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Advanced microbiome therapy engineered for serotonin production in vivo

A technology of microbes and drugs, applied in the field of advanced microbiome therapy engineered to produce serotonin in the body, can solve problems such as the spike in 5-HT levels

Pending Publication Date: 2022-05-06
DANMARKS TEKNISKE UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This has the undesired consequence of rapid uptake of the administered 5-HTP, resulting in a sudden spike in 5-HT levels

Method used

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  • Advanced microbiome therapy engineered for serotonin production in vivo
  • Advanced microbiome therapy engineered for serotonin production in vivo
  • Advanced microbiome therapy engineered for serotonin production in vivo

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0149] Example 1: Genetically Modified E. coli Cells Expressing Biosynthetic Pathways for 5-HT Production

[0150] The 5-HT biosynthetic pathway was introduced into cells of E. coli Nissl to establish a synthetic serotonin pathway to catalyze the two sequential metabolic steps of conversion of TRP to 5-HTP and then 5-HTP to 5-HT ( figure 1 ). Identification of the optimal combined enzymes for catalyzing these two metabolic steps was determined by expressing and determining the 5-HT yields obtainable from the combination of TPH and TDC genes.

[0151] 1.1 Method

[0152] 1.1.1 The modified E. coli cells are engineered as follows, refer to Table 1:

[0153] The host strain Escherichia coli Nissl 1917 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 (component BBa_J23101, Registry of Standard Biological Parts, www.parts.igem.org) and integrated into the...

example 2

[0161] Example 2: Enhancement of 5-HT and TRM production in genetically modified E. coli cells of the invention by increasing tryptophan pathway flux

[0162] Increased availability of the tryptophan pathway substrate tryptophan was shown to increase 5-HT production in the 5-HT producing strains of the invention ( image 3 ). Based on this observed increase, the endogenous pool of TRP available in the genetically engineered cells of the invention is increased by inactivation of the endogenous genes encoding the tryptophan repressor trpR and the tryptophanase tnaA , such as enhancing tryptophan pathway flux.

[0163] 2.1 Method:

[0164] Knockdown of trpR and tnaA from the host E. coli Nissl 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 double-strand breaks at desired gene loci in the host genome. The gRN...

example 3

[0168] Example 3: Genetically Modified E. coli Strains of the Invention Exhibit Stable 5-HT Production

[0169] When the 5-HT operon encoding the 5-HT biosynthetic pathway is cloned into a multi-copy plasmid in the host cells of the present invention, 5-HT production depends on the stability of the plasmid. Stability is preferably not dependent on plasmid genes conferring antibiotic resistance. Two solutions for conferring plasmid stability were compared; the pUC-based plasmid backbone (as in Examples 1 and 2) and the two native plasmids of E. coli Nissl pMUT1 (Blum-Oehler et al., 2003).

[0170] 3.1 Method:

[0171] A high copy number 5-HT producing 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 in order to generate E. coli Nissler strain NΔ3 (ΔtrpR, ΔtnaA, ΔinfA).

[0172] The ...

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Abstract

The present invention provides a composition for use as a medicament, the composition comprising a cell of a recombinant microorganism, the cell being capable of producing an increased amount of one or more of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT) and tryptamine (TRM) compared to a non-recombinant microorganism from which the cell is derived. The composition can be used for preventing and / or treating TRM, 5-HTP or 5-HT related conditions in mammals: central nervous system (CNS) conditions; an enteral nervous system (ENS) disorder; gastrointestinal (GI) disorders and metabolic disorders, and the compositions can be orally administered to mammals in need thereof. Furthermore, a composition comprising cells of a recombinant microorganism capable of producing melatonin is provided for use as a medicament, 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 medicament comprising cells of a recombinant microorganism capable of producing increased amounts of 5-hydroxytryptophan ( 5-HTP), 5-hydroxytryptamine (5-HT) and tryptamine (TRM) in one or more. The composition can be used for the prevention and / or treatment of TRM, 5-HTP or 5-HT-related disorders in mammals: central nervous system (CNS) disorders; enteric nervous system (ENS) disorders; gastrointestinal (GI) ) disorders and metabolic disorders, and the composition can be orally administered to a mammal in need thereof. In addition, there is provided a composition comprising cells of a recombinant microorganism capable of producing melatonin for use as a medicament. Background technique [0002] The biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT) is critical to neurotransmission and many other functions throughout the body. Up to 95% of the serotonin in the body is produced in the ga...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K35/742A61K35/744A61K35/745A61K35/747A61K35/741A61P25/00A61P25/22A61P25/24A61P1/00A61P35/00A61P3/00A61P1/16A61P3/10A61P29/00A61P37/02C12R1/185C12R1/01C12R1/145C12R1/225C12R1/46C12R1/07C12R1/44
CPCC12N9/0071C12N9/88C12N9/1029C12N9/1007C12N9/78A61K35/742A61K35/744A61K35/745A61K35/747A61K35/741A61P25/00A61P25/22A61P25/24A61P1/00A61P35/00A61P3/00A61P1/16A61P3/10A61P29/00A61P37/02C12Y114/16004C12Y401/01028C12Y401/99001C12Y203/01087C12Y201/01004C12Y305/04016A61K38/44A61K38/51A61K38/45C12N9/1003C12P17/10
Inventor M·O·A·萨默M·邦格斯H·H·王F·库西马诺
Owner DANMARKS TEKNISKE UNIV
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