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CN119120342B - 5-Hydroxytryptamine escherichia coli production strain and construction method and application thereof - Google Patents

5-Hydroxytryptamine escherichia coli production strain and construction method and application thereof Download PDF

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CN119120342B
CN119120342B CN202411629512.4A CN202411629512A CN119120342B CN 119120342 B CN119120342 B CN 119120342B CN 202411629512 A CN202411629512 A CN 202411629512A CN 119120342 B CN119120342 B CN 119120342B
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CN119120342A (en
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徐庆阳
刘韪玮
白玉
刘样豪
余子辰
刘莹
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Synthetic Biology Haihe Laboratory
Tianjin University of Science and Technology
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Synthetic Biology Haihe Laboratory
Tianjin University of Science and Technology
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Abstract

The invention provides a 5-hydroxytryptamine escherichia coli production strain, a construction method and application thereof, wherein the strain lacks tnaA, pykA, trpR, ldhA and trpL genes, trpE fbr、aroGfbr、serAfbr, tktA and prsA genes are upregulated, gdh and glnA fbr genes are heterologously expressed, pykF genes are dynamically regulated and controlled, and meanwhile, the production strain carries plasmid pACYC-FYSPD and plasmid pETDuet-TM2-RBS-AnTDC, the constructed strain has the characteristic of metabolic balance, and the expression intensity of key genes of all precursors is optimized, and simultaneously the expression quantity of a tryptophan hydroxylation-decarboxylation system and a coenzyme tetrahydrobiopterin synthesis regeneration system is regulated through a double-plasmid system, so that 5-hydroxytryptamine can be efficiently synthesized in a culture medium without byproducts.

Description

5-Hydroxytryptamine escherichia coli production strain and construction method and application thereof
Technical Field
The invention relates to the field of biotechnology production, in particular to a 5-hydroxytryptamine escherichia coli production strain, a construction method and application thereof.
Background
5-Hydroxytryptamine (5-HT) is a secondary metabolite of L-tryptophan by two steps of hydroxylation and decarboxylation, and was first extracted from serum in 1948, so is also known as serotonin. It is used as monoamine neurotransmitter, and has functions of regulating emotion, treating insomnia, scavenging free radicals, etc. In addition, 5-HT has important functions in multiple fields such as agriculture, medicine, scientific research and the like due to the functions of improving plant stress resistance, treating depression, promoting regeneration and repair of human tissues and the like.
At present, the production of 5-HT mainly depends on plant tissue extraction and chemical synthesis methods, but the two methods are limited by cost, raw materials and the like, so that large-scale production cannot be performed, and the price of 5-HT on the market is high. In addition, the synthesis route of 5-hydroxytryptamine is lengthy, various precursors involved are numerous, and how to balance the supply of each precursor is well, so that a microbial cell factory which has balanced metabolic flow and can fully utilize intracellular resources is constructed, and the technical problem which needs to be solved at present is urgent.
Disclosure of Invention
The invention aims to solve the technical problem of providing a 5-hydroxytryptamine escherichia coli production strain.
Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned strain of producing 5-hydroxytryptamine E.coli.
Another technical problem to be solved by the present invention is to provide an application of the above-mentioned 5-hydroxytryptamine E.coli producing strain.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a5-hydroxytryptamine E.coli producing strain designated as strain WW14, deleted tnaA, pykA, trpR, ldhA and trpL genes, upregulated trpE fbr、aroGfbr、serAfbr, tktA, prsA genes, heterologously expressed gdh and glnA fbr genes of B.subtilis 168, dynamically regulated pykF genes, and carried low copy plasmid pACYC-FYSPD and high copy plasmid pETDuet-TM2-RBS-AnTDC.
Preferably, the nucleotide sequence of the tnaA gene is shown in a sequence table SEQ ID NO.1, the nucleotide sequence of the trpR gene is shown in a sequence table SEQ ID NO.4, the nucleotide sequence of the pykA gene is shown in a sequence table SEQ ID NO.7, the nucleotide sequence of the ldhA gene is shown in a sequence table SEQ ID NO.11, and the nucleotide sequence of the trpL gene is shown in a sequence table SEQ ID NO. 31.
Preferably, the nucleotide sequence of the trpE fbr gene is shown in a sequence table SEQ ID NO.3, the nucleotide sequence of the aroG fbr gene is shown in a sequence table SEQ ID NO.5, the nucleotide sequence of the serA fbr gene is shown in a sequence table SEQ ID NO.10, the nucleotide sequence of the tktA gene is shown in a sequence table SEQ ID NO.9, the nucleotide sequence of the prsA gene is shown in a sequence table SEQ ID NO.8, the nucleotide sequence of the gdh gene is shown in a sequence table SEQ ID NO.12, the nucleotide sequence of the glnA fbr gene is shown in a sequence table SEQ ID NO.6, and the nucleotide sequence of the pykF gene is shown in a sequence table SEQ ID NO. 30.
Preferably, the nucleotide sequence of the plasmid pACYC-FYSPD is shown in a sequence table SEQ ID NO.27 of the 5-hydroxytryptamine escherichia coli production strain.
Preferably, the nucleotide sequence of the plasmid pETDuet-TM2-RBS-AnTDC is shown as a sequence table SEQ ID NO. 28.
The low copy plasmid pACYC-FYSPD expresses folE T198I and ygcM genes derived from escherichia coli and SPR, DHPR and PCD genes derived from human, the plasmid pACYC-FYSPD has a structure shown in figure 1, the plasmid pACYC-FYSPD carries plasmid elements such as p15A replication initiation site, chloramphenicol resistance, T7 promoter, terminator and the like, the plasmid pACYC-FYSPD carries folE fbr and ycgM genes derived from escherichia coli and SPR, DHPR and PCD genes derived from homo sapiens, the folE fbr, ycgM and SPR genes adopt M1-12 promoters for enhanced transcription, the PCD genes adopt M1-93 promoters for enhanced transcription, and the DHPR genes adopt trc promoters for enhanced transcription. The high copy plasmid pETDuet-TM2-RBS-AnTDC expresses a human TM2 gene and an Aspergillus niger AnTDC gene, the plasmid pETDuet-TM2-RBS-AnTDC has a structure shown in figure 2, the plasmid pETDuet-TM2-RBS-AnTDC carries ColE1 replication initiation site, ampicillin resistance, T7 promoter, terminator and other plasmid elements, and simultaneously carries a intellectual TM2 gene and an Aspergillus niger AnTDC gene, the TM2 gene and the AnTDC gene are connected through an RBS, the nucleotide sequence of the RBS is shown as a sequence table SEQ ID NO.29, and transcription is driven by the same T7 promoter, so that a miniature artificial gene cluster is constructed.
The nucleotide sequence of folE fbr gene is shown in sequence table SEQ ID NO.13, the nucleotide sequence of ycgM gene is shown in sequence table SEQ ID NO.14, the nucleotide sequence of SPR gene is shown in sequence table SEQ ID NO.15, the nucleotide sequence of PCD gene is shown in sequence table SEQ ID NO.16, the nucleotide sequence of DHPR gene is shown in sequence table SEQ ID NO.17, the nucleotide sequence of TM2 gene is shown in sequence table SEQ ID NO.18, the nucleotide sequence of AnTDC gene is shown in sequence table SEQ ID NO.19, the nucleotide sequence of M1-12 promoter is shown in sequence table SEQ ID NO.26, and the nucleotide sequence of T7 promoter is shown in sequence table SEQ ID NO. 21.
Preferably, the E.5-hydroxytryptamine E.coli producer strain described above uses E.coli W3110.DELTA. lacIZ wherein P XylF -T7RNAP (the strain E.coli HT01 obtained in example 2 of CN 116590210A) as the chassis strain, knocks out the tnaA, pykA, trpR, ldhA and trpL genes on the genome of the strain, enhances transcription of aroG fbr gene with the trc promoter and integrates into the tnaA gene locus, enhances transcription of trpE fbr gene with the trc promoter and integrates into trpLE gene locus, enhances transcription of glnA fbr gene derived from B.subtitle lis 168 with the trc promoter and integrates into the genome trpR gene locus, enhances transcription of prsA gene with the trc promoter and integrates into the ilvG gene locus, enhances transcription of tktA gene with the trc promoter and integrates into the pykA gene locus, replaces PpykF promoter (pykF gene promoter) with the flc promoter for dynamic downregulation of transcription of gene transcription of the flrF fbr gene, enhances transcription of glnA gene fbr gene from B.subtitle 168 with the trc promoter and integrates into the ild37. DBdBrF gene locus, and enhances transcription of hA gene transcription of hAdBrBrBrA37.
Preferably, the nucleotide sequence of the trc promoter is shown in a sequence table SEQ ID NO.20, the nucleotide sequence of the trpLE gene is shown in a sequence table SEQ ID NO.2, the nucleotide sequence of the PpykF promoter is shown in a sequence table SEQ ID NO.22, the nucleotide sequence of the fliC promoter is shown in a sequence table SEQ ID NO.23, the nucleotide sequence of the M1-93 promoter is shown in a sequence table SEQ ID NO.24, and the nucleotide sequence of the M1-37 promoter is shown in a sequence table SEQ ID NO. 25.
The construction method of the 5-hydroxytryptamine escherichia coli production strain comprises the following specific steps:
(1) Taking E.coli W3110 delta lacIZ as chassis strain P XylF -T7RNAP, knocking out tnaA gene, trpR gene and trpL gene, strengthening trpE fbr gene;
(2) Knocking out the pykA gene, strengthening the tktA gene, aroG fbr gene, serA fbr gene and prsA gene, introducing glnA fbr gene, replacing PpykF promoter (pykF gene promoter) with fliC promoter and dynamically regulating and controlling pykF gene;
(3) Knocking out the ldhA gene, introducing the gdh gene;
(4) pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC were introduced.
Preferably, the construction method of the 5-hydroxytryptamine escherichia coli production strain comprises the following specific steps:
(1) The tryptophan pathway is constructed by taking E.coli W3110 delta lacIZ as chassis strain P XylF -T7RNAP, knocking out gene tnaA encoding tryptophan degradation pathway, knocking out tryptophan repressor regulatory protein trpR, knocking out gene trpL encoding tryptophan leader peptide, and enhancing gene trpE fbr encoding anthranilate synthase mutant;
(2) The precursor supply balance comprises knocking out a gene pykA encoding pyruvate kinase II, reducing the conversion of phosphoenolpyruvate (PEP) into pyruvic acid, strengthening a gene tktA encoding transketolase I, improving the supply of 4-phosphoerythrose, strengthening a gene mutant aroG fbr encoding 3-deoxy-D-arabinoheptanoic acid-7-phosphate (DAHP) synthase, improving the supply of DAHP, strengthening a gene serA fbr encoding D-3-phosphoglycerate dehydrogenase, improving the supply of serine, strengthening a gene prsA encoding phosphoribosyl pyrophosphate synthase, improving the supply of phosphoribopyrophosphate as a key precursor of tryptophan, introducing a gene glnA fbr encoding glutamine synthase derived from B.subtilis 168, improving the supply of glutamine, replacing a PpykF promoter (pykF gene promoter) encoding pyruvate kinase I with a fliC promoter, and realizing the dynamic expression of gene pykF so as to further improve the accumulation;
(3) Optimizing NAD (P) H supply content by knocking out the lactate dehydrogenase-encoding gene ldhA to reduce NADH waste, introducing glucose dehydrogenase-encoding gene gdh derived from B.subtilis 168 for enhancing NAD (P) H production;
(4) pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC two-plasmid system, wherein pACYC-FYSPD plasmid carries plasmid elements such as p15A replication initiation site, chloramphenicol resistance, T7 promoter, terminator and the like, folE fbr, ygcM, SPR, PCD and DHPR genes are effectively expressed by the plasmid, folE fbr, ycgM and SPR genes are subjected to enhanced transcription by using M1-12 promoters, PCD genes are subjected to enhanced transcription by using M1-93 promoters, and DHPR genes are subjected to enhanced transcription by using trc promoters. The pETDuet-TM2-RBS-AnTDC plasmid carries ColE1 replication initiation site, ampicillin resistance, T7 promoter, terminator and other plasmid elements, and simultaneously carries TM2 gene from homo sapiens and AnTDC gene from Aspergillus niger, and the TM2 gene and AnTDC gene are connected through RBS and transcribed through the same T7 promoter to construct a miniature artificial gene cluster.
The application of the 5-hydroxytryptamine escherichia coli production strain in the aspect of fermenting and producing the 5-hydroxytryptamine.
Under suitable fermentation conditions, the 5-hydroxytryptamine E.coli producing strain is fermented in a medium, which may include, but is not limited to, carbon sources, nitrogen sources, inorganic salts, vitamins, etc., by means of a two-stage pH control. The fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen condition, fermentation pressure, fermentation time and the like. The culture medium can be obtained by a conventional method and used for producing 5-hydroxytryptamine, and the fermentation conditions can be adjusted to adapt to the production characteristics of the strain.
Preferably, the application of the 5-hydroxytryptamine escherichia coli production strain adopts shake flask fermentation culture, and the specific method comprises the following steps:
① Test tube culture, wherein the strain is inoculated in a test tube containing LB liquid medium, 220 rpm,37 ℃,12 h;
② Shake flask seed culture, inoculating test tube strain into shake flask seed culture medium for fermentation at 220 rpm and 34 deg.C for 12 hr at pH of 6.4-6.7,
③ Shake flask fermentation culture, wherein the fermentation inoculation amount is 20-25%, the temperature is 34 ℃, the temperature is 220 rpm, the culture time is 24-36h, and the pH is 6.4-6.7.
Preferably, the shake flask seed culture medium in the step ② is composed of 20-30g/L glucose, 4-6g/L yeast powder, 2-3g/L peptone, 4.7H2O 1-2g/L,KH2PO4 -4g/L MgSO, 1-2g/L ammonium sulfate and the balance water.
Preferably, the fermentation medium in the step ③ is 15-g/L glucose, 10-g/L xylose, 4.7H2 O2-4 g/L MgSO, 6-8g/L yeast powder, 4-6g/L peptone, 2-4g/L ammonium sulfate, 4-6g/L KH 2PO4, 2-g/L glutamic acid, 2-4mg/L FeSO 4.7H2O 40-80mg/L,MnSO45-8mg/L,VB1、VB3、VB5, 2-4mg/L biotin and the balance water.
Preferably, the application of the 5-hydroxytryptamine escherichia coli production strain adopts a fermentation tank for culture, and the specific method comprises the following steps:
(1) Slant culture, namely streaking and inoculating the strain on an activated slant containing chloramphenicol and ampicillin resistance, culturing for 12h at 34 ℃ and passaging for 2 times;
(2) Seed culture, namely eluting the activated thalli on the inclined plane by using sterilized distilled water, transferring the thalli into a fermentation tank to start seed culture, wherein the culture temperature is 37 ℃, the initial stirring rotation speed is 200rpm, the culture pH is maintained at 6.7+/-0.2, the dissolved oxygen value of a culture medium is maintained at 30%, and the OD 600nm is 25 as a seed culture solution maturation mark;
(3) And (3) fermenting and culturing, namely adding a fermentation culture medium into the seed culture for fermenting and culturing after the seed liquid is mature, wherein the culture temperature is 34 ℃, the culture pH is maintained at 6.4+/-0.2, the fermentation dissolved oxygen value of the culture medium is maintained at 20%, and the glucose concentration in the tank is controlled to be less than or equal to 1g/L.
Preferably, when the OD 600 reaches 10, the 5-hydroxytryptamine E.coli producing strain is induced by adding IPTG with a final concentration of 0.1 mM.
Preferably, the slant culture medium adopted in the step (1) comprises 1g/L glucose, 15mg/L chloramphenicol, 50 mg/L ampicillin, 5g/L peptone, 0.5g/L potassium dihydrogen phosphate, 5g/L yeast extract, 5g/L sodium chloride, 0.2g/L magnesium sulfate heptahydrate, 25g/L agar powder, and pH7.0.
Preferably, the seed culture medium adopted in the step (2) comprises 30g/L of glucose, 15mg/L of chloramphenicol, 50 mg/L of ampicillin, 4g/L of yeast extract, 2g/L of peptone, 2g/L of citric acid, 2g/L of ammonium sulfate, 3g/L of potassium dihydrogen phosphate, 2g/L of magnesium sulfate heptahydrate, 2g/L of glutamic acid, 0.5g/L of methionine and the balance of water.
Preferably, the fermentation medium adopted in the step (3) comprises 20g/L of glucose, 15mg/L of chloramphenicol, 50 mg/L of ampicillin, 6g/L of yeast extract, 4g/L of peptone, 2g/L of citric acid, 2g/L of ammonium sulfate, 4g/L of potassium dihydrogen phosphate, 3g/L of magnesium sulfate heptahydrate, 60mg/L of ferrous sulfate heptahydrate, 5mg/L of manganese sulfate monohydrate, 2g/L of monosodium glutamate, 1g/L of choline chloride, 1g/L of betaine and the balance of water.
The above culture medium can be prepared by standard method.
The beneficial effects are that:
The 5-hydroxytryptamine escherichia coli production strain has the characteristic of metabolic balance, is a tryptophan hydroxylation-decarboxylation system and a BH4 synthesis regeneration system which are stable and balanced, and can be used for efficiently synthesizing 5-hydroxytryptamine in a culture medium without byproducts by optimizing the expression intensity of key genes of all precursors and adjusting the expression quantity of the tryptophan hydroxylation-decarboxylation system and the coenzyme tetrahydrobiopterin (BH 4) synthesis regeneration system through a double-plasmid system, so that the 5-hydroxytryptamine production strain has stable performance and excellent industrial application prospect. Specifically:
(1) The balance of the supply of each precursor for tryptophan synthesis was maintained by adjusting the expression intensities of glnA fbr、prsA、serAfbr, tktA, pykA, pykF. (2) Knocking out the ldhA gene reduces NADH consumption while introducing a glucose dehydrogenase gdh encoding from B.subtilis 168 increases the supply of NAD (P) H. (3) The pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC double plasmid systems are constructed, on one hand, a tryptophan hydroxylation-decarboxylation system and a coenzyme tetrahydrobiopterin (BH 4) synthesis regeneration system are constructed to carry out hydroxylation decarboxylation on key precursors of tryptophan, and on the other hand, different plasmid vectors and the expression levels of all genes are selected and adjusted finely to maintain balance between the tryptophan hydroxylation-decarboxylation system and the BH4 synthesis regeneration system, so that intracellular resources are utilized to be used for synthesizing 5-hydroxytryptamine efficiently without byproducts to the maximum extent.
Drawings
FIG. 1 is a schematic diagram of the structure of plasmid pACYC-FYSPD.
FIG. 2 is a schematic representation of the structure of plasmid pETDuet-TM 2-RBS-AnTDC.
Detailed Description
In order to enable those skilled in the art to better understand the technical scheme of the present invention, the technical scheme of the present invention will be further described in detail below with reference to the specific embodiments.
The percentage "%" referred to in the examples is the mass percentage, the percentage of the solution is the gram of the solute contained in 100mL, and the percentage between the liquids is the volume ratio of the solution at 25 ℃.
The corresponding promoters and genes in the examples are shown in the sequence listing. The primers used in the construction of the related strains are shown in Table 1.
TABLE 1 primers involved in the construction of strains
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 CAACGTTGATATAATTGAGCCACTGGCTCGTAATTTATTGTTTAAACCAGGAAACAGCTAGAAAATTGGTTCGTTGGG
ldhA-M193-2 SEQ ID NO.55 GAGCCAGTGGCTCAATTATATCAACGTTGTTATCTCTTGTCAACACCGCCAGAGATAAATTGGCAGTTTTAGCGGTTTT
folE-T198I-F SEQ ID NO.56 CAACCAGTGCCACGACAACGATTTCTCTTGGTGGATTGTTC
folE-T198I-R SEQ ID NO.57 GAACAATCCACCAAGAGAAATCGTTGTCGTGGCACTGGTTG
line-pACYC-F SEQ ID NO.58 agactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaattgcggcacacggtcacactgc
line-pACYC-R SEQ ID NO.59 ccaaaagggctcaattatatcaacgttgttatctcttgtcaacaccgccagagataacagctcatttcagaatatttg
M112-floE-F SEQ ID NO.60 gatataattgagcccttttggtgcgtcagtcagtttaaaccaggaaacagctatgccatcactcagtaaagaagcg
M112-folE-R SEQ ID NO.61 gcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgtcagttgtgatgacgcacag
M112-SPR-F SEQ ID NO.62 gatataattgagcccttttggtgcgtcagtcagtttaaaccaggaaacagctatgcatcatcaccatcaccacgaagg
M112-SPR-R SEQ ID NO.63 caccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgttagatgtcgtagaagtcaacgtgag
M112-ygcM-F SEQ ID NO.64 gatataattgagcccttttggtgcgtcagtcagtttaaaccaggaaacagctatgatgtccaccacgttatttaaag
M112-ygcM-R SEQ ID NO.65 gttcaccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgtcattcgccgcgatagatacaac
M193-PCD-F SEQ ID NO.66 gatataattgagcccgtattgttagcatgtacgtttaaaccaggaaacagctatggctggtaaagctcaccgtctg
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 GCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACgtattacgaaagtggtggtgCTAGTATTATACCTAGGACTGAGCTAGCTG
pGRB-pykF-s SEQ ID NO.81 CAGCTAGCTCAGTCCTAGGTATAATACTAGcaccaccactttcgtaatacGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
pGRB-tnaA-a SEQ ID NO.82 CTATTTCTAGCTCTAAAACCTCTGTAGTATTAATTAAACCTAGTATTATACCTAGGACTG
pGRB-tnaA-s SEQ ID NO.83 CAGTCCTAGGTATAATACTAGGTTTAATTAATACTACAGAGGTTTTAGAGCTAGAAATAG
pGRB-trpL-a SEQ ID NO.84 CTATTTCTAGCTCTAAAACttcagtacgaaaattgctttCTAGTATTATACCTAGGACTG
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 atgactgatcaagctgaaaaaaagcactctggatggagtcaatgattactaacccg
trc-tktA-3 SEQ ID NO.92 GGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCatgtcctcacgtaaagagcttgc
trc-tktA-4 SEQ ID NO.93 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGttacagcagttcttttgctttcgcaac
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 CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCCCACGGCAATATTCCCAGCCTG
M193-serA-3 SEQ ID NO.98 CGTTGATATAATTGAGCCCGTATTGTTAGCATGTACGTTTAAACCAGGAAACAGCTATGGCAAAGGTATCGCTGGAGAAAG
M193-serA-8 SEQ ID NO.99 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGTACAGCAGACGGGCGCGAATGG
serA-4 SEQ ID NO.100 GGACGGTTTTCCGCGATGTGCATC
serA-5 SEQ ID NO.101 GATGCACATCGCGGAAAACCGTCC
serA-6 SEQ ID NO.102 GCGCGGCGATCGCGACGCCCTGCTCGG
serA-7 SEQ ID NO.103 CCGAGCAGGGCGTCGCGATCGCCGCGC
yeeL-1 SEQ ID NO.104 TTCATCGGGACGAGTGGAGA
yeeL-10 SEQ ID NO.105 CATTCCCTCTACAGAACTAGCCCT
yeeL-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
The terminology of the gene editing methods employed in the examples in reference to literature （Li Y,Lin Z,Huang C,et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering,2015,31：13-21.）,, to which the present invention relates, is explained in this article unless otherwise noted. The "knockout" refers to the inactivation of a target gene, and the "introduction" refers to the insertion of an exogenous gene into the genome of an engineering bacterium after the linkage of the exogenous gene with a promoter and a terminator.
Example 1
This example is intended to illustrate the specific construction procedure of strain WW 14. In particular, in the embodiment, if there is a method for operating the same type of gene, only 1 time is provided and annotation is made, and no redundant description is given.
① Knocking out tnaA and integrating aroG fbr genes at the locus, wherein an E.coli W3110 genome is used as a template, tnaA-1, tnaA-trc-2 and tnaA-trc-7 are respectively used for carrying out PCR amplification to obtain upstream and downstream homology arms, an E.coli W3110 genome is used as a template, and trc-aroG-3, aroG-4, aroG-5 and trc-aroG-6 are respectively used for carrying out PCR amplification to obtain aroG fbr fragments 1 and 2; then, using upstream and downstream homology arms and aroG fbr gene fragments 1 and 2 as templates, using tnaA-1 and tnaA-8 as primers, obtaining overlapped fragments through overlapped PCR amplification, using pGRB-tnaA-s and pGRB-tnaA-a as primers, annealing to obtain gRNA fragments, connecting the gRNA fragments with pGRB vector to obtain tnaA-pGRB, preparing E.coli W3110 delta lacIZ, electrically transforming competent cells of P XylF -T7RNAP (which is the strain E.coli HT01 obtained in CN116590210A example 2), electrically transforming target fragments into competent cells together with tnaA-pGRB, and screening to obtain positive transformants to obtain strain TRP01.
② Knocking out trpL gene and integrating trpE fbr gene at trpLE gene locus, using E.coli W3110 genome as template, using trpLE-1, trc-trpLE-2 and trpE-7, trpE-8 as template, respectively using E.coli W3110 genome as template, obtaining upper and lower homologous arms by PCR amplification, using trc-trpE-3, trpE-S40F-4 and trpE-S40F-5, trpE-6 as template, respectively using trc-trpE fbr fragment 1, 2 by PCR amplification, using upper and lower homologous arms, trpE fbr gene fragment 1, 2 as template, using trpLE-1, trpLE-8 as primer, obtaining overlapped fragment by overlapping PCR amplification, using pGRB-trpL-S, pGRB-trpL-a as primer, annealing to obtain gRNA fragment, and connecting it with pGRB carrier, obtaining trpL-pGRB, preparing electric transformed cell and obtaining electric transformed cell and electric transformed cell of pGRB, and obtaining electric transformed cell of interest by screening the electric transformed cell and positive cell transformed cell strain of interest.
③ Knocking out the trpR gene and integrating glnA fbr gene derived from B.subtilis 168 at the site, wherein the glnA fragments 1, 2 and 3 are obtained by PCR amplification by taking the B.subtilis 168 genome as a template and trpR-1, trpR-trc-2 and trpR-trc-9 and trpR-10 as primers respectively by taking trc-glnA-3, glnA-L159I-4 and glnA-L159I-5, glnA-E304A-6 and glnA-E304A-7 and trc-glnA-8 as primers, and the E.coliW3110 genome as a template; then, using the upstream and downstream homology arms and target gene fragments as templates, using trpR-1 and trpR-10 as primers, amplifying by overlapping PCR to obtain overlapped fragments, using pGRB-trpR-s and pGRB-trpR-a as primers, annealing to obtain gRNA fragments, connecting the gRNA fragments with pGRB vectors to obtain trpR-pGRB, preparing TRP02 electrotransformation competent cells, electrically transforming the target fragments and trpR-pGRB together into competent cells, and screening to obtain positive transformants to obtain strain TRP03.
④ The prsA gene was integrated by the same method as in ① except that the pseudogene sites ilvG, ilvG-trc-2, ilvG-trc-5, ilvG-6, trc-prsA-3, trc-prsA-4 were used. Competent cells were TRP03, resulting in strain TRP04.
⑤ The serA fbr gene was integrated using the same method as ① except that the pseudogene locus yeeL was used with the primers yeeL-1, yeeL-M193-2, yeeL-M193-9, yeeL-10, M193-serA-3, serA-4, serA-5, serA-6, serA-7, and M193-serA-8. Competent cells were TRP04, strain TRP05 was obtained.
⑥ The gene pykA was knocked out and the gene tktA was integrated at this site, with the same method as in ①, except that the primers used were pykA-1, pykA-trc-2, trc-pykA-5, pykA-6, trc-tktA-3 and trc-tktA-4. Competent cells were TRP05, strain TRP06 was obtained.
⑦ The gene ldhA was knocked out and the gdh gene derived from B.subtilis 168 was integrated at this site, with the same method as in ③, except that the primers used were ldhA-1, ldhA-M137-2, ldhA-M137-5, ldhA-6, M137-gdh-3 and M137-gdh-4. Competent cells were TRP06, resulting in strain TRP07.
⑧ Knocking out the promoter of the pykF gene (PpykF promoter) and replacing the promoter with fliC promoter, taking the E.coli W3110 genome as a template, respectively taking PfliC-F, pfliC-pykF-R as a primer to obtain fliC promoter fragments through PCR amplification, taking PfliC-pykF-F, pykF-R as a primer to obtain pykF gene fragments through PCR amplification, taking PfliC-F, pykF-R as a primer to obtain overlapped fragments through overlapped PCR amplification, taking pGRB-pykF-s and pGRB-pykF-a as primers, annealing to obtain gRNA fragments, connecting the gRNA fragments with a pGRB vector to obtain pykF-pGRB, preparing TRP07 electrotransformation competent cells, carrying out electrotransformation on the overlapped fragments and the pykF-pGRB into competent cells, and screening to obtain positive transformants to obtain the strain TRP08.
⑨ The plasmid pACYC-FYSPD and the plasmid pETDuet-TM2-RBS-AnTDC are transformed into a strain TRP08 to obtain a strain WW14, and the constructed and extracted complete plasmids pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC are transformed into TRP08 competent cells, and the transformation method adopts electric transformation to obtain the strain WW14.
Example 2
This example is directed to a method for constructing plasmid pACYC-FYSPD, comprising the following steps:
The E.coli W3110 genome is used as a template, M112-floE-F, folE-T198I-R, folE-T198I-F, M-folE-R is used as a primer to amplify folE gene fragments 1 and 2, folE gene fragments 1 and 2 are used as templates, primer M112-floE-F, M-folE-R is used to amplify folE fbr gene fragments, M112-ygcM-F, M-ygcM-R is used to amplify ygcM gene fragments, and SPR, PCR and DHPR gene fragments are obtained by PCR of M112-SPR-F, M112-SPR-R, M-PCD-F, M-193-PCD-R, trc-DHPR-F, trc-DHPR-R primers after synthesis of SPR, PCD and DHPR gene agents Jin Weizhi are respectively synthesized.
The linearized vector pACYCDuet-1 was obtained by PCR using the linearized vector primer line-pACYC-F, line-pACYC-R with plasmid pACYCDuet-1 (purchased from Ubbelopsis) as a template. Linearized vectors pACYCDuet-1 and folE fbr, ygcM, SPR, PCD and DHPR gene fragments were constructed using the ClonExpressMultiS One Step Cloning Kit kit from the biotechnology company limited of nanking nuozhen. Transferring the plasmid into DH5 alpha transformation competent cells, screening to obtain positive transformants, and extracting plasmids to obtain plasmids pACYC-FYSPD, wherein the nucleotide sequence of the plasmids pACYC-FYSPD is shown in a sequence table SEQ ID NO. 27.
Example 3
The present example is directed to a method for constructing plasmid pETDuet-TM2-RBS-AnTDC, comprising the following steps:
The TM2 gene and AnTDC gene are synthesized by Jin Weizhi biotechnology limited company, and then the T7-TM2-F, TM2-RBS-R, RBS-AnTDC-F, anTDC-R primers are used for PCR to obtain TM2 and AnTDC gene fragments, and the T7-TM2-RBS-AnTDC miniature gene cluster fragments are obtained by using the TM2 and AnTDC gene fragments as templates and using the T7-TM2-F, anTDC-R to carry out PCR.
The linearized vector pETDuet-1 was obtained by PCR using the linearized vector primer line-pETDuet-F, line-pETDuet-R as a template, from the organisms Ubbelopsis. The linearized vector pETDuet-1 and T7-TM2-RBS-AnTDC minigene cluster fragments were constructed by plasmid recombination using the ClonExpressMultiS One Step Cloning Kit kit from Nanjinouzan Biotechnology Co., ltd. Transferring the plasmid into DH5 alpha transformation competent cells, screening to obtain positive transformants, and extracting plasmids to obtain plasmids pETDuet-TM2-RBS-AnTDC, wherein the nucleotide sequence of the plasmids pETDuet-TM2-RBS-AnTDC is shown as a sequence table SEQ ID NO. 28.
Example 4
This example is intended to illustrate the shake flask fermentation application of strain WW14, comprising the specific steps of:
① Tube culture-80℃refrigerator deposited strain WW14 was inoculated in tubes containing 5mL LB liquid medium at 220rpm, 37℃and 12, 12 h.
② Shake flask seed culture, inoculating test tube strain into shake flask seed culture medium with glucose 25g/L, yeast powder 5g/L, peptone 2.5g/L, mgSO 4.7H2O 1.5g/L,KH2PO4 g/L, ammonium sulfate 1.5g/L, and water for fermentation at 220 rpm and 34 deg.C for 12 hr at pH 6.4-6.7.
③ Shake flask fermentation culture, wherein the fermentation inoculation amount is 20-25%, the temperature is 34 ℃, the fermentation inoculation amount is 220-rpm, the culture is 24-36h, the pH is 6.4-6.7, the fermentation culture medium is 15 g/L glucose, 3g/L MgSO 4.7H2 O, 7g/L yeast powder, 5g/L peptone, 3g/L ammonium sulfate, 2PO4 g/L KH, 2 g/L glutamic acid, 3mg/L FeSO 4.7H2O 60mg/L,MnSO47mg/L,VB1、VB3、VB5 respectively, 3mg/L biotin and the balance water.
The experiment uses E.coli W3110 delta lacIZ:: P XylF -T7RNAP as a control group, and through 28h fermentation verification, E.coli W3110 delta lacIZ:: P XylF -T7RNAP fails to accumulate 5-hydroxytryptamine, and strain WW14 accumulates 5.15 g/L of 5-hydroxytryptamine, thus proving the effectiveness of the strain.
Example 5
This example is intended to illustrate the comparison between the ability of strain WW14 and strain TRP08 to produce 5-hydroxytryptamine, tryptophan under the same culture conditions in the fermenter. The only difference between strain WW14 and strain TRP08 is that strain WW14 contains the dual plasmid systems pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC. The method comprises the following specific steps:
(1) Slant culturing, namely inoculating a preserved strain in a temperature of-80 ℃ to an activated slant containing chloramphenicol and ampicillin resistance by streaking, culturing for 12h at 34 ℃ and passaging for 2 times;
(2) Seed culture, namely eluting the activated thalli on the inclined plane by using sterilized distilled water, transferring the thalli into a 5L mechanical stirring type fermentation tank to start seed culture, wherein the culture temperature is 37 ℃, the initial stirring speed is 200rpm, the culture pH is maintained at 6.7+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of a culture medium is maintained at 30% by adjusting the stirring speed or ventilation quantity, and the OD 600nm is regarded as 25 as a seed culture medium maturation mark;
(3) After the seed solution is mature, 500mL of seed culture is reserved, and a fresh fermentation culture medium is immediately added, so that the final volume of fermentation is 2L, the fermentation culture is started, the culture temperature is 34 ℃, the pH value of the culture is maintained at 6.4+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the fermentation culture medium is maintained at 20% by adjusting the stirring speed or ventilation rate, the glucose concentration in a tank is controlled to be less than or equal to 1 g/L by feeding 80% glucose solution, and the fermentation period is 28h (strain WW14 needs to be induced by adding IPTG with the final concentration of 0.1 mM when OD 600 reaches 10).
The components of the slant culture medium adopted are 1g/L glucose, 15mg/L chloramphenicol, 50 mg/L ampicillin, 5g/L peptone, 0.5g/L potassium dihydrogen phosphate, 5g/L yeast extract, 5g/L sodium chloride, 0.2g/L magnesium sulfate heptahydrate, 25g/L agar powder, pH7.0, and 121 ℃ for sterilization for 20min, and then packaging into test tubes.
The seed culture medium comprises 30g/L of glucose, 15mg/L of chloramphenicol, 50 mg/L of ampicillin, 4g/L of yeast extract, 2g/L of peptone, 2g/L of citric acid, 2g/L of ammonium sulfate, 3g/L of monopotassium phosphate, 2g/L of magnesium sulfate heptahydrate, 2g/L of glutamic acid, 0.5g/L of methionine and the balance of water.
The components of the fermentation medium adopted are 20g/L glucose, 10 g/L xylose, 15mg/L chloramphenicol, 50 mg/L ampicillin, 6g/L yeast extract, 4g/L peptone, 2g/L citric acid, 2g/L ammonium sulfate, 4g/L potassium dihydrogen phosphate, 3g/L magnesium sulfate heptahydrate, 60mg/L ferrous sulfate heptahydrate, 5mg/L manganese sulfate monohydrate, 2g/L monosodium glutamate, 1g/L choline chloride, 1g/L betaine and the balance of water.
The total fermentation time period for strain TRP08 and strain WW14 was 28 h, and the final OD 600, tryptophan and 5-hydroxytryptamine yields were as shown in Table 2 below.
TABLE 2 fermentation comparison of strains TRP08 and WW14
TRP08 WW14
OD600 141.7 98.3
Tryptophan yield (g/L) 22.04 0.01
Yield of 5-hydroxytryptamine (g/L) 0 15.50
As a result, it was found that strain WW14 had the ability to produce 5-hydroxytryptamine, and the intermediate tryptophan was hardly accumulated. The method shows that each metabolic pathway in the strain WW14 is balanced, the intracellular resources are utilized to the maximum extent, and simultaneously, 15.50 g/L of 5-hydroxytryptamine is produced, so that the method is the highest yield of the 5-hydroxytryptamine reported at present.
The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be appreciated by those skilled in the art that variations and modifications of the invention and strain changes, which are carried out by or based on the methods of this invention, may be made without departing from the spirit of this invention.

Claims (9)

1. A strain of 5-hydroxytryptamine of escherichia coli, characterized by: coli W3110 Δ lacIZ: P XylF -T7RNAP is used as a chassis strain, tnaA, pykA, trpR, ldhA and trpL genes on the genome of the strain are knocked out, trpE fbr、aroGfbr、serAfbr, tktA, tRNA is upregulated, the prsA gene, the heterologous expression of the gdh and glnA fbr genes of B.subtilis 168, the dynamic downregulation of the pykF gene, and the production strain carrying a low copy plasmid pACYC-FYSPD and a high copy plasmid pETDuet-TM2-RBS-AnTDC; the nucleotide sequence of the trpA gene is shown in a sequence table SEQ ID NO.1, the nucleotide sequence of the trpR gene is shown in a sequence table SEQ ID NO.4, the nucleotide sequence of the pykA gene is shown in a sequence table SEQ ID NO.7, the nucleotide sequence of the ldhA gene is shown in a sequence table SEQ ID NO.11, the nucleotide sequence of the trpL gene is shown in a sequence table SEQ ID NO.31, the nucleotide sequence of the trpE fbr gene is shown in a sequence table SEQ ID NO.3, the nucleotide sequence of the aroG fbr gene is shown in a sequence table SEQ ID NO.5, the nucleotide sequence of the serA fbr gene is shown in a sequence table SEQ ID NO.10, the nucleotide sequence of the tktA gene is shown in a sequence table SEQ ID NO.9, the nucleotide sequence of the prsA gene is shown in a sequence table SEQ ID NO.8, the nucleotide sequence of the gdh gene is shown in a sequence table SEQ ID NO.12, the nucleotide sequence of the glnA fbr gene is shown in a sequence table SEQ ID NO.6, the nucleotide sequence of the trpF gene is shown in a sequence table SEQ ID NO.30, the nucleotide sequence of the plasmid pA is shown in a sequence table SEQ ID NO. 30-35, and the nucleotide sequence of the plasmid DNA-35.
2. A production strain of E.coli containing 5-hydroxytryptamine according to claim 1, wherein the aroG fbr gene is enhanced by the trc promoter and integrated into the tnaA gene locus, the trpE fbr gene is enhanced by the trc promoter and integrated into the trpLE gene locus, the glnA fbr gene derived from B.subtilis 168 is enhanced by the trc promoter and integrated into the genomic trpR gene locus, the prsA gene is enhanced by the trc promoter and integrated into the ilvG gene locus, the tktA gene is enhanced by the trc promoter and integrated into the pykA gene locus, the PpykF promoter is replaced with fliC promoter for dynamically down-regulating the expression of the pykF gene, the serA fbr gene is enhanced by the M1-93 promoter and integrated into the yeeL gene locus, and the gdh gene of B.subtilis 168 is enhanced by the M1-37 promoter and integrated into the ldhA gene locus.
3. The strain of E.coli producing 5-hydroxytryptamine of claim 2, wherein the trc promoter has a nucleotide sequence shown in SEQ ID No.20, ppykF promoter has a nucleotide sequence shown in SEQ ID No.22, fliC promoter has a nucleotide sequence shown in SEQ ID No.23, M1-93 promoter has a nucleotide sequence shown in SEQ ID No.24, and M1-37 promoter has a nucleotide sequence shown in SEQ ID No. 25.
4. A method for constructing a strain of E.coli producing 5-hydroxytryptamine as claimed in any one of claims 1 to 3, comprising the following steps:
(1) Taking E.coli W3110 delta lacIZ as chassis strain P XylF -T7RNAP, knocking out tnaA gene, trpR gene and trpL gene, reinforcing trpE fbr gene;
(2) Knocking out the pykA gene, strengthening the tktA gene, aroG fbr gene, serA fbr gene and prsA gene, introducing glnA fbr gene, replacing PpykF promoter with fliC promoter and dynamically regulating and controlling pykF gene;
(3) Knocking out the ldhA gene, introducing the gdh gene;
(4) pACYC-FYSPD and pETDuet-TM2-RBS-AnTDC were introduced.
5. Use of a strain of escherichia coli producing 5-hydroxytryptamine according to any one of claims 1 to 3 for the fermentative production of 5-hydroxytryptamine.
6. The use of the 5-hydroxytryptamine escherichia coli production strain as set forth in claim 5, wherein the shake flask fermentation culture is adopted, and the specific method is as follows:
① Test tube culture, wherein the strain is inoculated in a test tube containing LB liquid medium, 220 rpm,37 ℃,12 h;
② Shake flask seed culture, inoculating test tube strain into shake flask seed culture medium for fermentation at 220 rpm and 34 deg.C for 12 hr at pH of 6.4-6.7,
③ Shake flask fermentation culture, wherein the fermentation inoculation amount is 20-25%, the temperature is 34 ℃, the temperature is 220 rpm, the culture time is 24-36h, and the pH is 6.4-6.7.
7. The use of the strain of E.coli producing 5-hydroxytryptamine of claim 6, wherein the shake flask seed medium in step ② is glucose 20-30g/L, yeast powder 4-6g/L, peptone 2-3g/L, mgSO 4.7H2O 1-2g/L,KH2PO4 2-4g/L, ammonium sulfate 1-2g/L, and water in balance, the fermentation medium in step ③ is glucose 15-g g/L, xylose 10 g/L, mgSO 4.7H2 O2-4 g/L, yeast powder 6-8g/L, peptone 4-6g/L, ammonium sulfate 2-4g/L, KH 2PO4 -6g/L, glutamic acid 2-g/L, feSO 4.7H2O 40-80mg/L,MnSO45-8mg/L,VB1、VB3、VB5 each 2-4mg/L, biotin 2-4mg/L, and water in balance.
8. The use of the strain for producing 5-hydroxytryptamine colibacillus according to claim 5, wherein the strain is cultured by a fermentation tank, and the specific method is as follows:
(1) Slant culture, namely streaking and inoculating the strain on an activated slant containing chloramphenicol and ampicillin resistance, culturing for 12h at 34 ℃ and passaging for 2 times;
(2) Seed culture, namely eluting the activated thalli on the inclined plane by using sterilized distilled water, transferring the thalli into a fermentation tank to start seed culture, wherein the culture temperature is 37 ℃, the initial stirring rotation speed is 200rpm, the culture pH is maintained at 6.7+/-0.2, the dissolved oxygen value of a culture medium is maintained at 30%, and the OD 600nm is 25 as a seed culture solution maturation mark;
(3) And (3) fermenting and culturing, namely adding a fermentation culture medium into the seed culture for fermenting and culturing after the seed liquid is mature, wherein the culture temperature is 34 ℃, the culture pH is maintained at 6.4+/-0.2, the fermentation dissolved oxygen value of the culture medium is maintained at 20%, and the glucose concentration in the tank is controlled to be less than or equal to 1g/L.
9. The use of the strain of E.coli producing 5-hydroxytryptamine according to claim 8, wherein the slant culture medium used in the step (1) is glucose 1g/L, chloramphenicol 15mg/L, ampicillin 50 mg/L, peptone 5g/L, potassium dihydrogen phosphate 0.5g/L, yeast extract 5g/L, sodium chloride 5g/L, magnesium sulfate heptahydrate 0.2g/L, agar powder 25g/L, pH7.0; the seed culture medium adopted in the step (2) comprises 30g/L of glucose, 15mg/L of chloramphenicol, 50 mg/L of ampicillin, 4g/L of yeast extract, 2g/L of peptone, 2g/L of citric acid, 2g/L of ammonium sulfate, 3g/L of monopotassium phosphate, 2g/L of magnesium sulfate heptahydrate, 2g/L of glutamic acid, 0.5g/L of methionine and the balance of water, and the fermentation culture medium adopted in the step (3) comprises 20g/L of glucose, 15mg/L of chloramphenicol, 50 mg/L of ampicillin, 6g/L of yeast extract, 4g/L of peptone, 2g/L of citric acid, 2g/L of ammonium sulfate, 4g/L of monopotassium phosphate, 3g/L of magnesium sulfate heptahydrate, 60mg/L of ferrous sulfate heptahydrate, 5mg/L of manganese sulfate monohydrate, 1g/L of monosodium glutamate, 1g/L of choline chloride and the balance of betaine.
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