and integrated into the tnaA gene locus; the trc promoter was used to enhance the trpE fbr The gene was transcribed and integrated into the trpLE gene locus; the glnA gene from B. subtilis 168 was enhanced using the trc promoter fbrThe gene was transcribed and integrated into the trpR gene site of the genome; the prsA gene transcription was enhanced by the trc promoter and integrated into the ilvG gene site; the tktA gene transcription was enhanced by the trc promoter and integrated into the pykA gene site; the PpykF promoter (pykF gene promoter) was replaced with the fliC promoter to dynamically downregulate the expression of the pykF gene; the serA gene was enhanced by the M1-93 promoter fbr The gene was transcribed and integrated into the yeeL gene locus; the gdh gene transcription of B. subtilis 168 was enhanced using the M1-37 promoter and integrated into the ldhA gene locus.

[0016] Preferably, in the above-mentioned 5-HT Escherichia coli production strain, the nucleotide sequence of the trc promoter is shown in the sequence listing SEQ ID NO.20; the nucleotide sequence of the trpLE gene is shown in the sequence listing SEQ ID NO.2; the nucleotide sequence of the PpykF promoter is shown in the sequence listing SEQ ID NO.22; the nucleotide sequence of the fliC promoter is shown in the sequence listing SEQ ID NO.23; the nucleotide sequence of the M1-93 promoter is shown in the sequence listing SEQ ID NO.24; and the nucleotide sequence of the M1-37 promoter is shown in the sequence listing SEQ ID NO.25.

[0017] The method for constructing the above-mentioned 5-HT producing Escherichia coli strain comprises the following specific steps:

[0018] (1) E.coli W3110 ΔlacIZ::P XylF -T7RNAP was used as the chassis strain to knock out the tnaA gene, trpR gene and trpL gene; trpE was strengthened fbr Gene;

[0019] (2) Knock out the pykA gene; strengthen the tktA gene and aroG fbr gene, serA fbr Gene and prsA gene; introduction of glnA fbr Gene; replace the PpykF promoter (pykF gene promoter) with the fliC promoter to dynamically regulate the pykF gene;

[0020] (3) Knock out the ldhA gene and introduce the gdh gene;

[0021] (4) Introducing the pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC dual plasmid system.

[0022] Preferably, the method for constructing the above-mentioned 5-HT producing Escherichia coli strain comprises the following specific steps:

[0023] (1) Construction of tryptophan pathway: E.coli W3110 ΔlacIZ::P XylF -T7RNAP was used as the chassis strain to knock out the gene tnaA encoding the tryptophan decomposition pathway; knock out the tryptophan repressor regulatory protein trpR; knock out the gene trpL encoding the tryptophan leader peptide; and strengthen the gene trpE encoding the anthranilate synthase mutant fbr ;

[0024] (2) Balance of precursor supply: Knock out the pykA gene encoding pyruvate kinase II to reduce the conversion of phosphoenolpyruvate (PEP) into pyruvate; strengthen the tktA gene encoding transketolase I to increase the supply of erythrose 4-phosphate; strengthen the aroG gene mutant encoding 3-deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase fbr , improve the supply of DAHP; strengthen the gene encoding D-3-phosphoglycerate dehydrogenase mutant gene serA fbr , improve the supply of serine; strengthen the gene prsA encoding phosphoribosyl pyrophosphate synthase to improve the supply of phosphoribosyl pyrophosphate, a key precursor of tryptophan; introduce B.subtilis 168 The mutant gene encoding glutamine synthase glnA fbr , improve the supply of glutamine; replace the PpykF promoter (pykF gene promoter) encoding the pyruvate kinase I gene with the fliC promoter to achieve dynamic expression of the gene pykF to further improve PEP accumulation;

[0025] (3) Optimizing the supply of NAD(P)H: knocking out the lactate dehydrogenase gene ldhA to reduce the waste of NADH; introducing the glucose dehydrogenase gene gdh from B. subtilis 168 to increase the production of NAD(P)H;

[0026] (4) pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC dual plasmid system: pACYC-FYSPD plasmid carries plasmid elements such as p15A replication origin, chloramphenicol resistance, T7 promoter, and terminator. This plasmid effectively expresses folE fbr , ygcM, SPR, PCD and DHPR genes, folE fbrThe M1-12 promoter was used to enhance the transcription of ycgM and SPR genes, the M1-93 promoter was used to enhance the transcription of PCD gene, and the trc promoter was used to enhance the transcription of DHPR gene. The pETDuet-TM2-RBS-AnTDC plasmid carries the ColE1 replication initiation site, ampicillin resistance, T7 promoter, terminator and other plasmid elements, and also carries the TM2 gene from Homo sapiens and the AnTDC gene from Aspergillus niger. The TM2 gene and the AnTDC gene are connected through RBS and driven by the same T7 promoter to construct an artificial mini gene cluster.

[0027] Application of the above 5-HT Escherichia coli production strain in fermentation production of 5-HT.

[0028] Under suitable fermentation conditions, the 5-hydroxytryptamine Escherichia coli production strain obtains 5-hydroxytryptamine by fermentation in a culture medium through two-stage pH control, and the culture medium may include but is not limited to: a carbon source, a nitrogen source, an inorganic salt, vitamins, etc. Fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen conditions, fermentation pressure, fermentation time, etc. The culture medium can be obtained by conventional methods and used for 5-hydroxytryptamine production, and the fermentation conditions can be adjusted to adapt to the production characteristics of the strain.

[0029] Preferably, the application of the above-mentioned 5-HT producing Escherichia coli strain is carried out by shake flask fermentation culture, and the specific method is as follows:

[0030] ① Test tube culture: The strain was inoculated in a test tube containing LB liquid medium, 220 rpm, 37°C, 12 h;

[0031] ② Shake flask seed culture: Take the test tube bacteria and inoculate them into the shake flask seed culture medium for fermentation, 220 rpm, 34℃, 12h, pH6.4-6.7,

[0032] ③ Shake flask fermentation culture: fermentation inoculation amount is 20-25%, 34℃, 220 rpm, culture for 24-36h, pH 6.4-6.7.

[0033] Preferably, in the application of the above-mentioned 5-HT Escherichia coli production strain, the shake flask seed culture medium in step ② is: glucose 20-30 g / L, yeast powder 4-6 g / L, peptone 2-3 g / L, MgSO4.7H2O 1-2 g / L, KH2PO4 2-4 g / L, ammonium sulfate 1-2 g / L, and the rest is water.

[0034] Preferably, in the application of the above-mentioned 5-HT Escherichia coli production strain, the fermentation medium in step ③ is: glucose 15 g / L, xylose 10 g / L, MgSO4.7H2O 2-4 g / L, yeast powder 6-8 g / L, peptone 4-6 g / L, ammonium sulfate 2-4 g / L, KH2PO4 4-6 g / L, glutamate 2 g / L, FeSO4.7H2O 40-80 mg / L, MnSO4 5-8 mg / L, VB1, VB3, VB5 each 2-4 mg / L, biotin 2-4 mg / L, and the rest is water.

[0035] Preferably, the application of the above-mentioned 5-HT producing Escherichia coli strain is carried out by fermentation tank culture, and the specific method is as follows:

[0036] (1) Slant culture: The strain was streaked onto a slant containing chloramphenicol and ampicillin resistance activation medium, cultured at 34°C for 12 h, and subcultured twice;

[0037] (2) Seed culture: The activated bacteria on the slant were washed with sterilized distilled water and transferred to the fermenter to start seed culture. The culture temperature was 37°C, the initial stirring speed was 200 rpm, the culture pH was maintained at 6.7±0.2, the dissolved oxygen value of the culture medium was maintained at 30%, and the OD value was adjusted to 0. 600nm Reaching 25 is a sign of maturity of the seed culture medium;

[0038] (3) Fermentation culture: After the seed liquid matures, fermentation medium is added to the seed culture for fermentation culture. The culture temperature is 34°C, the culture pH is maintained at 6.4±0.2, the dissolved oxygen value of the culture medium is maintained at 20%, and the glucose concentration in the tank is controlled at ≤1g / L.

[0039] Preferably, the use of the above-mentioned 5-HT production strain, when OD 600 When the cell culture temperature reached 10, IPTG was added to a final concentration of 0.1 mM for induction.

[0040] Preferably, in the application of the above-mentioned 5-HT Escherichia coli production strain, the slant culture medium used in step (1) is: glucose 1 g / L, chloramphenicol 15 mg / L, ampicillin 50 mg / L, peptone 5 g / L, potassium dihydrogen phosphate 0.5 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, agar powder 25 g / L, pH 7.0.

[0041] Preferably, in the application of the above-mentioned 5-HT Escherichia coli production strain, the seed culture medium used in the step (2) is: 30 g / L glucose, 15 mg / L chloramphenicol, 50 mg / L ampicillin, 4 g / L yeast extract, 2 g / L peptone, 2 g / L citric acid, 2 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 2 g / L glutamic acid, 0.5 g / L methionine, and the rest is water.

[0042] Preferably, in the application of the above-mentioned 5-HT Escherichia coli production strain, the fermentation medium used in step (3) is: 20 g / L glucose, 15 mg / L chloramphenicol, 50 mg / L ampicillin, 6 g / L yeast extract, 4 g / L peptone, 2 g / L citric acid, 2 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 3 g / L magnesium sulfate heptahydrate, 60 mg / L ferrous sulfate heptahydrate, 5 mg / L manganese sulfate monohydrate, 2 g / L monosodium glutamate, 1 g / L choline chloride, 1 g / L betaine, and the remainder is water.

[0043] The above culture media can be prepared using standard methods.

[0044] Beneficial effects:

[0045] The above-mentioned 5-HT E. coli production strain has the characteristics of metabolic balance, and is a stable and balanced tryptophan hydroxylation-decarboxylation system and BH4 synthesis and regeneration system. By optimizing the expression intensity of each precursor key gene, and adjusting the expression amount of the tryptophan hydroxylation-decarboxylation system and the coenzyme tetrahydrobiopterin (BH4) synthesis and regeneration system through a dual plasmid system, it can efficiently synthesize 5-HT in the culture medium without by-products, has stable performance, and has excellent industrial application prospects. Specifically:

[0046] (1) By regulating glnA fbr 、prsA、serA fbr (1) The expression intensity of tktA, pykA, and pykF was adjusted to maintain the supply balance of various precursors for tryptophan synthesis. (2) The ldhA gene was knocked out to reduce NADH consumption and the glucose dehydrogenase gdh from B. subtilis 168 was introduced to increase the supply of NAD(P)H. (3) The pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC dual-plasmid system was constructed. On the one hand, the tryptophan hydroxylation-decarboxylation system and the coenzyme tetrahydrobiopterin (BH4) synthesis and regeneration system were constructed to hydroxylate and decarboxylate the key precursor tryptophan. On the other hand, different plasmid vectors were selected and the expression level of each gene was fine-tuned to maintain the balance between the tryptophan hydroxylation-decarboxylation system and the BH4 synthesis and regeneration system, thereby maximizing the utilization of intracellular resources for efficient and byproduct-free synthesis of 5-hydroxytryptamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the structure of plasmid pACYC-FYSPD.

[0048] Figure 2 Schematic diagram of the structure of plasmid pETDuet-TM2-RBS-AnTDC. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0050] The percentage sign "%" involved in the embodiments, unless otherwise specified, refers to mass percentage. The percentage of a solution refers to the number of grams of a solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0051] The corresponding promoters and genes in the examples are shown in the sequence table. The primers used in the construction of the strains involved are shown in Table 1.

[0052] Table 1 Primers involved in strain construction

[0053] Primer Name Sequence Number Primer Sequence (5’-3’) glnA-E304A-6 SEQ ID NO.32 cataacaaggtgcCGcatagccaggaacaag glnA-E304A-7 SEQ ID NO.33 cttgttcctggctatgCGgcaccttgttatg glnA-L159I-4 SEQ ID NO.34 cagttggagcAATgtcgaaatatccg glnA-L159I-5 SEQ ID NO.35 cggatatttcgacATTgctccaactg trc-glnA-3 SEQ ID NO.36 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCatggcaaagtacactagagaagatatcg trc-glnA-8 SEQ ID NO.37 CAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGttaatactgagacatatactgttcgcgttc trpR-1 SEQ ID NO.38 GCAGATTATGCCTGGTACAGC trpR-10 SEQ ID NO.39 CGTTGCCTGCGGTGATTCTG trpR-trc-2 SEQ ID NO.40 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGTAACCACTCCTGGTGACGC trpR-trc-9 SEQ ID NO.41 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCGTGGATCTAACAGCCTG ilvG-1 SEQ ID NO.42 CCGAGGAGCAGACAATGAATAACAG ilvG-6 SEQ ID NO.43 GAAGGCGCTGGCTAACATGAGG ilvG-trc-2 SEQ ID NO.44 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGGTGATGGCAACAACAGGG ilvG-trc-5 SEQ ID NO.45 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCTATCTACGCGCCGTTGTTG aroG-4 SEQ ID NO.46 catttcgttacgAacaggaagcag aroG-5 SEQ ID NO.47 ctgcttcctgtTcgtaacgaaatg trc-aroG-3 SEQ ID NO.48 GTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCatgaattatcagaacgacgatttacgcatc trc-aroG-6 SEQ ID NO.49 CAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGttacccgcgacgcgcttttac M137-gdh-3 SEQ ID NO.50 GTTGATATAATTGAGCCACTGGCTCGTAATTTATTGTTTAAACCAGGAAACAGCTatgggctataacagcctgaaagg M137-gdh-4 SEQ ID NO.51 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGttaaccgcggcccgc ldhA-1 SEQ ID NO.52 ACGGGTATTGTGGCATGTT ldhA-6 SEQ ID NO.53 GATGAAAGGTCATTGGGGA ldhA-M137-5 SEQ ID NO.54 CAACGTTGATATAATTGAGCCACTGGCTCGTAATTTATTGTTTAAACCAGGAAACAGCTAGAAAAATTGGTTCGTTGGG ldhA-M193-2 SEQ ID NO.55 GAGCCAGTGGCTCAATTATATCAACGTTGTTATCTCTTGTCAACACCGCCAGAGATAAATTGGCAGTTTTAGCGGTTTT folE-T198I-F SEQ ID NO.56 CAACCAGTGCCACGACAACGATTTCTCTTGGTGGATTGTTC folE-T198I-R SEQ ID NO.57 GAACAATCCACAAGAGAAATCGTTGTCGTGGCACTGGTTG line-pACYC-F SEQ ID NO.58 agactgggccttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaattgcggcacacggtcacactgc line-pACYC-R SEQ ID NO.59 ccaaaagggctcaattatatcaacgttgttatctcttgtcaacccgccagagataacagctcatttcagaatatttg M112-floE-F SEQ ID NO.60 gatataattgagccctttggtgcgtcagtcagtttaaaccaggaaacagctatgccatcactcagtaaagaagcg M112-fol-R SEQ ID NO.61 gcgttcaccgacaaacaacagataaaacgaaggcccagtctttcgactgagccttcgttttatttgtcagttgtgatgacgcacag M112-SPR-F SEQ ID NO.62 gatataattgagccctttggtgcgtcagtcagtttaaccaggaaacagctatgcatcatcaccatcaccacgaagg M112-SPR-R SEQ ID NO.63 caccgacaaacaagataaaacgaaaggcccagtctttcgactgagccttcgttttatttgttagatgtcgtagaagtcaacgtgag M112-ygcM-F SEQ ID NO.64 gatataattgagccctttggtgcgtcagtcagtttaaaccaggaaacagctatgatgtccaccacgttatttaaag M112-ygcM-R SEQ ID NO.65 gttcaccgacaaacaacagataaacgaaaggcccagtctttcgactgagccttcgttttatttgtcattcgccgcgatacaac M193-PCD-F SEQ ID NO.66 gatataattgagcccgtattgttagcatgtacgtttaaccaggaaacagctatggctggtaaagctcaccgtctg M193-PCD-R SEQ ID NO.67 ctcacaattccacacattatacgagccggatgattaattgtcaattaggtcatagaaacagcaacctgttcgatg trc-DHPR-F SEQ ID NO.68 gtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagaccatggctgctggtgaagctcgtcgtgttctgg trc-DHPR-R SEQ ID NO.69 caacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgttagaagtaagccggggtcagttcgg AnTDC-R SEQ ID NO.70 gctcagcggtggcagcagcgttagccgcgtttaatcacttcttccgc line-pETDuet-F SEQ ID NO.71 cgctgctgccaccgctgagc line-pETDuet-R SEQ ID NO.72 ctagaggggaattgttatccgctcacaattcccctatagtgagtcgtattaatcccaatacgcgtcaattcactggcc RBS-AnTDC-F SEQ ID NO.73 tttgtttaactttaagaaggagatataccatggatcgcgaacagtttcgcg T7-TM2-F SEQ ID NO.74 gagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatataccatgaaactggaagatgtaccgtg TM2-RBS-R SEQ ID NO.75 ggtatatctccttcttaaagttaaacaaattatgtatctttcaaaatttcgatactc PfliC-F SEQ ID NO.76 AGAGTATTTCGGCGACTAACAAAAAATG PfliC-pykF-F SEQ ID NO.77 GACTTGCAATATAGGATAACGAATCATGGTTTCAGCACTTTGGACTGTAG PfliC-pykF-R SEQ ID NO.78 GATTCGTTATCCTATATTGCAAGTC pykF-R SEQ ID NO.79 TTACAGGACGTGAACAGATGCGG pGRB-pykF-a SEQ ID NO.80 GCCTTATTTTTAACTTGCTATTTCTAGCTCTAAAACgtattacgaaagtggtggtgCTAGTATTATACCTAGGACTGAGCTAGCTG pGRB-pykF-s SEQ ID NO.81 CAGCTAGCTCAGTCCTAGGTATAATACTAGcaccaccactttcgtaatacGTTTTAGGCTAGAAATAGCAAGTTAAAATAAGGC pGRB-tnaA-a SEQ ID NO.82 CTATTTCTAGCTCTAAAACCTCTGTAGTATTAAATTAACCTAGTATTATACCTAGGACTG pGRB-tnaA-s SEQ ID NO.83 CAGTCCTAGGTATAATACTAGGTTTAATTAATACTACAGAGGTTTTAGAGCTAGAAATAG pGRB-trpL-a SEQ ID NO.84 CTATTTCTAGCTCTAAAAACttcagtacgaaaattgctttCTAGTATTATACCTAGGACTG pGRB-trpL-s SEQ ID NO.85 CAGTCCTAGGTATAATACTAGaaagcaattttcgtactgaaGTTTTAGAGCTAGAAATAG pGRB-trpR-a SEQ ID NO.86 CTATTTCTAGCTCTAAAACcaacgcttcgcgctcatctgCTAGTATTATACCTAGGACTG pGRB-trpR-s SEQ ID NO.87 CAGTCCTAGGTATAATACTAGcagatgagcgcgaagcgttgGTTTTAGAGCTAGAAATAG pykA-1 SEQ ID NO.88 cgttaccacgttaggcccag pykA-6 SEQ ID NO.89 cgcgtggtattagtagaacccacg pykA-trc-2 SEQ ID NO.90 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAActacgccaatcaacgccgc trc-pykA-5 SEQ ID NO.91 atgactgatcaagctgaaaaaaaagcactctggatggagtcaatgattactaacccg trc-tktA-3 SEQ ID NO.92 GGCTCGTATAATGTGTGGAATGTGTGAGCGGATAACAATTTCACACAGGAAACAGACCatgtcctcacgtaaagcttgc trc-tktA-4 SEQ ID NO.93 GACAAACAACAGATAAAACGAAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGttacagcagttcttttgctttcgcaac tnaA-1 SEQ ID NO.94 CCGGTGGTAATGGACTCCGC tnaA-8 SEQ ID NO.95 GTGAAAGCCAGATACAAGGGGTTG tnaA-trc-2 SEQ ID NO.96 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGCGACTTTATACAGCTCGCAGG tnaA-trc-7 SEQ ID NO.97 CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTATCTGTTGTCCCACGGCAATATTCCCAGCCTG M193-serA-3 SEQ ID NO.98 CGTTGATATAATGAGCCCGTATTGTTAGCATGTACGTTTAAACCAGGAAACAGCTATGGCAAAGGTATCGCTGGAGAAAG M193-serA-8 SEQ ID NO.99 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGTACAGCACGGGCGCGAATGG serA-4 SEQ ID NO.100 GGACGGTTTTCCGCGATGTGCATC serA-5 SEQ ID NO.101 GATGCACATCGCGGAAAAACCGTCC serA-6 SEQ ID NO.102 GCGCGGCGATCGCGACGCCCTGCTCGG serA-7 SEQ ID NO.103 CCGAGCAGGGCGTCGCGATCGCCGCGC and L-1 SEQ ID NO.104 TTCATCGGGACGAGTGGAGA and L-10 SEQ ID NO.105 CATTCCCTCTACAGAACTAGCCCT yeL-M193-2 SEQ ID NO.106 CGTTGATATAATTGAGCCCGTATTGTTAGCATGTACGTTTAAACCAGGAAACAGCTGCCATAGCATCGCCAATCTGATC yeeL-M193-9 SEQ ID NO.107 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCCAAAGGTGAAGATAAAGCCAGG trc-trpLE-2 SEQ ID NO.108 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGTCGATACCCTTTTTACGTGAACTTGCG trc-trpE-3 SEQ ID NO.109 atgactgatcaagctgaaaaaaagcactctgATGCAAACACAAAAACCGACTCTCG trpLE-1 SEQ ID NO.110 CTGATTGCGCCGTTCTGTCTG trpE-6 SEQ ID NO.111 GGATCACAATGTAGGTGTCGAG trpE-7 SEQ ID NO.112 CTCGACACCTACATTGTGATCC trpE-8 SEQ ID NO.113 CCGTCATGTTCAATGCTGGAGG trpE-S40F-4 SEQ ID NO.114 GTCACCTAAAACTGTAATGCGCAG trpE-S40F-5 SEQ ID NO.115 CTGCGCATTACAGTTTTAGGTGAC

[0054] The gene editing method used in the gene manipulation in the embodiment refers to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.), and the professional terms involved in the present invention can be explained in the article unless otherwise noted. "Knockout" as referred to in the present invention refers to the inactivation of the target gene, and "introduction" refers to the insertion of the exogenous gene into the genome of the engineered bacteria after connecting it to the promoter and terminator.

[0055] Example 1

[0056] This example is intended to illustrate the specific construction steps of strain WW14. In particular, if there is a method for the same type of gene manipulation in the example, it will only be provided once and annotated without further elaboration.

[0057] ① Knock out tnaA and integrate aroG at this site fbr Gene: E.coliThe W3110 genome was used as a template, and tnaA-1, tnaA-trc-2, tnaA-trc-7, and tnaA-8 were used to obtain the upstream and downstream homology arms. E.coli W3110 genome was used as template and aroG was obtained by PCR amplification using trc-aroG-3, aroG-4, aroG-5, and trc-aroG-6. fbr Fragments 1 and 2; then, upstream and downstream homology arms, aroG fbr Gene fragments 1 and 2 were used as templates, tnaA-1 and tnaA-8 were used as primers, and overlapping fragments were obtained by overlapping PCR amplification; pGRB-tnaA-s and pGRB-tnaA-a were used as primers, and gRNA fragments were annealed and connected with pGRB vector to obtain tnaA-pGRB; preparation E.coli W3110 ΔlacIZ::P XylF -T7RNAP (the strain E. coli HT01 obtained in Example 2 of CN116590210A) was electroporated into competent cells, the target fragment and tnaA-pGRB were electroporated into competent cells together, and positive transformants were screened to obtain strain TRP01.

[0058] ② Knock out the trpL gene and integrate trpE at the trpLE gene site fbr Gene: E.coli The W3110 genome was used as a template, and trpLE-1, trc-trpLE-2, trpE-7, and trpE-8 were used to obtain the upstream and downstream homology arms. E.coli The W3110 genome was used as a template to amplify trpE by PCR using trc-trpE-3, trpE-S40F-4, trpE-S40F-5, and trpE-6. fbr Fragments 1 and 2; then, upstream and downstream homology arms, trpE fbr Gene fragments 1 and 2 were used as templates, and trpLE-1 and trpLE-8 were used as primers to obtain overlapping fragments through overlapping PCR amplification; pGRB-trpL-s and pGRB-trpL-a were used as primers to anneal the gRNA fragment, which was connected to the pGRB vector to obtain trpL-pGRB; TRP01 electroporation competent cells were prepared, the target fragment and trpL-pGRB were electroporated into the competent cells together, and positive transformants were screened to obtain strain TRP02.

[0059] ③ Knock out the trpR gene and integrate glnA from B. subtilis 168 at this site fbrGene: Using the B. subtilis 168 genome as a template, trc-glnA-3, glnA-L159I-4, glnA-L159I-5, glnA-E304A-6, glnA-E304A-7, trc-glnA-8 were used to amplify glnA fragments 1, 2, and 3, respectively; using the E. coli W3110 genome as a template, trpR-1, trpR-trc-2, trpR-trc-9, trpR-10 were used as primers to amplify the upstream and downstream homology arms; Next, the upstream and downstream homology arms and the target gene fragments were used as templates, and trpR-1 and trpR-10 were used as primers to obtain overlapping fragments by overlapping PCR amplification; pGRB-trpR-s and pGRB-trpR-a were used as primers to anneal the gRNA fragments, which were connected to the pGRB vector to obtain trpR-pGRB; TRP02 electroporation competent cells were prepared, the target fragments and trpR-pGRB were electroporated into the competent cells together, and the positive transformants were screened to obtain strain TRP03.

[0060] ④ Integration of prsA gene: The method is the same as in ①, except that the pseudogene site used is ilvG, and the primers used are ilvG-1, ilvG-trc-2, ilvG-trc-5, ilvG-6, trc-prsA-3, and trc-prsA-4. The competent cell is TRP03, and the strain TRP04 is obtained.

[0061] ⑤Integration of serA fbr Gene: The same method as in ①, except that the pseudogene site used is yeeL, and the primers used are yeeL-1, yeeL-M193-2, yeeL-M193-9, yeeL-10, M193-serA-3, serA-4, serA-5, serA-6, serA-7, M193-serA-8. The competent cell is TRP04, and the strain TRP05 is obtained.

[0062] ⑥ Knockout of gene pykA and integration of gene tktA at the site: The same method as in ① is used, except that the primers used are pykA-1, pykA-trc-2, trc-pykA-5, pykA-6, trc-tktA-3 and trc-tktA-4. The competent cell is TRP05, and strain TRP06 is obtained.

[0063] ⑦ Knockout the gene ldhA and integrate the gdh gene from B. subtilis 168 at this site: The same method as in ③ is used, except that the primers used are ldhA-1, ldhA-M137-2, ldhA-M137-5, ldhA-6, M137-gdh-3 and M137-gdh-4. The competent cell is TRP06, and the strain TRP07 is obtained.

[0064] ⑧ Knock out the promoter of the pykF gene (PpykF promoter) and replace it with the fliC promoter: E.coli W3110 genome was used as a template, and the fliC promoter fragment was obtained by PCR amplification using PfliC-F and PfliC-pykF-R as primers, and the pykF gene fragment was obtained by PCR amplification using PfliC-pykF-F and pykF-R as primers; overlapping fragments were obtained by overlapping PCR amplification using PfliC-F and pykF-R as primers; gRNA fragments were annealed using pGRB-pykF-s and pGRB-pykF-a as primers, and connected with the pGRB vector to obtain pykF-pGRB; TRP07 electroporation competent cells were prepared, the overlapping fragments and pykF-pGRB were electroporated into the competent cells, and positive transformants were screened to obtain strain TRP08.

[0065] ⑨Transform plasmid pACYC-FYSPD and plasmid pETDuet-TM2-RBS-AnTDC into strain TRP08 to obtain strain WW14: Transform the constructed and extracted complete plasmids pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC into TRP08 competent cells by electroporation to obtain strain WW14.

[0066] Example 2

[0067] This example is intended to illustrate the construction method of plasmid pACYC-FYSPD, and the specific steps are as follows:

[0068] Respectively E.coli W3110 genome was used as template, and primers M112-floE-F, folE-T198I-R, folE-T198I-F, and M112-folE-R were used to amplify folE gene fragments 1 and 2. fbrGene fragments; the ygcM gene fragment was obtained by amplifying M112-ygcM-F and M112-ygcM-R; the SPR, PCD and DHPR genes were commissioned to be synthesized by Jinweizhi Biotechnology Co., Ltd. and then obtained by PCR using primers M112-SPR-F, M112-SPR-R, M193-PCD-F, M193-PCD-R, trc-DHPR-F and trc-DHPR-R, respectively.

[0069] Using plasmid pACYCDuet-1 (purchased from Ubao Biotech) as a template, linearized vector primers line-pACYC-F and line-pACYC-R were used to obtain linearized vector pACYCDuet-1 by PCR. fbr The ygcM, SPR, PCD and DHPR gene fragments were constructed by plasmid recombination using the ClonExpressMultiS One Step Cloning Kit of Nanjing Novogene Biotechnology Co., Ltd. They were transformed into DH5α competent cells, positive transformants were screened, and plasmids were extracted to obtain plasmid pACYC-FYSPD. The nucleotide sequence of the plasmid pACYC-FYSPD is shown in the sequence table SEQ ID NO.27.

[0070] Example 3

[0071] This example is intended to illustrate the construction method of plasmid pETDuet-TM2-RBS-AnTDC, and the specific steps are as follows:

[0072] The TM2 gene and AnTDC gene were commissioned to Jinweizhi Biotechnology Co., Ltd. for synthesis and then PCR was performed using primers T7-TM2-F, TM2-RBS-R, RBS-AnTDC-F, and AnTDC-R to obtain the TM2 and AnTDC gene fragments. Using the TM2 and AnTDC gene fragments as templates, PCR was performed using T7-TM2-F and AnTDC-R to obtain the T7-TM2-RBS-AnTDC small gene cluster fragment.

[0073] Plasmid pETDuet-1 (purchased from Ubao Bio) was used as a template, and linearized vector primers line-pETDuet-F and line-pETDuet-R were used to obtain linear vector pETDuet-1 by PCR. The linearized vector pETDuet-1 and the T7-TM2-RBS-AnTDC small gene cluster fragment were constructed by plasmid recombination using the ClonExpressMultiS OneStep Cloning Kit of Nanjing Novogene Biotech Co., Ltd. It was transformed into DH5α competent cells, positive transformants were screened, and plasmid pETDuet-TM2-RBS-AnTDC was obtained by extracting the plasmid. The nucleotide sequence of the plasmid pETDuet-TM2-RBS-AnTDC is shown in the sequence table SEQ ID NO.28.

[0074] Example 4

[0075] This example is intended to illustrate the application of shake flask fermentation of strain WW14, and the specific steps are:

[0076] ① Test tube culture: Take the strain WW14 stored in a -80℃ refrigerator and inoculate it into a test tube containing 5 mL of LB liquid culture medium, 220 rpm, 37℃, for 12 h.

[0077] ② Shake flask seed culture: Take the test tube bacteria and inoculate them into the shake flask seed culture medium for fermentation, 220 rpm, 34℃, 12h, pH6.4-6.7. The shake flask seed culture medium is 25g / L glucose, 5g / L yeast powder, 2.5g / L peptone, 1.5g / L MgSO4.7H2O, 3g / L KH2PO4, 1.5g / L ammonium sulfate, and the rest is water.

[0078] ③ Shake flask fermentation culture: the fermentation inoculation amount is 20-25%, 34℃, 220 rpm, culture time is 24-36h, pH6.4-6.7; the fermentation medium is: glucose 15 g / L, MgSO4.7H2O 3g / L, yeast powder 7g / L, peptone 5g / L, ammonium sulfate 3g / L, KH2PO45g / L, glutamate 2 g / L, FeSO4.7H2O 60mg / L, MnSO47mg / L, VB1, VB3, VB5 each 3mg / L, biotin 3mg / L, and the rest is water.

[0079] Experiment with E.coli W3110 ΔlacIZ::P XylF -T7RNAP was used as the control group and was verified by 28h fermentation. E.coli W3110 ΔlacIZ::P XylF-T7RNAP failed to accumulate 5-HT, while strain WW14 accumulated 5.15 g / L 5-HT, demonstrating the effectiveness of the strain.

[0080] Example 5

[0081] This example is intended to illustrate the comparison between the ability of strain WW14 and strain TRP08 to produce 5-hydroxytryptamine and tryptophan under the same culture conditions in a fermenter. The only difference between strain WW14 and strain TRP08 is that strain WW14 contains the dual plasmid system pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC. The specific steps are:

[0082] (1) Slant culture: Take the -80℃ preserved strain and inoculate it onto the activated slant containing chloramphenicol and ampicillin resistance, culture it at 34℃ for 12h, and subculture it twice;

[0083] (2) Seed culture: The activated bacteria on the slope were washed with sterilized distilled water and transferred to a 5 L mechanically stirred fermenter to start seed culture. The culture temperature was 37 °C, the initial stirring speed was 200 rpm, and the culture pH was maintained at 6.7 ± 0.2 by automatic flow addition of 25% ammonia solution. The dissolved oxygen value of the culture medium was maintained at 30% by adjusting the stirring speed or ventilation volume. The OD 600nm Reaching 25 is a sign of maturity of the seed culture medium;

[0084] (3) Fermentation culture: After the seed liquid matures, retain 500 mL of seed culture and immediately add fresh fermentation medium to make the final fermentation volume 2 L and start fermentation culture. The culture temperature is 34°C. The culture pH is maintained at 6.4±0.2 by automatically adding 25% ammonia solution. The dissolved oxygen value of the fermentation medium is maintained at 20% by adjusting the stirring speed or ventilation volume. The glucose concentration in the tank is controlled at ≤1 g / L by adding 80% glucose solution. The fermentation cycle is 28 h (strain WW14 needs to be adjusted when OD 600 When the concentration reached 10, IPTG was added to induce the mixture to a final concentration of 0.1 mM.

[0085] The slant culture medium used consisted of: 1 g / L glucose, 15 mg / L chloramphenicol, 50 mg / L ampicillin, 5 g / L peptone, 0.5 g / L potassium dihydrogen phosphate, 5 g / L yeast extract, 5 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 25 g / L agar powder, pH 7.0, sterilized at 121°C for 20 min and then dispensed into test tubes.

[0086] The components of the seed culture medium used are: glucose 30 g / L, chloramphenicol 15 mg / L, ampicillin 50 mg / L, yeast extract 4 g / L, peptone 2 g / L, citric acid 2 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 2 g / L, glutamic acid 2 g / L, methionine 0.5 g / L, and the rest is water.

[0087] The fermentation medium components used are: glucose 20g / L, xylose 10 g / L, chloramphenicol 15mg / L, ampicillin 50 mg / L, yeast extract 6g / L, peptone 4g / L, citric acid 2g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 4g / L, magnesium sulfate heptahydrate 3g / L, ferrous sulfate heptahydrate 60mg / L, manganese sulfate monohydrate 5mg / L, MSG 2g / L, choline chloride 1g / L, betaine 1g / L, and the rest is water.

[0088] The total fermentation time of strain TRP08 and strain WW14 was 28 h, and the final OD 600 The results of the production of 5-HT and 5-HT are shown in Table 2 below.

[0089] Table 2 Fermentation comparison of strains TRP08 and WW14

[0090] TRP08 WW14 <![CDATA[OD 600 ]]> 141.7 98.3 Tryptophan production (g / L) 22.04 0.01 5-Hydroxytryptamine production (g / L) 0 15.50

[0091] The results showed that strain WW14 has the ability to produce 5-hydroxytryptamine and hardly accumulates the intermediate product tryptophan. This shows that the metabolic pathways in strain WW14 are balanced, intracellular resources are maximized, and 15.50 g / L of 5-hydroxytryptamine is produced, which is the highest 5-hydroxytryptamine yield reported so far.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. A 5-HT producing Escherichia coli strain, characterized in that: by E. coli W3110 ΔlacIZ::P XylF -T7RNAP was used as the chassis strain to knock out the tnaA, pykA, trpR, ldhA and trpL genes on the strain genome, and up-regulated trpE fbr 、aroG fbr ,serA fbr , tktA, and prsA genes, and heterologously expressed gdh and glnA from B. subtilis 168 fbr gene, dynamically down-regulated the pykF gene, and the production bacteria carried the low-copy plasmid pACYC-FYSPD and the high-copy plasmid pETDuet-TM2-RBS-AnTDC; wherein the nucleotide sequence of the tnaA gene is shown in the sequence list SEQ ID NO.1; the nucleotide sequence of the trpR gene is shown in the sequence list SEQ ID NO.4; the nucleotide sequence of the pykA gene is shown in the sequence list SEQ ID NO.7; the nucleotide sequence of the ldhA gene is shown in the sequence list SEQ ID NO.11; the nucleotide sequence of the trpL gene is shown in the sequence list SEQ ID NO.31; the trpE fbr The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.3; aroG fbr The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.5; serA fbr The nucleotide sequence of the gene is shown in SEQ ID NO.10 in the sequence listing; the nucleotide sequence of the tktA gene is shown in SEQ ID NO.9 in the sequence listing; the nucleotide sequence of the prsA gene is shown in SEQ ID NO.8 in the sequence listing; the nucleotide sequence of the gdh gene is shown in SEQ ID NO.12 in the sequence listing; fbr The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.6; the nucleotide sequence of the pykF gene is shown in the sequence listing SEQ ID NO.30; the nucleotide sequence of the plasmid pACYC-FYSPD is shown in the sequence listing SEQ ID NO.27; the nucleotide sequence of the plasmid pETDuet-TM2-RBS-AnTDC is shown in the sequence listing SEQ ID NO.

28.

2. The 5-HT producing Escherichia coli strain according to claim 1, characterized in that: The trc promoter enhances aroG fbr The gene is transcribed and integrated into the tnaA gene locus; The trc promoter enhances trpE fbr The gene was transcribed and integrated into the trpLE gene locus; the glnA gene from B. subtilis 168 was enhanced using the trc promoter fbr The gene was transcribed and integrated into the trpR gene site of the genome; the prsA gene transcription was enhanced using the trc promoter and integrated into the ilvG gene site; The trc promoter was used to enhance tktA gene transcription and integrated into the pykA gene locus; the PpykF promoter was replaced with the fliC promoter to dynamically downregulate pykF gene expression; the M1-93 promoter was used to enhance serA fbr The gene was transcribed and integrated into the yeeL gene locus; the gdh gene transcription of B. subtilis 168 was enhanced using the M1-37 promoter and integrated into the ldhA gene locus.

3. The 5-HT producing Escherichia coli strain according to claim 2, characterized in that: The nucleotide sequence of the trc promoter is shown in the sequence listing SEQ ID NO.20; the nucleotide sequence of the PpykF promoter is shown in the sequence listing SEQ ID NO.22; the nucleotide sequence of the fliC promoter is shown in the sequence listing SEQ ID NO.23; the nucleotide sequence of the M1-93 promoter is shown in the sequence listing SEQ ID NO.24; and the nucleotide sequence of the M1-37 promoter is shown in the sequence listing SEQ ID NO.

25.

4. The method for constructing the 5-HT producing Escherichia coli strain according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: (1) E. coli W3110 ΔlacIZ::P XylF -T7RNAP was used as the chassis strain to knock out the tnaA gene, trpR gene and trpL gene; trpE was strengthened fbr Gene; (2) Knock out the pykA gene; strengthen the tktA gene and aroG fbr gene, serA fbr Gene and prsA gene; introduction of glnA fbr Gene; Replace the PpykF promoter with the fliC promoter to dynamically regulate the pykF gene; (3) Knock out the ldhA gene and introduce the gdh gene; (4) Introducing the pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC dual plasmid system.

5. Use of the 5-hydroxytryptamine-producing Escherichia coli strain according to any one of claims 1 to 3 in the fermentation production of 5-hydroxytryptamine.

6. The use of the 5-HT producing Escherichia coli strain according to claim 5, characterized in that: The shake flask fermentation culture was adopted, and the specific method was as follows: ① Test tube culture: The strain was inoculated in a test tube containing LB liquid medium, 220 rpm, 37°C, 12 h; ② Shake flask seed culture: Take the test tube bacteria and inoculate them into the shake flask seed culture medium for fermentation, 220 rpm, 34℃, 12h, pH6.4-6.7, ③ Shake flask fermentation culture: fermentation inoculation amount is 20-25%, 34℃, 220 rpm, culture for 24-36h, pH 6.4-6.

7.

7. The use of the 5-HT producing Escherichia coli strain according to claim 6, characterized in that: The shake flask seed culture medium in step ② is: 20-30 g / L glucose, 4-6 g / L yeast powder, 2-3 g / L peptone, 1-2 g / L MgSO4.7H2O, 2-4 g / L KH2PO4, 1-2 g / L ammonium sulfate, and the rest is water; the fermentation culture medium in step ③ is: 15 g / L glucose, 10 g / L xylose, 2-4 g / L MgSO4.7H2O, 6-8 g / L yeast powder, 4-6 g / L peptone, 2-4 g / L ammonium sulfate, 4-6 g / L KH2PO4, 2 g / L glutamic acid, 40-80 mg / L FeSO4.7H2O, 5-8 mg / L MnSO4, 2-4 mg / L each of VB1, VB3, and VB5, 2-4 mg / L biotin, and the rest is water.

8. The use of the 5-HT producing Escherichia coli strain according to claim 5, characterized in that: The fermentation tank is used for cultivation, and the specific method is as follows: (1) Slant culture: The strain was streaked onto a slant containing chloramphenicol and ampicillin resistance activation medium, cultured at 34°C for 12 h, and subcultured twice; (2) Seed culture: The activated bacteria on the slant were washed with sterilized distilled water and transferred to the fermenter to start seed culture. The culture temperature was 37°C, the initial stirring speed was 200 rpm, the culture pH was maintained at 6.7±0.2, the dissolved oxygen value of the culture medium was maintained at 30%, and the OD value was adjusted to 0. 600nm Reaching 25 is a sign of maturity of the seed culture medium; (3) Fermentation culture: After the seed liquid matures, fermentation medium is added to the seed culture for fermentation culture. The culture temperature is 34°C, the culture pH is maintained at 6.4±0.2, the dissolved oxygen value of the culture medium is maintained at 20%, and the glucose concentration in the tank is controlled at ≤1g / L.

9. The use of the 5-HT producing Escherichia coli strain according to claim 8, characterized in that: The slant culture medium used in step (1) is: 1 g / L glucose, 15 mg / L chloramphenicol, 50 mg / L ampicillin, 5 g / L peptone, 0.5 g / L potassium dihydrogen phosphate, 5 g / L yeast extract, 5 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 25 g / L agar powder, pH 7.0; the seed culture medium used in step (2) is: 30 g / L glucose, 15 mg / L chloramphenicol, 50 mg / L ampicillin, 4 g / L yeast extract, 2 g / L peptone, 2 g / L citric acid, 2 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 2 g / L glutamic acid, 0.5 g / L methionine, and the rest is water; the fermentation culture medium used in step (3) is: 20 g / L glucose, 15 mg / L chloramphenicol, 50 mg / L ampicillin mg / L, yeast extract 6g / L, peptone 4g / L, citric acid 2g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 4g / L, magnesium sulfate heptahydrate 3g / L, ferrous sulfate heptahydrate 60mg / L, manganese sulfate monohydrate 5mg / L, MSG 2g / L, choline chloride 1g / L, betaine 1g / L, and the rest is water.

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