CN112175890A - Genetically engineered bacterium for secreting alcohol dehydrogenase by using edible fungi - Google Patents

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

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Publication number
CN112175890A
CN112175890A CN201910588556.XA CN201910588556A CN112175890A CN 112175890 A CN112175890 A CN 112175890A CN 201910588556 A CN201910588556 A CN 201910588556A CN 112175890 A CN112175890 A CN 112175890A
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alcohol dehydrogenase
genetically engineered
dehydrogenase
engineered bacterium
alcohol
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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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • 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
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/231—Lactis

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Abstract

The invention discloses a genetic engineering bacterium for obligately secreting alcohol dehydrogenase, which is obtained by transferring plasmids for expressing alcohol dehydrogenase into host lactococcus lactis in a mode of expressing human proteins by the lactococcus lactis. The gene engineering bacteria provided by the invention can express ethanol dehydrogenase and secrete the ethanol dehydrogenase to the outside of cells of lactococcus lactis, and the ethanol dehydrogenase can catalyze ethanol into acetaldehyde. 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 antialcoholic food and antialcoholic health-care products.

Description

Genetically engineered bacterium for secreting alcohol dehydrogenase by using edible fungi
Technical Field
The invention relates to the field of gene recombination and enzyme engineering, in particular to application of an ethanol dehydrogenase secretion gene engineering bacterium in the aspects of antialcoholism food, health-care products and the like.
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 fully express the two dehydrogenases, ethanol is rapidly oxidized into acetaldehyde, then the acetaldehyde is rapidly oxidized into acetic acid, and then the acetic acid is metabolized and discharged out of the human body, so that the damage of the alcohol to the central nerve is reduced.
Generally, human alcohol dehydrogenase and acetaldehyde dehydrogenase can be fully expressed, and in the enzyme translated from the allele of single base mutation of acetaldehyde dehydrogenase, the glutamic acid at residue 487 is changed into lysine, so that the catalytic activity is basically lost, and the single base mutation of acetaldehyde dehydrogenase is basically appeared in the gene of Asian, so that the acetaldehyde which is produced by metabolism can not be metabolized into acetic acid in time, and the acetaldehyde poisoning called 'alcoholism' is caused. In recent years, acetaldehyde dehydrogenase has been successfully expressed, but since acetaldehyde dehydrogenase is not metabolized into the liver through blood when acetaldehyde dehydrogenase is taken, it is desirable to design a mixed microbial preparation of food grade alcohol dehydrogenase and acetaldehyde dehydrogenase so that alcohol is metabolized in the gastrointestinal tract in a large amount and the influence of alcohol on the liver and central nerve is reduced. Therefore, the food industrialization of the alcohol dehydrogenase product has important application value.
Disclosure of Invention
Therefore, the first object of the present invention is to provide a genetically engineered strain of lactococcus lactis with high ethanol dehydrogenase yield, which is obtained by genetic engineering techniques: lactococcus lactis (Lactococcus lactis) NZ3900_ pNZ8149-sig-ADH1A with the preservation number of CCTCC No: m2019090, 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 alcohol dehydrogenase, wherein the nucleotide sequence of the secretory alcohol 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 gene engineering bacterium, which is lactococcus lactis for expressing the secretory ethanol dehydrogenase gene.
In one embodiment of the invention, the Lactococcus lactis NZ3900 is used as a host, and the pNZ8149 is used as an expression vector to express a gene of secretory alcohol dehydrogenase.
The fifth purpose of the invention is to provide a production method of the gene engineering bacteria for producing the ethanol dehydrogenase, 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 expression of alcohol 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 alcohol dehydrogenase activity.
In one embodiment of the present invention, the fermentation medium is supplemented with oxidized coenzyme I (I)NAD+) It is used at a concentration of 0.1 to 50mmol/L to provide a hydrogen acceptor (hydride: H-) required for the reaction catalyzed by alcohol dehydrogenase.
The sixth purpose of the invention is to provide the application of the gene in preparing products containing alcohol 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 a genetically engineered bacterium capable of efficiently expressing and secreting alcohol dehydrogenase and a construction method of the genetically engineered bacterium. The invention realizes the mass expression of the alcohol dehydrogenase in the lactococcus lactis by a molecular biological technology, and can make the expression product alcohol dehydrogenase excreted out 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 alcohol 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 alcohol dehydrogenase to provide coenzyme zinc ion (Zn) required by the alcohol dehydrogenase2+) And adding oxidized coenzyme I (NAD) as a hydrogen ion acceptor+) The alcohol dehydrogenase has good biological activity. The construction of the gene engineering bacteria provides effective theoretical basis and application technology for the research and development of anti-alcoholism food and health care products.
Drawings
FIG. 1 shows the construction of recombinant plasmid for alcohol dehydrogenase.
FIG. 2 shows the restriction enzyme digestion verification of the recombinant plasmid pNZ8149-sig-ADH1A for alcohol dehydrogenase.
FIG. 3 is the SDS-PAGE gel of the recombinant lactococcus lactis alcohol dehydrogenase protein.
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 alcohol dehydrogenase gene (ADH 1A) shown in SEQ ID NO.1 by restriction enzymeNcoI andSpei, performing double enzyme digestion on a target gene fragment and a pNZ8149 vector fragment respectively, and connecting the target gene fragment and the pNZ8149 vector fragment 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 alcohol 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 alcohol 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 chart of the protein of alcohol dehydrogenase was shown in FIG. 3, using nisin before and after induction as a control.
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 alcohol dehydrogenase by using edible fungi
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1271
<212> DNA
<213> Artificaial Sequence
<400> 1
ccatggggag gaaaaattaa aaaagaacag ttatgaaaaa aaagattatc tcagctattt 60
taatgtctac agtgatactt tctgctgcag ccccgttgtc aggtgtttac gctgcatgca 120
tgagcacagc aggaaaagta atcaaatgca aagcagctgt gctatgggag ttaaagaaac 180
ccttttccat tgaggaggtg gaggttgcac ctcctaaggc ccatgaagtt cgtattaaga 240
tggtggctgt aggaatctgt ggcacagatg accacgtggt tagtggtact atggtgaccc 300
cacttcctgt gattttaggc catgaggcag ccggcatcgt ggagagtgtt ggagaagggg 360
tgactacagt caaaccaggt gataaagtca tcccactcgc tattcctcag tgtggaaaat 420
gcagaatttg taaaaacccg gagagcaact actgcttgaa aaacgatgta agcaatcctc 480
aggggaccct gcaggatggc accagcaggt tcacctgcag gaggaagccc atccaccact 540
tccttggcat cagcaccttc tcacagtaca cagtggtgga tgaaaatgca gtagccaaaa 600
ttgatgcagc ctcgccttta gagaaagtct gtctcattgg ctgtggattt tcaactggtt 660
atgggtctgc agtcaatgtt gccaaggtca ccccaggctc tacctgtgct gtgtttggcc 720
tgggaggggt cggcctatct gctattatgg gctgtaaagc agctggggca gccagaatca 780
ttgcggtgga catcaacaag gacaaatttg caaaggccaa agagttgggt gccactgaat 840
gcatcaaccc tcaagactac aagaaaccca tccaggaggt gctaaaggaa atgactgatg 900
gaggtgtgga tttttcattt gaagtcatcg gtcggcttga caccatgatg gcttccctgt 960
tatgttgtca tgaggcatgt ggcacaagtg tcatcgtagg ggtacctcct gattcccaaa 1020
acctctcaat gaaccctatg ctgctactga ctggacgtac ctggaaggga gctattcttg 1080
gtggctttaa aagtaaagaa tgtgtcccaa aacttgtggc tgattttatg gctaagaagt 1140
tttcattgga tgcattaata acccatgttt taccttttga aaaaataaat gaaggatttg 1200
acctgcttca ctctgggaaa agtatccgta ccattctgat gtttcaccac caccaccacc 1260
actgaactag t 1271
<210> 2
<211> 410
<212> PRT
<213> Artificaial Sequence
<400> 2
Met Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile Leu
1 5 10 15
Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Ala Cys Met Ser Thr
20 25 30
Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu Leu Lys
35 40 45
Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys Ala His
50 55 60
Glu Val Arg Ile Lys Met Val Ala Val Gly Ile Cys Gly Thr Asp Asp
65 70 75 80
His Val Val Ser Gly Thr Met Val Thr Pro Leu Pro Val Ile Leu Gly
85 90 95
His Glu Ala Ala Gly Ile Val Glu Ser Val Gly Glu Gly Val Thr Thr
100 105 110
Val Lys Pro Gly Asp Lys Val Ile Pro Leu Ala Ile Pro Gln Cys Gly
115 120 125
Lys Cys Arg Ile Cys Lys Asn Pro Glu Ser Asn Tyr Cys Leu Lys Asn
130 135 140
Asp Val Ser Asn Pro Gln Gly Thr Leu Gln Asp Gly Thr Ser Arg Phe
145 150 155 160
Thr Cys Arg Arg Lys Pro Ile His His Phe Leu Gly Ile Ser Thr Phe
165 170 175
Ser Gln Tyr Thr Val Val Asp Glu Asn Ala Val Ala Lys Ile Asp Ala
180 185 190
Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe Ser Thr
195 200 205
Gly Tyr Gly Ser Ala Val Asn Val Ala Lys Val Thr Pro Gly Ser Thr
210 215 220
Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Ala Ile Met Gly
225 230 235 240
Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Ala Val Asp Ile Asn Lys
245 250 255
Asp Lys Phe Ala Lys Ala Lys Glu Leu Gly Ala Thr Glu Cys Ile Asn
260 265 270
Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Lys Glu Met Thr
275 280 285
Asp Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu Asp Thr
290 295 300
Met Met Ala Ser Leu Leu Cys Cys His Glu Ala Cys Gly Thr Ser Val
305 310 315 320
Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn Pro Met
325 330 335
Leu Leu Leu Thr Gly Arg Thr Trp Lys Gly Ala Ile Leu Gly Gly Phe
340 345 350
Lys Ser Lys Glu Cys Val Pro Lys Leu Val Ala Asp Phe Met Ala Lys
355 360 365
Lys Phe Ser Leu Asp Ala Leu Ile Thr His Val Leu Pro Phe Glu Lys
370 375 380
Ile Asn Glu Gly Phe Asp Leu Leu His Ser Gly Lys Ser Ile Arg Thr
385 390 395 400
Ile Leu Met Phe His His His His His His
405 410

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 isLactococcus lactissubsp, NZ3900_ pNZ8149-sig-ADH1A, deposited in the China center for type culture Collection in 29 months 1 in 2019 with the preservation number of CCTCC No: m2019090.
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 alcohol dehydrogenase into a lactococcus lactis NZ3900 strain, and the gene fragment for expressing the secretory alcohol 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, obtaining a recombinant expression plasmid pNZ8149-sig-ADH1A by connection, and transforming the recombinant expression plasmid 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 segment expressing the secretory alcohol dehydrogenase gene further comprises a Ribosome Binding Site (RBS) and a sequence for expressing a Signal peptide (Signal peptide) for directing alcohol dehydrogenase to the outside of the lactococcus lactis cell.
5. A method for producing an ethanol dehydrogenase-producing genetically engineered bacterium, comprising inoculating the genetically engineered bacterium of claim 1 into M17 broth, and culturing at 25-37 ℃ for 8-36 hours.
6. The method according to claim 5, wherein nisin (nisin) is added to the fermentation medium at a final concentration of 0.1-100ng/ml to induce the promoter to start expression of alcohol dehydrogenase.
7. The method according to claim 5, wherein the fermentation medium is supplemented with Zn2+To a final concentration of 0.05-1g/ml of zinc gluconate solution to provide the coenzyme zinc ions required for the activity of the alcohol 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 alcohol dehydrogenase catalytic reaction-）。
9. The use of the genetically engineered bacterium of claim 1 in the preparation of a product containing alcohol 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.
CN201910588556.XA 2019-07-02 2019-07-02 Genetically engineered bacterium for secreting alcohol dehydrogenase by using edible fungi Pending CN112175890A (en)

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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

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

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