Advanced microbiome therapeutics designed to produce serotonin in vivo

Abstract

The present invention provides a composition for use as a pharmaceutical, comprising cells of a recombinant microorganism capable of producing one or more of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT), and tryptamine (TRM) in increased amounts compared to the non-recombinant microorganism from which it was derived. The composition finds use in the prevention and / or treatment of TRM, 5-HTP, or 5-HT-related disorders of the central nervous system (CNS), enteric nervous system (ENS), gastrointestinal (GI), and metabolism in mammals, and can be orally administered to a mammal in need thereof. Additionally, a composition comprising cells of a recombinant microorganism capable of producing melatonin is provided for use as a pharmaceutical, such as for the treatment of depression, dementia, cancer, and sleep disorders.

Description

[Technical field]

[0001] The present invention provides a composition for use as a medicine, comprising cells of a recombinant microorganism capable of producing one or more of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT) and tryptamine (TRM) in increased amounts compared to the non-recombinant microorganism from which it was derived. The composition finds use in the prevention and / or treatment of TRM, 5-HTP or 5-HT related disorders of the central nervous system (CNS), enteric nervous system (ENS), gastrointestinal (GI) and metabolism in mammals and may be orally administered to a mammal in need thereof. Additionally, a composition comprising cells of a recombinant microorganism capable of producing melatonin is provided for use as a medicine. [Background technology]

[0002] The biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT) is important for neurotransmission and many other functions throughout the body. Up to 95% of serotonin in the body is produced in the gastrointestinal tract. As shown in Figure 1, serotonin is derived from tryptophan (TRP) in a two-step reaction, first by tryptophan hydroxylase (TPH) to form a 5-hydroxytryptophan (5-HTP) intermediate, and then by tryptophan decarboxylase (TDC). In humans, tryptophan hydroxylase is the rate-limiting step in 5-HT biosynthesis and is made in two isoforms, TPH1 and TPH2. TPH2 is produced in neurons throughout the body, whereas TPH1 is expressed primarily in neuroendocrine cells in the gut.

[0003] In the gastrointestinal tract, serotonin regulates intestinal motility, cell turnover, and homeostasis. In the brain, imbalances in serotonin levels are associated with anxiety and depression. Treatment involves strategies to increase serotonin in patients suffering from a wide range of disorders associated with anxiety and depression.

[0004] Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for the treatment of depression. They are thought to increase extracellular levels of the neurotransmitter serotonin by limiting its reabsorption (reuptake) into presynaptic cells and increasing the levels of serotonin in the synaptic cleft available to bind to postsynaptic receptors. A subgroup of patients exhibits symptoms of SSRI treatment resistance (TRD). Treatment of patients with TRD, currently undergoing clinical trials in mammalian models, is based on combination therapy with an SSRI (serotonin reuptake inhibitor) and the serotonin precursor 5-hydroxytryptophan (5-HTP). This has the undesirable consequence of leading to a sudden increase in 5-HT levels due to the rapid uptake of administered 5-HTP. Efforts to address this issue are currently focused on combination therapy using sustained-release forms of 5-HTP.

[0005] Gut bacteria play an important role in gastrointestinal (GI) health and homeostasis. Gut microbes are thought to not only regulate gut homeostasis but also participate in bidirectional signaling pathways by the enteric nervous system (ENS) and central nervous system (CNS) along the gut-brain axis. Rather than providing a significant supply of 5-HT themselves, the resident gut microbiota are known to signal host mucosal endocrine cells to maintain intestinal 5-HT content and 5-HT plasma levels. More recently, oral ingestion of certain bacteria in animal models has been shown to result in changes in host physiology and behavior, suggesting a role for these microbes in gut-brain coupling.

[0006] In the hippocampus, striatum, cortex, and dentate gyrus of the brain, serotonin-based biosynthetic pathways, serotonin-based receptors, and serotonin-based signaling pathways are involved in memory, cognition / aging-related spatial learning, and memory formation. Serotonergic afferent neurons from the gastrointestinal tract modulate the dorsal motor nucleus of the vagus and the nucleus of the solitary tract via the vagus nerve, so that gut 5-HT is involved in neuronal memory, cognition, and learning.

[0007] Given the important role of TRM and 5-HT in mammalian homeostasis and pathologies resulting from insufficient 5-HT levels, new therapies are needed to increase 5-HT levels in patients, particularly those suffering from anxiety-related disorders, and TRD.

[0008] Melatonin, a signaling molecule, is a highly pleiotropic molecule that is released as a pineal hormone primarily at night. Melatonin secretion decreases with age, but reduced melatonin levels are also observed in various diseases, such as some types of dementia, some mood disorders, severe pain, cancer, and type 2 diabetes. Melatonin dysfunction is often associated with deviations in amplitude, phase, and coupling of circadian rhythms. Thus, there is a need for therapies that increase melatonin levels in patients who need it. Summary of the Invention

[0009] In a first embodiment, the present invention relates to a composition for use as a medicament, the composition comprising cells of a recombinant microorganism, the microorganism comprising: (a) tryptophan 5-hydroxylase (EC 1.14.16.4), (b) tryptophan decarboxylase (EC 4.1.1.28), and (c) a composition comprising one or more recombinant nucleic acid molecules encoding one or more proteins selected from tryptamine 5-hydroxylase (EC:1.14.-.-).

[0010] Thus, the cell is capable of producing increased amounts of one or more of 5-hydroxytryptophan, 5-hydroxytryptamine and tryptamine compared to the cell of the non-recombinant microbial organism from which it was derived.

[0011] In a second embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (a) tryptophan 5-hydroxylase (EC 1.14.16.4), (b) tryptophan decarboxylase (EC 4.1.1.28), and (c) providing a recombinant bacterial cell, the recombinant bacterial cell comprising one or more recombinant nucleic acid molecules or transgenes encoding one or more proteins selected from tryptamine 5-hydroxylase (EC:1.14.-.-); The microorganism lacks genes capable of expressing the trp operon repressor protein and tryptophanase (EC:4.1.99.1), and therefore the cells are capable of producing increased amounts of one or more of 5-hydroxytryptophan, 5-hydroxytryptamine, and tryptamine compared to cells of the non-recombinant microorganism from which they were derived.

[0012] Preferably, the recombinant bacterial cell is selected from among Escherichia, Bacteroides, Clostridium, Feacalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Staphylococcus and Bacillus.

[0013] In a third embodiment, the present invention provides a method for treating and / or preventing a TRM, 5-HTP, 5-HT, or melatonin related disorder in a subject, comprising administering to a subject diagnosed with a TRM, 5-HTP, 5-HT, or melatonin related disorder: (a) tryptophan 5-hydroxylase (EC 1.14.16.4), (b) tryptophan decarboxylase (EC 4.1.1.28), or (c) administering a recombinant bacterium engineered to express one or more of tryptamine 5-hydroxylase (EC:1.14.-.-).

[0014] Thus, the cell is capable of producing increased amounts of one or more of 5-hydroxytryptophan, 5-hydroxytryptamine and tryptamine relative to the cell of the non-recombinant microbial organism from which it is derived.

[0015] The TRM, 5-HTP, 5-HT, or melatonin related disorder can be a central nervous system (CNS), enteric nervous system (ENS), gastrointestinal (GI) or metabolic disorder in a mammal.

[0016] In a fourth embodiment, the present invention provides a recombinant microorganism comprising one or more recombinant nucleic acid molecules or transgenes, the microorganism comprising: (a) tryptophan 5-hydroxylase (EC 1.14.16.4) and tryptophan decarboxylase (EC 4.1.1.28), or (b) Tryptophan decarboxylase (EC 4.1.1.28) and tryptamine 5-hydroxylase (EC:1.14.-.-) and (c) serotonin acetyltransferase (AANAT) EC 2.3.1.87 and (d) comprising a recombinant nucleic acid molecule encoding acetylserotonin O-methyltransferase (ASMT) (EC 2.1.1.4); A recombinant microorganism is provided in which the cells are capable of producing melatonin, and thus the cells are capable of producing increased amounts of melatonin compared to the cells of the non-recombinant microorganism from which they were derived.

[0017] In a fifth embodiment, the present invention provides a composition for use as a medicament, the composition comprising cells of a recombinant microbial organism capable of producing melatonin according to the fourth embodiment, suitable for oral delivery to patients suffering from the following melatonin related disorders: circadian rhythm disorders, insomnia, jet lag, melatonin deficiency or melatonin related disorders such as autism, dementia, mood disorders, severe pain, cancer, and type 2 diabetes. [Brief description of the drawings]

[0018] Description of the invention BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Cartoon showing the catalytic activities of the 5-HT and melatonin biosynthetic pathways: TrpR (tryptophan operon repressor protein), TnaA (tryptophanase), FolE (encoding mutant GTP cyclohydrolase 1 (T198I)), TRP (tryptophan), TRM (tryptamine), 5-HTP (5-hydroxytryptophan), 5-HT (serotonin), TPH (tryptophan hydroxylase), TDC (tryptophan decarboxylase), T5H (tryptamine 5-hydroxylase), serotonin acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). [Diagram 2] [A] Histograms showing titers of TRM (tryptamine), 5-HTP (5-hydroxytryptophan), and 5-HT (serotonin) measured in medium derived from cultures of cells of six E. coli Nissle strains engineered to coexpress the TPH and TDC genes encoding human TPH1 and rice TDC (H1R), human THP2 (H2) and rice TDC (H2R), human THP2 (H2) and Cataranthus roseus TDC (H2C), human THP2 (H2) and guinea pig TDC (H2G), mouse TPH1 (M1) and rice TDC (M1R), and mouse THP2 (M2) and rice TDC (M2R), respectively. [B] Histograms showing titers of TRM (tryptamine), 5-HTP (5-hydroxytryptophan), and 5-HT (serotonin) measured in medium derived from 5-HTP-fed cultures of cells of three E. coli Nissle strains engineered to coexpress the TPH and TDC genes encoding human THP2 (H2) and rice TDC (H2R), human THP2 (H2) and Cataranthus roseus TDC (H2C), and human THP2 (H2) and guinea pig TDC (H2G), respectively. [Diagram 3]Histogram showing titers of TRM (tryptamine), 5-HTP (5-hydroxytryptophan), and 5-HT (serotonin) measured in medium after cultivation of E. coli Nissle strain (EcN△2+pUC-H2R) co-expressing TPH and TDC in growth medium supplemented with substrate in the range of 10 to 100 mg tryptophan / L. [Figure 4] [A] Graph showing cell density, measured as units of OD600 nm, of cultures of four E. coli Nissle strains. The strains are EcN△2+pUC-H2R, EcN△3+pUC-H2R, EcN△2+pMUT-H2R, each co-expressing human TPH2(H2) and rice TDC(R), and EcN +pMUT is an empty plasmid. EcN△2 has the deletions ΔtrpR, ΔtnaA, and EcN△3 has the deletions ΔtrpR, ΔtnaA, ΔinfA. [B] Graph showing cell density, measured as units of OD600 nm, of cultures of nine E. coli Nissle strains co-expressing human TPH2(H2) or mouse TPH1(M1) with rice TDC(R), and a control strain containing an empty plasmid. [C] Graph showing the percentage of kanamycin-resistant colonies detected in cultures of E. coli Nissle strains containing pUC- or pMUT-based serotonin-producing plasmids measured over a growth period of 100 generations without antibiotic selection. At each time point, cells were plated on LB agar plates + / - kanamycin and the percentage of colonies was calculated (n=6). [D] Histogram showing the titer for 0-100 cell generations of 5-HT (serotonin) measured in the medium after culture of E. coli Nissle strains co-expressing human TPH2 (H2) and rice TDC (R) cloned in pUC (pUC-H2R) or pMUT (pUC-H2R) plasmids. [Diagram 5][A] Histograms showing the titers of TRM (tryptamine) and 5-HT (serotonin) measured in the medium after cultivation of E. coli Nissle strains N, NΔ2 and "oN" co-expressing the human TPH2 and rice TDC (H2R) or mouse TPH1 and rice TDC (M1R) operons on pUC or pMUC plasmids or harboring the empty pMUT plasmid. [B] Histograms showing the titers of TRM (tryptamine), 5-HTP (hydroxytryptophan) and 5-HT (serotonin) measured in the medium after cultivation of E. coli Nissle "oN" strains expressing mouse TPH1 (oN10=oN+pMUTM1), rice TDC (oN11=oN+pMUTR), mouse TPH1 and rice TDC (oN14=oN+pMUTM1R) or harboring the empty plasmid (oN9=pMUT). All data shown are means with standard deviation error bars from n=3 biological replicates. [C] and [D] Left panel: Histograms showing the titers of TRP (tryptophan), TRM (tryptamine), 5-HTP (5-hydroxytryptophan) and 5-HT (serotonin) measured in the medium after cultivation of E. coli Nissle strains containing plasmids pMUT-H2R and pMUT-M1R operably linked to RBS sequences with a wide range of translation strengths. Right panel: Two-dimensional diagram showing the yields of 5-HT, 5-HTP and tryptamine in the culture medium measured for E. coli Nissle strains containing plasmid pMUT-H2R operably linked to RBS sequences with a range of translation strengths, where the x-axis corresponds to the RBS strength of the TPH gene and the y-axis corresponds to the RBS strength of the TDC, and the crossed-out boxes indicate combinations not tested. [E] Cartoon showing the genetic modifications into the E. coli Nissle genome and the genetic structure of the plasmids tested in [B]. [F] Histogram showing the 5-HTP titers measured in the medium after cultivation of E. coli Nissle strains NΔ2 (=folE WT) or oN (=folE(T1981I)), each containing the human TPH2pMUT plasmid. [Figure 6][A] Histograms showing 5-HT (serotonin) and TRM (tryptamine) titers measured in culture medium after cultivation of E. coli Nissle strain NΔ2 or strain oN co-expressing human TPH2 and rice TDC (H2R) genes, respectively, operably linked to RBS sequences of different translation strengths cloned into a pUC plasmid. [B] Histograms showing 5-HT (serotonin) and TRM (tryptamine) titers measured in culture medium after cultivation of E. coli Nissle strain NΔ2 or strain oN expressing human TPH2 or mouse TPH1 genes, respectively, in combination with rice TDC (H2R) genes operably linked to RBS sequences of different translation strengths cloned into a pMUT plasmid. [C] Graph showing theoretical 5-HT concentration in mouse gut versus abundance of 5-HT-producing bacteria measured as a percentage of the total gut microbiota. The theoretical physiological levels of 5-HT in the intestine are in the range of 5-30 mg / L, as shown. The relative abundance of 5-HT-producing bacteria required to produce 5-HT within the therapeutic range is plotted for bacteria with in vivo 5-HT production rates (P) between 0.1 and 10 mg / L*OD630*h. [Figure 7] [A] Images of thin sections of mesocolon from mice 24 hours after the last oral gavage of E. coli strains oN9, oN11, oN14, and control (PBS), where sections were stained with DAPI and anti-GFP antibodies. [B] Graph showing kanamycin-resistant GFP-positive bacterial concentrations in duodenum, jejunum ileocecal, cecum, proximal colon, distal colon, and feces from mice 24 hours after the last oral gavage with either E. coli strains oN9, oN11, or oN14, or control (PBS). Samples plated on LB agar plates and kanamycin, and colonies counted as colony forming units (CFU) per gram of feces (n=6). Data points indicated with y=0 were below the detection level of 103 CFU / gram. [Figure 8]Histograms showing [A] tryptophan and [B] 5-HT concentrations measured as mg / L in serum, mg / g biomass in washed homogenized E. coli tissues, and mg / g biomass in fecal material (n=7-8) from mice 24 h after the last oral gavage with either E. coli strains oN9, oN11, oN14, or control (PBS). All p-values ​​shown are from one-way ANOVA with Tukey's post-hoc correction for multiple comparison testing. All other comparisons not shown were not significant at p>0.05. Data are means with error bars as standard error of the mean. [C] Images of thin sections of mesocolon from mice 24 h after the last oral gavage with E. coli strains oN9, oN11, oN14, and control (PBS). Here, sections were stained with DAPI and anti-serotonin antibodies, and scale bars are 100 μm. [Figure 9] [A] Histograms showing relative expression of MUC2, TPH1, TPH2, SERT gene expression levels measured as relative mRNA by ΔΔCτ (n=6) in colonic tissue from mice 24 h after the last oral gavage of E. coli strains oN9, oN11, or oN14, or control (PBS). Data are means with error bars as standard error of the mean. [B] Images of mesocolon slices from mice 24 h after the last oral gavage with E. coli strains oN9, oN11, oN14, and control (PBS). Here, sections were stained with DAPI and anti-Muc-2 antibodies, and the scale bar is 400 μm. Arrows indicate increased MUC2 signal in oN14. [Figure 10]Histograms [(a)-(e)] showing relative expression of 5-HT receptors HTR1B HTR1D, HTR3, HTR4, and HTR7 in the colon by RT-PCR (n=6) in colonic tissue from mice 24 h after the last oral gavage with either E. coli strains oN9, oN11, oN14, or control (PBS). Histogram (f) showing total gastrointestinal transit time from mouth to anus using 6% carmine red solution (Methods) (n=7-8) in correspondingly treated cohorts of mice. All p-values ​​shown are from one-way ANOVA with Tukey's post-hoc correction for multiple comparison testing. All other comparisons not shown were not significant at p>0.05. Data are means with error bars as standard error of the mean. [Figure 11] Histograms showing (a) the time mice spent immobile during the last 4 min of a 6 min forced swim test (FST) (n=9-10), (b) the number of fecal pellets (fecal boli) produced during a 10 min open field test (OFT) (n=7-10), (c) the average total distance mice traveled during a 10 min OFT (n=7-10), (d) the time spent in both the inner 40 cm x 40 cm zone or the outer 10 cm ring during the OFT (n=7-10), and (e) representative tracking patterns of one mouse from each mouse cohort after oral gavage with either E. coli strains oN9, oN11, oN14, or control (PBS). All p values ​​shown are from one-way ANOVA with Tukey's post-hoc correction for multiple comparisons testing. All other comparisons not shown were not significant at p>0.05. Data are means with error bars as standard error of the mean. [Figure 12](a) Histogram showing the number of excretions produced during a 10-min open field test (OFT) (n=7-10), (b) the time mice spent in the 40 cm × 40 cm inner zone during the OFT (n=7-10), (c) the average distance mice traveled in the inner 40 cm × 40 cm zone during a 10-min OFT (n=7-10), (d) the number of mice entering the 40 cm × 40 cm inner zone during a 10-min OFT (n=7-10), (e) the average total distance mice traveled during a 10-min OFT (n=7- 10), (f) The time that mice stayed immobile during the last 4 min of a 6 min forced swim test (FST) (n=9-10) measured for each mouse cohort 21 days after gavage: PBS / IP:saline (negative control), gavage:PBS / IP:fluoxetine (SSRI positive comparator), gavage:E. coli strain oN14 / IP:saline (oN14 positive comparator), and gavage:E. coli strain oN14 / IP:fluoxetine (positive combination comparator). All p values ​​shown are from one-way ANOVA with Tukey's post-hoc correction for multiple comparisons testing. All other comparisons not shown were not significant at p>0.05. Data are means with error bars as standard error of the mean. [Figure 13] Histograms showing (A) 5-HT and (B) tryptophan concentrations in peripheral plasma samples and C) 5-HT and D) 5-HTP concentrations in urine samples in mice following oral gavage with either E. coli strain oN10 or an E. coli strain lacking TPH expression (EcN-control) (n=8, p<0.001 by two-tailed t-test for plasma 5-HT). [Figure 14] Cartoon showing the structure of plasmids for expression of the serotonin pathway in S. cerevisiae (pSc-1 to pSc-3). RTDC = tryptophan decarboxylase gene (rice), ck-TDC = tryptophan decarboxylase gene (Candidatus Koribacter versatilis Ellin345), TPH = tryptophan 5-hydroxylase 1 (mouse). [Figure 15](A) Histograms showing 5-HT (serotonin) titers measured in the culture medium after culturing S. cerevisiae with or without plasmids expressing mouse TPH1 and TDC genes from either rice (Sc-1 and Sc-2) or from Candidatus Koribacter versatilis Ellin345 (Sc-3). (B) Histograms showing 5-HT (serotonin) and TRM (tryptamine) titers measured in the culture medium after culturing E. coli Nissle strains co-expressing mouse TPH1 and TDC genes from either rice (oN14) or from Candidatus Koribacter versatilis Ellin345 (M1ck). (C) Graph showing cell density of cultures of the four E. coli Nissle strains measured as units of OD600 nm; oN control contained pMh-empty, oN14 contained pMUT14-5HT, M1R(trc) = mouse TPH1 and rice TDC expressed from the trc promoter, M1ck(trc) = mouse TPH1 and ck-TDC expressed from the trc promoter [SEQ ID NO: 112]. [Figure 16] Cartoon showing the structure of the plasmid for expression of the serotonin pathway in B. subtilis (pBS-M1Rf). R-TDC = tryptophan decarboxylase gene (rice), TPH = tryptophan 5-hydroxylase 1 gene (mouse), FolE = mutant GTP cyclohydrolase I gene (GCH1).

[0019] Abbreviations, Terms and Definitions: "Non-recombinant microorganism from which it is derived" means a microorganism that does not contain a recombinant nucleic acid molecule.

[0020] "Produces increased amounts" means compared to a non-recombinant organism that does not contain a recombinant nucleic acid molecule. For example, a recombinant microbial cell expressing a claimed recombinant enzyme is compared to a microbial cell that does not contain a nucleic acid molecule for the claimed enzyme. As a result of expression of an encoded nucleic acid that expresses an enzyme in a biosynthetic pathway according to Figure 1, the cell with the recombinant nucleic acid encoding the enzyme will produce increased amounts of one or more of 5-hydroxytryptophan, 5-hydroxytryptamine, and tryptamine.

[0021] gi number: (genInfo identifier) ​​is a unique integer that identifies a particular sequence regardless of database source, and is assigned by NCBI to all sequences processed by Entrez, including nucleotide sequences from DDBJ / EMBL / GenBank, protein sequences from SWISS-PROT, PIR and many others.

[0022] Amino acid sequence identity: The term "sequence identity" as used herein refers to a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length. The two sequences to be compared should be aligned to give the best possible match by inserting gaps or truncating the ends of protein sequences. Sequence identity can be calculated as ((Nref-Ndif)100) / (Nref), where Ndif is the total number of non-identical residues when the two sequences are aligned, and Nref is the number of residues in one sequence. Sequence identity calculation is preferably automated using a BLAST program, such as the BLASTP program (Pearson WR and DJ Lipman (1988)) (www.ncbi.nlm.nih.gov / cgi-bin / BLAST). Multiple alignments are performed using the sequence alignment method ClustalW using the default parameters described by Thompson J., et al 1994, available at http: / / www2.ebi.ac.uk / clustalw / .

[0023] Preferably, the number of one or more substitutions, insertions, additions or deletions of amino acid residues in a polypeptide compared to its reference polypeptide is limited, i.e., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions. Preferably, the substitutions are conservative amino acid substitutions and are limited to exchanges within members of Group 1: glycine, alanine, valine, leucine, isoleucine, Group 2: serine, cysteine, selenocysteine, threonine, methionine, Group 3: proline, Group 4: phenylalanine, tyrosine, tryptophan, Group 5: aspartic acid, glutamate, asparagine, glutamine.

[0024] Lacking a gene encoding a functional protein: A microorganism that lacks a gene capable of expressing a functional protein (e.g., tryptophan repressor or tryptophanase) is a microorganism that is unable to express the functional protein because it lacks the respective gene or the gene id is modified (e.g., inactivated). A series of genetic modifications are suitable for inactivating a gene, including deletion (knockout) of the gene from the genome of the microbial cell, deletion of its cognate regulatory sequence (e.g., promoter), substitution or addition of at least one nucleotide that results in loss of expression of the functional polypeptide encoded by the gene. If the encoded polypeptide is an enzyme, the genetic modification results in loss of detectable enzymatic activity of the respective polypeptide in the microbial cell.

[0025] Genome: The genetic material present in a cell or organism. The genome contains all the information necessary to build and maintain that cell or organism, and includes the genetic material in both chromosomes and plasmids present within the cell or organism.

[0026] Mutant GTP cyclohydrolase I (EC 3.5.4.16): abbreviated herein as mutant GCH1, should be understood as a mutant GCH1 that results in at least a 2-fold (e.g., at least a 3-fold, or a 4-fold, or a 5-fold, or a 6-fold, or a 7-fold, or a 8-fold, or a 9-fold, or a 10-fold) increased hydroxylation activity of tryptophan 5-hydroxylase (EC 1.14.16.4) as measured by 5-hydroxytryptophan yield, compared to non-mutated GCH1. The non-mutated GCH1 may be the parent enzyme from which the mutant is derived, whereby the mutation found in the mutant results in the increased activity.

[0027] Native gene: an endogenous gene within a microbial cell genome that is homologous to the host microorganism.

[0028] RBS: Ribosome binding site is a sequence of nucleotides upstream of the start codon of an mRNA transcript and is involved in the recruitment of ribosomes at the initiation of protein translation.

[0029] Ribosome Binding Site (RBS) Calculator: Provides a method to predict or control translation initiation rates (TIR) ​​in bacteria. When used to control the translation of a specific coding sequence, the RBS calculator generates synthetic DNA sequences that result in a defined translation initiation rate (and therefore protein expression strength). For any promoter sequence (which controls the transcription strength), many different RBS strengths can be designed to modulate the amount of protein produced from this transcript (Salis at al., 2009).

[0030] RBS intensity scale and units: Output values ​​are in the range of 1-1 million on a linear scale with arbitrary units experimentally validated using fluorescent protein abundance as a measure of expression intensity (Salis at al., 2009). A designed sequence of a particular intensity is not unique; i.e., different nucleotide sequences may code for RBSs with the same RBS intensity. Furthermore, RBS intensity is always context-dependent, so a designed sequence of defined intensity is only valid for the coding sequence for which it was calculated. The minimal nucleotide sequence that defines the RBS intensity is 35 base pairs upstream and 50 base pairs downstream of the start codon (ATG) of the coding sequence.

[0031] Transgene: A gene or genetic material that has been transferred from one organism to another, either naturally or by any of a number of genetic engineering techniques. The transgene transferred to the recipient can be from another individual of the same species or from an unrelated species. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] I: In a first aspect, the present invention relates to a composition for use as a medicament, the composition comprising cells of a recombinant microorganism, the microorganism comprising: (a) tryptophan 5-hydroxylase (EC 1.14.16.4), (b) tryptophan decarboxylase (EC 4.1.1.28), and (c) one or more transgenes or recombinant nucleic acid molecules encoding one or more proteins selected from tryptamine 5-hydroxylase (EC:1.14.-.-). Thus, the microbial cells are capable of producing increased amounts of one or more of 5-hydroxytryptophan (5-HT), 5-hydroxytryptamine (5-HTP) and tryptamine (TRM) compared to the non-recombinant microbial cells from which they were derived.

[0033] In a further embodiment thereof, the one or more recombinant nucleic acid molecules (a) tryptophan 5-hydroxylase (EC 1.14.16.4), (b) tryptophan decarboxylase (EC 4.1.1.28), (c) tryptamine 5-hydroxylase (EC:1.14.-.-), (d) tryptophan 5-hydroxylase (EC 1.14.16.4) and tryptophan decarboxylase (EC 4.1.1.28), and (e) encoding a protein selected from the group consisting of tryptophan decarboxylase (EC 4.1.1.28) and tryptamine 5-hydroxylase (EC: 1.14.-.-).

[0034] In a further embodiment thereof, the composition is for use as a therapeutic agent in preventing and / or treating TRM of the central nervous system (CNS), enteric nervous system (ENS), gastrointestinal (GI), 5-HTP or 5-HT related disorders, hormonal imbalance, metabolic disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), diabetes in a mammal.

[0035] More specifically, the TRM, 5-HTP or 5-HT homeostasis of the CNS that is treated by the composition of the present invention includes anxiety and depression-related behaviors, as well as memory disorders, cognitive disorders, and psychiatric disorders, particularly generalized anxiety disorder, phobic disorders, social anxiety disorder, panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, chronic stress disorder, separation and situational anxiety, age-related memory decline, and spatial memory formation, disorders of wakefulness, concentration, learning and cognition, autism, migraine, and immune-related disorders.The TRM or 5-HT-related therapies of the GI that are treated by the composition of the present invention include movement disorders, metabolic syndrome, obesity, weight control, inflammation-related diseases, inflammatory bowel disease, irritable bowel syndrome (IBS), celiac disease, diverticulosis, and colorectal cancer.

[0036] In another aspect, the present invention provides a composition for use as a medicament comprising a recombinant microorganism, the microorganism comprising: (a) tryptophan 5-hydroxylase (EC 1.14.16.4) and tryptophan decarboxylase (EC 4.1.1.28), or (b) one or more transgenes or recombinant nucleic acid molecules encoding tryptophan decarboxylase (EC 4.1.1.28) and tryptamine 5-hydroxylase (EC:1.14.-.-), moreover, (c) serotonin acetyltransferase (AANAT) EC 2.3.1.87 and (d) comprising a transgene or recombinant nucleic acid molecule encoding acetylserotonin O-methyltransferase (ASMT) (EC 2.1.1.4); Compositions are provided in which the cells are capable of producing melatonin, thus enabling the cells to produce increased amounts of melatonin compared to the cells of the non-recombinant microorganism from which they were derived.

[0037] In a further aspect thereof, the present invention provides a composition for use as a medicament, the composition comprising cells of a recombinant microbial organism capable of producing melatonin, suitable for oral delivery to patients suffering from the following melatonin-related disorders: circadian rhythm disorders, insomnia, jet lag, melatonin deficiency or melatonin-related disorders such as autism, dementia, mood disorders, severe pain, cancer, and type 2 diabetes.

[0038] In a further embodiment thereof, the composition is for oral administration to a mammal in need thereof.

[0039] In a further embodiment thereof, the microorganism in the composition is a live facultative anaerobic enterobacteria or yeast (Ianiro G et al., 2014), preferably a commensal or probiotic strain characterized by its ability to survive and / or colonize one or more regions of the mammalian intestine. For example, the bacteria is selected from Escherichia, Bacteroides, Clostridium, Feacalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Staphylococcus and Bacillus. Suitable species of bacteria include Escherichia coli (e.g., Nissle strain), Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, Bifidobacterium longum and Bifidobacterium adolescentis.Furthermore, suitable microorganisms in the composition can be selected from the microbial species identified by Li et al., 2014 and Zou et al., 2019 (the teachings of which are incorporated by reference).

[0040] In a further embodiment thereof, the microorganism in the composition comprises a nucleic acid sequence encoding tryptophan 5-hydroxylase (TPH) (EC 1.14.16.4), which catalyzes the conversion of L-tryptophan + tetrahydrobiopterin + O(2) => 5-hydroxy-L-tryptophan + 4a-hydroxytetrahydrobiopterin by hydroxylating tryptophan at the C5 position (Figure 1). Assays and quantification of 5-hydroxy-L-tryptophan yield are described in 1.1.2 and 1.1.3, respectively. Following oral administration to a mammal, the microorganism in the composition can express the TPH enzyme in the mammalian intestine, such as producing and secreting 5-HTP (Figure 2A). When expressed in the microorganism of the present invention, both isoforms of tryptophan hydroxylase, TPH1 and TPH2, are suitable for producing 5-HTP, and 5-HT when co-expressed with TDC having EC 4.1.1.28 (Figure 2A). However, in a preferred embodiment, the TPH enzyme expressed in the microorganism in the composition for administration to the gastrointestinal tract of a mammal is TPH1 due to the low oxygen environment of the intestine and the higher oxygen binding affinity of TPH1 compared to TPH2.

[0041] The microorganism in the composition may additionally or alternatively contain a nucleic acid molecule encoding tryptophan decarboxylase (TDC) (EC 4.1.1.28), which catalyzes the conversion of 5-hydroxy-L-tryptophan => 5-hydroxytryptamine + CO2 (Figure 1). Assays and quantification of 5-hydroxytryptamine yield are described in 1.1.2 and 1.1.3. TPH enzyme Basic Compositions containing microbial cells capable of expressing both TPH and TDC enzymes can produce and secrete 5-HT in the mammalian intestine (Figure 2A). In contrast, compositions containing microbial cells capable of expressing TDC enzymes can produce and secrete TRM in the mammalian intestine (Figures 1, 5B). Microbial 5HT productivity is dependent on the TPH enzyme expressed in the cells. Basicand TDC enzymes (Example 1). Thus, in some microorganisms, the combination of expressed enzymes is: (a) The tryptophan 5-hydroxylase has at least 80% sequence identity to SEQ ID NO: 6 or 12, and the amino acid sequence of the tryptophan decarboxylase has at least 80% sequence identity to SEQ ID NO: 18. .

[0042] In a further embodiment, the microorganism in the composition comprises a recombinant nucleic acid molecule or transgene encoding tryptamine 5-hydroxylase, which catalyzes the conversion of O2+ reduced [NADPH-heme protein reductase] + tryptamine => H++H2O+ oxidized [NADPH-heme protein reductase] + serotonin (Figure 1). Assay and quantification of tryptamine yield are described in 1.1.2 and 1.1.3. Preferably, the microorganism in the composition further comprises a nucleic acid sequence encoding TDC. Following oral administration to a mammal, the microorganism in the composition can express TPH and T5H enzymes in the mammalian intestine, such as producing and secreting 5-HT (Figure 1).

[0043] In a further embodiment, the microorganism in the composition comprises a nucleic acid molecule or transgene encoding either TPH and TDC, or TDC and T5H, and the following enzymes: - serotonin acetyltransferase (AANAT) EC 2.3.1.87 (which catalyzes the conversion of acetyl-CoA and serotonin to CoA and N-acetylserotonin), and -acetylserotonin O-methyltransferase (ASMT) (EC 2.1.1.4), which catalyzes the final reaction in the production of melatonin from L-tryptophan, the conversion of N-acetylserotonin and S-adenosyl-L-methionine (SAM) to melatonin and S-adenosyl-L-homocysteine ​​(SAH). SAH can then be recycled to SAM via the S-adenosyl-L-methionine cycle in microbial cells in which the S-adenosyl-L-methionine cycle is native (i.e., exogenously added) and constitutively expressed, e.g., E. coli (Figure 1).

[0044] In a further embodiment, the microorganism is genetically modified by the introduction of heterologous nucleic acid molecules encoding the 5-HT or melatonin pathway enzymes, including TPH, TDC, T5H, AANAT and ASMT enzymes, wherein each heterologous nucleic acid molecule is cognately linked to a promoter, such as a constitutive or inducible promoter. The heterologous nucleic acid molecules encoding the enzymes can be cloned into an operon linked to a common cognate promoter. The heterologous nucleic acid molecules can be cloned into a self-replicating episome introduced into the microorganism, or can be cloned into the chromosome of the microorganism. The episome can be a natural plasmid of the microorganism or a heterologous plasmid. Preferably, the plasmid lacks genetic elements that facilitate transduction into another microbial cell, or the plasmid contains a gene encoding a protein essential for the survival of the microbial cell (i.e., the essential gene described in Example 3).

[0045] In a further embodiment thereof, the microorganism is further genetically modified in that it lacks a gene capable of expressing a functional Trp operon repressor protein, and / or a functional tryptophanase (EC: 4.1.99.1) (FIG. 1). A microorganism that cannot express a functional Trp operon repressor protein cannot prevent the initiation of transcription of the tryptophan biosynthetic pathway because it lacks the repressor protein necessary to complex with L-tryptophan and bind to the operator region of the trp operon (e.g., 5'-ACTAGT-'3'). A microorganism that cannot express a functional tryptophanase (EC 4.1.99.1) cannot catalyze the reaction L-tryptophan + H(2)O<=>indole + pyruvate + NH(3). Microorganisms of the invention that lack a gene capable of expressing a functional Trp operon repressor protein and a functional tryptophanase (EC 4.1.99.1) produce enhanced levels of 5-HT and TRM (Examples 2, 4 and Figures 3, 6A) due to enhanced flux into the tryptophan pathway.

[0046] In a further embodiment thereof, the microorganism is further genetically modified to include a gene encoding mutant GTP cyclohydrolase I EC 3.5.4.16 (GCHI), and the mutant provides at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increased TPH hydroxylation activity as measured by 5HTP yield compared to native non-mutant GCH1 (e.g., parent GCH1). GCH1 catalyzes the regeneration of tetrahydrobiopterin cofactor from GTP, which is required for TPH synthesis (Figure 1). The microorganism of the present invention expressing mutant GCH1 enzyme lacking both trpR and tnaA genes produces increased amounts of 5-HT and TRM (Example 4, Figures 5A, 6A) due to enhanced flux to 5HTP synthesis.

[0047] In a further embodiment thereof, the microorganism further comprises a recombinant nucleic acid sequence encoding (a) tryptophan 5-hydroxylase (EC 1.14.16.4) and (b) tryptophan decarboxylase (EC: 4.1.1.28), and is further genetically modified to regulate the expression levels of the TPH and TDC enzymes of the 5-HT pathway, such as further increasing the amount of 5-HT produced while decreasing TRM production. By way of example, the expression of TPH and TDC is independently regulated by an RBS operably linked to each of their respective nucleic acid coding sequences (i.e. genes). As shown in Example 4 (Figures 5C, D, 6A), when the RBS of the TPH gene has a strength greater than that of the TDC gene (measured as the relative strength of the translation start), the 5-HT yield is increased and the TRM level is decreased. The 5-HT productivity of the microorganism of the invention lacking the trpR and tnaA genes is synergistically increased by virtue of their respective operably linked RBSs when the translation rate of the TPH gene transcript exceeds that of the TDC gene transcript (Figure 6A,B).

[0048] The composition according to the invention is for use as a medicine for animals, in particular mammals selected from the group consisting of humans, dogs, cats, pigs, cows, horses, goats and sheep, and poultry (e.g. chickens). In one embodiment, the composition is for use as a medicine for pregnant or lactating women.

[0049] In therapeutic applications, such as treating TRM, 5-HTP-, or HT-related disorders of the central nervous system (CNS), enteric nervous system (ENS), gastrointestinal (GI) and metabolic or melatonin-related disorders in mammals, the compositions according to the present invention are intended to be administered in an amount sufficient to at least partially cure or prevent the symptoms of the disease and its complications.An amount sufficient to achieve this is defined as a "therapeutically effective dose".An amount effective for this purpose will depend on many factors known to those skilled in the art, such as the severity of the disease, the weight and general condition of the patient.

[0050] Similarly, in prophylactic applications, the compositions of the present invention are intended to be administered to subjects susceptible to or otherwise at risk of a particular disease in an amount sufficient to at least partially reduce the risk of developing the disease. Such an amount is defined as a "prophylactically effective dose." Again, the exact amount depends on many factors specific to the patient, such as the patient's health and weight.

[0051] The microorganisms of the present invention may be provided in pure form or may be incorporated into a matrix. Such a matrix may advantageously protect the microorganism during passage through the gastrointestinal tract (including the acidic conditions of the stomach) and allow the viable cells of the microorganism to reach the intestine. Such a protective matrix may include sugar (such as maltodextrin), protein or fat components. In one embodiment, the protective matrix includes or is a vegetable oil. The microorganisms may be cultured according to any suitable method and prepared for encapsulation or addition to nutritional compositions, for example, by freeze-drying or spray-drying. Alternatively, they may be purchased already prepared in a form suitable for addition to food.

[0052] Suitable nutritional compositions include dairy products, beverage powders, dehydrated soups, dietary supplements, meal replacements, nutritional bars, cereals, confectionery products, animal feed supplements or dry pet foods.

[0053] The compositions of the present invention may further comprise protective hydrocolloids (gums, proteins, modified starches, etc.), binders, film formers, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, complexing compounds, dispersants, wetting agents, processing aids (solvents), flow agents, taste masking agents, weighting agents, jellifying agents, gel forming agents, antioxidants and antimicrobial agents. The compositions may also comprise conventional pharmaceutical additives such as water, gelatin of any origin, vegetable gums, lignin sulfonates, talc, sugars, starches, gum arabic, vegetable oils, polyalkylene glycols, flavors, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, fillers, as well as adjuvants, excipients and diluents, but are not limited thereto. In all cases, such additional components are selected having regard to their compatibility with the intended recipient.

[0054] In a preferred embodiment of the present invention, the composition further comprises at least one prebiotic.Therefore, prebiotic can promote the colonization of the microorganism of the present invention in the intestine of a subject after oral administration, thereby enhancing the effect of the microorganism contained in the composition according to the present invention.In addition, some prebiotics have a positive effect on digestion, etc.

[0055] Preferably, the prebiotic may be selected from the group consisting of oligosaccharides, and may optionally include fructose, galactose, mannose, soybean and / or inulin, dietary fiber, or mixtures thereof. The composition of the present invention may be provided in a powder form having a water activity of less than 0.2, for example in the range of 0.19 to 0.05, preferably less than 0.15.

[0056] After oral administration, the microorganisms of the present invention can survive and / or colonize one or more regions of the intestine, including the duodenum, small intestine, cecum, proximal and distal colon. As illustrated in Example 6, the orally administered microorganisms primarily colonized the cecum, proximal and distal colon.

[0057] Following intestinal colonization, the microorganisms of the present invention have been shown to produce detectable 5-HT in their feces, and importantly, accompanied by increased levels of 5-HT in the mucosal layer of the intestine (Example 6).In addition, it is believed that the colonizing microorganisms induce therapeutically beneficial changes in both gene expression and intestinal physiology.In particular, the observed changes in intestinal physiology include an increase in the mucosal layer, a maintenance of the levels of markers of intestinal barrier function, and a decrease in the levels of markers of intestinal inflammation, each of which is consistent with the therapeutic effect of 5-HT-related GI disorders.The expression of 5-HT-related receptors, HTR1B and HTR1D, in the intestine has also been observed after administration of the microorganisms of the present invention that produce high levels of 5-HT or TRM, which is consistent with the therapeutic effect of 5-HT-related CNS disorders, including migraine (Example 8).

[0058] Administration of the compositions of the present invention to a subject is demonstrated to have prophylactic and therapeutic effects against CNS-related disorders from preclinical studies conducted herein using well-established methods: the forced swim test (FST) and the open field test (OFT) (Examples 9 and 10).

[0059] II. In a second aspect, the present invention provides a method for producing a composition comprising: (a) tryptophan 5-hydroxylase (EC 1.14.16.4), (b) tryptophan decarboxylase (EC 4.1.1.28), and (c) providing a recombinant bacterium, the bacterium comprising one or more recombinant nucleic acid molecules or transgenes encoding one or more proteins selected from tryptamine 5-hydroxylase (EC:1.14.-.-), The microorganism lacks genes capable of expressing the trp operon repressor protein and tryptophanase (EC:4.1.99.1), and the cells are capable of producing increased amounts of one or more of 5-hydroxytryptophan, 5-hydroxytryptamine and tryptamine compared to cells of the non-recombinant microorganism from which they were derived.

[0060] In a further embodiment thereof, the one or more recombinant nucleic acid molecules or transgenes comprise: (a) tryptophan 5-hydroxylase (EC 1.14.16.4), (b) tryptophan decarboxylase (EC 4.1.1.28), (c) tryptamine 5-hydroxylase (EC:1.14.-.-), (d) tryptophan 5-hydroxylase (EC 1.14.16.4) and tryptophan decarboxylase (EC 4.1.1.28), and (e) encoding a protein selected from the group consisting of tryptophan decarboxylase (EC 4.1.1.28) and tryptamine 5-hydroxylase (EC: 1.14.-.-).

[0061] In a further embodiment, the bacterium is (a) tryptophan 5-hydroxylase (EC 1.14.16.4) and tryptophan decarboxylase (EC 4.1.1.28), or (b) a recombinant nucleic acid molecule encoding tryptophan decarboxylase (EC 4.1.1.28) and tryptamine 5-hydroxylase (EC:1.14.-.-), moreover, (c) serotonin acetyltransferase (AANAT) EC 2.3.1.87 and (d) comprising a recombinant nucleic acid molecule encoding acetylserotonin O-methyltransferase (ASMT) (EC 2.1.1.4); The cells are capable of producing increased amounts of melatonin as compared to the non-recombinant bacterial cells from which they were derived.

[0062] In a further embodiment thereof, the bacterium comprises a gene encoding a mutant GTP cyclohydrolase I (GCHI) whose amino acid sequence has at least 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to a wild-type GCHI having SEQ ID NO:2, and comprises one or more mutations, wherein the mutant provides increased hydroxylation activity of said TPH compared to native GCH1.

[0063] By way of example, the mutant GTP cyclohydrolase I (GCH1) has at least one or more mutations at amino acid residues selected from the group consisting of D97, M99, T101, V102, A125, K129, N170, V179, T196, T198, S199, L200, S207, H212, E213, F214, L215 and H221, wherein the N170 mutation is N170K, N170D or N170L, the V179 mutation is V179A, the H212 mutation is H212R or H212K, and the H221 mutation is H221R or H221K. Preferably, the mutation is a substitution selected from T198I, T198S, F214S, V179A, M99I and L200P.

[0064] One skilled in the art could test further mutants by co-expressing mutant GCH1 and tryptophan 5-hydroxylase in a microbial cell and testing for increased tryptophan hydroxylation activity to 5-hydroxytryptophan. Preferably, the mutant GCH1 increases the hydroxylation activity of tryptophan 5-hydroxylase by at least 2-fold, or 3-fold, or 4-fold, or 5-fold, 6-fold, or 7-fold, or 8-fold, or 9-fold, or 10-fold, as measured by the yield of 5-hydroxytryptophan, compared to the non-mutant GCH1 from which the mutant is derived.

[0065] In a further embodiment thereof, the bacterium is capable of expressing a protein having tryptophan 5-hydroxylase (EC 1.14.16.4) activity, wherein the amino acid sequence of the protein has at least 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 6, 8, or 12.

[0066] In a further aspect thereof, the bacterium is capable of expressing a protein having tryptophan decarboxylase (EC 4.1.1.28) activity, wherein the amino acid sequence of the protein has at least 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 14 or 18.

[0067] In a further embodiment thereof, the bacterium is capable of expressing a protein having tryptamine 5-hydroxylase (EC:1.14.-.-) activity, wherein the amino acid sequence of the protein has at least 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO:20.

[0068] In a further aspect thereof, the bacterium is capable of expressing a protein having serotonin acetyltransferase EC 2.3.1.87 activity, wherein the amino acid sequence of the protein has at least 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO:22.

[0069] In a further aspect thereof, the bacterium is capable of expressing a protein having acetylserotonin O-methyltransferase (EC 2.1.1.4) activity, wherein the amino acid sequence of the protein has at least 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO:24.

[0070] In a further embodiment thereof, the bacterium comprises a recombinant nucleic acid molecule encoding (a) a tryptophan 5-hydroxylase (EC 1.14.16.4), and (b) a tryptophan decarboxylase (EC 4.1.1.28), wherein the tryptophan 5-hydroxylase has at least 80% sequence identity to SEQ ID NO: 6 or 12, and the amino acid sequence of the tryptophan decarboxylase has at least 80% sequence identity to SEQ ID NO: 18.

[0071] In further embodiments thereof, the bacterium comprising recombinant nucleic acid molecules encoding (a) tryptophan 5-hydroxylase (EC 1.14.16.4) and (b) tryptophan decarboxylase (EC 4.1.1.28) is further genetically modified to upregulate the expression level of TPH relative to the TDC enzyme of the 5-HT pathway, e.g., to further increase the amount of 5-HT produced and reduce the production of TRM. By way of example, the nucleic acid molecules encoding tryptophan 5-hydroxylase and tryptophan decarboxylase are each operably linked to their respective RBSs, and the ratio of their intensities is at least 1.5:1.0, 1.75:1.0, 2.0:1.0, 2.5:1.0, 3.0:1.0, 3.5:1.0, 4.0:1.0, 4.5:1.0, 5.0:1.0, 5.5:1.0, 6.0:1.0, 7.0:1.0, 8.0:1.0, 9.0:1.0, 10.0:1.0, 11.0:1.0, 12.0:1.0, 13.0:1.0, 14.0:1.0, 15.0:1.0, 16.0:1.0, 17.0:1.0, 18.0:1.0, 19.0:1.0, 20.0:1.0, 21.0:1.0, 22.0:1.0, 23.0:1.0, 24.0:1.0, 25.0:1.0, 26.0:1.0, 27.0:1.0, 28.0:1.0, 29.0:1.0, 30.0:1.0, 3 0:1.0, 6.5:1.0, 7.0:1.0, 7.5:1.0, 8.0:1.0, 8.5:1.0, 9.0:1.0, 9.5:1.0, 10.0:1.0, 20:1, 40:1, 60:1.0, 80:1.0, 100:1.0, 150:1.0, 200:1.0, 250:1.0, and 300:1.0, more preferably at least 20.0:1.0, the RBS strength being measured as defined above with reference to Salis et al., 2009.

[0072] Alternatively, the bacterium comprising recombinant nucleic acid molecules encoding (a) tryptophan 5-hydroxylase (EC 1.14.16.4) and (b) tryptophan decarboxylase (EC 4.1.1.28) is further genetically modified to upregulate the expression level of TDC relative to the TPH enzyme of the 5-HT pathway, e.g., to further increase TRM production. To this end, the nucleic acid molecules encoding tryptophan decarboxylase and tryptophan 5-hydroxylase are each operably linked to their respective RBSs, and the ratio of their intensities is at least 1.5:1.0, 1.75:1.0, 2.0:1.0, 2.5:1.0, 3.0:1.0, 3.5:1.0, 4.0:1.0, 4.5:1.0, 5.0:1.0, 6.0:1.0, 7.0:1.0, 8.0:1.0, 9.0:1.0, 10.0:1.0, 11.0:1.0, 12.0:1.0, 13.0:1.0, 14.0:1.0, 15.0:1.0, 16.0:1.0, 17.0:1.0, 18.0:1.0, 19.0:1.0, 20.0:1.0, 21.0:1.0, 22.0:1.0, 23.0:1.0, 24.0:1.0, 25.0:1.0, 26.0:1.0, 27.0:1.0, 28.0:1.0, 29.0:1.0, 30.0:1.0, 31.0:1.0, 32.0:1.0, 33.0:1 0.5:1.0, 6.0:1.0, 6.5:1.0, 7.0:1.0, 7.5:1.0, 8.0:1.0, 8.5:1.0, 9.0:1.0, 9.5:1.0, 10.0:1.0, 20:1, 40:1, 60:1.0, 80:1.0, 100:1.0, 150:1.0, 200:1.0, 250:1.0, and 300:1.0, and more preferably at least 2.0:1.0.

[0073] In a further embodiment thereof, in the bacterium of the present invention, one or more recombinant nucleic acid molecules encoding (a) tryptophan 5-hydroxylase (EC 1.14.16.4) and / or (b) tryptophan decarboxylase (EC 4.1.1.28) are operably linked to a promoter that constitutively regulates expression or is inducible. When the promoter is a constitutive promoter, a suitable promoter may be selected from among the synthetic promoters listed at http: / / parts.igem.org / Promoters / Catalog / Anderson, preferably a strong promoter having a measured strength of 0.5 or more on the Anderson scale is selected.

[0074] In a further embodiment thereof, the bacterium is unable to express a functional trp operon repressor protein, the amino acid sequence of which has at least 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO:26.

[0075] In a further embodiment thereof, the bacterium is unable to express a protein having tryptophanase activity (EC:4.1.99.1), wherein the amino acid sequence of the protein has at least 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO:29.

[0076] In a further embodiment thereof, the bacteria is not antibiotic resistant to one or more clinically used antibiotics.

[0077] The recombinant bacterium according to the second aspect, in its various described forms, is suitable for use in compositions according to each of the further aspects of the invention. EXAMPLES

[0078] Example 1: Engineered Escherichia coli cells express a biosynthetic pathway for 5-HT production The 5-HT biosynthetic pathway was introduced into E. coli Nissle cells to establish a synthetic serotonin pathway that catalyzes two consecutive metabolic steps: converting TRP to 5-HTP and then converting 5-HTP to 5-HT (Figure 1). Identification of the optimal combination enzymes for catalyzing these two metabolic steps was determined by expressing and determining the 5-HT yields obtained from the combination of the TPH and TDC genes.

[0079] 1.1 Methodology 1.1.1 Modified E. coli cells were engineered as follows, with reference to Table 1. The host strain E. coli Nissle 1917 (Mutaflor®) was purchased from Ardeypharm GmbH, Germany. The gene for green fluorescent protein (GFP, GenBank: CAH64882.1) was placed under the control of a strong constitutive promoter (Part BBa_J23101, Registry of Standard Biological Parts, www.parts.igem.org) and integrated into the E. coli Nissle genome at the Tn7 attachment site using pGRG25 as described by McKenzie et al., (2006). Genes encoding TPH proteins from mammalian or plant sources: human TPH1 (H1), human THP2 (H2), mouse TPH1 (M1), mouse THP2 (M2), and TDC were derived from rice TDC (R), Cataranthus roseus (C), Guinea pig (G) and Candidatus Koribacter versatilis Ellin345 (ck) and were synthesized using codons optimized for E. coli using IDT's online tool (idtdna.com / CodonOpt). The tested combinations of TPH and TDC genes were cloned as operons under the control of the synthetic promoter BBa-J23107 in a self-replicating plasmid (using pUC as backbone) containing a kanamycin resistance gene. The plasmids were then transformed into the host strains by using standard cloning and transformation procedures known in the art.

[0080] 1.1.2 Growth and in vitro metabolite production were determined as follows. Strains were grown in modified M9 medium containing 1x M9 salts (M6030, Sigma Aldrich), 0.2% (w / v) glucose, 0.1% (w / v) casamino acids (catalog no. C2000, Teknova), 1 mM MgSO4, 50 μM FeCl3, 0.2% (v / v) 2YT medium (composition see below), and 50 mg / L L-tryptophan, unless otherwise stated. Kanamycin was added to a final concentration of 50 mg / L, unless otherwise stated. Three single colonies from each strain were picked and grown in 300 μl of medium in 96-deep-well plates, shaking at 250 rpm and 37 °C for 16 h. This preliminary culture was inoculated into the main culture by diluting 1:100 into fresh medium, and the cells were grown under the same conditions for 24 h. For substrate feeding, TRP and 5-HTP were added to the growth medium at 100 mg TRP or 5-HTP / L. Culture supernatants were then separated from the cells using 0.2 μm pore size filters and frozen at -20°C until analysis by LC-MS. All data shown are the mean + / - SD from at least three biological replicates.

[0081] 1.1.3 Metabolite production was quantified by LC-MS as follows. Detection of serotonin, tryptamine, tryptophan and 5-HTP was performed by liquid chromatography-mass spectrometry (LC-MS) measurements on a Dionex UltiMate 3000 UHPLC (Fisher Scientific, San Jose, CA) connected to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). The system used an Agilent Zorbax Eclipse Plus C18 2.1×100 mm, 1.8 μm column maintained at 35° C. The flow rate was 0.350 mL / min with mobile phases of 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). The gradient started at 5% B and followed a linear gradient to 35% B over 1.5 min. This solvent composition was held for 3.5 min, then immediately changed to 95% B and held for 1 min. Finally, the gradient changed to 5% B until 6 min. Samples (1 uL) were passed through an MS equipped with a heated electrospray ionization source (HESI) in positive ion mode with sheath gas set at 60 (au), auxiliary gas at 20 (au), and sweep gas at 2 (au). Cone and probe temperatures were 380 °C and 380 °C, respectively, and spray voltage was 3500 V. The scan range was 50-500 Da, and the time between scans was 100 ms. Detection of serotonin (160.07646 ion), tryptamine (144.08158 and 161.10775 ions) and tryptophan (205.09785 ion) was performed in full scan, while 5-HTP detection was performed after HCD fragmentation (221.09 > 162.05553, 25% HCD CE). Quantification of compounds was based on calculations from calibration standards analyzed before and after a set of 24 samples. All reagents used were of analytical grade.

[0082] 1.2 Results: E. coli Nissle, a genetically modified strain of the host engineered to express mouse TPH1 (M1) or human TPH2 (H2) in combination with the TDC of rice (R) or Cataranthus roseus (C), was found to produce measurable amounts of serotonin production (Figure 2A). When the strain was fed the substrate 5-HTP during cultivation, cells expressing the gene encoding rice TDC (R) produced the highest yield of 5-HT (79.5 ± 11.1% yield), indicating that rice TDC was the most efficient at catalyzing the 5-HTP decarboxylation step (Figure 2B). Therefore, E. coli Nissle, which expresses the "M1R" and "H2R" pathways and is a 5-HT producer, was selected for further optimization.

[0083] Feeding to TDC showed that all enzymes produced some serotonin, but the "R" enzyme from rice had the highest conversion rate (right panel).

[0084] Example 2. 5-HT and TRM production by genetically modified Escherichia coli cells of the present invention is enhanced by increasing tryptophan pathway flux Increased availability of tryptophan, a tryptophan pathway substrate, is shown to increase 5-HT production in the 5-HT producing strains of the invention (Figure 3). In light of this observation, the endogenous pool of available TRP was increased in the engineered cells of the invention, generated by inactivating the endogenous genes encoding the tryptophan repressor trpR and tryptophanase tnaA, thus enhancing tryptophan pathway flux.

[0085] 2.1 Methodology: Knockout of trpR and tnaA from the host E. coli Nissle genome was performed using CRISPR / Cas9 as described in (Mehrer et al., 2018). Briefly, a two-plasmid system consisting of an inducible cas9 / λ-Red expression plasmid and a guide RNA (gRNA) plasmid was used to introduce a double-stranded break at the desired locus in the host genome. gRNAs were designed using CRISPy-web (Blin et al., 2016) after uploading the EcN genome sequence (GenBank: CP007799.1). Templates for homologous recombination at the selected break sites were generated as follows: When possible, strains carrying the desired knockout (ΔtrpR::FRT-kan-FRT or ΔtnaA::FRT-kan-FRT, respectively) from the KEIO collection (Baba et al., 2006) were transformed with pSIJ8 (Jensen et al., 2015) and the FLP recombinase gene was induced to remove the kanamycin resistance gene. The resulting colonies were screened for kanamycin sensitivity. The ΔtrpR::FRT or ΔtnaA::FRT locus was amplified using oligos binding 500 bp upstream and downstream of the FRT site, generating PCR products of approximately 1 kb. CRISPR / Cas9 and gRNA expression plasmids were treated with the strains as previously described (Mehrer et al., 2018).

[0086] Growth of the host cells, their metabolite production and quantification were carried out as described in Examples 1.1.2 and 1.1.3.

[0087] 2.2 Results: To redirect the corresponding endogenous tryptophan flux into the 5-HT pathway, the strain of the present invention was further engineered by knocking out the genes encoding trpR and tnaA from the host genome to generate a host E. coli Nissle strain designated NΔ2. Exogenous supply of tryptophan is shown to further enhance 5HT and TRM production by such a genetically modified E. coli Nissle cell of the present invention (strain EcNΔ2+pUC-H2R in FIG. 3).

[0088] Example 3. Genetically modified E. coli strains of the present invention exhibit stable 5-HT production When the 5-HT operon encoding the 5-HT biosynthetic pathway is cloned into a multicopy plasmid in the host cell of the present invention, the production of 5-HT depends on the stability of the plasmid. Preferably, the stability does not depend on the plasmid genes that confer antibiotic resistance. Based on the plasmid backbone of pUC (as in Examples 1 and 2) and pMUT1, one of the two native plasmids in E. coli Nissle (Blum-Oehler et al., 2003), two solutions for conferring plasmid stability were compared.

[0089] 3.1 Methodology: A high copy number 5-HT production plasmid (pUC-H2R) was modified by introducing a synthetic copy of the essential bacterial gene infA downstream of the H2R pathway operon. The corresponding infA gene in the host genome was knocked out using the protocol described in Example 2.1 to generate E. coli Nissle strain NΔ3 (ΔtrpR, ΔtnaA, ΔinfA).

[0090] The operons constituting the H2R pathway were also cloned into the plasmid pMUT1 (GenBank: A84793.1) as follows: the pMUT1 plasmid was isolated from wild-type E. coli Nissle and amplified to serve as the pMUT backbone. Elements that could allow plasmid transfer were removed to generate pMUT (Δbp1-1323 and 1664-3117 from the circular sequence A84793.1), and the kanamycin resistance gene and the H2R or M1R operon (without infA) were inserted into the backbone to generate pMUT-H2R or pMUT-M1R, respectively.

[0091] The following additional versions of plasmids pMUT-H2R or pMUT-M1R were generated: the hok / sok toxin-antitoxin plasmid stability element (GenBank: MK134376, region 58002-58601) was inserted into the pMUT-M1R backbone to generate plasmid pMUT14-ser, the TPH gene was deleted from pMUT14-ser to generate pMUT11-trm, and both TPH and TDC were deleted to generate pMUT09-ctrl.

[0092] Measurement of cell proliferation: Cells were grown as described in Example 1.1.2, but after inoculation of the main culture, 200 μl was transferred to a flat-bottomed, clear, 96-well microtiter plate and then sealed with a gas-permeable membrane (Z380059, Sigma Aldrich). Cultures were grown at 37° C. with orbital shaking at 700 rpm and their proliferation was measured by OD 630nm Plates were recorded every 10 minutes on a BioTek™ ELx800 plate reader at RT.

[0093] Determination of plasmid stability: Six single colonies each from each test strain were picked and grown in 300 μl of 2YT medium (containing 1.6% (w / v) tryptone, 1% (w / v) yeast extract, 0.5% (w / v) NaCl) without kanamycin in 96 deep-well plates and shaken at 250 rpm at 37° C. for 24 h. The optical density (OD630) of the cultures was measured, the cultures were diluted 1:1000 in fresh medium and the cells were plated daily on LB agar plates with or without kanamycin, which corresponds to approximately 10-fold every 24 h. The colony forming units on the LB agar plates + / - kanamycin were counted daily. At time points t=0, 50 and 100 generations, the cultures were diluted 1:100 in the modified M9 medium (without kanamycin) described above and serotonin production was measured as described.

[0094] 3.2 Results: While stable, the pUC-based plasmid expressing both the H2R pathway and the infA gene caused undesirable growth defects in both NΔ2 and NΔ3 host cells (EcN harboring ΔtrpR, ΔtnaA, ΔinfA), presumably due to metabolic burden ( Figure 4A, B, and C ).

[0095] In contrast, pMUT-based plasmids (pMUT-H2R) were stably maintained in NΔ2 host cells (Figure 4A,B) and exhibited undetectable fitness defects in the form of antibiotic resistance for at least 100 generations in the absence of selective pressure (Figure 4A,B,C). Moreover, 5-HT production by EcNΔ2pMUT-H2R cells was stable over this period and comparable to the initial pUC-H2R titers (Figure 4D).

[0096] The hok / sok toxin-antitoxin module was introduced into the pMUT-M1R plasmid to generate pMUT14-ser and pMUT11-trm (Table 1 ) to further ensure stability in vivo.

[0097] Example 4. Optimization of 5-HT production by genetically modified E. coli strains of the present invention Tryptamine (TRM) is an important by-product detected among the metabolites produced by the engineered E. coli strains of the present invention expressing plant TDC enzymes (Figures 2A, 3). The genetic modifications designed to enhance the flux into 5-HTP synthesis were to increase the activity of GTP glycohydrolase (GCH1) activity, thereby enhancing the regeneration of the tetrahydrobiopterin cofactor required for TPH synthesis. To further enhance 5-HT production, further genetic modifications designed to fine-tune the relative expression strength of TPH and TDC were tested.

[0098] 4.1 Methodology The coding sequence of the E. coli NfolE gene was mutated by first amplifying the folE gene into two parts and then introducing the T198I coding mutation into the oligo used for overlap extension PCR. The resulting dsDNA fragment was purified and co-transformed with the gRNA plasmid to generate a markerless genetic mutation of the folE gene in the genome of EcN cells.

[0099] RBS strength libraries were designed using the "RBS calculator v1.1" (Salis et al., 2009) and library sizes of 4-6 variants within the theoretical strength range of 50-50000 arbitrary units for each co-expressed gene encoding TPH and TDC (M1R and H2R) were selected. RBS mutations were introduced upstream of each gene using oligos containing degenerate bases and Gibson assembly (Gibson et al., 2009). After testing the metabolite production profiles of the strains, transformants were randomly picked and RBS sequenced.

[0100] 4.2 Results The native folE gene in the host E. coli Nissle cells of the present invention was mutated to encode mutant GCH1 (T198I) to enhance regeneration of the tetrahydrobiopterin cofactor. In these optimized Nissle (oN) strains expressing the mutant GCH1 enzyme in combination with the 5-HT biosynthetic operon (oNpMUT1H2R or oNpMUTM1R), the production of 5-HT is increased compared to strains lacking this additional modification (NΔ2pMUT-H2R or NΔ2pMUT-M1R). Mutant GCH1 increases the hydroxylation of tryptophan to 5-hydroxytryptophan catalyzed by human tryptophan 5-hydroxylase by 4-fold in strains containing pMUT10-5htp (Figure 5F).

[0101] oN strains expressing mutant GCH1 enzymes in combination with either the 5-HTP biosynthetic pathway (oN10 = oN pMUTM1) or the TRM pathway (oN11 = oN pMUTR) produced 5-HTP and TRM, respectively, as the only detectable products of the pathway (Fig. 5B ).

[0102] Modulation of the relative expression strength of TPH and TDC using selected combinations of the ribosome binding sites (RBS) of each gene is shown to enhance 5-HT production and reduce TRM accumulation (Figure 5C and D). Increased 5-HT production was obtained when the relative strength of the RBS operably linked to the TPH gene exceeded the relative strength of the RBS operably linked to the TDC gene.

[0103] The synergistic effects of genetic modifications introduced into E. coli Nissle strains containing genes of the 5-HT pathway are shown in Figure 6 on the respective 5-HT productivity and 5-HT:TRM ratio. Knocking out the trpR and tnaA genes in the E. coli genome led to synergistic effects on 5-HT yield and 5-HT:TRM ratio in E. coli cells, thanks to the functionally linked RBS, provided that the relative expression of the TPH genes exceeded that of the TDC genes (Figure 6A). Introduction of the folE genomic mutation (encoding the T198I substitution) further increased the 5-HT yield and 5-HT:TRM ratio when combined with the aforementioned mutations in E. coli Nissle strains (Figure 6B).

[0104] Example 5 Oral administration of the genetically modified E. coli strains of the present invention is predicted to produce therapeutic 5-HT levels in subjects The therapeutically effective level of 5-HT in the intestine has been reported to be in the range of 5-30 mg / L (Liu, Q. et al., 2008). The relative abundance of 5-HT-producing bacteria required to produce 5-HT in the therapeutic range in the mouse intestine is 0.1-10 mg / L. * O.D. 630 * h (Figure 6C). This is based on basic assumptions regarding gut bacterial density, which is predicted to range from 1E2 to E11 / g (Sender et al., 2016), depending on location (Casteleyn et al., 2010), as shown below. [Table 1]

[0105] In vitro, specific productivity (P) is expressed as the bacterial load per hour (OD 630nmSerotonin titer (mg / L) can be measured. The depletion rate of 5-HT is unknown and therefore is set equal to the productivity to determine the physiologically relevant concentration (mg / L). It is predicted that the genetically modified E. coli strains of the present invention (e.g., oN14 strain) can produce 5-HT in vivo when administered at near physiologically relevant levels.

[0106] Example 6 Oral administration of the genetically modified E. coli strain of the present invention enhances 5-HT production in mouse intestine Oral delivery of the genetically modified E. coli Nissle strains of the present invention and their localization to the intestine and enhanced 5-HT production has been demonstrated in mice.

[0107] 6.1 Methodology 6.1.1 Oral dosing: Male mice (C57BL / 6) aged 6-8 weeks, supplied by Taconic Biosciences or Jackson Laboratories, were group-housed with free access to food and water in a specific pathogen-free (SPF) facility with a 12-h light / dark cycle (lights on 07:00-19:00), constant temperature. Upon transport, mice were given one week to adapt to their new location, followed by a 7-day, three-rotation microbiota normalization protocol. On days 1, 3, and 5 of the normalization protocol, mice were randomly mixed. On day 6, mice were mixed one last time and separated into respective cohorts with 2-3 cages per cohort, with 3-5 mice in each cage. The exact number varied by experiment. On day 7, mice were tagged for identification and housed in groups of 3-5 mice in bottle-fed cages, starting from day zero of the experimental protocol, set in designated racks and opened only under a laminar flow hood. Fecal samples were collected on day zero of the experimental protocol. Mice were then administrated with 200 μl of sterile PBS or 10 freshly grown PBS in sterile PBS at the same time every 24 h for 10 days. 9The indicated E. coli Nissle strains of cells: oN14 (E. coli Nissle strain oN containing plasmid pMUT14-ser), oN11 (E. coli Nissle strain oN containing plasmid pMUT11-trm), and oN9 (E. coli Nissle strain oN containing plasmid control plasmid pMUT9-ctrl) were orally administered (Figure 5E). 24 h after the last oral gavage, fecal pellets were collected and mice were euthanized for serum, intestinal tissue, and luminal feces collection or used for locomotor or behavioral testing.

[0108] Intestinal localization of 6.1.2oN strains was determined by plating digests from different intestinal regions on LB agar containing 50 mg / L kanamycin and quantifying the number of GFP-positive kanamycin-resistant (KanR) colonies.

[0109] 6.1.3 Immunodetection in thin sections: Colon samples were collected and 5 mm sections were cut in mid-colon and fixed for 24 h in methanol-Carnoy (60% methanol, 30% chloroform, 10% acetic acid) solution or 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Fixed samples were then washed three times and stored in 70% ethanol. Samples, kept intact in the lumen with the digesta, were embedded in paraffin, sectioned at 4 μm in the lumen, and prepared for immunohistochemistry or subjected to hematoxylin and eosin staining. For immunohistochemistry: PFA-fixed slides were used for serotonin (5-HT) antibody staining, and methacan fixed samples were used for Muc2, green fluorescent protein (GFP), and chromogranin A (CgA) antibody staining. All slides were baked at 57°C for 2 hours, deparaffinized in xylene and ethanol-water gradients, rehydrated, and then treated with antigen retrieval solution of 10 mM sodium citrate, pH 6.0, at 90°C for 30 minutes. Slides were blocked with 5% fetal bovine serum (FBS) in PBS for 30 minutes at room temperature, then incubated with primary antibodies in 5% FBS in PBS at 4°C overnight. Slides were washed 3 times with PBS, incubated with secondary antibodies in PBS for 2 hours at room temperature, washed 3 times with PBS, incubated with 10 μg / ml DAPI (4',6-diamidino-2-phenylindole) for 5 minutes, washed 3 times with PBS, and mounted with glycerol. Slides stained with primary antibodies pre-conjugated with Alexa fluorophores did not undergo secondary antibody staining. Antibody used: anti-GFP rabbit IgG with Alexa Fluor 594 conjugate (Life Technologies A21312). Anti-Muc2 polyclonal rabbit (Santa Cruz Technologies sc-15334). Anti-chromagranin A polyclonal rabbit (Abcam ab15160). Anti-serotonin polyclonal goat (Abcam ab66047). Secondary anti-rabbit Alexa Fluor 568 (Thermo-Scientific Novex a11011). Secondary anti-goat IgG Alexa Fluor 488 (Abcam ab150077).All images were taken with a Nikon Eclipse Ti2-E inverted microscope system, and image conversion and cell size (particle) analysis were performed using NIS-Elements, Advanced Research Software and ImageJ (FIJI) for Macbook version 2.0.0-rc-69 / 1.52i.

[0110] 6.1.4 In vivo metabolite analysis. Serum, tissue samples, and fecal samples were analyzed for serotonin, 5-hydroxytryptophan, tryptamine, and tryptophan. For serum: After euthanasia, blood samples were collected from the inferior vena cava and samples were mixed 1:1 with 0.9% NaCl, 0.2% ascorbic acid. Samples were kept at room temperature for 10 minutes and then placed on ice until centrifuged at 2000g for 10 minutes at 4°C. Supernatants were saved for analysis and immediately frozen at -20°C. For tissue analysis: Colon tissue was weighed, frozen in liquid nitrogen immediately after collection, and freeze-fractured by tapping for 30 seconds against stainless steel microvials (Biospec 2007) with 6.34 mm stainless steel beads (Biospec 11079635ss) in a BeadBeater (Biospec 112011). Tissues were then resuspended in 0.9% NaCl, 0.1% ascorbic acid and centrifuged at 2000g for 10 minutes at 4°C. The supernatant was saved for analysis and immediately frozen at -20°C. For fecal samples: Samples were weighed and placed in Corning Cryovials containing 0.5ml of 0.9% NaCl with 0.1% ascorbic acid containing 200ml zirconia / silica beads (Biospec 11079101z) and agitated in a BeadBeater for 5 minutes at 4°C. Samples were centrifuged at 2000g for 10 minutes at 4°C. The supernatant was saved for analysis and immediately frozen at -20°C.

[0111] 6.2 Results Mice were inoculated with 10 mL of E. coli Nissle strains ON14, ON1, and the control strain oN9 (Figure 5E). 9Ten days after oral administration of oN cells, the administered cells were found to accumulate primarily in their large intestine. The highest concentration of oN cells was found in the proximal colon, >10 8 CFU / g (Fig. 7A), whereas the smallest number of oN bacteria was detected in the small intestine (<10 4 In the central colon, oN cells were localized along the mucosal layer based on their visualization by immunohistochemistry using an anti-GFP antibody (FIG. 7B).

[0112] Levels of TRP, 5-HTP, 5-HT and TRM were also measured in serum, colonic tissue and fecal samples from orally dosed mice. Tryptophan levels were elevated in colonic tissue and feces, but not in serum, only in mice orally dosed with E. coli Nissle strain oN14 (Figure 8A). 5-HT levels were not statistically different in serum and colonic tissue between cohorts (p>0.4 by ANOVA). However, 5-HT levels in feces were significantly increased in mice dosed with E. coli Nissle strain oN14 compared to the other groups (13.4-fold increase, p<0.0001 by ANOVA) (Figure 8B). Tryptamine and 5-HTP levels were below the detection limit in all samples.

[0113] Although 5-HT levels were not elevated in the colonic tissue itself (after washing to remove residual digesta), elevated levels of serotonin were specifically detected along the mucosal layer of the colon in mice administered E. coli Nissle strain oN14 (Figure 8C). These results indicate that orally administered E. coli Nissle strain oN14 cells accumulate and produce 5-HT in vivo along the mucosa of the mouse colon.

[0114] Example 7 Oral administration of genetically modified E. coli strains of the invention induces changes in gene expression and physiology in the intestine Oral administration of E.coli Nissle strain oN14 to mice has been shown to upregulate the expression of Muc2, increase mucosal thickening, and then improve mucosal integrity. Excessive serotonin derived from enterochromaffin cells may stimulate intestinal inflammation. However, even though increased levels of 5-HT were detected in the mucosal layer of the colon of treated mice (Example 6), this did not result in changes in the expression of markers of intestinal inflammation, intestinal turnover, or intestinal barrier function (not shown).

[0115] Expression of host (mouse) TPH2, but not TPH1, was increased in the colon of mice treated with E. coli Nissle strain oN14, indicating that bacterially derived 5-HT can induce TPH2-mediated neuronal 5-HT biosynthesis in the mouse ENS. However, the total number or size of EC cells (the major producers of host 5-HT in the intestine) in these mice was not affected.

[0116] Although tryptophan and serotonin metabolism can also be affected in the kynurenine pathway, whose dysfunction is associated with many neurological and metabolic disorders, no changes were detected in the expression of genes encoding the rate-limiting enzymes that convert L-tryptophan to N-formylkynurenine in the colons of mice treated with E. coli Nissle strain oN14.

[0117] 7.1 Methodology 7.1.1 Oral Administration: As described in 6.1.1.

[0118] 7.1.2 Gene Expression Analysis: Tissues were freeze-fractured according to the protocol in Example 6.1.4. Tissues were then resuspended in Trizol (Invitrogen) according to the manufacturer's instructions for RNA isolation. Total RNA was resuspended in 30 μL of RNase-free water. RNA was converted to cDNA using the iScript gDNA Clear cDNA Synthesis Kit (BioRad 1725035) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qPCR) was performed in triplicate according to the manufacturer's instructions in 384-well plates on an Applied Biosystems QuantStudio6 Flex Real-Time PCR System. The primers used are listed in Table 3. Cyclophilin was used as a housekeeping gene. Relative mRNA levels were quantified using the ΔΔCτ method.

[0119] 7.1.3 Immunodetection in thin sections: As described in Example 6.1.3.

[0120] 7.2 Results Expression of the MUC2 gene in colonic tissue from mice administered E. coli Nissle strain oN14 was increased approximately two-fold compared to the control group (p<0.05 by ANOVA) (Figure 9A). Similarly, sections of colonic tissue immunohistochemically stained with anti-Muc2 antibody showed a more prominent mucosal layer in mice administered E. coli Nissle strain oN14 (Figure 9B). Hematoxylin and eosin staining of colonic sections further confirmed the lack of histopathological changes associated with intestinal inflammation (not shown).

[0121] However, no differences were observed in the expression of goblet cell marker CDX2 or intestinal stem cell markers BM1 and LGR5 (not shown), indicating that Muc2-producing goblet cells and general intestinal turnover were not altered. In contrast, no changes in gene expression levels by qPCR were detected for the inflammatory markers IL6, IL1β, NFKβ, IL17A, and TNFα in the colonic tissue of mice administered E. coli Nissle strain oN14 (not shown). Furthermore, no changes in colon length were observed (p=0.66 by ANOVA), another indicator of chronic intestinal inflammation if it was found to be shortened (not shown). Expression of TJP1, TJP2, or OCLN, markers for maintaining the colonic intestinal barrier, was also not altered in mice administered E. coli Nissle strain oN14 (data not shown).

[0122] No differences in TPH1 and SERT expression were observed, but an unexpected 2.8-fold increase in TPH2 expression was detected in the colon of mice treated with E. coli Nissle strain oN14 compared to the other groups (p<0.001 by ANOVA) (Figure 9A). However, no changes in the total number or size of EC cells (the major producers of host 5-HT in the intestine) were detected, as quantified by qPCR and immunohistochemistry for the EC cell marker chromogranin A (not shown).

[0123] Furthermore, no changes in the expression of IDO1, IDO2, and TDO2 were detected in mice administered E. coli Nissle strain oN14, which encodes the rate-limiting enzyme that converts L-tryptophan to N-formylkynurenine (not shown).

[0124] Example 8 Oral administration of the genetically modified E. coli strain of the present invention enhances the expression of serotonin receptors in the intestine and related effects Serotonin is the ligand for many 5-HT receptors, GPCRs that mediate excitatory and inhibitory neurotransmission throughout the ENS, CNS, and peripheral nervous system. Ligand activation of 5HT1b receptors in the CNS has been associated with reduced aggression (de Almeida et al., 2002) and, together with 5HT1d, is the target receptor for tryptamine-based treatment of migraine (Tepper et al., 2002). Oral administration of both E. coli Nissle strains oN11 and oN14 to mice has been shown to increase gene expression of HTR1B and HTR1D, suggesting a possible tryptamine-mediated effect.

[0125] 5HT3 receptors have various physiological roles related to emesis, irritable bowel syndrome (IBS), schizophrenia, anxiety, learning, memory and addiction, and are clinically modulated by antiemetic 5HT3 antagonists (Thompson et al., 2007). 5HT4 receptors induce neurogenesis in the enteric nervous system and are associated with regulation of gastrointestinal peristalsis, stress-induced feeding behavior, altered learning and memory, and depression in the CNS (Gershon et al., (2007), Lucas et al., (2007), Lamirault et al., (2001)). 5HTr7 stimulation can improve cognition and memory (Meneses et al., 2015), and 5HTr7 antagonism can reverse the actions of antidepressants and antipsychotics in the CNS (Roth, Blet al., 1994), but its function in the intestine is poorly understood, despite having been linked to IBS and inflammatory bowel disease (IBD) (Guseva, D. et al., 2014). Given the variety and magnitude of changes detected in 5-HT receptor gene expression, it is plausible that E. coli strain oN14 induces a physiological response in the host.

[0126] Upregulated expression of HTR3, HTR4, and HTR7 genes was detected only in the oN14 cohort, indicating a specific serotonin-mediated response.

[0127] Furthermore, oral administration of E. coli Nissle strain oN14 was shown to reduce total gastrointestinal transit, consistent with the knowledge that serotonin regulates gastrointestinal motility by inducing both the intraluminal pressure that triggers the peristaltic reflex and the passage of intestinal motor complexes through the small intestine during the interdigestive interval.

[0128] Although changes in gut motility and biogenic amines produced by bacteria could potentially affect the gut microbiota, we did not detect any significant changes in gut microbiota between mouse cohorts as a result of administration of E. coli Nissle strains oN14, oN11, oN9 or PBS alone based on 16S metagenomic sequencing of feces from the mice cohorts (not shown). At the same time, administration of E. coli Nissle strains oN14 and oN11 induces many physiological responses in the gut, including host 5-HT receptor gene expression and gastrointestinal motility, but leaves the resident microbial community unaffected.

[0129] 8.1 Methodology 8.1.1 Oral Administration: As described in 6.1.1.

[0130] 8.1.2 Gene Expression Analysis: As described in 7.1.1.

[0131] 8.1.3 Total Gastrointestinal Transit: Total gastrointestinal transit time was studied using carmine red, which cannot be absorbed from the intestinal lumen. A 6% solution of carmine red (300 μl, Sigma-Aldrich) suspended in 0.5% methylcellulose (Sigma-Aldrich) was administered orally by gavage through a 21-gauge blunt-tip feeding needle. The time at which gavage was administered was recorded as TO. After gavage, fecal pellets were monitored for the presence of carmine red at 10-minute intervals. Total gastrointestinal transit time was considered as the interval between TO and the time at which carmine red was first observed in the stool.

[0132] 8.1.4 Metagenomic 16S Sequencing and Analysis: Fecal pellets were collected from mice on days 0 and 10 and immediately stored at -20 °C until paired-end sequencing. Genomic DNA was extracted from fecal pellets using the Epicentre Gram-positive kit plus an initial bead-beating step with 0.1 mm zirconia beads. PCR amplification and multiplex barcoding of the 16S rRNA V4 region of samples was performed according to a previous protocol. The V4 region of the 16S rRNA gene was amplified with 1x NEBNext q5 Hot Start HiFi PCR Master Mix using custom primers according to the method of Kozich et al., (2013). Sequencing was performed using an Illumina MiSeq system (300V2 kit). Sequenced paired reads were prepared using USEARCHv10.0.240_i86osx64. Forward and reverse reads were paired and filtered to a minimum length of 240 with a maximum expected error of 1. Sequences were dereplicated, clustered with a minimum cluster size of 2 and mapped to OTUs at 97% identity58. OTU taxonomy was assigned with the RDP classifier59. Sequences aligned with QIIME1, python 2.7, matplotlib1.4.3 using PyNAST in Conda. Tree constructed with FastTree on reads with RStudio package ape. Preformed OTU data analysis and visualization using Graphpad Prism and RStudio with gglot2 and phyloseq packages. Statistical significance performed by ANISOM using RStudio with phyloseq and vegan packages.

[0133] 8.2 Results Increased expression of HTR1B and HTR1D genes was detected in mice administered E. coli strains oN11 and oN14 (both p<0.05 by ANOVA) (Fig. 10a, b), whereas increased expression of HTR3, HTR4, and HTR7 genes was observed in the colon of mice administered E. coli strain oN14 (all p<0.01 by ANOVA) (Fig. 10c-e).

[0134] Furthermore, administration of E. coli strain oN14 specifically reduced total gastrointestinal transit time by 15% in treated mice compared to the control cohort (p<0.05 by ANOVA) (Figure 10). 16S metagenomic sequencing of feces from mouse cohorts following administration of E. coli Nissle strains oN14, oN11, oN9 or PBS alone on days 0 and 11 revealed no significant changes in gut microbiota between cohorts from day 0 to day 11 (R: 0.07403, significance: 0.155 by ANISOM) (not shown).

[0135] Example 9 Oral administration of the genetically modified E. coli strain of the present invention reduced anxiety in mice Oral administration of the genetically modified E. coli oN strain of the present invention is shown to induce behavioral changes in treated mice, which may be mediated through the brain-gut axis or an increase in peripheral and / or brain 5-HT levels. Two established methods, the forced swim test (FST) and the open field test (OFT), were employed to demonstrate these behavioral changes. The FST, commonly used to evaluate the efficacy of anxiolytic and antidepressant drugs in rodents, quantifies behavioral resignation by recording the time spent immobile in a water-filled container as a measure of desperation to escape a stressful environment. The OFT measures anxiety levels and willingness to explore in a stressful environment by detecting total excretion, total distance traveled, and the time spent exploring and staying in the inner and outer zones of a 50 x 50 cm white, well-lit open field.

[0136] The results of these studies indicate that oral administration of E. coli strain oN14 to mice has therapeutic effects against anxiety-related disorders.

[0137] 9.1 Methodology 9.1.1 Oral Administration: As described in 6.1.1.

[0138] 9.1.2 Forced swim test: Each mouse was placed in a clear cylindrical tank (30 cm high × 20 cm) containing 15 cm of water at 23–25 °C for 6 min and recorded as described by Ferguson (2001). Mice were then towel-dried and placed under a heat lamp to prevent hypothermia, then returned to their original cage. Recordings were anonymized and blinded observers scored the time spent immobile during the last 4 min of the 6 min test, as described by Can et al., (2012).

[0139] 9.1.3 Open field test: Each mouse was placed in a well-lit 50 cm (length) × 50 cm (width) × 38 cm (height) chamber with a white open roof and recorded for 10 min, according to previously described methods (Spohn, SNet et al., 2016). The mouse was then returned to its original cage. Fecal pellets produced during the 10 min test were counted. Recordings were then post-processed and analyzed using BehaviorCloud as previously described (Spohn, SNet et al., 2016). The mouse tracking software recorded the total path traveled by each mouse, the total distance traveled, the time spent in the inner zone, defined as the inner 40 cm square and the outer zone, defined as the outer 10 cm border closest to the wall, the number of entries into the inner zone, and the distance traveled within the inner zone.

[0140] 9.2 Results FST and OFT were performed on cohorts of mice after administration of E. coli Nissle strains oN14, oN11, oN9 or PBS alone.

[0141] In the FST, the cohort of mice treated with E. coli Nissle strain oN14 showed a statistically significant reduction in immobility time during the FST by up to 17% compared to the control cohort treated with E. coli Nissle strain oN (p=0.004 by ANOVA) (FIG. 11a), a result that was robustly replicated in another independent cohort of animals (not shown).

[0142] In the OFT, the mouse cohort administered E. coli Nissle strain oN14 showed a reduced number of excretions during the 10 min test (Fig. 11b), which may be associated with reduced stress or reduced total gastrointestinal transit (Fig. 10f). While wild-type mice naturally prefer closed, dark, and narrow spaces, the mouse cohort administered E. coli Nissle strain oN14 spent more time exploring the inner 40 × 40 cm zone and less time near the edge along the 10 × 10 cm outer zone (Fig. 11d, e), with no change in total distance traveled (Fig. 11c). These effects observed in the OFT were confirmed by OFT in another cohort of mice (not shown). An additional OFT in mice obtained from a different supplier (Jackson Laboratory) with different initial gut microbiota also produced similar physiological and behavioral results as above (not shown).

[0143] Example 10 Efficacy of oral administration of genetically modified E. coli strains of the present invention is consistent with SSRIs in mice Fluoxetine, sold under the brand names Prozac and Sarafem, among others, is a commonly used antidepressant of the selective serotonin reuptake inhibitor (SSRI) class. It is used to treat major depressive disorder, post-traumatic stress disorder (PTSD), and other indications. The efficacy of oral administration of the genetically modified E. coli oN strain of the present invention is shown to correspond to administration of fluoxetine by its ability to induce behavioral changes in treated mice that indicate a therapeutic reduction in anxiety or stress.

[0144] 10.1 Methodology: 10.1.1 Dosing: Oral dosing was performed as described in 6.1.1 with the following modifications: Mice were dosed with either sterile PBS alone (PBS) or freshly grown 10 9 Cells of E. coli Nissle strain designated oN14 (E. coli Nissle strain oN containing pMUT14-ser) were administered by oral gavage. Additionally, mice were administered a 200 μl intraperitoneal (IP) injection of saline or the SSRI, fluoxetine in saline at 10 mg / kg body weight. Mice received concurrent oral and IP treatments at the same time every 24 h for 21 days. Thus, four mouse treatment groups were given IP:saline / gavage:PBS (negative control), IP:fluoxetine / gavage:PBS (SSRI positive comparator), IP:saline / gavage:oN14 (oN14 positive comparator), and IP:fluoxetine / gavage:oN14 (positive combination comparator). 24 h after the last administration, mice were subjected to motility and behavioral testing.

[0145] 10.2 Results The phenotype exhibited by mice after treatment with oN14 (IP: saline / gavage: oN14) for 21 days is very similar to that of those treated with fluoxetine (IP: fluoxetine / gavage). As shown in Figure 12, A) a decrease in feces produced during the OFT is observed with both oN14, fluoxetine or combination treatment (IP: fluoxetine / gavage: oN14), indicating that a decrease in stress levels leads to fewer defecations. Furthermore, in Figure 12B)-E), the time spent in the medial zone and the distance traveled, as well as the total number of incursions into the medial zone, are all increased by oN14 or fluoxetine treatment. An increase in these parameters is interpreted as a decrease in anxiety or stress, indicating an increased willingness to explore in an unknown environment. In the forced swim test (Figure 12F), the time spent immobile is measured as an index of despair / depression. Treatment with oN14 and fluoxetine both significantly reduce the time of immobility, showing similar effects. Mice treated with oN14 showed similar effects on time spent in the medial zone, entry into the medial zone, and distance in the medial zone compared to mice treated with fluoxetine (Figure 12). The combination of oN14 and fluoxetine treatments resulted in a slight reduction in the total effect when compared to the individual groups treated with oN14 or fluoxetine alone.

[0146] Example 11 Oral administration of the genetically modified E. coli strain of the present invention increases plasma and urinary serotonin Oral administration of 5-HTP-producing E. coli Nissle strain oN10 to mice was shown to increase plasma and urinary serotonin and 5-HTP concentrations in mice.

[0147] 11.1 Methodology 11.1.1 Oral dosing: As described in 6.1.1 with the following modifications. Animals received streptomycin (5 g / L) in drinking water from 3 days prior to gavage and throughout the experiment. A single oral gavage of either 108 cells of oN10 or a control EcN strain lacking tryptophan hydroxylase (EcN_control). Animals were treated with the TDC inhibitor carbidopa by intraperitoneal injection (IP) every 24 hours and fresh fecal samples were collected daily for 7 days after which the animals were euthanized. Plasma samples were taken on days 2 and 7 after gavage.

[0148] 11.1.2 In vivo metabolite analysis: Plasma, tissues, gut contents, urine and feces samples were analyzed for serotonin, 5-hydroxytrytophan, tryptamine, 5-hydroxyindoleacetic acid (5-HIAA) and tryptophan. For plasma: Blood samples were taken from the submandibular vein on the second day after gavage (and on the eighth day after cardiac puncture) and kept on ice for 10 min in Li-Heparin Microtainers, then centrifuged at 10000g, 4°C for 90 s to separate plasma, which was flash frozen at -80°C. After thawing the samples, an internal standard buffer containing 0.9% NaCl, 0.2% ascorbic acid and 20 mg / L C-13 labeled tryptophan was added and proteins were precipitated using methanol extraction. After drying the samples using a vacuum centrifuge, they were reconstituted in 50 μl ddH2O for LC-MS / MS analysis. For tissue analysis: colonic tissues were weighed, frozen in liquid nitrogen immediately after collection, and freeze-fractured by tapping for 30 seconds with a stainless steel microvial (Biospec 2007) containing a single 6.34 mm stainless steel bead (Biospec 11079635ss) in a BeadBeater (Biospec 112011). Urine was collected on days 2 and 6 after gavage and immediately flash frozen at -80°C. After thawing the samples, an internal standard buffer containing 0.9% NaCl, 0.2% ascorbic acid, and 20 mg / L C-13 labeled tryptophan was added, and proteins were precipitated using methanol extraction. After drying the samples using a vacuum centrifuge, they were reconstituted in 50 μl of ddH2O for LC-MS / MS analysis.

[0149] 11.2 Results A significant increase in 5-HT concentrations was observed in plasma containing oN10 compared to the control strain lacking TPH expression (p<0.001 by two-tailed t-test), indicating successful oN10-derived production of 5-HTP in vivo (Figure 13A). The amount of 5-HTP produced by oN10 was sufficient to increase peripheral plasma serotonin concentrations without affecting tryptophan concentrations (Figure 13B), indicating a specific effect of oN10 on serotonin metabolism within physiologically relevant levels. Plasma concentrations of 5-HTP were below the quantification level in all samples. However, since 5-HTP is known to cross the blood-brain barrier, peripherally produced 5-HTP can increase serotonin biosynthesis in the brain, with potential therapeutic effects on mood, sleep, anxiety and other disorders (Turner et al., 2006). The oN10 strain represents a mechanism for delivering 5-HTP from the gastrointestinal tract in a constant dose, avoiding the undesirable effects of dose-dependent fluctuations in neurotransmitters such as 5-HTP and 5-HT.

[0150] Urinary 5-HT and 5-HTP concentrations were also increased in oN10 compared to controls (FIGS. 13C and D, respectively), indicating increased serotonin metabolism in oN10.

[0151] Example 12: Genetically engineered Saccharomyces cerevisiae cells express a biosynthetic pathway for 5-HT production Saccharomyces cerevisiae (S. cerevisiae) was genetically modified by the introduction of recombinant genes to establish a synthetic serotonin pathway that includes enzymes catalyzing two consecutive metabolic steps: converting TRP to 5-HTP and then converting 5-HTP to 5-HT (Figure 1). Identification of the optimal combination of enzymes catalyzing these two metabolic steps was determined by expressing and determining the 5-HT yields obtained from combinations of the TPH and TDC genes.

[0152] 12.1 Methodology The modified S. cerevisiae cells were engineered as follows, with reference to Table 1. The host strain, S. cerevisiae, was obtained from Mans et al., (2015). The genes encoding the TPH proteins from mammalian, plant or bacterial sources, mouse TPH1 (M1) and rice TDC (R), were the same as in 1.1.1. Different TDC genes from the bacterium Candidatus Koribacter versatilis Ellin345 (ck) were also tested. The pathway genes were cloned into either the yeast 2μ plasmid backbone or the ARS / CEN backbone (see FIG. 14). The M1 gene was cloned under the control of the PKG1 promoter, and the TDC genes (either rice TDC or ck TDC) were controlled by the TEF1 promoter. The plasmids were then transformed into the host strain by using standard cloning and transformation procedures known in the art.

[0153] Growth and in vitro metabolite production were determined as follows: Yeast strains were grown in synthetic complete dropout (SC) medium (6.7 g / L yeast nitrogen base without amino acids, 20 g / L glucose, 2 g / L dropout mix). The dropout mix used maintained the plasmid by lacking histidine. A negative control (no plasmid) was grown with 20 mg / L histidine. Three single colonies from each strain were picked and grown in 2 ml μl medium in 24 deep-well plates, shaking at 250 rpm at 30°C for 48 hours. The main cultures were grown in shake flasks at OD 200 in 25 ml SC medium + 500 mg / L tryptophan. 600 = 0.05 and cells were grown under the same conditions for 72 h. Culture supernatants were then separated from the cells by centrifugation at 17000 x g for 2 min and frozen at -20°C until analysis by HPLC. All data shown are the mean + / - SD from at least three biological replicates.

[0154] Metabolite production was quantified by LC-MS as in 1.1.3.

[0155] 12.2 Results: Genetically modified strains of the host S. cerevisiae engineered to express mouse TPH1 (M1) in combination with the bacterial TDC gene from rice (R) [or Candidatus Koribacter versatilis Ellin345 (ck_TDC)] produced significant amounts of serotonin (Figure 15A). In the control strain without TPH or TDC gene expression (Sc_Control), no serotonin production was detected (p<0.0018, ANOVA), while strains expressing mouse TPH1 and R-TDC (pSc-1 and pSc-2) or ck-TDC (pSc-3) (see Table 1) produced approximately 0.38+ / -0.07mg / L and 0.52+ / -0.06mg / L of serotonin, respectively. The ck-TDC gene can also functionally replace the R-TDC gene, allowing the production of serotonin in the E. coli construct (Figure 15B), but strong overexpression of ck-TDC causes cytotoxicity in oN (Figure 15C), resulting in reduced growth rate and final biomass yield compared to R-TDC expression.

[0156] Example 13: Genetically engineered Bacillus subtilis 168 cells express a biosynthetic pathway for 5-HT production Bacillus subtilis (B. subtilis) was genetically modified by the introduction of recombinant genes to establish a synthetic serotonin pathway that includes enzymes that catalyze two consecutive metabolic steps: converting TRP to 5-HTP and then converting 5-HTP to 5-HT (Figure 1). Identification of the optimal combination enzymes that catalyze these two metabolic steps will be determined by expressing and determining the 5-HT yields obtained from the combination of the TPH and TDC genes.

[0157] 13.1 Methodology The host strain B. subtilis 168 obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) [NC_000964] was genetically engineered as follows, with reference to Table 1. The genes encoding the TPH proteins derived from mammalian or plant sources, mouse TPH1 (M1) and rice TDC (R), were the same as in 1.1.1. The tested combinations of TPH and TDC genes were cloned into an integrative plasmid (derived from pDG364, see Table 1) together with the T198I variant of the E. coli folE gene to facilitate genomic integration of the pathway operon. The genes were placed in a synthetic operon consisting of a strong Bacillus-specific promoter (Ps11), a strong RBS of M1 (R01), a medium-strength RBS of TDC (RBS50 in Table 1), and a strong RBS of folE (T198I) (R11). Plasmid pBs-M1Rf contained the mouse TPH1, rice TDC, and folE(T198I) genes (FIG. 16).

[0158] Cloning was performed in E. coli Top10 by adopting standard molecular biology techniques. The plasmid was then transformed into the host strain by using standard cloning and transformation procedures known in the art. Correct pathway integration into the B. subtilis 168 genome at the amyE locus was verified by Sanger sequencing.

[0159] Growth and in vitro metabolite production were determined as follows. B. subtilis 168 strains were grown in Lysogeny Broth (LB) medium containing 0.2% (w / v) glucose and 50 mg / L L-tryptophan. Three single colonies from each strain were picked and grown in 300 μl of medium in 96-deep-well plates, shaking at 250 rpm at 37°C for 16 h. This preculture was inoculated into the main culture by diluting 1:100 into fresh medium, and the cells were grown under the same conditions for 24 h. The culture supernatant was then separated from the cells using a 0.2 μm pore size filter and frozen at -20°C until analysis by LC-MS. All data shown are the mean + / - SD from at least three biological replicates.

[0160] Metabolite production was quantified by LC-MS as in 1.1.3.

[0161] 13.2 Results: A host genetically modified strain, B. subtilis 168, engineered to express mouse TPH1 (M1), is expected to produce serotonin and 5-HTP in combination with TDCs derived from rice (R) and E. coli folE (T198I).

[0162] Although 5-HTP or 5-HT is not produced using the wild-type Bacillus subtilis 168 strain, expression of tryptophan hydroxylase M1 together with folE(T198I) would allow for the production of 5-HTP. Expression of M1_TPH, folE(T198I) and R_TDC is expected to result in the production of serotonin.

[0163] [Table 2-1] [Table 2-2] [Table 3]

Table 4

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Casteleyn,C.,Rekecki,A.,Van der Aa,A.,Simoens,P.& Van den Broeck,W.Surface area assessment of the murine intestinal tract as a prerequisite for oral dose translation from mouse to man.Lab.Anim.44,176-83(2010). de Almeida,R.M.& Miczek,K.A.Aggression escalated by social instigation or by discontinuation of reinforcement(“frustration”)in mice:inhibition by anpirtoline:a 5-HT1B receptor agonist.Neuropsychopharmacology 27,171-181(2002). Ferguson,J.M.SSRI Antidepressant Medications:Adverse Effects and Tolerability.Prim Care Companion J Clin Psychiatry 3,22-27(2001). Gershon,M.D.& Liu,M.T.Serotonin and neuroprotection in functional bowel disorders.Neurogastroenterol Motil 19 Suppl 2,19-24(2007). Gibson,D.G.et al.,Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods 6,343-345(2009). Guiziou,S.et al.,A part toolbox to tune genetic expression in Bacillus subtilis.Nucleic Acids Res.44,7495-7508(2016). 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Claims

1. 1. A composition for use as a pharmaceutical, said composition comprising recombinant Escherichia Bacterium cells, said cells each comprising one or more recombinant nucleic acid molecules encoding: (a) tryptophan 5-hydroxylase (EC 1.14.16.4), and (b) tryptophan decarboxylase (EC 4.1.1.28) The Escherichia Bacterium lacks a gene capable of expressing: i. Trp operon repressor protein, and / or ii. Tryptophanase (EC 4.1.99.1) the cell comprises a nucleic acid molecule encoding a mutant GTP cyclohydrolase I (EC 3.5.4.16), the mutant providing an increased hydroxylation activity of the tryptophan 5-hydroxylase of at least 2-fold compared to a non-mutated GCH1; The composition is intended to be administered orally.

2. The composition for use according to claim 1, wherein the mutant GTP cyclohydrolase I has amino acids having at least 80% sequence identity to SEQ ID NO:2 and includes substitutions selected from T198I, T198S, F214S, V179A, M99I and L200P.

3. The composition for use described in claim 1, wherein the amino acid sequence of the tryptophan 5-hydroxylase has at least 80% sequence identity to SEQ ID NO: 6 or 12, and the amino acid sequence of the tryptophan decarboxylase has at least 80% sequence identity to SEQ ID NO:

18.

4. The Escherichia strain is Further included are recombinant nucleic acid molecules encoding: (c) serotonin acetyltransferase (EC 2.3.1.87), and (d) acetylserotonin O-methyltransferase (EC 2.1.1.4); The composition for use according to claim 1 , wherein the cells are capable of producing melatonin.

5. A composition for use according to any one of claims 1 to 4, wherein the nucleic acid molecules encoding the tryptophan 5-hydroxylase and the tryptophan decarboxylase are operably linked to a first ribosome binding site and a second ribosome binding site, respectively, and the translation initiation strength of the first ribosome binding site is at least 10 times greater than that of the second ribosome binding site.

6. A composition for use according to any one of claims 1 to 4, wherein the nucleic acid molecules encoding the tryptophan 5-hydroxylase and the tryptophan decarboxylase are operably linked to a first ribosome binding site and a second ribosome binding site, respectively, and the translation initiation strength of the first ribosome binding site is at least two times lower than that of the second ribosome binding site.

7. The composition for use according to any one of claims 1 to 6, wherein the Escherichia Bacterium is an Escherichia coli strain.

8. A composition for use according to any one of claims 1 to 7 for use in the prevention and / or treatment of TRM, 5-HTP, 5-HT related disorders of the central nervous system (CNS) and enteric nervous system (ENS) in an animal.

9. 9. The composition for use according to claim 8, wherein the disorder of the central nervous system is selected from the group consisting of behavioral, memory, cognitive and psychiatric disorders associated with anxiety and depression, generalized anxiety disorder, phobic disorders, social anxiety disorder, panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, chronic stress disorder, separation and situational anxiety, age-related memory decline, dementia and disorders in sleep, spatial memory formation, vigilance, concentration, learning and cognition, autism and migraine.

10. The composition for use according to any one of claims 1 to 9, wherein said composition is for oral administration to a mammal in need thereof.

11. 1. A recombinant cell of Escherichia Bacterium comprising one or more recombinant nucleic acid molecules or transgenes encoding one or more proteins selected from: (a) tryptophan 5-hydroxylase (EC 1.14.16.4), and (b) tryptophan decarboxylase (EC 4.1.1.28) The Escherichia Bacterium lacks a gene capable of expressing: i. trp operon repressor protein and ii. Tryptophanase (EC: 4.1.99.1) A recombinant cell, wherein the cell comprises a nucleic acid sequence encoding a mutant GTP cyclohydrolase I (EC 3.5.4.16), the mutant providing an increased hydroxylation activity of the tryptophan 5-hydroxylase of at least 2-fold compared to non-mutated GCH1.

12. The recombinant cell of Escherichia Bacterium of claim 11, wherein the mutant GTP cyclohydrolase I has an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 and includes substitutions selected from T198I, T198S, F214S, V179A, M99I and L200P.

13. 13. The recombinant Escherichia Bacterium cell of claim 11 or 12, wherein the amino acid sequence of the tryptophan 5-hydroxylase has at least 80% sequence identity to SEQ ID NO:6 or 12, and the amino acid sequence of the tryptophan decarboxylase has at least 80% sequence identity to SEQ ID NO:

18.

14. The nucleic acid molecule encoding tryptophan 5-hydroxylase and tryptophan decarboxylase is operably linked to a first ribosome binding site and a second ribosome binding site, respectively; 14. The recombinant Escherichia Bacterium cell of claim 11, wherein the translation initiation strength of the first ribosome binding site is at least 10 times greater than the second ribosome binding site.

15. A method for producing a nucleic acid molecule comprising the steps of: (a) detecting a tryptophan 5-hydroxylase and a tryptophan decarboxylase in a mammalian cell; and (b) detecting a tryptophan 5-hydroxylase and a tryptophan decarboxylase in a mammalian cell.

15. The recombinant Escherichia Bacterium cell of claim 11, wherein the translation initiation strength of the first ribosome binding site is at least two-fold lower than the second ribosome binding site.

16. The method of claim 1, further comprising the steps of: (c) serotonin acetyltransferase (EC 2.3.1.87), and (d) acetylserotonin O-methyltransferase (EC 2.1.1.4), the recombinant cell of Escherichia Bacterium of claim 11, wherein the cell is capable of producing melatonin.

17. The recombinant bacterial cell of any one of claims 11 to 16, wherein the cell is not antibiotic resistant to one or more clinically used antibiotics.

18. A recombinant cell of Escherichia Bacterium according to any one of claims 11 to 17, wherein the cell is an Escherichia coli strain.

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