Alcoholism relieving engineering bacteria, construction and use thereof

Abstract

The application discloses an alcoholism-relieving engineering bacterium and application. Specifically, the application provides an ethanol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) engineering bacterium, wherein the engineering bacterium is Escherichia coli, and an exogenous ADH and ALDH expression cassette is integrated into the genome of the Escherichia coli.

Description

A hangover-detoxifying engineered bacterium and its construction and use Technical Field

[0001] The present invention relates to the fields of biotechnology / synthetic biology / bioengineering / genetic engineering / enzyme engineering; in particular, the present invention relates to an alcohol-detoxifying engineered bacterium and its construction and use.

[0002] Sequence Listing

[0003] This application contains an electronic sequence listing (L240042CN-sequencelisting.xml; size: 33KB; creation time: 2025.01.08), the contents of which are incorporated herein by reference in their entirety. Background Art

[0004] After the human body ingests alcohol, it is first rapidly absorbed by the stomach, then diffuses freely into the bloodstream, and then circulates into the liver and intestines. Alcohol in the human body is primarily metabolized into acetaldehyde by alcohol dehydrogenase (ADH) in the liver, which is then metabolized into harmless acetic acid by acetaldehyde dehydrogenase (ALDH).

[0005] A large proportion of Asians carry a variant of the aldehyde dehydrogenase enzyme. Mutations in the Aldh2 allele reduce the activity of the enzyme, making the conversion of acetaldehyde to acetic acid inefficient. This leads to the accumulation of this harmful substance in the body, causing symptoms such as flushing, rapid heartbeat, nausea, blurred vision, shortness of breath, and liver damage. Furthermore, acetaldehyde is a known carcinogen, and its accumulation in the body can increase the risk of acetaldehyde-related cancers, such as liver cancer, esophageal cancer, and digestive tract cancer.

[0006] One of the effective strategies to deal with the accumulation of acetaldehyde after drinking is to use engineered microorganisms to efficiently metabolize the ingested alcohol and the intermediate product acetaldehyde in the digestive tract, thereby alleviating the harm caused by acetaldehyde accumulation in the human body.

[0007] Currently, the hangover-relieving products on the market include natural products, natural probiotics and genetically engineered bacteria. Among them, natural products such as curcumin and puerarin, and natural probiotics such as Pediococcus pentosaceus and Lactobacillus rhamnosus, have not yet had clear in vivo evidence to prove their hangover-relieving effects. The ZB183 strain sold by Zbiotics in the United States is a strain that overexpresses acetaldehyde dehydrogenase by modifying Bacillus subtilis. Its in vitro acetaldehyde metabolism capacity is limited. Considering the complex environment in the intestine, whether ZB183 can play an effective role in vivo has not yet been supported by data. The existing public technology (publication number CN110592125A) expresses alcohol dehydrogenase and acetaldehyde dehydrogenase in Bacillus subtilis. Its in vitro experiments show that the genetically engineered bacteria can effectively degrade ethanol, but do not show its metabolic effect on acetaldehyde. In addition, the expensive cofactor NAD+ is added to its reaction system, which greatly increases the production cost.

[0008] The existing technology still has certain limitations in terms of the effects of sobering up and / or alleviating liver damage after drinking.

[0009] Therefore, the purpose of the present invention is to develop a genetically engineered bacterium that can degrade ethanol and / or acetaldehyde more cheaply, safely and efficiently, so as to alleviate the harm caused by the accumulation of ethanol and / or acetaldehyde in the human body. Summary of the Invention

[0010] In a first aspect of the present invention, an engineered bacterium producing alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) is provided. The engineered bacterium is Escherichia coli Nissle 1917, and the Escherichia coli has or has exogenous ADH and ALDH expression cassettes integrated into its genome, and the ADH and ALDH expression cassettes are used to express alcohol dehydrogenase and acetaldehyde dehydrogenase.

[0011] In one embodiment, the source of the exogenous ADH includes: Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Kluveromyces lactis.

[0012] In one embodiment, the sources of the exogenous ALDH include: Saccharomyces cerevisiae, Escherichia coli, Cupriavidus necator, and Pichia terricola.

[0013] In one embodiment, the exogenous ADH and / or ALDH is derived from Saccharomyces cerevisiae; preferably, the exogenous ADH and / or ALDH is derived from Saccharomyces cerevisiae S288c.

[0014] In one embodiment, the ADH is Saccharomyces cerevisiae ADH2.

[0015] In one embodiment, the Saccharomyces cerevisiae ADH2 has the amino acid sequence shown in SEQ ID NO: 1, or its amino acid sequence is shown in SEQ ID NO: 1.

[0016] In one embodiment, the gene encoding the alcohol dehydrogenase has been codon-optimized for expression in E. coli.

[0017] In one embodiment, the gene encoding Saccharomyces cerevisiae ADH2 has a nucleotide sequence as shown in SEQ ID NO: 8, or the nucleotide sequence of the gene encoding it is shown in SEQ ID NO: 8.

[0018] In one embodiment, the ALDH is Saccharomyces cerevisiae ALDH1.

[0019] In one embodiment, the Saccharomyces cerevisiae ALDH1 has the amino acid sequence shown in SEQ ID NO: 2, or its amino acid sequence is shown in SEQ ID NO: 2.

[0020] In one embodiment, the gene encoding the acetaldehyde dehydrogenase has been codon-optimized for expression in E. coli.

[0021] In one embodiment, the Saccharomyces cerevisiae ALDH1 encoding gene has a nucleotide sequence as shown in SEQ ID NO: 9, or the nucleotide sequence of the encoding gene thereof is shown in SEQ ID NO: 9.

[0022] The present invention also provides an engineered bacterium expressing alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), characterized in that the engineered bacterium is Escherichia coli, and the Escherichia coli has or has exogenous ADH and ALDH expression cassettes integrated into its genome, and the ADH and ALDH expression cassettes are used to express alcohol dehydrogenase and acetaldehyde dehydrogenase, wherein the ADH has the amino acid sequence shown in SEQ ID NO: 1, and the ALDH has the amino acid sequence shown in SEQ ID NO: 2.

[0023] In one embodiment, the E. coli is E. coli Nissle 1917.

[0024] In one embodiment, the gene encoding ADH has a nucleotide sequence as shown in SEQ ID NO: 8, and the gene encoding ALDH has a nucleotide sequence as shown in SEQ ID NO: 9.

[0025] In one embodiment, the expression of the genes encoding ADH and ALDH is controlled by one promoter, or respectively controlled by different promoters.

[0026] In one embodiment, the genes encoding the ADH and the ALDH are linked in tandem.

[0027] In one embodiment, the genes encoding the ADH and the ALDH are linked in tandem via a ribosome binding site (RBS).

[0028] In one embodiment, the RBS has the nucleotide sequence shown in SEQ ID NO:3.

[0029] In one embodiment, the engineered bacteria is expressed on a vector or integrated into the genome for expression.

[0030] In one embodiment, the exogenous ADH and ALDH expression cassette has the structure shown in Formula I: Z1-Z2-Z3-Z4 (I)

[0031] Wherein, “-” is each independently a bond or a nucleotide linking sequence;

[0032] Z1 is a promoter or a 5′-UTR element containing a promoter;

[0033] Z2 is absent or is an optional enhancer;

[0034] Z3 is a nucleotide sequence encoding ADH and ALDH, wherein the nucleotide sequence encoding ADH and the nucleotide sequence encoding ALDH are connected in tandem via a ribosome binding site (RBS); and

[0035] Z4 is absent or is a 3'-UTR element.

[0036] In one embodiment, the promoter is a constitutive promoter or an inducible promoter.

[0037] In one embodiment, the promoter is an endogenous promoter or an exogenous promoter.

[0038] In one embodiment, the promoter is the tac promoter.

[0039] In a second aspect, the present invention provides a method for constructing an engineered bacterium expressing alcohol dehydrogenase and acetaldehyde dehydrogenase as described above, comprising the following steps:

[0040] (s1) Obtaining exogenous ADH and ALDH genes;

[0041] (s2) connecting the ADH and ALDH genes to a plasmid, and connecting the ADH and ALDH genes via RBS, thereby constructing a recombinant plasmid containing the ADH and ALDH genes; and

[0042] (s3) transforming Escherichia coli Nissle 1917 strain with the recombinant plasmid, and screening transformants on a selective medium to obtain the engineered bacteria.

[0043] In one embodiment, the source of the exogenous ADH includes: Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Kluveromyces lactis.

[0044] In one embodiment, the sources of the exogenous ALDH include: Saccharomyces cerevisiae, Escherichia coli, Cupriavidus necator, and Pichia terricola.

[0045] In one embodiment, the exogenous ADH is an alcohol dehydrogenase selected from the group consisting of Saccharomyces cerevisiae ADH1, Saccharomyces cerevisiae ADH2, Candida albicans ADH1, Kluyveromyces marxianus ADH4, Kluyveromyces lactis ADH4, or a combination thereof.

[0046] In one embodiment, the exogenous ALDH is an acetaldehyde dehydrogenase selected from the group consisting of Saccharomyces cerevisiae ALDH1, Saccharomyces cerevisiae ALDH2, Saccharomyces cerevisiae ALDH3, Escherichia coli ADHE, Cupriavidus necrotizingus AcoD, Pichia trichoderma ALDH, or a combination thereof.

[0047] In one embodiment, the exogenous ADH is derived from Saccharomyces cerevisiae; preferably, from Saccharomyces cerevisiae S288c.

[0048] In one embodiment, the exogenous ALDH is derived from Saccharomyces cerevisiae; preferably, from Saccharomyces cerevisiae S288c.

[0049] In one embodiment, the exogenous ADH is Saccharomyces cerevisiae ADH2.

[0050] In one embodiment, the exogenous ALDH is Saccharomyces cerevisiae ALDH1.

[0051] In one embodiment, the Saccharomyces cerevisiae ADH2 has the amino acid sequence shown in SEQ ID NO: 1.

[0052] In one embodiment, the gene encoding the alcohol dehydrogenase has been codon-optimized for expression in E. coli.

[0053] In one embodiment, the gene encoding ADH2 has the nucleotide sequence shown in SEQ ID NO:8.

[0054] In one embodiment, the Saccharomyces cerevisiae ALDH1 has the amino acid sequence shown in SEQ ID NO:2.

[0055] In one embodiment, the gene encoding the acetaldehyde dehydrogenase has been codon-optimized for expression in E. coli.

[0056] In one embodiment, the gene encoding ALDH1 has the nucleotide sequence shown in SEQ ID NO:9.

[0057] In one embodiment, in the recombinant plasmid, the encoding genes of ADH and ALDH are expressed under the control of one promoter, or are expressed under the control of different promoters.

[0058] In one embodiment, the RBS has the nucleotide sequence shown in SEQ ID NO:3.

[0059] In one embodiment, the recombinant plasmid contains the structure shown in Formula I: Z1-Z2-Z3-Z4 (I)

[0060] Wherein, “-” is each independently a bond or a nucleotide linking sequence;

[0061] Z1 is a promoter or a 5′-UTR element containing a promoter;

[0062] Z2 is absent or is an optional enhancer;

[0063] Z3 is a nucleotide sequence encoding ADH and ALDH, wherein the nucleotide sequence encoding ADH and the nucleotide sequence encoding ALDH are connected in tandem via a ribosome binding site (RBS); and

[0064] Z4 is absent or is a 3'-UTR element.

[0065] In one embodiment, the promoter is a constitutive promoter or an inducible promoter.

[0066] In one embodiment, the promoter is an endogenous promoter or an exogenous promoter.

[0067] In one embodiment, the promoter is the tac promoter.

[0068] In a third aspect, the present invention provides a method for producing alcohol dehydrogenase and acetaldehyde dehydrogenase, wherein the engineered bacteria producing alcohol dehydrogenase and acetaldehyde dehydrogenase as described in the first aspect of the present invention are cultured to obtain alcohol dehydrogenase and acetaldehyde dehydrogenase.

[0069] In a fourth aspect, the present invention provides a composition for promoting acetaldehyde degradation and / or hangover relief, comprising: (a) a safe and effective amount of engineered bacteria producing alcohol dehydrogenase and acetaldehyde dehydrogenase as described in the first aspect of the present invention; and (b) a food or pharmaceutically acceptable carrier.

[0070] In one embodiment, the composition is selected from the group consisting of a food composition, a health product composition, a pharmaceutical composition, or a combination thereof.

[0071] In one embodiment, the composition is an oral formulation.

[0072] In one embodiment, the composition is in the form of: (i) a liquid; (ii) a solid; or (iii) a semisolid.

[0073] In one embodiment, the dosage form of the composition is selected from the group consisting of powders, powders, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

[0074] In one embodiment, the food composition comprises an emulsion product, a solution product, a powder product, or a suspension product.

[0075] In one embodiment, the food composition comprises milk, milk powder, or emulsion.

[0076] In one embodiment, the liquid preparation is selected from the group consisting of a solution preparation or a suspension preparation.

[0077] In one embodiment, the composition contains 1×10-1×10 20 cfu / mL or cfu / g of the engineered bacteria of the present invention, preferably 1×10 4 -1×10 15 cfu / mL or cfu / g of the engineered bacteria of the present invention, based on the total volume or weight of the composition.

[0078] In one embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt% of the engineered bacteria of the present invention, based on the total weight of the composition.

[0079] In one embodiment, the composition is in unit dosage form (a tablet, a capsule or a vial), and the mass of the composition in each unit dosage form is 0.05-5 g, preferably 0.1-1 g.

[0080] In one embodiment, the composition further comprises other probiotics and / or prebiotics.

[0081] In one embodiment, the probiotics are selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof.

[0082] In one embodiment, the prebiotic is selected from the group consisting of fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylooligosaccharides (XOS), lactofructooligosaccharides (LACT), soy oligosaccharides (SOS), inulin, or a combination thereof.

[0083] In one embodiment, the composition is in the form of a solid powder.

[0084] In one embodiment, the composition is in the form of a lyophilized powder.

[0085] In a fifth aspect, the present invention provides a use of the engineered bacteria of the present invention or the composition of the present invention in preparing a drug, wherein the drug is used for

[0086] (a) degrading ethanol and acetaldehyde; and / or

[0087] (b) Alleviate alcoholic liver damage.

[0088] In one embodiment, the alcoholic liver injury is acute alcoholic liver injury.

[0089] In a sixth aspect, the present invention provides a use of the engineered bacteria of the present invention or the composition of the present invention in the preparation of a medicament, wherein the medicament is used for (a) reducing the amount of hepatocyte swelling; and / or

[0090] (b) Improvement of inflammatory infiltration.

[0091] In a seventh aspect, the present invention provides a method for preparing the composition of the present invention, comprising the steps of:

[0092] The engineered bacteria of the present invention are mixed with a food or pharmaceutically acceptable carrier to form the composition of the present invention.

[0093] In an eighth aspect, the present invention provides a method for reducing ethanol and / or acetaldehyde levels in a subject in need thereof, comprising administering to the subject an effective amount of the engineered bacteria as described in the present invention or the composition as described in the present invention.

[0094] In one embodiment, the administering comprises oral administration.

[0095] In one embodiment, the administration dosage is 0.01-5 g / 50 kg body weight / day, preferably, 0.1-2 g / 50 kg body weight / day.

[0096] In one embodiment, the subject comprises a mammal, such as a human.

[0097] In one embodiment, the method is non-diagnostic and non-therapeutic.

[0098] In a ninth aspect, the present invention provides a method for preventing and / or treating alcoholic liver injury in a subject in need thereof, comprising administering to the subject an effective amount of the engineered bacteria of the present invention or the composition of the present invention.

[0099] In one embodiment, the administering comprises oral administration.

[0100] In one embodiment, the administration dosage is 0.01-5 g / 50 kg body weight / day, preferably, 0.1-2 g / 50 kg body weight / day.

[0101] In one embodiment, the subject comprises a mammal, such as a human.

[0102] In one embodiment, the method is non-diagnostic and non-therapeutic.

[0103] In a tenth aspect, the present invention provides a method for reducing the content of ethanol and / or acetaldehyde in vitro, comprising co-culturing the engineered bacteria of the present invention or the composition of the present invention with a sample containing ethanol and / or acetaldehyde.

[0104] After extensive and in-depth research and extensive screening, the inventors discovered that Escherichia coli Nissle 1917, which overexpresses exogenous alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) genes, has a very strong ability to metabolize ethanol and / or acetaldehyde, significantly exceeding that of wild-type E. coli. Therefore, the engineered bacteria of the present invention can not only be used to increase the production of alcohol dehydrogenase and acetaldehyde dehydrogenase, but can also be used to metabolize ethanol and acetaldehyde.

[0105] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG1 shows a schematic diagram of the construction of the engineered bacteria of the present invention. The coding sequences of ADH and ALDH are connected in series through a ribosome binding site (RBS), and their expression is regulated by a promoter and a terminator to form an expression cassette.

[0107] Figure 2 shows the level of acetaldehyde metabolism in vitro by the engineered bacterium Ec1101 (blank control; WT: wild-type EcN1917; ALD01: engineered EcN 1917, overexpressing only scALDH1 from Saccharomyces cerevisiae; Ec1101: engineered EcN 1917, overexpressing scADH2 from Saccharomyces cerevisiae and scALDH1 from Saccharomyces cerevisiae), n=3.

[0108] FIG3 shows the level of ethanol metabolism in vitro by the engineered bacteria Ec1101 (blank control; WT: wild type EcN1917; Ec1101: overexpressing ADH2 and ALDH1 from Saccharomyces cerevisiae), n=3.

[0109] Figure 4 shows the level of acetaldehyde metabolism in vitro by engineered bacteria overexpressing acetaldehyde dehydrogenases from different sources (blank control; AL2-AL4: engineered EcN 1917, only overexpressing acetaldehyde dehydrogenases from different sources; ALD01: engineered EcN 1917, only overexpressing scALDH1 from Saccharomyces cerevisiae), n=3.

[0110] Figure 5 shows the level of ethanol metabolism in vitro by engineered bacteria overexpressing alcohol dehydrogenases from different sources (blank control; A2-A4: engineered EcN 1917, overexpressing alcohol dehydrogenases from different sources; ADH01: engineered EcN 1917, overexpressing scADH2 from Saccharomyces cerevisiae), n=3.

[0111] Figure 6 shows the results of an in vitro experiment. Ethanol metabolism levels in the ileum, cecum, and colon of mice after oral administration of the engineered bacteria Ec1101 (blank control; Ec1101: overexpressing scADH2 and scALDH1 from Saccharomyces cerevisiae), n = 3.

[0112] Figure 7 shows the results of an in vitro experiment. The levels of acetaldehyde metabolized in the intestinal contents of the ileum, cecum, and colon of mice after oral administration of the engineered bacteria Ec1101 (WT: wild-type EcN 1917; Ec1101: overexpressing scADH2 and scALDH1 from Saccharomyces cerevisiae), n = 3.

[0113] Figure 8 shows the results of in vivo experiments. Ethanol (Figure 8A) or acetaldehyde (Figure 8B) concentrations in the blood of mice two hours after oral administration of the engineered bacteria Ec1101 (model: blank control; EcN: wild-type EcN 1917 administered orally after model establishment; GMO: engineered bacteria Ec1101 administered orally after model establishment). n = 10.

[0114] Figure 9 shows the results of the in vivo experiment. The proportion of mice that regained consciousness after oral administration of the engineered bacteria Ec1101 (model: blank control group; EcN: wild-type EcN 1917 after model establishment; GMO: engineered bacteria Ec1101 after model establishment), n=10.

[0115] FIG10 shows images of liver tissue and its sections after oral administration of engineered bacteria Ec1101 to mice (model: blank control group).

[0116] the term

[0117] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0118] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0119] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0120] As used herein, the term "effective amount" or "pharmaceutically effective amount" refers to the amount and / or dosage and / or dosage regimen of one or more agents necessary to produce the desired result, such as an amount sufficient to alleviate in a subject one or more symptoms associated with the condition or disease for which the subject is receiving therapy or composition, or an amount sufficient to lessen the severity of the condition or delay its progression in a subject (e.g., a therapeutically effective amount), an amount sufficient to reduce the risk of developing or delay its onset, and / or reduce the ultimate severity of a disease or condition in a subject (e.g., a prophylactically effective amount).

[0121] As used herein, the term "encodes / encoded / encoding" refers to a sequence that is capable of being transcribed into mRNA and / or translated into a peptide or protein. The term "coding sequence" or "gene" refers to a polynucleotide sequence that encodes a peptide or protein. These two terms can be used interchangeably in the present invention. In some embodiments, the coding sequence is a complementary DNA (cDNA) sequence that is reverse transcribed from a messenger RNA (mRNA). In some embodiments, the coding sequence is mRNA.

[0122] As used herein, the terms "nucleotide sequence", "nucleic acid" or "polynucleotide" include oligonucleotides (i.e., short polynucleotides). They also refer to synthetic and / or non-naturally occurring nucleic acid molecules (e.g., backbone residues or bonds comprising nucleotide analogs or modifications). The terms also refer to deoxyribonucleotides or ribonucleotide oligonucleotides in single-stranded or double-stranded form. The terms encompass nucleic acids containing natural nucleotide analogs. The terms also encompass nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues.

[0123] As used herein, "engineered bacteria of the present invention", "engineered bacteria described in the present invention", "alcohol dehydrogenase and acetaldehyde dehydrogenase engineered bacteria", "ADH and ALDH engineered bacteria of the present invention", and "genetically engineered bacteria of the present invention" are used interchangeably and all refer to Escherichia coli having or integrating exogenous alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) encoding genes in its genome.

[0124] The present invention provides an engineered bacterium producing alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). The engineered bacterium is Escherichia coli Nissle 1917, and the E. coli has, or has exogenous ADH and ALDH expression cassettes integrated into its genome. The ADH and ALDH expression cassettes are used to express alcohol dehydrogenase and acetaldehyde dehydrogenase. In particular, the engineered bacterium is capable of overexpressing alcohol dehydrogenase and acetaldehyde dehydrogenase.

[0125] In one embodiment, the exogenous ADH is derived from: Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Kluveromyces lactis.

[0126] In one embodiment, the exogenous ALDH is derived from: Saccharomyces cerevisiae, Escherichia coli, Cupriavidus necator, or Pichia terricola.

[0127] In one embodiment, the exogenous ADH is an alcohol dehydrogenase selected from the group consisting of Saccharomyces cerevisiae ADH1, Saccharomyces cerevisiae ADH2, Candida albicans ADH1, Kluyveromyces marxianus ADH4, and Kluyveromyces lactis ADH4.

[0128] In one embodiment, the exogenous ALDH is an acetaldehyde dehydrogenase selected from the group consisting of Saccharomyces cerevisiae ALDH1, Saccharomyces cerevisiae ALDH2, Saccharomyces cerevisiae ALDH3, Escherichia coli ADHE, Cupriavidus necrotizingus AcoD, and Pichia terricola ALDH.

[0129] In one embodiment, the exogenous ADH is Saccharomyces cerevisiae ADH2.

[0130] In one embodiment, the exogenous ALDH is Saccharomyces cerevisiae ALDH1.

[0131] In one embodiment, the Saccharomyces cerevisiae ADH2 has the amino acid sequence shown in SEQ ID NO: 1.

[0132] In one embodiment, the gene encoding the alcohol dehydrogenase has been codon-optimized for expression in E. coli.

[0133] In one embodiment, the gene encoding ADH2 has the nucleotide sequence shown in SEQ ID NO:8.

[0134] In one embodiment, the Saccharomyces cerevisiae ALDH1 has the amino acid sequence shown in SEQ ID NO:2.

[0135] In one embodiment, the gene encoding the acetaldehyde dehydrogenase has been codon-optimized for expression in E. coli.

[0136] In one embodiment, the gene encoding ALDH1 has a nucleotide sequence as shown in SEQ ID NO:9.

[0137] Composition

[0138] The present invention also provides a composition, preferably a pharmaceutical composition. The composition comprises an effective amount of the alcohol dehydrogenase and acetaldehyde dehydrogenase engineered bacteria of the present invention, i.e., Escherichia coli (EcN 1917) having or having ADH and ALDH encoding genes integrated into its genome. In one embodiment, the composition further comprises a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof; and / or a prebiotic selected from the group consisting of fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylooligosaccharides (XOS), lactofructooligosaccharides (LACT), soy oligosaccharides (SOS), inulin, or a combination thereof.

[0139] In one embodiment, the composition is a liquid preparation, a solid preparation, or a semisolid preparation.

[0140] In one embodiment, the liquid preparation is selected from the group consisting of a solution preparation or a suspension preparation.

[0141] In one embodiment, the dosage form of the composition is selected from the group consisting of powders, powders, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

[0142] The pharmaceutical composition of the present invention can be administered in the form of tablets, injections or capsules. The pharmaceutical preparation includes excipients, pharmaceutically acceptable media and carriers, which can be selected according to the route of administration. The pharmaceutical preparation of the present invention can further include auxiliary active ingredients.

[0143] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil can be used as carriers, excipients or diluents of the pharmaceutical composition of the present invention.

[0144] In addition, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings. The pharmaceutical composition of the present invention can be produced as an enteric coating formulation by various well-known methods so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass smoothly through the stomach without being destroyed by gastric acid.

[0145] Alternatively, the microorganisms of the present invention can be used in the form of capsules prepared by conventional methods. For example, standard excipients can be mixed with the freeze-dried microorganisms of the present invention to form pellets, which can then be filled into gelatin capsules. Furthermore, the microorganisms of the present invention can be mixed with pharmaceutically acceptable excipients such as liquid glue, cellulose, silicates, or mineral oil to form suspensions or dispersions, which can be filled into soft gelatin capsules.

[0146] The pharmaceutical composition of the present invention can be prepared as enteric-coated tablets for oral administration. The term "enteric coating" as used herein includes all conventional pharmaceutical coatings that are not degraded by gastric acid but fully decompose in the small intestine to rapidly release the microorganisms of the present invention. The enteric coating of the present invention can be maintained in synthetic gastric acid, such as a pH 1 HCl solution, at 36-38°C for more than 2 hours and preferably decomposes within 1.0 hour in synthetic intestinal fluid, such as a pH 7.0 buffer.

[0147] The enteric coating of the present invention is coated with about 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The thickness of the enteric coating of the present invention is 5-100 μm, and ideally 20-80 μm. The enteric coating ingredients are selected from conventional polymers known in the art.

[0148] The preferred enteric coating of the present invention is prepared from a copolymer of cellulose acetate phthalate polymer or trimellitate polymer and methacrylic acid (for example, a copolymer of methacrylic acid containing more than 40% and methylcellulose hydroxypropyl phthalate or its ester derivatives).

[0149] The cellulose acetate phthalate used in the casing of the present invention has a viscosity of approximately 45-90 cp, an acetyl content of 17-26%, and a phthalic acid content of 30-40%. The cellulose acetate trimellitate used in the casing has a viscosity of approximately 5-21 cs and an acetylphthalate content of 17-26%. Cellulose acetate trimellitate is manufactured by Eastman Coda and can be used as the casing material of the present invention.

[0150] The hydroxypropyl methylcellulose phthalate used in the enteric coating of the present invention generally has a molecular weight of 20,000-130,000 Daltons, an ideal molecular weight of 80,000-100,000 Daltons, a hydroxypropyl content of 5-10%, a methoxy content of 18-24%, and a phthaloyl content of 21-35%.

[0151] The hydroxypropyl methylcellulose phthalate used in the enteric coating of the present invention is HP50, manufactured by Shin-Etsu Chemidnl Co. Ltd. of Japan. HP50 contains 6-10% hydroxypropyl groups, 20-24% methoxy groups, 21-27% propyl groups, and has a molecular weight of 84,000 Daltons. Another enteric coating material is HP55, which contains 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxy groups, 27-35% phthalic acid, and has a molecular weight of 78,000 Daltons.

[0152] The enteric coating of the present invention is prepared as follows: the enteric coating solution is sprayed onto the core using conventional methods. In this enteric coating method, all solvents are alcohols (such as ethanol), ketones (such as acetone), halogenated hydrocarbon compounds (such as dichloromethane), or combinations thereof. Softeners such as di-n-butyl phthalate and triacetin are added to the enteric coating solution in a ratio of 1 part of coating to about 0.05 part or about 0.3 part of softener. The spraying method is preferably carried out continuously, and the amount of material sprayed can be controlled according to the conditions used for coating. The spray pressure can be adjusted at will, and generally speaking, ideal results can be obtained at an average pressure of 1-1.5 bar.

[0153] In the specification, "pharmaceutically effective amount" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals. For example, in the present invention, a drug containing 1×10-1×10 20 cfu / ml or cfu / g (especially, can contain 1×10 4 -1×10 15 cfu / ml or cfu / g; more specifically, it may contain 1×10 6 -1×10 11 cfu / ml or cfu / g) of the alcohol dehydrogenase and acetaldehyde dehydrogenase engineering bacteria of the present invention.

[0154] When used to prepare pharmaceutical compositions, the effective dosage of the alcohol dehydrogenase and acetaldehyde dehydrogenase engineered bacteria of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The dosage form suitable for oral administration comprises about 1×10-1×10 20 cfu / ml or cfu / g (especially, can contain 1×10 4 -1×10 15 cfu / ml or cfu / g; more specifically, it may contain 1×10 6 -1×10 11 cfu / ml or cfu / g) of the active alcohol-detoxifying engineered bacteria of the present invention or the active ingredient produced by fermentation. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, depending on the exigencies of the therapeutic condition, several divided doses can be given daily, or the dose can be reduced proportionally.

[0155] The engineered alcohol dehydrogenase and acetaldehyde dehydrogenase bacteria of the present invention can be administered orally, among other routes. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include culture media, polyethylene glycol, nonionic surfactants, and edible oils (such as corn oil, peanut oil, and sesame oil), as long as they are suitable for the characteristics of the engineered alcohol dehydrogenase and acetaldehyde dehydrogenase bacteria of the present invention and the specific administration method desired. Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavorings, pigments, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.

[0156] From the standpoint of ease of preparation and administration, preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. Oral administration is preferred.

[0157] The composition of the present invention is administered to the individual once or more per day. The dosage unit represents a dosage that can be divided in form and is suitable for humans or all other mammalian individuals. Each unit contains a pharmaceutically acceptable carrier and an effective therapeutic amount of the microorganism of the present invention. The dosage varies with the patient's weight and severity of obesity, the supplementary active ingredients included and the microorganisms used. In addition, if possible, the administration can be separated and, if necessary, continuous. Therefore, the dosage does not limit the present invention. In addition, the "composition" in the present invention not only means a medicine but also means a functional food and a health supplement food. In a preferred embodiment, the composition includes: beverages, food, medicines, animal feed, etc.

[0158] In one embodiment, the present invention also provides a food composition comprising an effective amount of the alcohol dehydrogenase and acetaldehyde dehydrogenase engineered bacteria of the present invention, and a balance of a food-acceptable carrier, wherein the dosage form of the food composition is selected from solid, dairy, solution, powder, or suspension products.

[0159] In one embodiment, the composition is formulated as follows:

[0160] 1×10-1×10 20 cfu / mL of the alcohol dehydrogenase and acetaldehyde dehydrogenase engineering bacteria of the present invention; and a food or pharmaceutically acceptable carrier, and / or excipient.

[0161] In one embodiment, the composition is formulated as follows:

[0162] 1×10 6 -1×10 11 cfu / mL of the alcohol dehydrogenase and acetaldehyde dehydrogenase engineering bacteria of the present invention; and a food or pharmaceutically acceptable carrier, and / or excipient.

[0163] Escherichia coli Nissle 1917

[0164] Escherichia coli (Escherichia coli) is widely used as a host in biotechnology due to its advantages, including rapid growth, ease of genetic manipulation, and rapid recombinant protein synthesis. Among them, the probiotic Escherichia coli Nissle 1917 (also known as EcN 1917) has a long history of use as a probiotic. As a host, it boasts advantages such as rapid growth, ease of genetic manipulation, and rapid recombinant protein synthesis.

[0165] Alcohol dehydrogenase and acetaldehyde dehydrogenase

[0166] Alcohol dehydrogenase and acetaldehyde dehydrogenase are widely distributed in prokaryotes and eukaryotes. Alcohol dehydrogenase catalyzes the reversible reaction between ethanol and acetaldehyde, using nicotinamide adenine dinucleotide (NAD+) as a coenzyme. Acetaldehyde dehydrogenase, using NAD+ as a cofactor, catalyzes the oxidation of acetaldehyde to acetic acid.

[0167] The main advantages of the present invention include:

[0168] (a) In the present invention, Escherichia coli Nissle 1917 is used as a host to simultaneously express exogenous alcohol dehydrogenase and acetaldehyde dehydrogenase. The resulting engineered bacteria has a significantly stronger ability to metabolize ethanol and / or acetaldehyde.

[0169] (b) In vitro experiments showed that the engineered bacteria can degrade most acetaldehyde in a short period of time, producing harmless acetic acid molecules and less toxic ethanol without the need for additional cofactors.

[0170] (c) In vitro experiments showed that the engineered bacteria can degrade ethanol in a short period of time, producing harmless acetic acid molecules without producing the intermediate product acetaldehyde, and without the need for additional cofactors.

[0171] (d) Animal experiments have shown that the engineered bacteria can effectively relieve alcoholism and significantly alleviate alcoholic liver damage. The engineered bacteria can be used to develop related functional foods and pharmaceuticals (alcoholism products).

[0172] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0173] Example 1. Synthesis of alcohol dehydrogenase and acetaldehyde dehydrogenase and construction of recombinant plasmids

[0174] The alcohol dehydrogenase scADH2 (SEQ ID NO: 1) and acetaldehyde dehydrogenase scALDH1 (SEQ ID NO: 2) from Saccharomyces cerevisiae S288c were codon-optimized by GenScript Biotech Co., Ltd. based on amino acid sequences from the NCBI database (Table 1). The codon-optimized scAdh2 and scAldh1 gene sequences (SEQ ID NOs: 8 and 9, respectively) were linked by a RBS (SEQ ID NO: 3) and synthesized by GenScript Biotech Co., Ltd. into the commercial pET-21a(+) vector, designated pET-21a(+)_ScADH2_ScALDH1.

[0175] Two sets of primers were used to introduce the tac promoter and rrnB1 terminator sequences: Primer 1 (SEQ ID NO:4) and Primer 2 (SEQ ID NO:5) were used to amplify the DNA fragment from ScADH2 to ScALDH1 in pET-21a(+)_ScADH2_ScALDH1. Primers 3 (SEQ ID NO:6) and Primer 4 (SEQ ID NO:7) were used to amplify the gene expression cassette fragment from pET-21a(+)_ScADH2_ScALDH1, replacing the T7 promoter and T7 terminator in the original expression cassette with the tac promoter and rrnB1 terminator. The two amplified fragments were constructed into the plasmid pET-21a(+)_Ptac_ScADH2_ScALDH1_TrrnB1 using the Gibson assembly method.

[0176] Recombinant plasmids for overexpressing alcohol dehydrogenase scADH2 or acetaldehyde dehydrogenase scALDH1 were synthesized by GenScript Biotech Co., Ltd. based on the sequences in the NCBI database using the commercial pMAL-c4x vector (named pMAL-c4x_ScADH2 and pMAL-c4x_ScALDH1, respectively).

[0177] In addition, the same method was used to construct recombinant plasmids that overexpressed alcohol dehydrogenase (hereinafter referred to as ADH2, ADH3 and ADH4) or acetaldehyde dehydrogenase (hereinafter referred to as ALDH2, ALDH3 and ALDH4) from other sources.

[0178] The relevant amino acid and nucleotide sequences are shown in Table 1. The schematic diagram of the expression cassette construction is shown in Figure 1.

[0179] Table 1

[0180] The amino acid sequence and encoding nucleotide sequence of ADH2 are shown in SEQ ID NOs: 10 and 11, respectively;

[0181] The amino acid sequence and encoding nucleotide sequence of ADH3 are shown in SEQ ID NOs: 12 and 13, respectively;

[0182] The amino acid sequence and encoding nucleotide sequence of ADH4 are shown in SEQ ID NOs: 14 and 15, respectively;

[0183] The amino acid sequence and encoding nucleotide sequence of ALDH2 are shown in SEQ ID NOs: 16 and 17, respectively;

[0184] The amino acid sequence and encoding nucleotide sequence of ALDH3 are shown in SEQ ID NOs: 18 and 19, respectively;

[0185] The amino acid sequence and encoding nucleotide sequence of ALDH4 are shown in SEQ ID NOs: 20 and 21, respectively.

[0186] Example 2. Construction of recombinant Escherichia coli

[0187] The recombinant plasmid in Example 1 was transformed into wild-type EcN 1917 using electroporation. The specific steps are as follows:

[0188] (1) A single colony of EcN 1917 was inoculated into 1 mL of LB liquid medium and shaken overnight at 37°C and 200 rpm to obtain an activated seed solution;

[0189] (2) Inoculate the seed solution into 10 mL of LB liquid medium at a volume ratio of 1:100 and culture in a shaker at 37°C and 200 rpm until the OD600 of the bacterial solution is approximately 0.5;

[0190] (3) Collect the cells by centrifugation at 6,000 rpm and 4°C for 5 min and wash three times with 1 mL of pre-cooled sterile water;

[0191] (4) Resuspend the cells in 50 μL of pre-chilled sterile water, add 1 μg of plasmid, incubate on ice for 5 min, then place in a 0.1 cm electroporation cuvette and electroporate at 1.8 kV;

[0192] (5) Add 1 mL of LB liquid medium to resuspend the electroporated cells. Transfer the resuspended liquid to a shaker tube and resuscitate at 37°C, 200 rpm, for 1 h. Concentrate and spread the liquid onto an LB medium plate supplemented with 100 mg / L ampicillin and culture overnight at 37°C.

[0193] (6) The transformants were used as templates, and positive transformants were identified by colony PCR and sequencing. The positive transformants were frozen in 25% glycerol by volume and stored at -80°C.

[0194] The names of the obtained transformants (i.e., recombinant bacteria) are shown in Table 2. Although different plasmid vectors (pMAL-c4x and pET-21a(+) after replacing the promoter and terminator) were used, it did not affect the results, and the results were still comparable.

[0195] Table 2

[0196] Example 3: Cultivation of recombinant Escherichia coli and detection of its ability to metabolize ethanol or acetaldehyde in vitro

[0197] Under in vitro conditions, the metabolic capacity of the recombinant bacteria obtained in Example 2 for ethanol and acetaldehyde was detected using a whole-cell catalytic system. The specific steps are as follows:

[0198] (1) Pick a single colony and inoculate it into 1 mL of LB liquid medium. Shake the culture in a shaker at 37°C and 200 rpm overnight to obtain an activated seed solution.

[0199] (2) Inoculate the seed solution into a shake flask at a volume ratio of 1:100, and culture at 37°C at 200 rpm until the OD600 of the bacterial solution reaches about 0.5. Add 0.1 mM IPTG for overnight induction.

[0200] (3) The next day, collect 2 mL of bacterial suspension by centrifugation at 6,000 rpm for 5 min. Discard the supernatant and resuspend the bacteria in 2 mL of PBS. Add the resuspended bacteria to a 15 mL shake tube and add ethanol or acetaldehyde to a final concentration of 2 g / L as a substrate. Take 2 mL of PBS solution without the bacteria but containing ethanol or acetaldehyde to a final concentration of 2 g / L and place it in the same 15 mL shake tube as a blank control.

[0201] (4) The shaking tube was placed in a shaker at 37°C and 200 rpm for 2 h. The upper reaction liquid was centrifuged at 4°C and 10,000 rpm. The contents of ethanol, acetaldehyde, and acetic acid in the reaction system were detected by liquid chromatography.

[0202] The results of acetaldehyde metabolism are shown in Table 3 and Figure 2. After two hours of reaction in the whole-cell catalytic system, wild-type EcN 1917 consumed 36% of the acetaldehyde, recombinant strain Ec1101 consumed 83%, and recombinant strain ALD01, which only expresses acetaldehyde dehydrogenase, consumed 40%. Compared to wild-type EcN 1917, recombinant strain Ec1101 exhibited significantly enhanced acetaldehyde metabolism, converting acetaldehyde primarily into harmless acetic acid and a smaller amount into less toxic ethanol, thereby minimizing the acetaldehyde concentration in the reaction system. Notably, despite using the same scALDH1 coding sequence, the recombinant strain Ec1101 consumed significantly more acetaldehyde than ALD01 (83% vs 40%, residual amount 0.21 g / L vs 0.73 g / L). This suggests that overexpressing ADH2 (SEQ ID NO: 1) in addition to overexpressing ALDH1 (SEQ ID NO: 2) in EcN 1917 significantly enhanced the engineered strain's acetaldehyde metabolism capacity, converting more acetaldehyde to acetate. This result was highly surprising.

[0203] Table 3

[0204] The results of ethanol metabolism are shown in Figure 3. The recombinant strain Ec1101 consumed 30% of the ethanol. Compared to the wild-type strain EcN 1917, the recombinant strain Ec1101 had a stronger ethanol metabolism capacity, efficiently converting ethanol to acetic acid without the accumulation of the harmful intermediate acetaldehyde.

[0205] The results of Figures 4 and 5 show that the engineered bacteria expressing other acetaldehyde dehydrogenases (ALDH2-4) or alcohol dehydrogenases (ADH2-4) from different sources alone all have a certain ability to metabolize acetaldehyde or ethanol.

[0206] Example 4. In vitro experiments on the metabolism of ethanol and acetaldehyde by engineered bacteria

[0207] After mice were gavage-fed with live bacteria, their intestinal contents were separated and the metabolic activity of the live bacteria in the intestine was tested. The specific steps were as follows:

[0208] (1) Nine C57BL / 6J mice were divided into three groups: A, B, and C, with three mice in each group.

[0209] (2) The mice in group A were dissected and the contents of the ileum, cecum, and colon were obtained.

[0210] (3) Mice in group B were orally administered with 5e10 cfu of wild-type EcN 1917. Half an hour later, the mice were dissected and the contents of the ileum, cecum, and colon were obtained and weighed.

[0211] (4) Mice in group C were gavaged with 5e10 cfu of recombinant bacteria Ec1101. Mice were dissected half an hour later, and two portions of the ileum, cecum, and colon were collected and weighed.

[0212] (5) Add ethanol in PBS at a final concentration of 2 g / L to the intestinal contents of mice in groups A and C, and add acetaldehyde in PBS at a final concentration of 2 g / L to the intestinal contents of mice in groups B and C. The mixture was reacted at 37°C for two hours. Group A served as the blank control group.

[0213] (6) After the reaction is completed, the upper layer of the reaction solution is centrifuged at 4°C and 10,000 rpm, and the contents of ethanol, acetaldehyde, and acetic acid in the reaction system are detected by liquid chromatography.

[0214] As shown in FIG6 , the ileal contents of mice orally administered with Ec1101 were able to metabolize 25% ethanol.

[0215] As shown in Table 4 and FIG7 , the ileal contents of mice orally administered with Ec1101 were able to metabolize 95% of acetaldehyde, and had a significantly stronger acetaldehyde metabolism ability than the wild-type EcN 1917.

[0216] The above results show that oral live bacteria can maintain significant metabolic activity in the intestine.

[0217] Table 4

[0218] Example 5. Animal experiments

[0219] Live bacteria were orally administered to mice that had consumed excessive amounts of alcohol, and the concentrations of ethanol and acetaldehyde in their bodies and the duration of their sobriety were measured. The specific steps are as follows:

[0220] (1) Thirty C57BL / 6J mice were divided into three groups: A, B, and C, with 10 mice in each group.

[0221] (2) Each mouse was gavaged with 50% ethanol at a dose of 10 mL / kg. Five minutes later, mice in Groups B and C were gavaged with 5e10 cfu of wild-type EcN 1917 or recombinant Ec1101, respectively. The same dose of live bacteria was repeated every hour thereafter in Groups B and C, respectively designated as the EcN and GMO groups. Mice in Group A received no treatment and served as the model group.

[0222] (3) One hour after oral administration of live bacteria, the mice were tested for righting reflex every half hour. Specifically, the mice in each group were turned over and the number of mice that could right themselves within 30 seconds was counted. The percentage of mice in each group that could right themselves was recorded as the awake percentage. The results are shown in Figure 9.

[0223] (4) Two hours after oral administration of live bacteria, blood was collected from the orbital vein of each mouse and the ethanol and acetaldehyde concentrations in the blood were measured using gas chromatography. The results are shown in Figures 8 and 9, respectively.

[0224] Figure 8A shows that the blood alcohol concentration in mice gavaged with Ec1101 (approximately 2.68 g / L) was significantly lower than that in the model group (approximately 5.84 g / L) and the mice gavaged with wild-type EcN 1917 (approximately 5.42 g / L), indicating that oral administration of Ec1101 has a significant effect in lowering blood alcohol concentration.

[0225] Figure 8B shows that the blood aldehyde concentration in mice gavaged with Ec1101 (approximately 0.25 mg / L) was significantly lower than that in the model group (approximately 15 mg / L) and mice gavaged with wild-type EcN 1917 (approximately 9 mg / L), once again verifying that Ec1101 has significantly better acetaldehyde metabolism ability.

[0226] As shown in Figure 9, starting from 1.5 hours, the sober proportion of mice gavaged with Ec1101 was generally higher than that of the model group and the mice gavaged with wild-type EcN 1917, indicating that oral administration of Ec1101 has a significant sobering effect.

[0227] Example 6. Using engineered bacteria to alleviate alcoholic liver damage

[0228] Following Example 5, C57BL / 6J mice carrying the ALDH2 mutation were gavaged with ethanol and live bacteria. On the first day, mice were gavaged with 50% ethanol at a dose of 10 mL / kg to establish the model. Five minutes later, the intervention group was gavaged with 5e10 cfu of the recombinant Ec1101 bacteria. The model group did not receive live bacteria. The intervention group was then gavaged with the same dose of live bacteria every hour for a total of six times. One day later, the ethanol and live bacteria gavages from the first day were repeated on the third day. On the fifth day, mice were observed for behavior and liver tissue was obtained for pathological sections.

[0229] On the fifth day, mice in the intervention group were significantly more active than those in the model group. Liver pathology sections are shown in Figure 10. In the liver sections, the model group showed marked swelling of stem cells, disorganized hepatic cords, and inflammatory cell infiltration. In the intervention group, hepatocyte swelling was reduced, and inflammatory infiltration was significantly improved. These results demonstrate that the engineered bacteria Ec1101 can significantly alleviate alcoholic liver damage caused by excessive ethanol intake.

[0230] discuss

[0231] The present invention utilizes EcN 1917 as a host bacterium and, through genetic engineering techniques, highly expresses exogenous alcohol dehydrogenase and acetaldehyde dehydrogenase, resulting in a strong metabolic capacity for ethanol and acetaldehyde. Furthermore, in animal experiments, the engineered bacterium was able to effectively reduce blood alcohol content, shorten the duration of intoxication, and alleviate alcoholic liver damage, thereby effectively alleviating the flushing reaction caused by drinking and the discomfort caused by intoxication. It has significant alcohol sobering and liver protection effects and can be used in the fields of food and medicine, such as for the development of related functional foods and drugs (alcohol sobering products). Furthermore, the results in the examples show that the simultaneous overexpression of scALDH1 (SEQ ID NO: 2) and scADH2 (SEQ ID NO: 1) in EcN 1917 significantly enhances acetaldehyde metabolism, a result that is particularly surprising compared to engineered bacteria expressing only acetaldehyde dehydrogenase (scALDH1).

[0232] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An engineered bacterium of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), characterized in that, The engineered bacterium is Escherichia coli Nissle 1917, and exogenous ADH and ALDH expression cassettes are present in or integrated into the genome of the Escherichia coli, and the exogenous ADH and ALDH expression cassettes are used to express alcohol dehydrogenase and aldehyde dehydrogenase.

2. The engineered bacterium according to claim 1, wherein The sources of the exogenous ADH include: Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Kluyveromyces lactis.

3. The engineered bacterium according to claim 1, characterized in that, The sources of the exogenous ALDH include: Saccharomyces cerevisiae, Escherichia coli, Cupriavidus necator, Pichia terricola.

4. The engineered bacterium according to any one of claims 1-3, characterized in that, The ADH has the amino acid sequence shown in SEQ ID NO:

1.

5. The engineered bacterium according to any one of claims 1 to 3, characterized in that, The ALDH has the amino acid sequence shown in SEQ ID NO:

2.

6. The engineered bacterium according to any one of claims 1-5, characterized in that, The coding genes of the ADH and the ALDH are tandemly linked through a Ribosome binding site (RBS).

7. The engineered bacterium according to claim 6, wherein, The exogenous ADH and ALDH expression cassettes have the structure shown in Formula I: Z1-Z2-Z3-Z4 (I) In the formula, each "-" is independently a bond or a nucleotide linker sequence; Z1 is a promoter or a 5'-UTR element containing a promoter; Z2 is absent or is an optional enhancer; Z3 is the nucleotide sequence encoding the exogenous ADH and ALDH, wherein the nucleotide sequence encoding ADH and the nucleotide sequence encoding ALDH are tandemly linked through a Ribosome binding site (RBS); and Z4 is absent or is a 3'-UTR element.

8. A method for constructing an engineered bacterium expressing alcohol dehydrogenase and aldehyde dehydrogenase as described in any one of claims 1-7, characterized in that, It includes the following steps: (s1) Obtain exogenous ADH and ALDH genes; (s2) Link the ADH and ALDH genes to a plasmid, and the ADH and ALDH genes are tandemly linked through an RBS, thereby constructing a recombinant plasmid containing the ADH and ALDH genes; and (s3) Transform an Escherichia coli strain with the recombinant plasmid, and screen for transformants on a selective medium, thereby obtaining the engineered bacterium.

9. A method for producing alcohol dehydrogenase and aldehyde dehydrogenase, characterized in that, Cultivate the engineered bacterium producing alcohol dehydrogenase and aldehyde dehydrogenase as described in any one of claims 1-7 to obtain alcohol dehydrogenase and aldehyde dehydrogenase.

10. A composition for promoting acetaldehyde degradation and / or relieving hangover, characterized in that, The composition comprises: (a) a safe and effective amount of the engineered bacterium expressing alcohol dehydrogenase and aldehyde dehydrogenase as described in any one of claims 1-7; and (b) a food-grade or pharmaceutically acceptable carrier.

11. Use of the engineered bacterium according to any one of claims 1-7, or the composition according to claim 10, in the preparation of a drug, characterized in that, The drug can be used for (a) degrading ethanol and / or acetaldehyde; and / or (b) alleviating alcoholic liver injury.

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