Engineering bacterium with alcohol degrading capacity and preparation and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and mainly relates to an escherichia coli Nissle 1917 genetically engineered bacterium with an alcohol degradation effect as well as a preparation method and application of the escherichia coli Nissle 1917 genetically engineered bacterium. The engineering bacteria are obtained by modifying an adhp gene segment and a puuc gene segment in a host strain. Chitosan and sodium alginate are coated on the surface of a genetic engineering strain through an electrostatic layer-by-layer self-assembly technology, so that the tolerance of the strain to gastric acid, alcohol and cholate is effectively improved. After being coated with two layers of chitosan and sodium alginate, the genetically engineered bacterium constructed by the invention can be used for preparing food or medicines with an alcohol degrading function, and has important practical significance and economic value in the application of treating and preventing the alcoholic liver disease of a human body.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and mainly relates to a genetic engineering bacterium of Escherichia coli Nissle 1917 capable of degrading alcohol, and a preparation method and application thereof. Background Art

[0002] Alcohol has existed in the history of human civilization for thousands of years and has played an important role in artistic creation, entertainment, communication and many other aspects. However, excessive drinking without control in situations such as relieving stress, social occasions and various festival celebrations may develop into alcoholic liver disease. The development process of alcoholic liver disease is fatty liver, alcoholic hepatitis, liver fibrosis, cirrhosis and hepatocellular carcinoma. Most of the alcohol is absorbed in the intestines, then circulates to the liver through the blood and completes alcohol metabolism. More than 90% of ethanol is oxidized and metabolized in the liver to produce acetaldehyde and acetic acid.

[0003] There are many metabolic mechanisms for the liver to eliminate alcohol, mainly including the hepatocyte cytoplasmic alcohol dehydrogenase (ADH) system and the microsomal ethanol oxidation system (MEOS). In the process of alcohol dehydrogenase catalyzing ethanol oxidation dehydrogenation, it will produce too much reduced coenzyme I (NADH), resulting in an increase in the NADH / NAD+ ratio, further affecting fatty acid oxidation and leading to an increase in triglyceride synthesis. In the MEOS system, the most important enzyme is cytochrome P450 2E1 (CYP2E1), and its protein and mRNA expression levels will be upregulated with long-term drinking. However, in the process of CYP2E1 metabolism of alcohol, not only acetaldehyde is produced, but also more reactive oxygen species (ROS) are induced. Excessive ROS in the liver will react with macromolecular substances such as phospholipids, enzymes and membrane receptors of biological membranes to form lipid peroxidation products, thereby destroying the fluidity and permeability of cell membranes, and ultimately affecting the structure and function of cells. ROS will react with macromolecules such as phospholipids, enzymes and membrane receptors of biological membranes, side chains of polyunsaturated fatty acids, and nucleic acids to form lipid peroxidation products, thereby changing the fluidity and permeability of cell membranes, and ultimately leading to changes in cell structure and function.

[0004] Among various treatment strategies for alcoholic liver disease, abstinence is still the most effective. Some drugs can speed up the metabolism of alcohol. For example, dandelion cypermethrin can be used to increase the catalytic activity of alcohol dehydrogenase and acetaldehyde dehydrogenase. Injection of alcohol dehydrogenase and acetaldehyde dehydrogenase encapsulated in red blood cells can provide additional catalytic activity. However, intravenous injection of exogenous proteases has disadvantages such as easy inactivation, high cost, and easy to cause immune response. At present, intestinal microbial enzymes can be used to degrade ethanol and acetaldehyde into acetic acid to reduce the alcohol metabolism pressure in the liver. However, probiotics that have not been genetically engineered have low enzyme activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in their bodies and cannot effectively degrade alcohol to alleviate the development of alcoholic liver disease. Therefore, in-depth research on alleviating the development of alcoholic liver disease is needed. Summary of the invention

[0005] The invention aims to provide a genetically engineered bacterium Escherichia coli Nissle 1917 capable of degrading alcohol, and a preparation method and application thereof.

[0006] To achieve the above purpose, the present invention adopts the following technical solution:

[0007] An engineered bacterium capable of degrading alcohol, wherein the engineered bacterium is a host strain in which an adhp gene fragment and a puuc gene fragment are modified.

[0008] The host strain is Escherichia coli Nissle 1917;

[0009] The adhp gene fragment and the puuc gene fragment are derived from the adhp gene and the puuc gene of Escherichia coli K12; wherein the nucleotide sequence of the adhp gene fragment is the nucleotide sequence shown in SEQ ID NO.1; and the nucleotide sequence of the puuc gene fragment is the nucleotide sequence shown in SEQ ID NO.2.

[0010] The engineering bacteria contains an arabinose-inducible promoter.

[0011] A method for preparing the genetically engineered bacteria is to transform a recombinant plasmid capable of overexpressing adhp and puuc genes under the control of an arabinose-inducible promoter into Escherichia coli Nissle 1917 by electroporation. The genetically engineered bacteria is named ECN@ALDH.

[0012] The recombinant plasmid comprises a kanamycin resistance gene sequence, an arabinose regulatory protein araC gene sequence, a ParaBAD promoter sequence, and adhp and puuc gene sequences.

[0013] An application of the engineered bacteria, and an application of the genetically engineered bacteria in the preparation of medicines or foods for preventing and treating alcoholic liver disease.

[0014] A preparation with the function of degrading alcohol, comprising the preparation.

[0015] The preparation is a biocompatible material coated on the surface of genetically engineered bacteria, wherein the biocompatible material is chitosan and sodium alginate, wherein the mass ratio of chitosan to sodium alginate is 1:1 to 1:2, and the mass ratio of the sum of chitosan and sodium alginate to the engineered bacteria is 1:1 to 4:1.

[0016] The genetically engineered bacteria uses electrostatic layer-by-layer self-assembly technology to electrostatically adsorb chitosan and sodium alginate in sequence to form protection, namely ECN@ALDH(C / A)2.

[0017] The preparation is in the form of powder, granules, tablets or capsules.

[0018] Unless otherwise specified, all professional terms and technical concepts involved in this article are well known to those skilled in the art. Although there are a variety of similar or equivalent methods that can be used to construct the Escherichia coli Nissle 1917 genetically engineered bacteria of the present invention, a suitable method and material will be described below. These materials, methods and examples are only used as examples and do not limit the present invention to any extent.

[0019] The advantages of the present invention are:

[0020] The invention introduces the adhp gene and the puuc gene from the specifically matched Escherichia coli K12 into the Escherichia coli Nissle 1917, and places the two genes under the control of an arabinose-inducible promoter. The obtained Escherichia coli Nissle 1917 genetic engineering bacteria has good alcohol degradation ability. Subsequently, the chitosan and the sodium alginate are electrostatically adsorbed in sequence by the electrostatic layer-by-layer self-assembly technology to form two layers of protection, which effectively improves the survival rate of the Escherichia coli Nissle 1917 genetic engineering bacteria under the conditions of simulated gastric acid, bile salt and high concentration alcohol, and effectively improves the alcohol degradation ability of the Escherichia coli Nissle 1917 genetic engineering bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the determination of ethanol and acetaldehyde dehydrogenase activities of the genetically engineered bacterium Escherichia coli Nissle 1917 in Example 2 of the present invention are shown in Table 1. a: ethanol dehydrogenase activity, b: acetaldehyde dehydrogenase activity.

[0022] Figure 2 The results of the alcohol degradation ability test of the genetically engineered Escherichia coli Nissle 1917 bacteria in Example 3 of the present invention. a: the in vitro alcohol degradation rate of the recombinant strain at different ethanol concentrations. b, c: the ethanol concentration and acetaldehyde concentration in the serum of alcoholic mice treated with PBS, ECN and ECN@ALDH, respectively (n=6).

[0023] Figure 3 The present invention provides the results of measuring the alcohol degradation efficiency and acetaldehyde concentration of the recombinant strains that overexpress different combinations of alcohol dehydrogenase and acetaldehyde dehydrogenase.

[0024] Figure 4 a is the scanning electron microscopy image of ECN@ALDH and ECN@ALDH preparations.

[0025] Figure 4 b is the surface zeta potential results of ECN@ALDH and chitosan / sodium alginate layer coatings with different coatings during the preparation of ECN@ALDH preparation.

[0026] Figure 5 The tolerance results of the Escherichia coli Nissle 1917 preparation prepared in Example 4 of the present invention to different concentrations of alcohol, simulated gastric acid, and different concentrations of simulated bile salt solutions are shown.

[0027] Figure 6 This is the determination of the alcohol degradation activity of the Escherichia coli Nissle 1917 genetically engineered bacteria preparation in vivo as mentioned in Example 5 of the present invention. Wherein a: determination of blood ethanol content at different times, b: determination of blood acetaldehyde content at different times.

[0028] Figure 7 The effect of the Escherichia coli Nissle 1917 genetically engineered bacteria preparation on alcoholic fatty liver disease in Example 6 of the present invention. a: The treatment process of the alcoholic fatty liver disease model. b, c: The weight changes of mice in different treatment groups during the 84-day treatment course (n=6). d: Comparison of HE staining images of different treatment groups.

[0029] Figure 8 Determination of blood biochemical indexes of alcoholic fatty liver disease according to the sixth embodiment of the present invention. a, b: Comparison of organ coefficients (liver, fat) of different treatment groups. c: Statistics of adipocyte area. dj: Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglycerides (TG), total bile acid (TBA), total cholesterol (T-CHO) of mice (n=6). DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with specific embodiments. It should be pointed out that the specific implementation methods described here are only for illustrating and explaining the present invention, and are not intended to limit the present invention.

[0031] The present invention transforms Escherichia coli Nissle 1917 through genetic engineering technology, realizes rapid alcohol degradation while reducing the enrichment of the intermediate product acetaldehyde, which is beneficial to reducing the damage of ethanol and acetaldehyde to the liver. At the same time, chitosan and sodium alginate are further coated on the surface of the genetically engineered strain through electrostatic layer-by-layer self-assembly technology, which effectively improves the strain's tolerance to gastric acid, alcohol, and bile salts. After being coated with two layers of chitosan and sodium alginate, the genetically engineered bacteria constructed by the present invention can be used to prepare food or medicine with the function of degrading alcohol, and its application in the treatment and prevention of human alcoholic liver disease has important practical significance and economic value.

[0032] The primer sequences involved in the examples are shown in Table 1

[0033] Table 1: Primer sequences

[0034]

[0035]

[0036] Example 1 Construction of recombinant strains:

[0037] The adhp or puuc sequence annotated in the whole genome sequencing results of strain Escherichia coli K-12 was amplified. ADHP-F, ADHP-R, PUUC-F, and PUUC-R in Table 1 were used respectively. PCR reaction conditions: 94°C denaturation for 5 min, followed by 30 cycles of (94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 2 min), and 72°C extension for 10 min.

[0038] Using the commercial pBAD18 plasmid as a template, araC_P araBAD Fragment and P araBAD The fragments were amplified using P1-F, P1-R, P2-F, and P2-R in Table 1, respectively.

[0039] The ADHP fragment obtained above was mixed with araC_P araBAD The fragments were subjected to overlap PCR to obtain fusion fragment 1, using Overlap-F1 and Overlap-R1 in Table 1.

[0040] The PUUC fragment was combined with P araBAD The fragments were subjected to overlap PCR to obtain fusion fragment 2, using Overlap-F2 and Overlap-R2 in Table 1.

[0041] Overlap PCR was performed on fusion fragment 1 and fusion fragment 2 to obtain fusion fragment 3, using Overlap-F1 and Overlap-R2 in Table 1.

[0042] The fusion fragment 3 obtained above and pBBR1-MCS2 were double-digested with Sac I and Xba I restriction endonucleases, respectively, and the recombinant vector was connected after gel recovery and purification; the obtained recombinant expression vector was transformed into Escherichia coli Nissle 1917 to obtain Escherichia coli Nissle 1917 genetically engineered bacteria ECN@adhp-puuc (named ECN@ALDH).

[0043] Example 2 Determination of enzyme activity of Escherichia coli Nissle 1917 genetically engineered bacteria:

[0044] The recombinant strain obtained in Example 1 was cultured overnight at 37° C. in LB medium (yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, pH 7.0) as a seed culture. Then 1 wt% of the seed culture was cultured in LB medium containing 100 μg / mL kanamycin at 37° C.

[0045] When the cells reach OD 600 The expression of E. coli Nissle 1917 was induced at 37°C for 12 hours to obtain E. coli Nissle 1917 genetically engineered bacteria (ECN@ALDH). The samples were diluted and coated on LB solid medium containing kanamycin resistance, and the total number of bacterial colonies per milliliter of sample was determined by colony counting.

[0046] To determine the alcohol dehydrogenase and acetaldehyde dehydrogenase activities of Escherichia coli Nissle 1917 genetically engineered bacteria, the cells were resuspended in 50 mM Tris-HCl buffer (pH 7.5) and adjusted to 10 9 The CFU bacteria were then lysed by ultrasound, and the ultrasonic disruption conditions were as follows: ultrasound for 3 seconds, stop for 6 seconds, and a net ultrasound time of 5 minutes. The crude enzyme was obtained by centrifugation at 8500 rpm for 10 minutes at 4°C.

[0047] The reaction solution for measuring the catalytic activity of alcohol dehydrogenase or acetaldehyde dehydrogenase includes 400 μL 10% ethanol or 400 μL 0.3% acetaldehyde, 400 μL 2% NAD + , 2100 μL 50 mM Tris-HCl buffer (pH 7.4). Finally, 100 μL of the crude enzyme solution prepared as above was added to each system, and the absorbance change of the reaction solution at a wavelength of 340 nm was detected at 37°C. The amount of enzyme required to produce 1 μmol NADH per minute was defined as one enzyme activity unit (U).

[0048] The results showed that the catalytic activity of alcohol dehydrogenase from Escherichia coli Nissle 1917 was 15.49 U / 1×109 CFUs( Figure 1 A), the catalytic activity of acetaldehyde dehydrogenase is 2.68U / 1×10 9 CFUs( Figure 1 B). Compared with the wild-type E. coli Nissle 1917, the activity of alcohol dehydrogenase increased by 4 times, and the activity of acetaldehyde dehydrogenase increased by 3 times.

[0049] Example 3 Determination of alcohol degradation ability of Escherichia coli Nissle 1917 genetically engineered bacteria:

[0050] Escherichia coli Nissle 1917 genetically engineered bacteria (ECN@ALDH) were prepared according to the method of Example 2. The ethanol degradation reaction solution included 50 mM Tris-HCl buffer (pH 7.4), 2% NAD + and different concentrations of ethanol (1%, 5%, 10%, 20% (v / v)). 10 CFUs were resuspended in 10 ml of ethanol degradation reaction solution and incubated at 37°C and 150 rpm for different time periods. After the reaction was completed, the cells were lysed by ultrasonication and then centrifuged at 10,000 rpm for 5 min to collect the supernatant for ethanol and acetaldehyde content determination.

[0051] To verify the alcohol degradation efficiency of the recombinant strain in vivo, 18 male C57BL / 6 mice aged 6 to 8 weeks were divided into three groups (further experiments were performed after 1 week of acclimatization) and intragastrically administered with PBS, ECN (1×10 10 CFU / mL) and ECN@ALDH (1×10 10 CFU / mL). 30 minutes later, alcohol (5 mg / g) was intragastrically administered. 1 hour after administration, 30 μL of tail vein blood was collected to detect blood ethanol and acetaldehyde levels.

[0052] The results showed that the degradation rate of Escherichia coli Nissle 1917 was 58.54% when the ethanol concentration was 1%; the degradation rate was 44.11% when the ethanol concentration was 5%; the degradation rate was 18.02% when the ethanol concentration was 10%. Figure 2 a). The results of in vivo alcohol degradation efficiency test showed that compared with wild-type E. coli Nissle 1917, the recombinant strain can effectively reduce the ethanol and acetaldehyde content in the blood ( Figure 2 b, 2c).

[0053] Comparative Example 1

[0054] According to Example 1, recombinant strains of different combinations of alcohol dehydrogenase and acetaldehyde dehydrogenase were prepared. Among them, the alcohol dehydrogenase genes were adhp (accession numbers: P39451) and yqhd (accession numbers: Q46856). The acetaldehyde dehydrogenase genes were puuc (accession numbers: P23883), aldA (accession numbers: P25553) and aldB (accession numbers: P37685). The ethanol degradation reaction solution included 50 mM Tris-HCl buffer (pH 7.4), 2% NAD + and 5% (v / v) ethanol. Different recombinant strains were added at 1×10 10 CFUs were resuspended in 10 ml of ethanol degradation reaction solution and incubated at 37°C and 150 rpm for 6 hours. After the reaction was completed, the cells were lysed by ultrasonication and then centrifuged at 10,000 rpm for 5 min to collect the supernatant for ethanol and acetaldehyde content determination.

[0055] The results showed that the alcohol degradation rates of the three recombinant strains, ECN@ALDH, ECN@adhp-aldA and ECN@adhp-aldB, were all higher than 40% when the alcohol concentration was 5%. Figure 3 a). However, ECN@adhp-aldA and ECN@adhp-aldB produced higher concentrations of acetaldehyde during the alcohol degradation process ( Figure 3 b). If used as a therapeutic agent to degrade alcohol in the intestine, excessive acetaldehyde will enter the blood through the intestinal barrier and cause harm to the body, causing uncomfortable symptoms such as flushing, palpitations and decreased blood pressure. However, the alcohol degradation rates of the three recombinant bacteria ECN@yqhd-puuc, ECN@yqhd-aldA and ECN@yqhd-aldB were all lower than 23% when the alcohol concentration was 5%. In summary, overexpression of adhp and puuc genes in E. coli Nissle1917 not only has a high alcohol degradation ability, but also does not produce excessive acetaldehyde that has potential harm to the body.

[0056] Example 4 Preparation and Characterization of Escherichia coli Nissle 1917 Genetically Engineered Bacteria Preparation:

[0057] The preparation process of Escherichia coli Nissle 1917 genetically engineered bacteria preparation is as follows:

[0058] 2 mg / mL sodium alginate and 2 mg / mL soluble chitosan were dissolved in 0.5 M NaCl solution (pH 6.0) respectively and set aside.

[0059] The recombinant strain ECN@ALDH was induced to express with 0.2% L-arabinose, and its fermentation quality was verified by enzyme activity assay. The activity of alcohol dehydrogenase reached 15U / 1×10 9 CFUs, acetaldehyde dehydrogenase activity reached 2.5U / 1×10 9 CFUs.

[0060] After the assay, the cell pellet was resuspended in the chitosan solution prepared above and adjusted to 1 mg weight of bacteria / ml. After incubation at 25°C for 1 h, it was washed 3 times with 0.5 M NaCl solution. The precipitate was collected by centrifugation at 10,000 rpm for 2 minutes and resuspended in the sodium alginate solution above and adjusted to 2 mg weight / ml. After incubation for 1 h, the first layer was applied to the ECN surface and named ECN@ALDH(C / A) 1 Then, the coating was repeated according to the above operation to obtain ECN@ALDH(C / A) 2 .

[0061] ECN@ALDH and ECN@ALDH(C / A) 2 Resuspend the sample in physiological saline. Add an appropriate amount of glutaraldehyde (final concentration 2%) and fix at 4°C for 6 hours. Take an appropriate amount of bacterial suspension on a clean cover paper, spread it evenly, dry it, and fix it under a flame. Soak the cells in 40, 70, 90, and 100% ethanol for 15 minutes respectively. After soaking, place the specimen under a scanning electron microscope and take a picture (see Figure 4 ).

[0062] ECN@ALDH and ECN@ALDH(C / A) 2 The samples were placed in 30% alcohol, 50% alcohol, pH 2.5 gastric acid or 0.1%, 0.2%, 0.3% bile salts at 37°C and 50 rpm for 30 minutes and 1 hour. 2 ) is fixed at 1×10 9 CFUs. Then, samples were collected at different time points, centrifuged at 9000 rpm for 5 minutes, and the bacteria in each sample were collected, washed three times with PBS, diluted and plated on solid agar plates containing kanamycin. After incubation at 37°C for 48 h, the number of single colonies was counted to determine the protective effect of chitosan / sodium alginate coating on the recombinant strain ECN@ALDH (see Figure 5 ).

[0063] The results show that: Figure 4 The change in the surface zeta potential of b shows that chitosan and sodium alginate are adsorbed layer by layer on the surface of ECN@ALDH through electrostatic interaction to encapsulate probiotics, eventually forming a coating on the surface of the bacteria ( Figure 4a). Figure 5 As shown, the chitosan / sodium alginate coating of ECN@ALDH improved the survival rate in the presence of 30% and 50% alcohol, gastric acid, and bile salts.

[0064] Example 5 Determination of alcohol degradation activity in vivo of Escherichia coli Nissle 1917 genetically engineered bacterial preparation:

[0065] To determine the in vivo ethanol degradation ability of Escherichia coli Nissle 1917 genetically engineered bacteria preparations, 18 6-8 week old male C57BL / 6 mice were divided into three groups (PBS, ECN@ALDH, ECN@ALDH(C / A) 2 Each mouse was first intragastrically administered with 5 mg / g alcohol (ALD), and 15 min later, 10 μL / g PBS, ECN@ALDH (1×10 10 CFU / mL) and ECN@ALDH(C / A) 2 (1×10 10 CFU / mL). 30 μL of blood samples were collected from the rat tail vein at 0h, 1h, 2h, 3h, and 4h after administration to detect the levels of ethanol and acetaldehyde in the blood.

[0066] The results showed that within the first 2 hours of alcohol administration, ECN@ALDH(C / A) 2 The blood alcohol content of mice in the ECN@ALDH group was lower than that in the ECN@ALDH group, but there was no significant difference ( Figure 6 a). However, there were significant differences in blood ethanol and acetaldehyde levels between the two groups at the 3rd and 4th hours ( Figure 6 b) This may be related to the improved tolerance of chitosan / sodium alginate preparations to high alcohol concentrations produced by recombinant bacteria. The improved tolerance of the recombinant strain can ensure continuous alcohol metabolism and is expected to effectively relieve liver metabolic pressure.

[0067] Example 6 Effect of Escherichia coli Nissle 1917 genetically engineered bacterial preparation on alcoholic fatty liver induced by high-fat diet combined with alcohol:

[0068] In the alcohol-induced fatty liver model induced by high-fat diet combined with alcohol, 24 mice were divided into 4 groups and fed with high-fat diet (HFD, 60% kcal% fat; catalog no. D12492; Research Diet, Beijing HFK Bio-Technology.co., LTD) for 84 days. The normal group was fed with maintenance feed. 5 mg / g alcohol was given by gavage at 10 am every day, and the normal group was replaced with PBS solution. 30 minutes after alcohol feeding, ECN@ALDH (C / A) 2 +ALC+HFD group mice were given ECN@ALDH(C / A)2 (1×10 8 CFUs / g mouse). This regimen was repeated daily for 84 consecutive days.

[0069] After the experiment, the mice were dissected, and liver and epididymal adipose tissue samples were collected and fixed in 4% paraformaldehyde solution. Hematoxylin-eosin staining (H&E) was commissioned to Wuhan Saiwei Biotechnology Co., Ltd. The biochemical indicators aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglycerides (TG), total bile acid (TBA), and total cholesterol (T-CHO) were determined according to the protocol developed by the kit manufacturer (Nanjing Jiancheng Bioengineering Institute).

[0070] The results showed that a high-fat diet could induce obesity in mice, while the weight of the HFD+ALC group stopped increasing from day 28 to day 56 ( Figure 7 b,c). However, ECN@ALDH(C / A) 2 The body weight changes of the treated group during this period were not disturbed by alcohol, which indicates that ECN@ALDH(C / A) 2 It has a protective effect on nutrient absorption in the body. The organ index of liver tissue in the HFD+ALC group was significantly increased ( Figure 8 a), and the pathological results showed obvious fat droplet aggregation, cell ballooning, hepatocyte ballooning and inflammatory infiltration. However, under the treatment of Escherichia coli Nissle 1917 genetically engineered bacteria preparation, only part of the fat cavitation occurred, showing a good therapeutic effect.

[0071] Organ index and pathological analysis of epididymal adipose tissue showed that ECN@ALDH(C / A) 2 The fat content of the treatment group ( Figure 8 b) and adipocyte size ( Figure 8 c) was significantly lower than that in the HFD+ALC group. The enlargement of adipocytes indicates liver fat accumulation and impaired metabolic regulation. The AST( Figure 8 d) and ALT( Figure 8 e) The level was significantly higher than that of the Normal group and ECN@ALDH (C / A) 2 Treatment group. The serum LDL content in the HFD+ALC group was 2.23 times that of the Normal group, which was ECN@ALDH(C / A) 2 1.58 times of the treated group ( Figure 8 f). The HDL level in the HFD+ALC group was not significantly different from that in the Normal group, but under ECN@ALDH(C / A)2 treatment, its content increased by 1.65 times compared with that in the HFD+ALC group ( Figure 8g). In addition, the TG content in the HFD group was significantly higher than that in the HFD+ALC group, which may be related to the effect of alcohol on intestinal nutrient absorption ( Figure 8 h). T-CHO in the HFD group and HFD+ALC group ( Figure 8 i) and TBA( Figure 8 j) There is no significant difference in content. In summary, ECN@ALDH(C / A) 2 After the intervention, the abnormal blood indicators caused by alcohol and high-fat diet were effectively alleviated.

[0072] SEQ ID NO.1

[0073] atgaaggctg cagttgttac gaaggatcat catgttgacg ttacgtataa aacactgcgc 60

[0074] tcactgaaac atggcgaagc cctgctgaaa atggagtgtt gtggtgtatg tcataccgat 120

[0075] cttcatgtta agaatggcga ttttggtgac aaaaccggcg taattctggg ccatgaaggc 180

[0076] atcggtgtgg tggcagaagt gggtccaggt gtcacctcat taaaaccagg cgatcgtgcc 240

[0077] agcgtggcgt ggttctacga aggatgcggt cattgcgaat actgtaacag tggtaacgaa 300

[0078] acgctctgcc gttcagttaa aaatgccgga tacagcgttg atggcgggat ggcggaagag 360

[0079] tgcatcgtgg tcgccgatta cgcggtaaaa gtgccagatg gtctggactc ggcggcggcc 420

[0080] agcagcatta cctgtgcggg agtcaccacc tacaaagccg ttaagctgtc aaaaattcgt 480

[0081] ccagggcagt ggattgctat ctacggtctt ggcggtctgg gtaacctcgc cctgcaatac 540

[0082] gcgaagaatg tctttaacgc caaagtgatc gccattgatg tcaatgatga gcagttaaaa 600

[0083] ctggcaaccg aaatgggcgc agatttagcg attaactcac acaccgaaga cgccgccaaa 660

[0084] attgtgcagg agaaaactgg tggcgctcac gctgcggtgg taacagcggt agctaaagct 720

[0085] gcgtttaact cggcagttga tgctgtccgt gcaggcggtc gtgttgtggc tgtcggtcta 780

[0086] ccgccggagt ctatgagcct ggatatccca cgtcttgtgc tggatggtat tgaagtggtc 840

[0087] ggttcgctgg tcggcacgcg ccaggattta actgaagcct tccagtttgc cgccgaaggt 900

[0088] aaagtggtgc cgaaagtcgc cctgcgtccg ttagcggaca tcaacaccat ctttactgag 960

[0089] atggaagaag gcaaaatccg tggccgcatg gtgattgatt tccgtcacta a 1011

[0090] SEQ ID NO.2

[0091] atgaattttc atcatctggc ttactggcag gataaagcgt taagtctcgc cattgaaaac 60

[0092] cgcttattta ttaacggtga atatactgct gcggcggaaa atgaaacctt tgaaaccgtt 120

[0093] gatccggtca cccaggcacc gctggcgaaa attgcccgcg gcaagagcgt cgatatcgac 180

[0094] cgtgcgatga gcgcagcacg cggcgtattt gaacgcggcg actggtcact ctcttctccg 240

[0095] gctaaacgta aagcggtact gaataaactc gccgatttaa tggaagccca cgccgaagag 300

[0096] ctggcactgc tggaaactct cgacaccggc aaaccgattc gtcacagtct gcgtgatgat 360

[0097] attcccggcg cggcgcgcgc cattcgctgg tacgccgaag cgatcgacaa agtgtatggc 420

[0098] gaagtggcga ccaccagtag ccatgagctg gcgatgatcg tgcgtgaacc ggtcggcgtg 480

[0099] attgccgcca tcgtgccgtg gaacttcccg ctgttgctga cttgctggaa actcggcccg 540

[0100] gcgctggcgg cgggaaacag cgtgattcta aaaccgtctg aaaaatcacc gctcagtgcg 600

[0101] attcgtctcg cggggctggc gaaagaagca ggcttgccgg atggtgtgtt gaacgtggtg 660

[0102] acgggttttg gtcatgaagc cgggcaggcg ctgtcgcgtc ataacgatat cgacgcatt 720

[0103] gcctttaccg gttcaacccg taccgggaaa cagctgctga aagatgcggg cgacagcaac 780

[0104] atgaaacgcg tctggctgga agcgggcggc aaaagcgcca acatcgtttt cgctgactgc 840

[0105] ccggatttgc aacaggcgg aagcgccacc gcagcaggca tttctacaa ccagggacag 900

[0106] gtgtgcatcg ccggaacgcg cctgttgctg gagagagca tcgccgatga attcttagcc 960

[0107] ctgttaaaac agcaggcgca aaactggcag ccgggccatc cacttgatcc cgcaaccacc1020

[0108] atgggcacct taatcgactg cgcccacgcc gactcggtcc atagctttat tcgggaaggc1080

[0109] gaagcaag ggcaactgtt gttggatggc cgtaacgccg ggctggctgc cgccatcggc 1140 ccgaccatct ttgtggatgt ggacccgaat gcgtccttaa gtcgcgaaga gattttcggt 1200 ccggtgctgg tggtcacgcg tttcacatca gaaacagg cgctacagct tgccaacgac 1260 agccagtacg gccttggcgc ggcggtatgg acgcgcgacc tctcccgcgc gcaccgcatg 1320 agccgacgcc tgaaagccgg ttccgtcttc gtcaataact acaacgacgg cgatatgacc 1380 gtgccgtttg gcggctataa gcagagcggc aacggtcgcg acaaatccct gcatgccctt 1440 gaaaaattca ctgaactgaa aaccatctgg ataagcctgg aggcctga 1488

Claims

1. An engineered bacterium capable of degrading alcohol, characterized in that: The engineered bacteria are modified adhp gene fragments and puuc gene fragments in the host strain.

2. The engineered bacteria according to claim 1, characterized in that: The host strain is Escherichia coli Nissle 1917; The adhp gene fragment and the puuc gene fragment are derived from the adhp gene and the puuc gene of Escherichia coli K12; wherein the nucleotide sequence of the adhp gene fragment is the nucleotide sequence shown in SEQ ID NO.1; and the nucleotide sequence of the puuc gene fragment is the nucleotide sequence shown in SEQ ID NO.

2.

3. The engineered bacteria according to claim 1 or 2, characterized in that: The engineering bacteria contains an arabinose-inducible promoter.

4. A method for preparing the genetically engineered bacteria according to claim 1, characterized in that: The recombinant plasmid capable of overexpressing adhp and puuc genes under the control of an arabinose-inducible promoter was transformed into Escherichia coli Nissle 1917 by electroporation, and the genetically engineered bacteria was named ECN@ALDH.

5. An application of the engineered bacteria according to claim 1, characterized in that: The genetically engineered bacteria are used in preparing medicines or foods for preventing and treating alcoholic liver disease.

6. A preparation having the function of degrading alcohol, characterized in that: The preparation comprises the preparation according to claim 1.

7. The preparation according to claim 6, characterized in that: The preparation is a biocompatible material coated on the surface of genetically engineered bacteria, wherein the biocompatible material is chitosan and sodium alginate.

8. The preparation according to claim 7, characterized in that: The genetically engineered bacteria described in claim 1 electrostatically adsorb chitosan and sodium alginate in sequence through electrostatic layer-by-layer self-assembly technology to form protection, namely ECN@ALDH(C / A)2.

9. The preparation according to claim 6, characterized in that: The preparation is in the form of powder, granules, tablets or capsules.

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