Search

CN-121971491-A - Engineered propionic acid-producing microbial cells and uses thereof

CN121971491ACN 121971491 ACN121971491 ACN 121971491ACN-121971491-A

Abstract

The present invention relates to engineered microbial cells producing propionic acid and uses thereof. In particular, the invention provides an engineered microbial cell that produces propionic acid comprising specific genetic modifications of (1) increased activity of an enzyme associated with the propionic acid synthesis pathway, and optionally (2) blocking or reducing the acetic acid synthesis pathway, which has increased propionic acid production. Also provided are methods of constructing the engineered microbial cells that produce propionic acid, and uses in the production of propionic acid. Also provided is the use of the propionic acid-producing engineered microbial cells or pharmaceutical compositions thereof in the treatment of alzheimer's disease.

Inventors

  • WU YILI
  • HUANG HE
  • SONG WEIHONG

Assignees

  • 瓯江实验室

Dates

Publication Date
20260505
Application Date
20260205

Claims (16)

  1. 1. Use of an engineered microbial cell producing propionic acid in the manufacture of a medicament for preventing and/or treating a neurodegenerative disease, such as Alzheimer's disease, Wherein the engineered microbial cell comprises: (1) Increased activity of the propionic acid synthesis pathway-related enzyme, e.g., heterologous expression of a gene encoding the propionic acid synthesis pathway-related enzyme; Preferably, the gene encoding the enzyme involved in the propionic acid synthesis pathway is selected from 1,2 or 3 of the group consisting of a gene encoding propionyl-CoA transferase (pct), a gene encoding lactyl-CoA dehydratase (lcd), a gene encoding acrylyl-CoA reductase (acr); optionally, the composition may be in the form of a gel, (2) Blocking or reducing the acetate synthesis pathway, for example by knocking out, deleting or knocking down an endogenous acetate synthesis pathway-related gene; Preferably, the acetate synthesis pathway related genes are selected from 1, 2 or 3 of the group consisting of a gene encoding pyruvate formate lyase (pflB), a gene encoding pyruvate oxidase (poxB), a gene encoding acetate kinase (ackA).
  2. 2. The use of claim 1, wherein the engineered microbial cell comprises any one selected from the group consisting of: (1) Heterologous expression of exogenous pct gene, exogenous lcd gene and exogenous acr gene; (2) (i) heterologous expression of an exogenous pct gene, an exogenous lcd gene, and an exogenous acr gene, and (ii) deletion, knock-out, or knock-down of an endogenous pflB gene; (3) (i) heterologous expression of an exogenous pct gene, an exogenous lcd gene, and an exogenous acr gene, and (ii) deletion, knock-out, or knock-down of an endogenous pflB gene and a poxB gene; (4) (i) heterologous expression of exogenous pct gene, exogenous lcd gene, and exogenous acr gene, and (ii) deletion, knock-out, or knock-down of endogenous pflB gene, poxB gene, and ackA gene.
  3. 3. Use according to claim 1 or 2, wherein the enzyme related to the propionic acid synthesis pathway is derived from clostridium propionicum (Clostridium propionicum), acetobacter thermophilus (Moorella thermoacetica), preferably clostridium propionicum (Clostridium propionicum).
  4. 4. The use of any one of claims 1-3, wherein the engineered microbial cell is derived from a lactic acid producing microbial cell; The engineered microbial cells are derived from escherichia coli, yeast, probiotics, clostridium propionicum, lactobacillus, bifidobacterium, or actinomycetes; Preferably, the E.coli is selected from ESCHERICHIA COLI NISSLE 1917.
  5. 5. Use of a pharmaceutical composition in the manufacture of a medicament for preventing and/or treating a neurodegenerative disease, such as alzheimer's disease, in a subject; wherein the pharmaceutical composition comprises engineered microbial cells that produce propionic acid, and optionally a pharmaceutically acceptable carrier and/or excipient; Wherein the engineered microbial cell that produces propionic acid is selected from the engineered microbial cell of any one of claims 1-4.
  6. 6. Use of a fermentation product in the manufacture of a medicament for preventing and/or treating a neurodegenerative disease, such as alzheimer's disease, in a subject; Wherein the fermentation product is obtained by fermentation production of the engineered microbial cell of any one of claims 1-4.
  7. 7. An engineered microbial cell that produces propionic acid, comprising: (1) Increased activity of the propionic acid synthesis pathway-related enzyme, e.g., heterologous expression of a gene encoding the propionic acid synthesis pathway-related enzyme; Wherein the gene encoding the enzyme involved in the propionic acid synthesis pathway is selected from 1,2 or 3 of the group consisting of a gene encoding propionyl-CoA transferase (pct), a gene encoding lactyl-CoA dehydratase (lcd), and a gene encoding acrylyl-CoA reductase (acr); wherein the propionic acid synthesis pathway-related enzyme is derived from clostridium propionicum (Clostridium propionicum); optionally, the composition may be in the form of a gel, (2) The engineered microbial cells also comprise a blocking or reducing acetate synthesis pathway, for example by knocking out, deleting or knocking down an endogenous acetate synthesis pathway-related gene; Wherein the acetate synthesis pathway related genes are selected from 1, 2 or 3 of a gene encoding pyruvate formate lyase (pflB), a gene encoding pyruvate oxidase (poxB), a gene encoding acetate kinase (ackA); wherein the engineered microbial cells are derived from lactic acid producing microbial cells, preferably the engineered microbial cells are derived from E.coli, yeast, probiotic bacteria, clostridium propionicum, lactobacillus, bifidobacterium, or actinomycetes, preferably the E.coli is selected from ESCHERICHIA COLI NISSLE 1917.
  8. 8. The engineered microbial cell of claim 7, comprising any one selected from the group consisting of: (1) Heterologous expression of exogenous pct gene, exogenous lcd gene and exogenous acr gene; (2) (i) heterologous expression of an exogenous pct gene, an exogenous lcd gene, and an exogenous acr gene, and (ii) deletion, knock-out, or knock-down of an endogenous pflB gene; (3) (i) heterologous expression of an exogenous pct gene, an exogenous lcd gene, and an exogenous acr gene, and (ii) deletion, knock-out, or knock-down of an endogenous pflB gene and a poxB gene; (4) (i) heterologous expression of exogenous pct gene, exogenous lcd gene, and exogenous acr gene, and (ii) deletion, knock-out, or knock-down of endogenous pflB gene, poxB gene, and ackA gene.
  9. 9. The method of constructing an engineered microbial cell of claim 7 or 8, comprising the steps of: (1) Introducing into a host cell the exogenous gene encoding the enzyme involved in the propionic acid synthesis pathway (e.g., exogenous pct gene, exogenous lcd gene, and exogenous acr gene), and (2) Knocking out, deleting or knocking down the endogenous acetate synthesis pathway related genes (e.g., 1, 2 or 3 of the endogenous pflB gene, poxB gene and ackA gene) described in the host cell; Preferably, the host cell is selected from the group consisting of a lactic acid producing microbial cell, preferably, the host cell is selected from the group consisting of E.coli, yeast, probiotic bacteria, clostridium propionicum, lactobacillus, bifidobacterium, and actinomycetes; Preferably, the E.coli is selected from ESCHERICHIA COLI NISSLE 1917.
  10. 10. A method of producing propionic acid comprising culturing the engineered microbial cell of claim 7 or 8 in a medium; Preferably, the medium comprises glucose at a concentration of 10 g/L.
  11. 11. The method of claim 10, further comprising recovering propionic acid from the culture.
  12. 12. A method for increasing the yield or conversion of propionic acid in propionic acid biosynthesis, comprising introducing into a host cell a genetic modification as defined in any one of claims 7 or 8; Preferably, the host cell is selected from the group consisting of a lactic acid producing microbial cell, preferably, the host cell is selected from the group consisting of E.coli, yeast, probiotic bacteria, clostridium propionicum, lactobacillus, bifidobacterium, and actinomycetes; Preferably, the E.coli is selected from ESCHERICHIA COLI NISSLE 1917.
  13. 13. A fermentation product obtained by fermentation production of the engineered microbial cell of claim 7 or 8.
  14. 14. Use of the engineered microbial cell of claim 7 or 8, or the fermentation product of claim 13, in the biosynthesis of propionic acid.
  15. 15. A pharmaceutical composition comprising the engineered microbial cell of claim 7 or 8 or the fermentation product of claim 13, and optionally a pharmaceutically acceptable carrier and/or excipient.
  16. 16. Use of an engineered microbial cell according to claim 7 or 8, a fermentation product according to claim 13 or a pharmaceutical composition according to claim 15 for the manufacture of a medicament for the prevention and/or treatment of a neurodegenerative disease, such as alzheimer's disease, in a subject.

Description

Engineered propionic acid-producing microbial cells and uses thereof Technical Field The invention relates to the technical fields of biomedicine and probiotics, in particular to an engineering microbial cell for producing propionic acid, a construction method thereof and application of the engineering microbial cell for producing propionic acid in the production of propionic acid and the treatment of Alzheimer disease. Background Alzheimer's Disease (AD) is the most common form of dementia, and presents a tremendous social and economic burden. Senile plaques are considered to be a pathological feature of AD, consisting of amyloid β protein (aβ), which may lead to secondary pathological changes such as Tau protein hyperphosphorylation, neuroinflammation, oxidative stress and neurite degeneration, ultimately leading to dementia. The intestinal brain axis brain and intestinal tract are regulated by nerves, endocrine, immunity and metabolism. Studies have shown that the intestinal flora of AD patients is markedly deregulated, including a reduction in beneficial bacteria and an increase in harmful bacteria (Zhang T, et al Gut Microbes 2023, 15 (2): 2271613.). Probiotics have been shown to regulate intestinal flora structure, short chain fatty acids being the main metabolite of probiotics. Can be used for effectively treating AD. However, because of the imbalance in intestinal flora in AD patients, there is a reduction in short chain fatty acid producing bacteria, propionic acid being one of the short chain fats, has anti-inflammatory and antioxidant properties, protects the integrity of the blood brain barrier, regulates mitochondrial division and autophagy (Wang Z, et al Microbiome 2025, 13 (1)), and thus reduces cognitive impairment. However, the exogenous supplementation of propionic acid has the defects of rapid absorption of the proximal small intestine after oral administration, high first pass ratio of the liver and insufficient effective concentration of the colon, slow release of enteric capsules/pellets, low bioavailability and possibly excessive transient blood concentration. The lack of a 'viable bacteria continuous factory' effect by pure chemical supplementation, fecal propionic acid is recovered to a baseline after drug withdrawal, and the general basic imbalance of 'propionic acid-producing deficiency-proinflammatory bacteria expansion' of AD patients cannot be corrected. The natural propionic acid-producing bacteria are supplemented, and the natural propionic acid-producing bacteria have inherent limitations, namely, human-derived culturable propionic acid-producing bacteria mainly comprise AKKERMANSIA MUCINIPHILA, phascolarctobacterium succinatutens, roseburia inulinivorans and the like, but have the defects that ① genome is large, a large number of mobile elements are contained, genetic background is unclear, ② is strictly anaerobic, is extremely sensitive to oxygen, bile salts and gastric acid, oral survival rate is low, ③ in-vitro propionic acid yield is low, and acetic acid, succinic acid and other byproducts cannot be supplied in a 'precise-sufficient quantity', and ④ partial strains carry virulence islands (such as ALISTIPES spp. LPS synthetic clusters) and have potential infection risks. Therefore, it is difficult for natural bacteria to be developed directly as "next-generation living organism medicines". Coli Nissle 1917 (EcN) is a WHO approved probiotic, and has been matured for genetic manipulation and has been used in clinical trials for inflammatory bowel disease (Luo R, et al, nature Communications 2026.), diabetes (Luo J, et al, canadian Journal of Infectious DISEASES AND MEDICAL Microbiology 2025, 2025 (1): 6675676.), and the like. Therefore, development of propionic acid-producing probiotics is highly needed to provide innovative strategies for clinical control of alzheimer's disease. Disclosure of Invention The invention uses a synthetic biological method and takes escherichia coli Nissle 1917 as chassis bacteria to construct an engineering microbial cell for producing propionic acid, wherein the engineering microbial cell has improved propionic acid yield. In addition, the double advantages of the engineering microorganism cells combined with the propionic acid and the probiotics show remarkable synergistic effect, can effectively improve the working memory capacity and the space learning memory capacity of the Alzheimer disease model mice, provides an innovative strategy for clinical prevention and treatment of the Alzheimer disease, and further shows wide prospects of the engineering probiotics in treatment of neurodegenerative diseases. Specifically, the invention provides the following technical scheme. I. Use of engineered microbial cells producing propionic acid in the treatment of alzheimer's disease Thus, in a first aspect, the present invention provides the use of an engineered microbial cell producing propionic acid in the manufacture of a medicament for the preven