CN112210519A - Genetically engineered bacterium for secreting acetaldehyde dehydrogenase by using edible fungi - Google Patents

Genetically engineered bacterium for secreting acetaldehyde dehydrogenase by using edible fungi Download PDF

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Publication number
CN112210519A
CN112210519A CN201910613094.2A CN201910613094A CN112210519A CN 112210519 A CN112210519 A CN 112210519A CN 201910613094 A CN201910613094 A CN 201910613094A CN 112210519 A CN112210519 A CN 112210519A
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acetaldehyde dehydrogenase
genetically engineered
engineered bacterium
ala
acetaldehyde
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韩倩
李明
莫翠萍
方崇洲
周华涛
周海
周光前
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Shenlun Biotech Shenzhen Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/99—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with other acceptors (1.2.99)
    • C12Y102/99003—Aldehyde dehydrogenase (pyrroloquinoline-quinone) (1.2.99.3)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention discloses a genetic engineering bacterium for obligately secreting acetaldehyde dehydrogenase, which is obtained by transferring plasmids for expressing the acetaldehyde dehydrogenase into host lactococcus lactis in a mode of expressing human proteins by the lactococcus lactis. The genetic engineering bacteria provided by the invention can express acetaldehyde dehydrogenase and secrete the acetaldehyde dehydrogenase to the outside of the cells of lactococcus lactis, and the acetaldehyde dehydrogenase can catalyze acetaldehyde into acetic acid. The strains, plasmids and reagents used in the invention are all food-grade materials, can be used in food fermentation processing industry, can enhance the effective metabolism of alcohol in human body, and has a far-reaching application prospect in the aspects of antialcoholism food and health care products.

Description

Genetically engineered bacterium for secreting acetaldehyde dehydrogenase by using edible fungi
Technical Field
The invention relates to the field of gene recombination and enzyme engineering, in particular to application of acetaldehyde-secreting dehydrogenase gene engineering bacteria in antialcoholism foods and health-care products.
Background
Ingestion of large amounts of alcohol causes a shift in nervous system excitation to high inhibition, severely disrupting the normal functioning of the human nervous system. Alcohol metabolism in humans depends mainly on the catalytic metabolism of Alcohol Dehydrogenase (ADH) and Acetaldehyde dehydrogenase (ALDH) in the liver. After the alcohol is taken into a human body, the alcohol is quickly absorbed into the blood through mucous membranes at various parts of the oral cavity, the esophagus, the stomach and the intestinal tract, and circulates to the liver for metabolism. When the human body can sufficiently express the two dehydrogenases, ethanol is rapidly oxidized into acetaldehyde, then the acetaldehyde is rapidly oxidized into acetic acid, and then the acetaldehyde is metabolized and discharged, so that the damage of the central nerve caused by the alcohol is reduced.
In general, human alcohol dehydrogenase and acetaldehyde dehydrogenase can be sufficiently expressed, and in the enzyme translated from the allele of single base mutation of acetaldehyde dehydrogenase, the glutamic acid at residue 487 is changed to lysine, resulting in substantial loss of catalytic activity. Such single base mutation of acetaldehyde dehydrogenase occurs basically in asian genes, so that acetaldehyde which has been produced by metabolism is not metabolized into acetic acid in time, resulting in acetaldehyde poisoning called "alcoholism". In recent years, acetaldehyde dehydrogenase has been successfully expressed, but because acetaldehyde dehydrogenase is not metabolized through blood into liver by taking acetaldehyde dehydrogenase preparation, it is desirable to design a microbial mixture of food grade alcohol dehydrogenase and acetaldehyde dehydrogenase so that alcohol is metabolized in gastrointestinal tract in large amount to reduce damage of alcohol to liver and central nerve. Therefore, the food industrialization of the acetaldehyde dehydrogenase product has important application value.
Disclosure of Invention
Therefore, the first purpose of the invention is to provide a lactococcus lactis gene engineering strain with high acetaldehyde dehydrogenase yield obtained by gene engineering technologyLactococcus lactisNZ3900-pNZ8149-sig-ALDH2 with the preservation number of CCTCC No: m2019091, the preservation date is 2019, 1 month and 29 days, the preservation unit is China center for type culture Collection, and the address is Wuhan university Collection No. 299 in the Wuhan district, Wuhan city, Hubei province.
The second purpose of the invention is to provide a food-grade genetically engineered bacterium for enhancing alcohol metabolism in human gastrointestinal tracts, which is to provide a genetically engineered bacterium for coding secretory acetaldehyde dehydrogenase, wherein the nucleotide sequence of the secretory acetaldehyde dehydrogenase is shown in SEQ ID NO. 1.
The third purpose of the invention is to provide a vector and a host cell line carrying the gene.
The fourth purpose of the invention is to provide a lactococcus lactis genetically engineered bacterium, which is lactococcus lactis for expressing the secretory acetaldehyde dehydrogenase gene.
In one embodiment of the present invention, the lactococcus lactis is lactococcus lactisLactococcus lactisNZ3900 is used as a host, and pNZ8149 is used as an expression vector to express a gene of secretory acetaldehyde dehydrogenase.
The fifth purpose of the invention is to provide a production method of the acetaldehyde dehydrogenase gene engineering bacteria, which is to inoculate the engineering bacteria into M17 broth culture medium and culture the bacteria for 8 to 36 hours at 25 to 37 ℃.
In one embodiment of the present invention, nisin (nisin) is added to the fermentation medium at a final concentration of 0.1-100ng/ml to induce the promoter to start the expression of acetaldehyde dehydrogenase.
In one embodiment of the present invention, the fermentation medium should be supplemented with Zn2+A zinc gluconate solution at a final concentration of 0.05-1g/ml to provide the zinc ions required for acetaldehyde dehydrogenase activity.
In one embodiment of the present invention, oxidized coenzyme I (NAD) is added to the fermentation medium+) The concentration of the hydrogen acceptor (hydride; H) is 0.1-50mmol/L to provide hydrogen acceptor (hydride; H) for acetaldehyde dehydrogenase catalytic reaction-）。
The sixth purpose of the invention is to provide the application of the gene in preparing products containing acetaldehyde dehydrogenase.
In one embodiment of the invention, the product should comprise food and health care products.
The technical effects are as follows: the invention provides an engineering bacterium capable of efficiently expressing and secreting acetaldehyde dehydrogenase genes and a construction method thereof. The invention realizes the mass expression of acetaldehyde dehydrogenase in lactococcus lactis by a molecular biological technology, and can secrete the expressed product acetaldehyde dehydrogenase to the outside of lactic acid bacteria. The bacterial species and plasmids used in the present invention are food grade materials without antibiotic selection, and the inducer nisin inducing the expression of acetaldehyde dehydrogenase is the only bacteriocin allowed to be used as a food additive in the world. Finally, a zinc gluconate solution is added to the lactococcus lactis culture solution capable of secreting acetaldehyde dehydrogenase to provide coenzyme zinc ion (Zn) required by the acetaldehyde dehydrogenase2+) And adding oxidized coenzyme I (NAD) as a hydrogen ion acceptor+) So that the acetaldehyde dehydrogenase has good biological activity. Provides effective theoretical basis and application technology for the research and development of anti-inebriation food and health care products.
Drawings
FIG. 1 shows the construction of acetaldehyde dehydrogenase recombinant plasmid.
FIG. 2 shows the restriction enzyme digestion verification of acetaldehyde dehydrogenase recombinant plasmid pNZ8149-sig-ALDH 2.
FIG. 3 is an SDS-PAGE gel of the protein of the recombinant lactococcus lactis acetaldehyde dehydrogenase.
Detailed Description
The invention provides a genetically engineered bacterium for alcohol metabolism in human gastrointestinal tracts, and the invention is further described in detail below in order to make the purpose and technical scheme of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Artificially synthesizing a secretory acetaldehyde dehydrogenase gene (sig-ALDH 2) shown in SEQ ID NO.1 by using restriction enzymeNcoI andSpecarrying out double enzyme digestion on a target gene fragment and a pNZ8149 vector fragment, and connecting by using T4 ligase to obtain a recombinant plasmid; and (2) electrically transforming the recombinant plasmid into a lactococcus lactis NZ3900 expression strain, screening by using an M17 broth standard culture medium (containing lactose), selecting positive clones, and extracting plasmid enzyme digestion verification (figure 2) or sequencing verification to obtain the genetic engineering bacteria for expressing the secretory acetaldehyde dehydrogenase.
Example 2
The recombinant genetically engineered bacterium obtained in example 1 was inoculated into M17 broth standard medium (solid), and strain activation was performed at 30 ℃. The activated strain was inoculated into M17 broth standard medium (liquid), cultured at 30 ℃ and 180rpm for 8-36 hours to prepare seed culture liquid. Respectively taking 2ml of activated seed liquid, adding the activated seed liquid into 200ml of M17 broth standard culture medium, culturing at 30 ℃ and 180rpm until OD600 is approximately equal to 0.5, adding nisin solution to the final concentration of 25ng/ml, and beginning to induce the recombinant genetically engineered bacteria to express the target protein. The amino acid sequence of the obtained acetaldehyde dehydrogenase is SEQ ID NO. 2.
Example 3
The recombinant genetically engineered bacterium of example 1 was subjected to the measurement of the expression level of the foreign protein, and the SDS-PAGE gel electrophoresis pattern of the protein of acetaldehyde dehydrogenase was shown in fig. 3, using nisin as a control before induction.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the above teachings, and that all such modifications and variations are intended to be within the scope of the invention as defined in the appended claims.
Sequence listing
<110> Shenzhen Biotech (Shenzhen) Limited
<120> a genetically engineered bacterium for secreting acetaldehyde dehydrogenase by using edible fungi
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taatgtctac agtgatactt tctgctgcag ccccgttgtc aggtgtttac gctgcatgca 120
tgttgcgcgc tgccgcccgc ttcgggcccc gcctgggccg ccgcctcttg tcagccgccg 180
ccacccaggc cgtgcctgcc cccaaccagc agcccgaggt cttctgcaac cagattttca 240
taaacaatga atggcacgat gccgtcagca ggaaaacatt ccccaccgtc aatccgtcca 300
ctggagaggt catctgtcag gtagctgaag gggacaagga agatgtggac aaggcagtga 360
aggccgcccg ggccgccttc cagctgggct caccttggcg ccgcatggac gcatcacaca 420
ggggccggct gctgaaccgc ctggccgatc tgatcgagcg ggaccggacc tacctggcgg 480
ccttggagac cctggacaat ggcaagccct atgtcatctc ctacctggtg gatttggaca 540
tggtcctcaa atgtctccgg tattatgccg gctgggctga taagtaccac gggaaaacca 600
tccccattga cggagacttc ttcagctaca cacgccatga acctgtgggg gtgtgcgggc 660
agatcattcc gtggaatttc ccgctcctga tgcaagcatg gaagctgggc ccagccttgg 720
caactggaaa cgtggttgtg atgaaggtag ctgagcagac acccctcacc gccctctatg 780
tggccaacct gatcaaggag gctggctttc cccctggtgt ggtcaacatt gtgcctggat 840
ttggccccac ggctggggcc gccattgcct cccatgagga tgtggacaaa gtggcattca 900
caggctccac tgagattggc cgcgtaatcc aggttgctgc tgggagcagc aacctcaaga 960
gagtgacctt ggagctgggg gggaagagcc ccaacatcat catgtcagat gccgatatgg 1020
attgggccgt ggaacaggcc cacttcgccc tgttcttcaa ccagggccag tgctgctgtg 1080
ccggctcccg gaccttcgtg caggaggaca tctatgatga gtttgtggag cggagcgttg 1140
cccgggccaa gtctcgggtg gtcgggaacc cctttgatag caagaccgag caggggccgc 1200
aggtggatga aactcagttt aagaagatcc tcggctacat caacacgggg aagcaagagg 1260
gggcgaagct gctgtgtggt gggggcattg ctgctgaccg tggttacttc atccagccca 1320
ctgtgtttgg agatgtgcag gatggcatga ccatcgccaa ggaggagata ttcgggccag 1380
tgatgcagat cctgaagttc aagaccatag aggaggttgt tgggagagcc aacaattcca 1440
cgtacgggct ggccgcagct gtcttcacaa aggatttgga caaggccaat tacctgtccc 1500
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cctttggtgg ctacaagatg tcggggagtg gccgggagtt gggcgagtac gggcttcagg 1620
catacactga agtgaaaact gtcacagtca aagtgcctca gaagaactca caccaccacc 1680
accaccacta aactagt 1697
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Thr Ala Gly Ala Ala Ile Ala Ser His Glu Asp Val Asp Lys Val Ala
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Asn Ile Ile Met Ser Asp Ala Asp Met Asp Trp Ala Val Glu Gln Ala
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Arg Thr Phe Val Gln Glu Asp Ile Tyr Asp Glu Phe Val Glu Arg Ser
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Claims (10)

1. A food-grade genetically engineered bacterium for enhancing alcohol metabolism in human gastrointestinal tracts is characterized in that the genetically engineered bacterium isLactococcuslactisNZ3900-pNZ8149-sig-ALDH2, which is preserved in China center for type culture Collection in 29 months 1 in 2019, and the preservation number is CCTCC No: m2019091.
2. The food-grade genetically engineered bacterium for alcohol metabolism according to claim 1, wherein the genetically engineered bacterium is obtained by transferring a gene fragment for expressing secretory acetaldehyde dehydrogenase into a lactococcus lactis NZ3900 strain, and the gene fragment for expressing the secretory acetaldehyde dehydrogenase is a nucleotide sequence in SEQ ID No. 1.
3. The genetically engineered bacterium for alcohol metabolism of claim 2, wherein the nucleotide sequence of SEQ ID NO.1 and the expression vector pNZ8149 are constructed by enzyme cleavage siteNcoI andSpei, connecting to obtain a recombinant expression vector pNZ8149-sig-ALDH2, and transforming the recombinant expression vector into host bacteria to obtain the genetic engineering bacteria for alcohol metabolism.
4. The genetically engineered bacterium for alcohol metabolism according to claim 2, wherein the fragment expressing the secretory acetaldehyde dehydrogenase gene further comprises a Ribosome Binding Site (RBS) and a sequence for expressing a Signal peptide (Signal peptide) for directing acetaldehyde dehydrogenase to the outside of lactococcus lactis cells.
5. A method for producing acetaldehyde dehydrogenase gene engineering bacteria, which is characterized in that the gene engineering bacteria of claim 1 are inoculated into M17 broth culture medium and cultured for 8-36 hours at 25-37 ℃.
6. The method of claim 5, wherein nisin is added to the fermentation medium to induce the promoter to start the expression of acetaldehyde dehydrogenase at a final concentration of 0.1-100 ng/ml.
7. The method according to claim 5, wherein the fermentation medium is supplemented with Zn2+A zinc gluconate solution with a final concentration of 0.05-1g/ml to provide the coenzyme zinc ions required for the activity of the acetaldehyde dehydrogenase.
8. The method of claim 5, wherein oxidized coenzyme I (NAD) is added to the fermentation medium+) The final concentration is 0.1-50mmol/L to provide hydrogen acceptor (hydride: H) for acetaldehyde dehydrogenase catalytic reaction-）。
9. The use of the genetically engineered bacterium of claim 1 in the preparation of a product containing acetaldehyde dehydrogenase.
10. The use of the gene of claim 2 in the preparation of anti-hangover food or health product containing alcohol dehydrogenase and acetaldehyde dehydrogenase.
CN201910613094.2A 2019-07-09 2019-07-09 Genetically engineered bacterium for secreting acetaldehyde dehydrogenase by using edible fungi Pending CN112210519A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113647632A (en) * 2021-08-23 2021-11-16 江苏汉肽生物医药有限公司 Application of lactococcus lactis capable of dispelling effects of alcohol
CN114426942A (en) * 2022-01-25 2022-05-03 中国科学院动物研究所 Recombinant lactococcus lactis, microcapsule and application thereof
CN114891808A (en) * 2022-06-21 2022-08-12 珠海丽凡达生物技术有限公司 mRNA molecule for encoding ALDH2 polypeptide, application and mRNA medicament
WO2023141744A1 (en) * 2022-01-25 2023-08-03 中国科学院动物研究所 Recombinant lactococcus lactis, microcapsule and use thereof
CN114426942B (en) * 2022-01-25 2024-07-09 中国科学院动物研究所 Recombinant lactococcus lactis, microcapsule and application thereof

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WO2017035141A2 (en) * 2015-08-24 2017-03-02 White Dog Labs, Inc. Microbial organisms for converting acetyl-coa into crotyl alcohol and methods for producing crotyl alcohol

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CN101921785A (en) * 2010-07-05 2010-12-22 浙江工业大学 Aldehyde dehydrogenase gene, vector, engineering bacteria and application thereof
WO2017035141A2 (en) * 2015-08-24 2017-03-02 White Dog Labs, Inc. Microbial organisms for converting acetyl-coa into crotyl alcohol and methods for producing crotyl alcohol

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113647632A (en) * 2021-08-23 2021-11-16 江苏汉肽生物医药有限公司 Application of lactococcus lactis capable of dispelling effects of alcohol
CN114426942A (en) * 2022-01-25 2022-05-03 中国科学院动物研究所 Recombinant lactococcus lactis, microcapsule and application thereof
WO2023141744A1 (en) * 2022-01-25 2023-08-03 中国科学院动物研究所 Recombinant lactococcus lactis, microcapsule and use thereof
CN114426942B (en) * 2022-01-25 2024-07-09 中国科学院动物研究所 Recombinant lactococcus lactis, microcapsule and application thereof
CN114891808A (en) * 2022-06-21 2022-08-12 珠海丽凡达生物技术有限公司 mRNA molecule for encoding ALDH2 polypeptide, application and mRNA medicament
CN114891808B (en) * 2022-06-21 2023-10-27 珠海丽凡达生物技术有限公司 mRNA molecule encoding ALDH2 polypeptide, application and mRNA medicament

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