An engineered 5-hydroxytryptamine strain and its application

Abstract

This invention discloses an engineered serotonin-producing strain and its applications. The strain has an insertion of a tryptophan hydroxylase (TPH1) gene expression sequence and a tryptophan deacidase (tdc) gene expression sequence into its chromosome. The TPH1 gene expression sequence is inserted at the lacZ gene locus, with a size of 2000-3000 bp, and the tdc gene expression sequence is inserted at the malEK gene locus, with a size of 1500-3000 bp. This engineered strain grows well and can stably express the target genes. In particular, the insertion of the humanized tryptophan hydroxylase (TPH1) gene and the rice tryptophan deacidase (tdc(R)) gene into *E. coli* with the tnaA gene knocked out significantly increases 5-HT production to 82 mg / L. The engineered serotonin-producing strain provided by this invention shows broad application prospects in improving intestinal dysfunction and / or anxiety-depression-like behaviors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to an engineered 5-hydroxytryptamine strain and its applications. Background Technology

[0002] Slow transit constipation (STC) is a common type of functional constipation, accounting for 10.3% to 45.5% of all functional constipation cases. It is likely caused by dysfunction of the colonic smooth muscle or its innervation, leading to abnormal colonic motility. Epidemiological studies show that STC has a high prevalence worldwide and has become a factor affecting people's quality of life.

[0003] Currently, the main drugs used to treat STC disease are laxatives, mild laxatives, and prokinetic agents. While laxatives and mild laxatives are effective, long-term use can easily lead to drug dependence and, to some extent, exacerbate patients' anxiety and depression. Existing prokinetic agents work by activating 5-HT receptor 4 (5-HT4R), such as mosapride and prucalopride. By acting on 5-HT4R in the intestine, they stimulate intramuscular cholinergic neurons, inducing the release of acetylcholine, substance P, and calcitonin gene-related peptide, resulting in coordinated contraction and relaxation of gastrointestinal smooth muscle. However, these prokinetic agents are generally expensive, and most have a short duration of effect. Genetically engineered strains have become a new direction for drug research in the treatment of STC disease.

[0004] Currently, some genetically engineered strains are used to prepare prokinetic agents. For example, the earlier patent application CN202111267670.6, "A Modified Strain, Its Application in the Preparation of Prokinetic Agents and Products," can be used to prepare prokinetic agents to relieve constipation. However, this strain produces a low yield of serotonin (5-HT). Patent CN202080051128.4, "Engineering for Advanced Microbiome Therapy to Produce Serotonin in Vivo," provides an engineered strain that can increase serotonin production, but the plasmids expressing both the H2R pathway and the infA gene cause undesirable growth defects in the host cell. Therefore, there is an urgent need for a genetically engineered strain that grows well and produces high levels of 5-HT. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides an engineered 5-HT strain and its application. The purpose is to insert a tryptophan hydroxylase TPH1 gene expression sequence into the lacZ gene locus of the *E. coli* strain and a tryptophan deacidase tdc gene expression sequence into the malEK gene locus, particularly inserting a humanized tryptophan hydroxylase TPH1 gene and a rice tryptophan deacidase tdc(R) gene, thereby solving the technical problems of defective growth and / or low 5-HT yield in engineered strains.

[0006] To achieve the above objectives, according to one aspect of the present invention, an engineered 5-hydroxytryptamine strain is provided, wherein the strain's chromosome is inserted with an expression sequence comprising a tryptophan hydroxylase TPH1 gene and a tryptophan deacidase tdc gene, wherein the tryptophan hydroxylase TPH1 gene expression sequence is inserted at the lacZ gene site and is 2000-3000 bp in size, and the tryptophan deacidase tdc gene expression sequence is inserted at the malEK gene site and is 1500-3000 bp in size.

[0007] Preferably, the engineered 5-hydroxytryptamine strain has a tryptophan hydroxylase TPH1 gene sequence size of 2000-2500 bp; and the tryptophan deacidase tdc gene sequence size after codon optimization and addition of the His6 tag has a size of 1500-2000 bp.

[0008] Preferably, the engineered 5-hydroxytryptamine strain has a humanized tryptophan hydroxylase TPH1 gene expression sequence and a rice tryptophan deacidase tdc gene expression sequence.

[0009] Preferably, the engineered 5-hydroxytryptamine strain has the following expression sequence: the tryptophan hydroxylase TPH1 gene expression sequence is shown in SEQ TD NO.1; and the tryptophan deacidase tdc gene expression sequence is shown in SEQ TD NO.2.

[0010] Preferably, the engineered 5-hydroxytryptamine strain comprises Escherichia coli, wherein the Escherichia coli lacks the expression gene for the key enzyme in tryptophan production.

[0011] Preferably, the engineered 5-hydroxytryptamine strain is an EcN strain with the tnaA gene knocked out.

[0012] Preferably, in the engineered 5-hydroxytryptamine strain, the promoter in the recombinant gene is a constitutive promoter, and the constitutive promoter includes a pBAD promoter with the araC regulatory element removed.

[0013] According to another aspect of the invention, a prokinetic formulation is also provided, wherein the prokinetic formulation comprises engineered 5-hydroxytryptamine strains as described in the present invention.

[0014] According to another aspect of the invention, the use of the engineered 5-hydroxytryptamine strains described herein in improving intestinal dysfunction and / or anxiety-depression-like behaviors is also provided.

[0015] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects:

[0016] The engineered serotonin strain provided by this invention, by inserting the entire target genes of tryptophan hydroxylase TPH1 and tryptophan deacidase tdc into the lacZ and malEK gene loci on the E. coli chromosome, respectively, can stably express the target genes without affecting the growth of the engineered strain. In particular, the engineered strain obtained by inserting humanized tryptophan hydroxylase TPH1 and rice tryptophan deacidase tdc(R) genes exhibits good growth, stable expression of the target genes, and a significant increase in 5-HT production, reaching up to 82 mg / L. Therefore, the engineered serotonin strain provided by this invention shows broad application prospects in improving intestinal dysfunction and / or anxiety-depression-like behaviors. Attached Figure Description

[0017] Figure 1 These are the PCR detection results of EcN 5-HTP engineered bacteria;

[0018] Figure 1 M: 250bp DNA marker; 1–15: candidate genetically engineered strain EcNΔtnaA lacZ::PBAD-tph(H1); N: EcNΔtnaA strain;

[0019] Figure 2 These are the PCR detection results of the EcN 5-HT strain construction;

[0020] Figure 2 M: DL5000 DNA Marker; 1–16: Candidate 5-HT genetically engineered strains; N: EcN 5-HTP strains;

[0021] Figure 3 These are the results of growth defect determination for engineered bacteria EcN 5-HT;

[0022] Figure 3 EcNΔtnaA is a tnaA gene knockout strain; EcN 5-HT is a 5-HT-producing genetically engineered strain.

[0023] Figure 4 These are the results of growth defect determination for the EcN 5-HTP(PBAD-tdc-His) strain;

[0024] Figure 4 The EcN 5-HTP(vector) is an EcN 5-HTP strain containing an empty vector plasmid;

[0025] Figure 5 These are the PCR detection results of the EcN 5-HTP(PBAD-tdc-His) strain construction;

[0026] Figure 5 Medium M: 250bp DNA Marker; 1: strain EcN 5-HTP (PBAD-tdc(C)-His); 2: strain EcN 5-HTP (PBAD-tdc(R)-His); 3: strain EcN 5-HTP (PBAD-tdc(B)-His);

[0027] Figure 6 The results are quantitative analysis of 5-HT production by different EcN 5-HTP (PBAD-tdc-His) strains;

[0028] Figure 7 The results are a quantitative comparative analysis of 5-HT production by EcN 5-HTP engineered bacteria and EcN 5-HT engineered bacteria.

[0029] Figure 8 The effect of engineered probiotic EcN 5-HT on the number of feces in STC mice;

[0030] Figure 9 The effect of engineered probiotic EcN 5-HT on the total movement distance of STC mice in the open field experiment;

[0031] Figure 10 The effect of engineered probiotic EcN 5-HT on the time spent in the central region of an open field in STC mice;

[0032] Figure 11 These are the movement trajectories of mice in each group during the open field experiment. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0034] Studies have shown that 5-HT plays a crucial role in the regulation of intestinal motility. In mammals, 5-HT is derived from tryptophan (Trp) through a two-step reaction. First, tryptophan is converted to 5-hydroxytryptophan (5-HTP) intermediate under the catalysis of tryptophan hydroxylase (TPH), which is the rate-limiting step in 5-HT synthesis. Second, 5-HTP is converted to 5-HT by tryptophan decarboxylase (TDC). Therefore, genetically engineered strains that promote 5-HT production can be obtained by transferring tryptophan hydroxylase (TPH) and / or tryptophan decarboxylase (TDC) into probiotics, especially genetically engineered strains obtained by simultaneously transferring tryptophan hydroxylase (TPH) and tryptophan decarboxylase (TDC) into probiotics.

[0035] For example, patent CN202080051128.4 selected partial fragments from the human tryptophan hydroxylase TPH2 gene and the rice tryptophan decarboxylase gene tdc(R) and inserted them into Escherichia coli strains to obtain recombinant engineered bacteria. However, although the expression of the target gene in the obtained recombinant engineered bacteria was relatively stable, it caused undesirable growth defects in the host cells.

[0036] To ensure that the obtained genetically engineered strains can stably express the target genes without affecting their normal growth and significantly promote 5-HT production in vivo, we found that inserting a 2000-3000 bp tryptophan hydroxylase 1 (TPH1) gene at the lacZ gene locus on the chromosome of wild-type Escherichia coli Nissle 1917 (EcN WT) and inserting a 1500-3000 bp tryptophan decarboxylase gene tdc at the malEK gene locus can stably express these two target genes without affecting the normal growth of the strain. In particular, inserting the human tryptophan hydroxylase 1 (TPH1) gene and the rice tryptophan decarboxylase gene tdc(R) (GenBank AK069031) can significantly promote 5-HT production. Especially after knocking out the tnaA gene in EcN and then inserting the target genes, it is even more beneficial to increase the yield of 5-HT.

[0037] This invention provides an engineered 5-hydroxytryptamine strain whose chromosome contains an inserted tryptophan hydroxylase TPH1 gene expression sequence and a tryptophan deacidase tdc gene expression sequence. The tryptophan hydroxylase TPH1 gene expression sequence is 2000-3000 bp in size, and the tryptophan deacidase tdc gene expression sequence is 1500-3000 bp in size.

[0038] Inserting these two target genes can significantly increase the production of 5-HT. However, inserting target gene fragments that are too small or too large can easily affect the stable expression of the target genes. Experimental results show that selecting these two sizes of target genes for insertion into the strain chromosome can ensure the stable expression of the target genes TPH1 and tdc in the strain.

[0039] The tryptophan hydroxylase TPH1 gene expression sequence is inserted into the lacZ gene site, and the tryptophan deacidase tdc gene expression sequence is inserted into the malEK gene site. Since the inserted genes are foreign genes, selecting appropriate insertion sites is one of the key factors in order not to affect the normal physiological function of the strain. Experiments have confirmed that inserting the target genes into these two sites can ensure that the normal physiological function of the strain is not affected.

[0040] Preferably, the expression sequence of the tryptophan hydroxylase TPH1 gene has a sequence size of 2000-2500 bp, and the expression sequence of the tryptophan deacidase tdc gene, after codon optimization and addition of a His6 tag, has a sequence size of 1500-2000 bp.

[0041] More preferably, the expression sequence of the tryptophan hydroxylase TPH1 gene is a humanized tryptophan hydroxylase TPH1 gene sequence, as shown in sequence SEQ TD NO.1; the expression sequence of the tryptophan deacidase tdc gene is the sequence of rice tryptophan deacidase tdc(R) after codon optimization and addition of the His6 tag, as shown in sequence SEQ TD NO.2; the insertion of these two target genes can significantly increase the yield of 5-HT, and can stably express and produce 5-HT without affecting the growth of the strain itself. It can be continuously expressed in multiple experiments, and the yield of 5-HT is significantly improved;

[0042] The engineered 5-hydroxytryptamine strain includes Escherichia coli, which lacks the expression gene for the key enzyme in tryptophan production. Preferably, it is an EcN strain with the tnaA gene knocked out, such as wild-type Escherichia coli Nissle 1917 (EcNWT). Knocking out the tnaA gene can prevent the metabolic breakdown of tryptophan or 5-hydroxytryptamine, thereby increasing the accumulation of the substrate tryptophan, which is beneficial for increasing the yield of 5-HT without affecting the normal growth of the strain.

[0043] The engineered 5-hydroxytryptamine strain preferably has a constitutive promoter, including the pBAD promoter with the araC regulatory element removed. Constitutive promoters do not require induction, can initiate gene expression in all tissues, and the regulation is not affected by external conditions. The expression of the initiated gene is continuous, which is beneficial for the continuous and stable expression of the target gene.

[0044] In addition, the present invention provides a prokinetic formulation comprising the engineered 5-hydroxytryptamine strain described in the present invention, and preferably further comprising an intestinal solvent.

[0045] In addition, the present invention also provides the application of engineered 5-hydroxytryptamine strains as described herein in improving intestinal dysfunction and anxiety-depression-like behaviors.

[0046] The following is an example:

[0047] The strains and plasmids used in the following examples are as follows:

[0048]

[0049]

[0050] The primers used in the following examples are as follows:

[0051]

[0052] Example 1: Engineered 5-hydroxytryptamine strain 1

[0053] The engineered 5-hydroxytryptamine strain 1 was constructed using wild-type Escherichia coli Nissle 1917 (EcN WT) as the chassis cell. The tnaA gene was first knocked out, and the human tryptophan hydroxylase 1 tph(H1) gene was inserted into the lacZ gene locus, while the rice tryptophan decarboxylase gene tdc(R) (GenBank AK069031) was inserted into the malEK gene locus. The specific preparation method is as follows:

[0054] (1) Construction of engineered bacteria EcN 5-HTP

[0055] (1-1) Obtaining EcNΔtnaA strain

[0056] The pCas plasmid was electroporated into EcN-WT competent cells. Positive clones were screened on LB plates containing 50 μg / mL kanamycin to obtain strain EcN-pCas. EcN-pCas was then used to prepare competent cells. During the preparation process, when the OD reached 6000.4, 0.2% L-arabinose was added to induce the expression of Cas9 protein. The pTargetF-ΔtnaA sgRNA plasmid was constructed. The homologous ligation product and pTargetF-ΔtnaA sgRNA were simultaneously electroporated into EcN-pCas competent cells. Positive clones were screened on antibiotic plates (streptomycin 100 μg / mL, kanamycin 50 μg / mL, spectinomycin 100 μg / mL). After the pTargetF and pCas plasmids were eliminated, the resulting strain was named EcNΔtnaA.

[0057] (1-2) Construction of engineered strain EcN 5-HTP

[0058] Using the CRISPR / Cas9 dual plasmid system, the target human tryptophan hydroxylase 1tph(H1) gene was introduced into the lacZ gene locus on the EcNΔtnaA chromosome. PCR detection of chromosome construction was performed as follows. Figure 1 As shown, by obtaining the EcN 5-HTP strain and inserting the tph(H1) gene into the lacZ gene locus, the tph(H1) gene can be stably expressed in the EcN strain; the details are as follows:

[0059] EcNΔtnaA-pCas competent cells and pTargetF-sgRNAlacZ::PBAD-tph(H1) plasmid were prepared and electroporated into EcNΔtnaA-pCas competent cells. To induce Cas9 expression, L-arabinose with a final concentration of 0.2% was added to triple-antibiotic solid plates (streptomycin 100 μg / mL, spectinomycin 100 μg / mL, kanamycin 50 μg / mL), and X-gal 40 μg / mL was added for blue-white screening. Positive clones were purified on triple-antibiotic solid plates, and pTargetF plasmid and pCas plasmid were eliminated. The strain after plasmid elimination was named EcN 5-HTP and then expanded and preserved.

[0060] (2) Construction of engineered bacteria EcN 5-HT

[0061] The rice tryptophan decarboxylase gene tdc(R) (GenBank AK069031) was introduced into the malEK gene locus on the EcN5-HTP chromosome to construct the EcN 5-HT strain. Inserting the tdc(R) gene into the malEK gene locus ensured stable expression of the tdc(R) gene in the EcN strain. Details are as follows:

[0062] Preparation of EcN 5-HTP-pKD46 competent cells: Plasmid pKD46 was extracted and electroporated into EcN5-HTP competent cells. Positive clones were screened on 100 μg / mL ampicillin-resistant LB agar plates, and the positive clones were purified by three-zone streak to obtain strain EcN 5-HTP(pKD46). During the preparation of EcN 5-HTP(pKD46) competent cells, the bacterial culture was cultured to OD6000.4, and 0.2% L-arabinose was added.

[0063] Plasmids pKD3 and pACYC-PBAD-tdc(R)-His6 were extracted. Using pKD3 as a template, primers PKD3-malEKinsertCmF and PKD3-malEKinsertCmR were used to amplify the chloramphenicol resistance fragment. The 5' end of this fragment has a homology of approximately 60 bp with the malEK gene locus on the EcN genome, and the 3' end carries a sequence of approximately 20 bp homology with the target gene tdc(R). Using plasmid pACYC-PBAD-tdc(R)-His6 as a template, the target tdc(R) gene fragment was amplified. Primers pACYC-malEK::PBAD-tdc(R)F and pACYC-malEK::PBAD-tdc(R)R were used. The 5' end of this fragment carries a homology of approximately 20 bp with the chloramphenicol fragment, and the 3' end carries a homology of approximately 60 bp with the malEK locus on the EcN genome.

[0064] Fusion PCR homologous recombination: Chloramphenicol fragment and tdc(R) gene fragment were used as templates, 30 ng each. PKD3-malEKinsert Cm-F and pACYC-malEK::PBAD-tdc(R)R were used as upstream and downstream primers, respectively. The two target fragments were ligated by fusion PCR. The ligation results were verified by agarose gel electrophoresis, and the purified ligation product was recovered and its concentration was quantified using a microplate reader.

[0065] Construction of EcN 5-HTP malEK::PBAD-tdc(R): Approximately 1 μg of the Cm-tdc(R) ligation product was electroporated into EcN 5-HTP(pKD46) competent cells. After incubation at 30°C for 1 h, the cells were plated on LB agar plates (containing 100 μg / mL ampicillin, 15 μg / mL chloramphenicol, 100 μg / mL streptomycin, and 0.2% L-arabinose). The plates were incubated overnight at 30°C. Positive clones were screened by PCR. Viability testing was performed using primers EcN malEK::PBAD-tdc(R)check F and EcN malEK::PBAD-tdc(R)check R to obtain the engineered strain EcN 5-HTP malEK::PBAD-tdc(R). The genome of the purified engineered strain was extracted and further analyzed by PCR. The strain was then sequenced for confirmation and preserved.

[0066] Elimination of pKD46 plasmid: The frozen strain *EcN5-HTP malEK::PBAD-tdc(R)* was revived on LB agar plates supplemented with 15 μg / mL chloramphenicol and 100 μg / mL streptomycin. A single colony was picked and inoculated into 5 mL of LB medium containing 15 μg / mL chloramphenicol and 100 μg / mL streptomycin resistance, and cultured overnight at 42°C. 10 μL of the overnight culture was plated onto double-antibiotic plates (chloramphenicol 15 μg / mL, streptomycin 100 μg / mL) and triple-antibiotic plates (chloramphenicol 15 μg / mL, streptomycin 100 μg / mL, ampicillin 100 μg / mL) to verify the elimination of pKD46 plasmid and to preserve the strain. Strains with successfully eliminated pKD46 plasmid did not grow on ampicillin-containing plates.

[0067] Elimination of chloramphenicol resistance and PCP20 plasmid: pCP20 is an ampicillin and CmR plasmid with temperature-sensitive replication and heat-induced FLP, used for the elimination of chloramphenicol genes on the strain chromosome. Competent cells were prepared from the *EcN 5-HTP malEK::PBAD-tdc(R)* strain with pKD46 plasmid elimination. The pCP20 plasmid was electroporated into the competent cells to obtain strain *EcN 5-HTP malEK::PBAD-tdc(R)* (pCP20). The strain was inoculated into LB flasks containing 100 μg / mL streptomycin and cultured overnight at 43°C. 10 μL of the overnight culture was plated onto monoclonal antibody plates (streptomycin 100 μg / mL), double antibody plates 1 (chloramphenicol 15 μg / mL, streptomycin 100 μg / mL), and double antibody plates 2 (streptomycin 100 μg / mL, ampicillin 100 μg / mL) to verify chloramphenicol resistance and the elimination of the PCP20 plasmid. Theoretically, the strain should simultaneously lose the chloramphenicol resistance gene and the FLP helper plasmid pCP20, and it did not grow on double antibody plates 1 and 2. The genome of the strain with both the resistance gene and plasmid eliminated was extracted and sequenced for verification, resulting in an engineered 5-hydroxytryptamine strain, designated EcN 5-HT. The PCR results are shown below. Figure 2 As shown; the growth of EcN 5-HT strain is as follows. Figure 3 As shown.

[0068] Comparative Example 1: Engineered 5-hydroxytryptamine strain 2

[0069] The engineered 5-hydroxytryptamine strain 2 was constructed using wild-type Escherichia coli Nissle 1917 (EcN WT) as the chassis cell. The tnaA gene was first knocked out, and the human tryptophan hydroxylase 1tph(H1) gene was inserted into the lacZ gene locus, while the vinca tryptophan decarboxylase gene tdc(C) (GenBank: MG748691.1) was inserted into the malEK gene locus. The engineered strain was prepared according to the following method:

[0070] (1) Knock out tnaA in EcN and name the obtained strain EcNΔtnaA: Same as in Example 1;

[0071] (2) The human tryptophan hydroxylase 1tph(H1) gene was introduced into the EcNΔtnaA chromosome, and the resulting strain was named EcN 5-HTP: same as in Example 1;

[0072] (3) Replace the tdc(R) gene in Example 1 with the tdc(C) gene and introduce it into the malEK gene locus on the EcN 5-HTP chromosome. Other operations are the same as in Example 1 to construct the EcN 5-HT strain.

[0073] Comparative Example 2: Engineered 5-hydroxytryptamine strain 3

[0074] The engineered 5-hydroxytryptamine strain 3 was constructed using wild-type Escherichia coli Nissle 1917 (EcN WT) as the chassis cell. The tnaA gene was first knocked out, and the human tryptophan hydroxylase 1tph(H1) gene was inserted into the lacZ gene locus. The L-amino acid decarboxylase gene tdc(B) from Bacillus atrophaeus strain C89 (GenBank JQ400024.1) was inserted into the malEK gene locus. The specific preparation method is as follows:

[0075] (1) Knock out tnaA in EcN and name the obtained strain EcNΔtnaA: Same as in Example 1;

[0076] (2) The human tryptophan hydroxylase 1tph(H1) gene was introduced into the EcNΔtnaA chromosome, and the resulting strain was named EcN 5-HTP: same as in Example 1;

[0077] (3) Replace the tdc(R) gene in Example 1 with the tdc(B) gene and introduce it into the malEK gene locus on the EcN 5-HTP chromosome. Other operations are the same as in Example 1 to construct the EcN 5-HT strain.

[0078] Example 3: Identification of growth defects in engineered 5-hydroxytryptamine strains

[0079] The recombinant plasmids pACYC-PBAD-tdc(C)-His6, pACYC-PBAD-tdc(R)-His6, and pACYC-PBAD-tdc(B)-His6 were introduced into the EcN 5-HTP strain. As in Example 1, the successful introduction of the recombinant plasmids was detected by PCR, yielding strains EcN 5-HTP(PBAD-tdc(C)-His), EcN5-HTP(PBAD-tdc(R)-His), and EcN 5-HTP(PBAD-tdc(B)-His). Growth defect assays were then performed, as detailed below:

[0080] Identification of bacterial growth defects: Resuscitate the strain on a plate, pick a single colony and add it to 5 mL of LB medium. Add 100 μg / mL streptomycin and incubate overnight at 37°C. Transfer the culture to 2 mL centrifuge tubes at a 1:100 ratio, mix well, and aliquot 200 μL into 96-well plates. Set the microplate reader program to: absorption wavelength 600 nm, 30°C or 37°C, shake the medium every 20 min, and monitor the growth of the strain until the plateau phase. Analyze the growth based on the strain's OD value. 600 Values ​​were used to plot growth curves for different strains and to assess growth defects, such as... Figure 4 As shown, the results indicate that the three EcN5-HTP(P) compounds... BAD All strains (-tdc-His) grew well.

[0081] Example 4: Quantitative analysis of 5-HT production from tdc genes of different sources

[0082] The following genes were synthesized in vitro: *Catharanthus roseus* tryptophan decarboxylase gene *tdc(C)* (GenBank: MG748691.1); *Rice* tryptophan decarboxylase gene *tdc(R)* (GenBank: AK069031); and *Bacillus atrophaeus* strain C89 L-amino acid decarboxylase gene *tdc(B)* (GenBank: JQ400024.1). After codon optimization and the addition of a His6 tag, the genes were cloned into the plasmid *pACYC-PBAD-p15A* to prepare recombinant plasmid DNA. Three different recombinant plasmids were identified as pACYC-PBAD-tdc(C)-His6, pACYC-PBAD-tdc(R)-His6, and pACYC-PBAD-tdc(B)-His6. These were electroporated into EcN5-HTP competent cells, and positive clones were detected by PCR, yielding strain EcN5. EcN 5-HTP (PBAD-tdc(C)-His), EcN 5-HTP (PBAD-tdc(R)-His), and EcN 5-HTP (PBAD-tdc(B)-His) were constructed and preserved. The PCR detection results of the three EcN 5-HTP (PBAD-tdc-His) strains are as follows: Figure 5 As shown.

[0083] To quantify the yield of 5-HT catalyzed by tdc from different sources in EcN 5-HTP ((PBAD-tdc-His)), overnight cultured strains EcN 5-HTP (PBAD-tdc(C)-His), EcN 5-HTP (PBAD-tdc(R)-His), and EcN 5-HTP (PBAD-tdc(B)-His) were transferred at a 1% inoculum to 15 ml of fresh LB medium supplemented with 100 μg / mL streptomycin and 100 μg / mL ampicillin. The culture was incubated until the OD reached approximately 6000.6, then 0.2% arabinose was added and the culture was induced at 30°C for 2.5 h. 14 mL of the induced bacterial culture was then incubated at 80°C. Centrifuge at 000 rpm / min for 10 min, discard the supernatant, and transfer all cells to 5 mL of 5-HT fermentation medium. Ferment at 0℃. Take the bacterial solution after 24 h of fermentation, sterilize it through a 0.22 μm filter membrane, and detect the yield of 5-HT by LC-MS. The 5-HT fermentation medium was resistant to streptomycin (100 μg / mL) and kanamycin (50 μg / mL).

[0084] In this embodiment, the strain is prepared by cloning the target gene in pACYC-P. BAD Recombinant plasmid DNA was prepared on the p15A ori plasmid, introduced into the strain, and the 5-HT yield of different tryptophan decarboxylases was tested in the EcN 5-HTP strain. The results are as follows: Figure 6 .

[0085] Depend on Figure 6 It was found that the three engineered strains EcN 5-HTP (PBAD-tdc-His) produced different concentrations of 5-HT, all of which were significantly higher than those of the strain EcN 5-HTP (vector) containing an empty plasmid. Among them, strain EcN 5-HTP (PBAD-tdc(R)-His) had the highest yield, about 1495 mg / L, strain EcN 5-HTP (PBAD-tdc(B)-His) had the second highest yield, about 474 mg / L, and strain EcN 5-HTP (PBAD-tdc(C)-His) had the lowest yield, about 60 mg / L. The results indicate that the rice tryptophan decarboxylase gene tdc(R) is the best source of tdc gene.

[0086] Example 5: Quantitative analysis of 5-HT production by engineered strains EcN 5-HTP and EcN 5-HT

[0087] As shown in Example 4, the rice tryptophan decarboxylase gene tdc(R) is the optimal source of tdc genes. Although it has a high yield, expression in recombinant plasmids is unstable. To avoid the instability caused by plasmid expression, the target tdc gene was constructed into the malEK gene locus on the chromosome of the EcN5-HTP strain using the λRed homologous recombination system for stable expression. This resulted in the engineered 5-hydroxytryptamine strain described in this invention. Quantitative analysis of the product was performed as follows:

[0088] Quantitative analysis of 5-HT production by EcNΔtnaA, EcN 5-HTP, and EcN 5-HT strains in Comparative Example 1 was performed. 5-HT production was detected using LC-MS. The 5-HT production of the genetically engineered strains was quantified based on the 5-HT standard curve. The 5-HT fermentation medium was used with streptomycin resistance (100 μg / mL). The results are as follows: Figure 7 As shown.

[0089] In this experiment, approximately 82 mg / L of 5-HT was detected in the engineered strain EcN 5-HT from Example 1, which was significantly higher than that of EcNΔtnaA and EcN 5-HTP. This indicates that the introduction of the rice tryptophan decarboxylase tdc(R) gene significantly increased the production of 5-HT in the strain.

[0090] Example 6: Engineered bacteria EcN 5-HT improves constipation in mice

[0091] Establishment of a mouse constipation model: Male C57BL / 6J mice (SPF, 6 weeks old) were selected. The mice were kept at a room temperature of 22±3℃ with a 12-hour light-dark cycle. All mice had free access to food and water. Except for the control group (Normal, n=8), the remaining 24 mice were intraperitoneally injected twice daily with loperamide (0.8 mg / mL, National Drug Approval Number H10910085) for 7 consecutive days to induce constipation. On day 8, the 24 mice were randomly divided into a constipation model group (Model, n=8).

[0092] The effects of the tnaA knockout group (EcNΔtnaA, n=8) and the 5-HT engineered probiotic intervention group (EcN5-HT, n=8) on improving constipation in mice were compared. Mice in the EcNΔtnaA and EcN5-HT groups were administered 200 μL of 1×10⁹ CFU / mL bacterial suspension (resuspended in 2% NaHCO₃ solution) by gavage, while mice in the Normal and Model groups were administered 200 μL of 2% NaHCO₃ solution by gavage. This was repeated every 1 day for 14 consecutive days. After day 17, under the same conditions and time period, each mouse was placed individually in a sterile empty cage, and feces were collected. The number of feces excreted by the mice within 1 hour was recorded.

[0093] The results are as follows Figure 8 As shown, compared with the normal group, mice in the model group had reduced defecation. After oral treatment with EcN 5-HT engineered bacteria, mice defecation increased and basically returned to normal levels, with no difference from the normal group.

[0094] Example 7: Engineered bacteria EcN 5-HT improve anxiety and depressive symptoms.

[0095] A mouse model of depression was established; mice were randomly assigned to groups, and an open field test was used to compare the effects of oral administration of EcNΔtnaA and the EcN5-HT engineered bacteria from Example 1 on anxiety and depression behaviors in mice. The results are shown in […]. Figure 9-10 ;

[0096] The open field test can measure anxiety and exploration willingness in mice under stress. By tracking the performance of mice in the open field test, recording the total movement distance and the time spent exploring the central area, it was found that the model group mice had significantly reduced movement. Figure 9 The time spent in the central area was significantly reduced, see Figure 10 ;like Figure 11 As shown in the motion trajectory diagram, it can be observed that the mice in the model group prefer to stay in the corner of the open field and have a weaker willingness to explore new environments. This reflects the anxiety state of the mice in the STC model group. However, there is no significant difference between the EcN 5-HT group and the normal group, indicating that the EcN 5-HT engineered bacteria can prevent anxiety and depression in mice.

[0097] Depend on Figure 9 and Figure 10 It can be seen that oral treatment with EcN5-HT engineered bacteria significantly increased the movement distance of STC mice in the open field and the exploration time in the central area, while also showing positive exploration of the unfamiliar environment, indicating that oral treatment with EcN5-HT engineered bacteria can improve the anxiety behavior of STC mice.

[0098] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An engineered 5-hydroxytryptamine strain, characterized in that, The strain is an engineered Escherichia coli strain with an insertion on its chromosome including a tryptophan hydroxylase TPH1 gene expression sequence and a tryptophan deacidase tdc gene expression sequence. The tryptophan hydroxylase TPH1 gene expression sequence, as shown in SEQ TD NO.1, is inserted at the lacZ gene site, and the tryptophan deacidase tdc gene expression sequence, as shown in SEQ TD NO.2, is inserted at the malEK gene site.

2. The engineered 5-hydroxytryptamine strain as described in claim 1, characterized in that, The expression sequence of the tryptophan hydroxylase TPH1 gene is the humanized tryptophan hydroxylase TPH1 gene sequence; the expression sequence of the tryptophan deacidase tdc gene is the rice tryptophan deacidase tdc(R).

3. The engineered 5-hydroxytryptamine strain according to any one of claims 1 or 2, characterized in that, The engineered 5-hydroxytryptamine strain includes Escherichia coli, which lacks the expression gene for the key enzyme in tryptophan production.

4. The engineered 5-hydroxytryptamine strain as described in claim 3, characterized in that, The engineered 5-hydroxytryptamine strain is an EcN strain with the tnaA gene knocked out.

5. The engineered 5-hydroxytryptamine strain as described in claim 4, characterized in that, The engineered 5-hydroxytryptamine strain has a constitutive promoter in its recombinant gene, which includes a pBAD promoter with the araC regulatory element removed.

6. A formulation for promoting intestinal motility, characterized in that, The intestinal motility agent comprises the engineered 5-hydroxytryptamine strain as described in any one of claims 1 to 5.

7. The use of the engineered 5-hydroxytryptamine strain according to any one of claims 1 to 5 in the preparation of a medicament for improving intestinal dysfunction and / or anxiety-depression-like behavior, characterized in that, The intestinal dysfunction is functional constipation.

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