WO2019195592A1

WO2019195592A1 - Genetically-engineered microbes and compositions thereof

Info

Publication number: WO2019195592A1
Application number: PCT/US2019/025855
Authority: WO
Inventors: Xiaoyang Wu
Original assignee: The University Of Chicago
Priority date: 2018-04-04
Filing date: 2019-04-04
Publication date: 2019-10-10

Abstract

Provided herein are genetically-engineered microbes, compositions containing the microbes, and methods of their use, wherein the microbes are capable of reducing acetaldehyde levels and/or the effects of such alcohol metabolites in individuals. The invention provides a genetically-engineered microbe that includes a recombinant gene encoding an aldehyde dehydrogenase ALDH2. The invention provides a methods of treating an individual for elevated levels of acetaldehyde, the method includes administering to the individual a genetically-engineered microbe that includes a recombinant gene encoding an aldehyde dehydrogenase ALDH2, thereby reducing the levels of acetaldehyde in the individual.

Description

GENETICALLY-ENGINEERED MICROBES AND COMPOSITIONS THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/652,688, filed April 4, 2018, which is incorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant number R01 OD023700 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

[0003] This disclosure relates to genetically-engineered microbes, and compositions thereof, capable of metabolizing byproducts of ethanol metabolism.

Description of Related Art

[0004] Alcohol consumption is common throughout the world. In the U.S., excessive drinking is problematic for as many 7.2% or 17 million adults aged 18 and older who abuse alcohol and have an alcohol use disorder (“AUD”) according to data from 2012. There are several diseases that can develop in association with alcohol abuse, including heart disease, high blood pressure, liver disease, and cancer, among others. Mechanistically, alcohol abuse can contribute to the development of certain diseases through the formation of toxic metabolites of alcohol (ethanol). A major toxic metabolite of alcohol is acetaldehyde, which has been shown to be a strong carcinogen and can cause many different types of tissue damage.

[0005] Ethanol is metabolized by cytochrome P450 and alcohol dehydrogenases (ADH) into acetaldehyde, which is further metabolized to acetate, which is harmless, by aldehyde dehydrogenase (ALDH). In certain populations, especially those of Asian descent, variants of the ADH genes ADH1B or ADH1C result in metabolism of ethanol to acetaldehyde at a much higher efficiency (up to 40-100-fold greater rate of conversion). Individuals can be either homozygous or heterozygous for the variant genes. This higher rate of metabolism results in a much faster exposure to elevated acetaldehyde concentrations upon alcohol consumption. This phenotype (sometimes called Asian flush) is exacerbated in a subset of the populations carrying the ADH1B or ADH1C variants where an ALDH gene variant (allele) is also present. The variant of mitochondrial aldehyde dehydrogenase (expressed from the ALDH2 gene) breaks down acetaldehyde to acetate at an abnormally slow rate. Individuals can be either homozygous or heterozygous for the variant ALDH2 gene. Therefore, those individuals with either ADH1B or ADH1C gene variant in combination with mALDH2 gene variant experience an even greater exposure to acetaldehyde when consuming alcohol and are at greater risk for cancer and tissue damage associated with elevated acetaldehyde levels. Therefore, approaches for improving both alcohol and acetaldehyde metabolism are required.

[0006] Nosova et al. have proposed that many human aerobic colonic bacteria possess significant aldehyde dehydrogenase activity and can metabolize acetaldehyde to acetate under conditions similar to those at the colonic mucosal surface. Alcohol Alcohol. 1996 Nov;3l(6):555-64. However, it was also suggested that individuals can vary by the capability of their colonic flora to remove toxic acetaldehyde.

[0007] Similarly, a probiotic, Lactobacillus GG ATCC 53103, has been reported to metabolize ethanol and acetaldehyde in vitro and may be beneficial for individuals with high gastrointestinal acetaldehyde levels following alcohol intake. Nosova et al. Alcohol Alcohol. 2000 Nov-Dec;35(6):56l-8. Other probiotics with acetaldehyde dehydrogenase activity have been proposed for combatting side-effects of alcohol metabolism, such as Lactobacillus plantarum. As well, approaches to combat hangovers due to excess alcohol consumption include administration of mixed probiotics containing Bacillus and Bifidobacterium species and Lactobacillus acidophilus. Other probiotic compositions for accelerating alcohol catabolism include include mixtures of Pedoiococcus species and Lactobacillus plantarum. Still other probiotic compositions use mixtures of Lactobacillus and Bifidobacterium species to reduce blood alcohol content following alcohol consumption. However, there remains a need for improved therapies that prevent and/or reduce the harmful effects of alcohol metabolism in individuals.

SUMMARY OF THE INVENTION

[0008] Provided herein are genetically-engineered microbes, compositions containing the microbes, and methods of their use, wherein the microbes are capable of reducing acetaldehyde levels and/or the effects of such alcohol metabolites in individuals. [0009] In a first aspect, the invention provides a genetically-engineered microbe that includes a recombinant gene encoding an aldehyde dehydrogenase. In one embodiment of the first aspect, the aldehyde dehydrogenase is ALDH2. The ALDH2 can be a human or yeast ALDH2. The ALDH2 can include an amino acid sequence that is at least 85% identical to SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment of the first aspect, the genetically- engineered microbe is a bacterium or a yeast. The bacterium can be one or more of a Lactobacillus, a Bifidobacterium, an Enterococcus, a Streptococcus, a Pediococcus, a Leuconostoc, a Bacillus, an Escherichia, or a lactic acid bacterium. The Lactobacillus can be Lactobacillus GG, /.. acidophilus, L. bulgaricus, L. jugurti, L. helveticus, L. salivarius, L. casei, L. plantarum, L. rhamnosus, L. paracasei, L. lactis, L. infantis, L. reuteri, or L. brevis. The Bifidobacterium can be Bifidobacterium AN AHP 16467, B. thermophilum, B. indicum, B. asteroids, B. lactis, B. longum, B. coagulans, B. dentium, B. infantis, or B. bifldum. The Enterococcus can be Enterococcus faecium. The Streptococcus can be Streptococcus thermophilus . The Pediococcus can be Pediococcus acidilactici . The yeast can be a Saccharomyces . The Saccharomyces can be Saccharomyces boulardii.

[0010] In a second aspect, the invention provides a composition for reducing acetaldehyde concentration in an individual, the composition including a genetically-engineered microbe according to any of the preceding aspects or embodiments, and one or more vehicles, carriers, or additives. In one embodiment of the second aspect, the genetically-engineered microbe is a live cell, a disrupted cell wall fraction, a dead cell, a dried bacterium, a cell culture, or a fermentation broth. In another embodiment of the second aspect, the vehicle or carrier includes a culture medium, a protein, a carbohydrate, a fat, an oil, a flavoring agent, a seasoning agent, a food, water, or mixtures thereof. The carbohydrate can be a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide. The flavoring agent can be a natural flavor or a synthetic flavor. The additive can be a vitamin, a mineral, an antioxidant, a colorant, a preservative, caffeine, or mixtures thereof. In another embodiment of the second aspect, the composition is a finished food product, a powder, a granule, a tablet, a capsule, or a liquid. In a further embodiment of the second aspect, the composition includes about 0.01 to about 99.9% by weight genetically-engineered microbe.

[0011] In a third aspect, the invention provides a method of treating an individual for elevated levels of acetaldehyde, the method includes administering to the individual a genetically-engineered microbe according to the first aspect and embodiments thereof and reducing the levels of acetaldehyde in the individual.

[0012] In a fourth aspect, the invention provides a method of treating an individual for Asian flush, the method including administering to the individual a genetically-engineered microbe according to the first aspect and embodiments thereof and reducing the levels of acetaldehyde in the individual.

[0013] In a fifth aspect, the invention provides a method of preventing elevated levels of acetaldehyde in an individual, the method including administering to the individual a composition including a genetically-engineered microbe expressing recombinant ALDH2 and preventing the manifestation of elevated levels of acetaldehyde in the individual upon consumption and metabolism of ethanol.

[0014] In some embodiments of the third, fourth, or fifth aspects, the individual is deficient in one or more alcohol dehydrogenases. In some embodiments, the individual is a carrier of ADH1B and/or ADH1C variant alleles.

[0015] In some embodiments of the third, fourth, or fifth aspects, the individual is deficient in aldehyde dehydrogenase. In some embodiments, the individual is a carrier of an ALDH2 variant allele.

[0016] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

DESCRIPTION OF DRAWINGS

[0017] FIGS. 1A-1B. Establishment of transformed E. coli. FIG. 1A. E.coli transformed with different plasmids encoding human ALDH2 (hi ALDH2) or yeast ALDH2 ( yALDH2 ) were lysed for SDS-PAGE analysis. Arrows denote bands for huALDH2 and yALDH2, respectively. FIG. IB. Clearance of acetaldehyde (referred to as“aldehyde”) in solution with genetically- engineered E. coli versus control.

[0018] FIGS. 2A-2C. Decrease of acetaldehyde concentration in animals fed with genetically-engineered bacteria. Mice were treated with different E. coli as indicated. Serum (A), colon feces (B), and colon epithelium (C) acetaldehyde (referred to as “aldehyde”) concentrations were determined at different time points as indicated after oral gavage of ethanol.

[0019] FIG 3. Decrease of acetaldehyde concentration in serum of ALDH2 knockout animals fed with genetically-engineered bacteria. ALDH2 knockout (KO) animals were treated with E. coli expressing yeast ALDH2 (\AIJ)H2). Serum acetaldehyde (referred to as “aldehyde”) concentrations were determined at 30 and 60 minutes after oral gavage of ethanol.

DETAILED DESCRIPTION

[0020] All publications, patents, and patent applications cited herein are hereby -expressly incorporated by reference in their entirety for all purposes.

[0021] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. For example, reference to“a metabolite” means one or more metabolites.

[0022] It is noted that terms like“preferably,”“commonly,” and“typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0023] For the purposes of describing and defining the present invention it is noted that the term“substantially” as used herein represents the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term“substantially” is also used herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0024] Methods well known to those skilled in the art can be used to construct genetic expression constructs, targeting vectors, and genetically-engineered cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, polymerase chain reaction (PCR) techniques, and others. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al, 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et ah, 1990, Academic Press, San Diego, CA).

[0025] As used herein, the terms“polynucleotide,”“nucleotide,”“oligonucleotide,” and “nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.

[0026] As used herein, the term“genetically-engineered” refers to the genetic manipulation of one or more cells, whereby the genome of the one or more cells has been augmented by at least one DNA sequence. Candidate DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed”), and other genes or DNA sequences which one desires to introduce into the one or more cells. It will be appreciated that typically the genome of genetically-engineered cells described herein is augmented through transient or stable introduction of one or more recombinant genes.

[0027] Generally, introduced DNA is not originally resident in the genetically-engineered cell that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given genetically-engineered cell, and to subsequently introduce one or more additional copies of that DNA into the same genetically-engineered cell, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination, site-directed mutagenesis, and/or genome editing technology, including CRISPR (clustered regularly-interspaced short palindromic repeats), and/or mammalian transposon technology, such as by using the piggyBac™ transposon. In some instances, the introduced DNA is introduced into the recipient via viral vectors, including vectors derived from retrovirus, lentivirus, and adeno-associated virus. In some instances, the introduced DNA is introduced into the recipient cell directly with electroporation.

[0028] As used herein, the term“recombinant gene” refers to a gene or DNA sequence that is introduced into a genetically-engineered cell, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host.“Introduced,” or“augmented” in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species, or can be a DNA sequence that originated from or is present in the same species, but has been incorporated into a cell by methods to form a genetically-engineered cell. It will be appreciated that a recombinant gene that is introduced into a cell can be identical to a DNA sequence that is normally present in the cell being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. Recombinant genes can also be introduced with different driving promoters or associated sequences that can alter the gene’s expression level or pattern. Such recombinant genes are particularly-encoded by cDNA. Non-coding sequences, such as short hairpin RNAs, microRNAs, or long non-coding RNAs, may also be included.

[0029] It is further contemplated that recombinant genes can be codon optimized to maximize protein expression in genetically-engineered cells by increasing the translation efficiency of a particular gene. Codon optimization can be achieved, for example, by transforming nucleotide sequences of one species into the genetic sequence of a different species. Optimal codons help to achieve faster translation rates and high accuracy. As a result of these factors, translational selection is expected to be stronger in highly-expressed genes. However, while optimal codon usage is contemplated herein for expression of disclosed proteins, all possible codons are contemplated for use herein for nucleic acids encoding any disclosed protein.

[0030] As used herein, the phrase “individual deficient in alcohol dehydrogenase,” “individual deficient in one or more alcohol dehydrogenases,”“ADH deficient individual,” or “ADH deficient person” refers to an individual with a variant in an alcohol dehydrogenase (ADH) gene, such as ADH1B or ADH1C, that results in metabolism of ethanol to acetaldehyde with greater efficiency than a non-variant ADH gene and causes a greater rate of conversion of ethanol to acetaldehyde. The ADH deficient individual can be a carrier of ADH1B and/or ADH1C variant alleles and can convert a given amount of consumed alcohol into acetylaldehyde at a faster rate than a comparable person (e.g., similar size, age, health) with a non-variant ADH gene who consumes the same amount of alcohol.

[0031] As used herein, the phrase “individual deficient in aldehyde dehydrogenase,” “ALDH deficient individual,” or“ALDH deficient person” refers to an individual with a variant in an aldehyde dehydrogenase (ALDH) gene such as ALDH2, that results in breakdown of acetaldehyde to acetate at an abnormally slow rate by the ALDH deficient person compared to a comparable person (e.g., similar size, age, health) with a non-variant ALDH gene. The ALDH deficient individual can be either homozygous or heterozygous for the variant ALDH2 gene.

[0032] As used herein, the term“about” refers to ±10% of any particular value. [0033] As used herein, the terms “or” and“and/or” are utilized to describe multiple components in combination or exclusive of one another. For example,“x, y, and/or z” can refer to“x” alone,“y” alone,“z” alone,“x, y, and z,”“(x and y) or z,”“x or (y and z),” or“x or y or z.”

OVERVIEW

[0034] Aldehyde accumulation can have a negative impact on human health. Acetaldehyde, specifically, is a known strong carcinogen and can lead to tissue damage and disease. Drinking alcohol can cause significant elevation of acetaldehyde concentration in vivo. Further, individuals with particular ADH and/or ALDH alleles are at elevated risk for prolonged exposure to acetaldehyde when consuming alcohol. The present disclosure is directed to genetically-engineered probiotics (bacteria, yeast, or other microbes, collectively referred to herein as microbes) and compositions thereof that can metabolize ethanol and/or acetaldehyde. Both immediate and long-term negative effects of alcohol consumption can be reduced or avoided by the genetically-engineered microbes and compositions of the present disclosure.

[0035] In one embodiment, to protect tissues or organs from acetaldehyde-induced damage, this disclosure is directed to genetically-engineered strains of probiotic bacteria. In another embodiment, to protect tissues or organs from acetaldehyde-induced damage, this disclosure is directed to genetically-engineered strains of probiotic yeast. Such microbes can be transformed to carry recombinant ALDH genes, such as, for example, ALDH2 from human and/or yeast.

[0036] In one embodiment, the present disclosure is directed to genetically-engineered microbes capable of increasing the rate of acetaldehyde metabolism in an individual administered the genetically-engineered microbes. In another embodiment, the present disclosure is directed to reducing or prevent tissue damage from elevated levels of acetaldehyde in individuals administered microbes genetically-engineered to metabolize acetaldehyde at increased rates compared to control.

[0037] In another embodiment, the present disclosure is directed to providing one or more probiotic supplements that when administered to an individual increase the rate of acetaldehyde metabolism in the individual’s gastrointestinal tract over the individual’s innate rate of acetaldehyde metabolism. For example, a normal acetaldehyde metabolizer (i.e., an individual with normally functioning ALDH) could self-administer the probiotic supplement (e.g., by taking a pill or other dosage form and/or consuming a food product containing the probiotic) before or after consumption of ethanol to increase their rate of clearance of acetaldehyde and thereby reduce the time of exposure to acetaldehyde. In this way, the individual can reduce acetaldehyde-induced damage and associated complications (e.g., development of cancer) from ethanol consumption.

[0038] In another embodiment, the present disclosure is directed to preventing or reducing Asian flush in an individual harboring one or more alleles causing the condition by administering the genetically-engineered microbes of the present disclosure.

[0039] It is contemplated that effective reduction in acetaldehyde levels in an individual can be achieved by administering the genetically-engineered microbes of the present disclosure either before, during, or after consumption of alcohol by the individual.

[0040] In another embodiment, it is contemplated that a probiotic supplement of the present disclosure can help remove ethanol from an individual’s system at an accelerated rate. For example, a probiotic supplement can include a gentically-engineered microbe that expresses one or more alcohol dehydrogenases to supplement an individual’s natural rate of ethanol metabolism and can optionally contain one or more aldehyde dehydrogenases to clear acetaldehyde levels.

[0041] It is further contemplated that recombinant ADH and/or ALDH enzymes expressed in the microbes of the present disclosure can be genetically manipulated and/or modified to at least one of increase their expression levels, increase their metabolic efficiency, and increase their functional lifespan. Similarly, the genetially-engineered microbes can otherwise be selected and/or otherwise genetically modified to increase ADH and/or ALDH enzyme expression levels, increase their metabolic efficiency, and increase their functional lifespan.

[0042] Additional characteristics and advantages of the present invention are described below.

[0043] Microbe Selection and Growth

[0044] Suitable microbes that can be used in the present disclosure include, but are not limited to, bacteria or yeast. Examples of bacteria for use herein include one or more strains of the following genera: Lactobacillus, Bifidobacterium, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, Escherichia, as well as lactic acid bacteria. Contemplated species of Lactobacillus include Lactobacillus GG, /.. acidophilus, L. bulgaricus, L. jugurti, L. helveticus, L. salivarius, L. casei, L. plantarum, L. rhamnosus, L. paracasei, L. lactis, L. infantis, L. reuteri, and/or L. brevis. Contemplated species of Bifidobacterium include Bifidobacterium AN AHP 16467, B. thermophilum, B. indicum, B. asteroids, B. lactis, B. longum, B. coagulans, B. dentium, B. infantis, and/or B. bifldum. Contemplated species of Enterococcus include Enterococcus faecium. Contemplated species of Streptococcus include Streptococcus thermophilus. Contemplated species of Pediococcus include Pediococcus acidilactici . Contemplated species of Escherichia include Escherichia coli. Any other bacteria useful as a probiotic is contemplated for use herein.

[0045] Examples of yeast contemplated for use herein include Saccharomyces species and others. In one specific example, Saccharomyces boulardii is contemplated for use herein.

[0046] Genetic constructs

[0047] In some embodiments, genetically-engineered microbes of the present invention can include one or more recombinant genes. Genetic constructs contemplated for use herein can be transiently expressed or permanently expressed in a recombinant host cell. In one particular embodiment, a genetically-engineered microbe can include one or more alcohol dehydrogenase genes and/or aldehyde dehydrogenase genes. For example, the common (non variant) allele of ADH1B or ADH1C can be used. In another example, a human A DH 2 (fmALDHI) and/or yeast ALDH2 (yALDH2) can be used. Alternatively, it is contemplated that other variants of ADH and/or ALDH genes can be used.

[0048] The ALDH gene is highly conserved during evolution and has been cloned in many species, which can be potentially used for genetically engineering probiotic microbes. Examples of species harboring an ALDH gene that can be used herein include bovine, sheep, horse, mouse, rat, yeast, fungus, and others. The ALDH gene family also has many different members. For example, the human ALDH family includes ALDH1-10, SSDH (succinic semialdehyde dehydrogenase), and MMSDH (methylmalonate semialdehyde dehydrogenase). These other genes can also be potentially used for genetic engineering probiotic microbes. In some embodiments, contemplated probiotic microbes can be transformed with and express multiple ALDH genes from multiple species, which can include different ALDH gene family members.

[0049] In one embodiment, a recombinant ALDH enzyme contemplated for use here includes the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, a recombinant ALDH enzyme contemplated for use here includes an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, or 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4. In a further embodiment, a contemplated ALDH enzyme for use herein includes an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4.

[0050] To engineer target microbe strains, multiple approaches can be used. A plasmid encoding ALDH2 (and/or an ADH) can be prepared and transformed (chemically and/or electrically) into the target cells. Transformed cells can be selected with intrinsic selection markers in the plasmid. Alternatively or in addition, recombinant bacteria phages can be prepared to encode ALDH2, and then be used to deliver the gene to target cells. A BAC (bacteria artificial chromosome) or YAC (yeast artificial chromosome) can be engineered to express ALDH2 and delivered to target cells to guide exogenous ALDH2 expression. In addition, a chromosome targeting construct can be prepared to deliver an expression cassette encoding ALDH2 to the endogenous chromosomes of target cells, which can drive exogenous ALDH2 expression.

[0051] In some embodiments, genetically-modified microbes of the present disclosure encoding one or more ALDH genes and/or one or more ADH genes can be induced to express the one or more ALDH genes and/or one or more ADH genes. For example, an isopropyl b-D-l- thiogalactopyranoside (IPTG)-inducible promoter can be used to drive expression. Other inducible promoters are also contemplated for use herein.

[0052] One of skill in the art will recognize that additional methods for producing the genetically-engineered microbes of the present disclosure are readily available. For example, all procedures for preparing genetically-engineered bacteria and genetically-engineered yeast (or any other useful microbe) are contemplated herein.

[0053] Compositions

[0054] Compositions contemplated for use herein include one or more genetically-modified microbes (a live cell, a disrupted cell wall fraction, a dead cell, a dried bacterium, a cell culture, or a fermentation broth), as described herein, and one or more vehicles or carriers. Contemplated vehicles or carriers include a culture medium, a protein, a carbohydrate, a fat, an oil, a flavoring agent, a seasoning agent, a food, water, a candy, a dissolvable breath freshener, a chewing gum, or mixtures thereof. Examples of carbohydrates include monosaccharides, e.g., glucose, fructose, and the like; disaccharides, such as maltose and sucrose, oligosaccharides and the like; and a polysaccharide, such as dextrin, sugar alcohol such as a conventional sugar and xylitol, sorbitol, erythritol, such as cyclodextrins. Contemplated flavoring agents include natural flavors such as thaumatin, stevia extract, and synthetic flavors such as saccharin, aspartame, etc.

[0055] In one embodiment, a composition (also referred to as a probiotic supplement) can be a finished food product (e.g., a dairy product, a protein drink, an energy drink, yogurt, ice cream, an energy bar, a mixer for an alcoholic beverage, a condiment, a spread, or any other commonly consumed food product), a powder or granule that can be added to a food or drink for consumption, a tablet, a capsule, or a liquid that can be either directly consumed or added to a food or drink for consumption.

[0056] When formulated as a powder, granule, tablet, or capsule form, compositions can further comprise suitable carriers, excipients, and diluents used in food manufacturing such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil. Additional additives can include fillers, extenders, binders, wetting agents, disintegrating agents, surface active agents used in food applications.

[0057] Additional additives of compositions contemplated herein include nutrient supplements, like vitamins and minerals, antioxidants, colorants, preservatives, caffeine, or mixtures thereof.

[0058] Compositions of the present disclosure can include about 0.01 to about 99.9%, or about 1% to about 99%, or about 2% to about 98%, about 5% to about 95%, about 10% to about 90%, about 20% to about 80%, or about 30%, or about 40%, or about 50% by weight genetically-engineered microbes. Alternative amounts are also contemplated. For example, amounts of genetically-engineered microbes of about 1 ^c 10⁵ to about 1 ^c 10¹⁰ CFU/g are contemplated.

[0059] Treatment Methodologies

[0060] In some embodiments of the present disclosure, methods of treating an individual for elevated acetaldehyde levels are contemplated.

[0061] In one embodiment, a method of preventing elevated levels of acetaldehyde in an individual or treating an individual for elevated levels of acetaldehyde includes administering to the individual a genetically-engineered microbe expressing a recombinant ALDH2 gene and reducing the levels of acetaldehyde in the individual.

[0062] In another embodiment, a method of treating an individual for Asian flush includes administering to the individual a genetically-engineered microbe expressing a recombinant ALDH2 gene and reducing the levels of acetaldehyde in the individual.

[0063] In one embodiment, a method of preventing elevated levels of acetaldehyde in an individual includes administering to the individual a composition including a genetically- engineered microbe expressing recombinant ALDH2 prior to, concomitant with, or after ethanol consumption and preventing the manifestation of elevated levels of acetaldehyde in the individual upon consumption and metabolism of ethanol.

[0064] In some embodiments, methods of increasing the rate of ethanol metabolism in an individual are contemplated including administering to the individual a genetically-engineered microbe expressing a recombinant ADH gene and increasing the rate of conversion of ethanol to acetaldehyde in the individual.

[0065] In some embodiments, combinatorial methods of increasing the rate of metabolism of ethanol and its metabolites in an individual are contemplated. For example, an individual can be administered genetically-engineered microbes expressing a recombinant ADH gene to increase the rate of conversion of ethanol to acetaldehyde in the individual and a recombinant ALDH2 gene to increase the conversion of acetaldehyde to acetate. In some embodiments, the recombinant ADH gene and recombinant ALDH2 gene are expressed in the same microbe. In some embodiments, the recombinant ADH gene and recombinant ALDH2 gene are expressed from the same constructs in the microbe. In some embodiments, the recombinant ADH gene and recombinant ALDH2 gene are expressed from different constructs in the same microbe. In some embodiments, the recombinant ADH gene and recombinant ALDH2 gene are expressed in different microbes.

EXAMPLES

[0066] The Examples that follow are illustrative of specific embodiments of the invention and various uses thereof. They are set forth for explanatory purposes only and are not to be taken as limiting the invention.

Example No. 1: Genetically-engineered E. coli Introduction

[0067] In this example, genetically-engineered E. coli were established. A strong T7 prokaryotic promoter was used to drive gene expression, and codon optimized the human and yeast ALDH2 sequences were used to enhance their expression in prokaryotic hosts.

Experimental Procedures

[0068] Genetically-engineered microbes expressing the human ALDH2 enzyme were established by transformation of E. coli K12 bacteria with a recombinant plasmid encoding a human ALDH2 gene (codon optimized for prokaryotic expression). Transformants were selected with corresponding antibiotics and expanded. Expression of human ALDH2 was confirmed by SDS-PAGE.

[0069] Genetically-engineered microbes carrying the yeast ALDH2 enzyme were established by transformation of E. coli K12 bacteria with a recombinant plasmid encoding yeast ALDH2 gene (codon optimized for prokaryotic expression). Transformants were selected with corresponding antibiotics and expanded. Expression of yeast ALDH2 was confirmed by SDS-PAGE.

[0070] Clearance of acetaldehyde in solution with transformed E. coli was measured by using a Sigma aldehyde quantification kit. Briefly, control (unengineered) E. coli or E. coli with exogenous expression of human or yeast ALDH2 were incubated in PBS solution with acetaldehyde (5 mM). An aliquot of the mixture was collected at different time points, and the concentration of acetaldehyde in the aliquots was determined by the quantification kit. Control bacteria show residual activity of acetaldehyde clearance, whereas engineered E. coli can remove almost all the acetaldehyde in solution within 30 minutes.

Results

[0071] FIG. 1 shows E. coli transformed with different plasmids encoding human ALDH2 (huALDH2) or yeast ALDH2 (yALDH2) were lysed for SDS-PAGE analysis (FIG. 1A). Arrows denote bands for huALDH2 and yALDH2, respectively. Clearance of acetaldehyde in solution with genetically-engineered E. coli versus control is shown in FIG. IB.

Example No. 2: Genetically-engineered Lactobacillus Introduction

[0072] In this example, a genetically-engineered Lactobacillus reuteri is established.

Experimental Procedures

[0073] For L. reuteri engineering, the shuttle plasmid is designed to contain a strong Erm promoter to express foreign genes, lactobacillus ribosome binding sites are included in the plasmid to ensure proper translation, L. reuteri codon optimized sequences are used to modify human and yeast ALDH2.

Example No. 3: In vivo Administration of Genetically-engineered Microbes to CD1 Mice

Introduction

[0074] In this example, engineered bacteria were adminstered to test animals, and the effect to reduce acetaldehyde upon ethanol consumption was examined in vivo.

Experimental Procedures

[0075] CD1 adult animals were fed with engineered E. coli (from Example No. 1) solution through oral gavage for two consecutive days. Subsequently, the mice were orally administered ethanol at a dose of 0.1 mL/l0 g body weight. Serum, proximal colon content, and proximal colon epithelium were harvested at 1 hour and 2 hours, respectively. Proximal colon content and proximal colon epithelium were quickly harvested and sealed in a small volume of water and later meshed and adjusted in water at the ratio of 50 mg/200 pl PBS. After brief centrifugation, supernatant was harvested for acetaldehyde concentration detection using Sigma Aldehyde detection kit. The acetaldehyde concentration detection in serum, proximal colon content, and proximal colon epithelium was done on the same day in order to avoid the variations caused by acetaldehyde evaporation. Results are shown in triplicate (FIGS. 2A-2C).

Example No. 4: In vivo Administration of Genetically-engineered Microbes to ALDH2 Knockout (KO) Mice

Introduction [0076] In this Example, ALDH2 KO mice were used to study the preventive/therapeutic effect of engineered probiotics carrying ALDH2 enzyme for human patients carrying non functional ALDH2 alleles.

Experimental Procedures

[0077] Adult AI.DH2 KO mice were fed engineered E. coli expressing yeast ALDH2 for 2-7 days. Treated and untreated ALDH2 KO mice were orally administered ethanol at a dose of 0.1 mL/l0 g body weight. Serum samples were harvested at 30 minutes and 60 minutes respectively. After brief centrifugation, supernatant was harvested for acetaldehyde concentration detection using the Sigma Aldehyde detection kit.

Results

[0078] Absence of ALDH2 leads to a significant increase of acetaldehyde concentration in blood (compare “Control” bars of FIG. 3 to FIG. 2, noting y axis scale). However, administration of engineered E.coli expressing yeast ALDH2 significantly reduced the serum level of acetaldehyde indicating that engineered probiotics can protect the animals from acetaldehyde-induced toxicity systemically (FIG. 3).

Example No. 5: In vivo Administration of Genetically-engineered Microbes to APC Min Mice

Introduction

[0079] Colonic accumulation of acetaldehyde is recognized as a strong carcinogen leading to CRC (colorectal cancer). In humans, most CRCs exhibit dysregulation of Wnt signaling pathway, caused by mutational inactivation of APC or activation of b-catenin. Because of its usefuless as a model for tumorigenesis of FAP (familial adenomatous polyposis) and CRC, APC Min mice have been widely used to model CRC pathogenesis. These mice are predisposed to spontaneous intestinal adenomas and carcinomas in an autosomal dominant fashion with full penetrance. This model is useful for exploring the potential protective effect of engineered probiotics in CRC tumorigenesis.

Experimental Procedures [0080] Adult APC min mice are fed engineered probiotics ( E . coli and/or L. reuteri ) that express human or yeast ALDH2 for 2-7 days. Treated and untreated mice are exposed to long term ethanol administration (fed with alcohol liquid diet containing 4.5% v/v ethanol, for 10 weeks). The treated group continues to receive oral gavage feeding of enginereed bacteria, whereas the untreated group receives saline as control. The incidence and size of intestinal lesions are analyzed and monitored in the control group and the group administered with engineered bacteria. The number and size of intestinal polyps are quantified using whole-mount staining of intestine epithelium with methylene blue, which has been routinely used to highlight both micro- and macroscopic lesions in mouse intestinal tract. The macroscopic tumors of APC''¹'” mice usually display a pleomorphic histology ranging from tubular adenomas of low- grade dysplasia to carcinoma in situ. By histopathological analysis, progression of intestinal tumors are graded and quantified in different groups.

Example No. 6: In vivo Administration of Genetically-engineered Microbes to Mice Treated with a Colon Carcinogen

Introduction

[0081] Treatment with colon carcinogens, such as AOM (azoxymethane), has also been extensively used to model colorectal carcinogenesis. After injection, AOM is metabolized in cells to produce MAM (methylazoxymethanol glycoside) with very high intracellular stability. In colonic cells, MAM further produces a methyldiazonium ion that can lead to alkylation damage of chromosomal DNA. Although chemical carcinogens may target a large number of genes for mutation, it has been shown in mice that AOM treatment frequently causes mutation in K-Ras and Wnt pathway genes.

Experimental Procedures

[0082] To study the effect of engineered probiotics, adult C57/BL6J mice are fed with engineered probiotics E. coli and/or L. reuteri) that express human or yeast ALDH2 for 2-7 days. Treated and untreated mice are treated with 6 weekly injections of 10 mg/kg of AOM plus long term ethanol administration (alcohol liquid diet as above in Example No. 5, for 10 weeks). The treated group continues to receive oral gavage feeding of genetically-enginereed bacteria, whereas the untreated group receives saline as control. Mice are sacrified 24, 36, and 48 weeks after the last injection. Large intestine tissue is collected and examined as described above to quantify the incidence and size of tumors. Progression of colonic neoplasms is also determined by pathological analysis.

Sequence IDs of genes used in Examples.

[0083] Having described the invention in detail and by reference to specific aspects and/or embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention may be identified herein as particularly advantageous, it is contemplated that the present invention is not limited to these particular aspects of the invention.

Claims

WHAT IS CLAIMED IS:

1. A genetically-engineered microbe, comprising:

a recombinant gene encoding an aldehyde dehydrogenase.

2. The genetically-engineered microbe of claim 1, wherein the aldehyde dehydrogenase is ALDH2.

3. The genetically-engineered microbe of claim 2, wherein the ALDH2 is a human or yeast ALDH2.

4. The genetically-engineered microbe of claim 3, wherein the ALDH2 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 2 or SEQ ID NO: 4.

5. The genetically-engineered microbe of claim 1, wherein the genetically- engineered microbe is a bacterium or a yeast.

6. The genetically-engineered microbe of claim 5, wherein the bacterium is one or more of a Lactobacillus , a Bifidobacterium, an Enterococcus, a Streptococcus, a Pediococcus, a Leuconostoc, a Bacillus, an Escherichia, or a lactic acid bacteria.

7. The genetically-engineered microbe of claim 6, wherein the Lactobacillus is Lactobacillus GG, /.. acidophilus, L. bulgaricus, L. jugurti, L. helveticus, L. salivarius, L. casei, L. plantarum, L. rhamnosus, L. paracasei, L. lactis, L. infantis, L. reuteri, or L. brevis.

8. The genetically-engineered microbe of claim 6, wherein the Bifidobacterium is Bifidobacterium AN AHP 16467, B. thermophilum, B. indicum, B. asteroids, B. lactis, B. longum, B. coagulans, B. dentium, B. infantis, or B. bifldum.

9. The genetically-engineered microbe of claim 6, wherein the Enterococcus is Enterococcus faecium.

10. The genetically-engineered microbe of claim 6, wherein the Streptococcus is Streptococcus thermophilus.

11. The genetically-engineered microbe of claim 6, wherein the Pediococcus is Pediococcus acidilactici .

12. The genetically-engineered microbe of claim 5, wherein the yeast is a Saccharomyces.

13. The genetically-engineered microbe of claim 12, wherein the Saccharomyces is Saccharomyces boulardii.

14. A composition for reducing acetaldehyde concentration in an individual, comprising:

a genetically-engineered microbe according to any of the preceding claims; and one or more of a vehicle, a carrier, and an additive.

15. The composition of claim 14, wherein the genetically-engineered microbe is a live cell, a disrupted cell wall fraction, a dead cell, a dried bacterium, a cell culture, or a fermentation broth.

16. The composition of claim 14, wherein the vehicle or the carrier comprises a culture medium, a protein, a carbohydrate, a fat, an oil, a flavoring agent, a seasoning agent, a food, water, or mixtures thereof.

17. The composition of claim 16, wherein the carbohydrate comprises a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide.

18. The composition of claim 16, wherein the flavoring agent comprises a natural flavor or a synthetic flavor.

19. The composition of claim 14, wherein the additive is a vitamin, a mineral, an antioxidant, a colorant, a preservative, caffeine, or a mixture thereof.

20. The composition of claim 14, wherein the composition is a finished food product, a powder, a granule, a tablet, a capsule, or a liquid.

21. The composition of claim 14, wherein the composition comprises about 0.01 to about 99.9% by weight genetically-engineered microbe.

22. A method of treating an individual for elevated levels of acetaldehyde, comprising:

administering to the individual a genetically-engineered microbe according to any of claims 1-13; and

reducing the levels of acetaldehyde in the individual.

23. A method of treating an individual for Asian flush, comprising:

reducing the levels of acetaldehyde in the individual.

24. A method of preventing elevated levels of acetaldehyde in an individual, comprising:

administering to the individual a composition including a genetically-engineered microbe expressing recombinant ALDH2 and

preventing the manifestation of elevated levels of acetaldehyde in the individual upon consumption and metabolism of ethanol.

25. The method of any of claims 22-24, wherein the individual is deficient in one or more alcohol dehydrogenases.

26. The method of claim 25, wherein the individual is a carrier of ADH1B and/or ADH1C variant alleles.

27. The method of any of claims 22-24, wherein the individual is deficient in aldehyde dehydrogenase.

28. The method of claim 27, wherein the individual is a carrier of an ALDH2 variant allele.