CN114561333B - Engineering bacterium for converting branched chain amino acid and application thereof in preparation of products for treating maple diabetes - Google Patents

Engineering bacterium for converting branched chain amino acid and application thereof in preparation of products for treating maple diabetes Download PDF

Info

Publication number
CN114561333B
CN114561333B CN202210170368.7A CN202210170368A CN114561333B CN 114561333 B CN114561333 B CN 114561333B CN 202210170368 A CN202210170368 A CN 202210170368A CN 114561333 B CN114561333 B CN 114561333B
Authority
CN
China
Prior art keywords
gene
chain amino
branched
amino acid
dehydrogenase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210170368.7A
Other languages
Chinese (zh)
Other versions
CN114561333A (en
Inventor
曹广祥
付加芳
马欣
王杰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong First Medical University and Shandong Academy of Medical Sciences
Original Assignee
Shandong First Medical University and Shandong Academy of Medical Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shandong First Medical University and Shandong Academy of Medical Sciences filed Critical Shandong First Medical University and Shandong Academy of Medical Sciences
Priority to CN202210170368.7A priority Critical patent/CN114561333B/en
Publication of CN114561333A publication Critical patent/CN114561333A/en
Application granted granted Critical
Publication of CN114561333B publication Critical patent/CN114561333B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
    • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
    • A23G3/364—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • A23G3/366—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • 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)
    • 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)
    • C12Y101/0103—3-Hydroxybutyrate dehydrogenase (1.1.1.30)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01009—Leucine dehydrogenase (1.4.1.9)
    • 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
    • A23V2400/11—Lactobacillus
    • A23V2400/113—Acidophilus
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Inorganic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention relates to engineering bacteria for converting branched chain amino acid and application thereof in preparing products for treating maple diabetes. The invention constructs a vector containing genes LeuDH and MorA, and transfers the vector into lactobacillus by homologous recombination. Proved by verification, the recombinant strain can effectively express leucine dehydrogenase and alpha-hydroxy acid dehydrogenase, realize the degradation of branched chain amino acid, and is expected to be used as engineering bacteria for preparing drugs for treating maple diabetes, thereby having important significance for clinical treatment of maple diabetes.

Description

Engineering bacterium for converting branched chain amino acid and application thereof in preparation of products for treating maple diabetes
Technical Field
The invention belongs to the technical field of biological medicines, relates to engineering bacteria for converting branched chain amino acid into alpha-hydroxy acid, a bacterial agent of the engineering bacteria, a pharmaceutical composition and application of the engineering bacteria as a branched chain amino acid degradation agent, and particularly relates to application of the engineering bacteria in preparation of products for treating maple diabetes.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Maple diabetes (MSUD) also known as ketoroluria is recorded in the first few diseases catalog issued in 2018. Maple diabetes is an autosomal recessive genetic disease that results in a blocked catabolism of Branched Chain Amino Acids (BCAAs) due to congenital defects in the branched chain ketoacid dehydrogenase complex. Branched-chain amino acids and corresponding keto acids accumulate in urine and blood of patients. Clinical manifestations mainly invade the nervous system, multiple manifestations of progressive brain damage symptoms, and low intelligence in children.
Branched-chain amino acids are essential amino acids for the human body, including leucine, isoleucine and valine, and MSUD patients need to obtain proper amounts of branched-chain amino acids from foods. The treatment methods of hemodialysis, total intravenous nutrition, insulin, vitamin B1 and the like are adopted for patients with acute metabolic crisis, and the daily treatment scheme of MSUD patients is low protein diet, so that the branched chain amino acid intake of the patients is limited. However, long-term low protein diet therapy affects the growth and mental health of MSUD patients, and patients have poor quality of life.
Both phenylketonuria and MSUD are diseases of amino acid metabolism disorders, and defects in phenylalanine hydroxylase in phenylketonuria patients lead to phenylalanine metabolism disorders. At present, the treatment of phenylketonuria by using engineering probiotics of Escherichia coli has entered clinical experiments, and phenylalanine intake of patients is reduced by constructing engineering probiotics of Escherichia coli which can degrade phenylalanine in food in intestinal tracts of patients. However, no research report on the treatment of MSUD engineering probiotics exists at present.
Disclosure of Invention
Based on the technical background, the invention aims to provide a recombinant probiotic engineering bacterium for treating maple diabetes, wherein leucine dehydrogenase genes and alpha-hydroxy acid dehydrogenase genes are expressed through recombinant strains. Proved by verification, the recombinant strain can be planted at the gastrointestinal part, degrade and convert branched chain amino acids from food protein sources, reduce accumulation of the branched chain amino acids and keto acids thereof in the organism, and is expected to be applied to clinical treatment of maple diabetes.
Therefore, the invention provides the following technical scheme:
in a first aspect of the invention, there is provided an engineered bacterium for converting branched-chain amino acids, the engineered bacterium having been modified to have enhanced leucine dehydrogenase and alpha-hydroxy acid dehydrogenase gene expression compared to a wild-type strain, the starting strain being lactobacillus.
The engineering bacteria can express two enzymes of leucine dehydrogenase and alpha-hydroxy acid dehydrogenase. Wherein, the leucine dehydrogenase degrades branched-chain amino acid into alpha-keto acid, the alpha-hydroxy acid dehydrogenase continuously converts the alpha-keto acid into the alpha-hydroxy acid, and the alpha-hydroxy acid is a nutrient substance which is harmless to human body. The wild microorganism has no two enzymes, can only convert branched-chain amino acid into alpha-keto acid through an amino transfer way, cannot be further converted after being absorbed by human body, and can accumulate in the body of a patient and cause diseases. Therefore, the invention designs the method for converting the branched-chain amino acid which cannot be metabolized originally into the nutrient substance through the cooperation relationship of the two enzymes, thereby realizing the treatment effect.
Considering that the engineering bacteria are applied to the treatment of MSUD, the probiotics are firstly adopted as an initial strain so as to ensure the safety of the engineering bacteria. Lactobacillus belongs to lactic acid bacteria and is an important probiotic. A large number of researches show that the lactobacillus can regulate normal flora of gastrointestinal tract, maintain microecological balance, inhibit growth of putrefying bacteria and pathogenic bacteria in intestinal tract, improve immunity of organism, and the like. The engineering bacteria constructed by using lactobacillus as an initial strain is expected to obtain better biocompatibility.
Further, the starting strain is one of Lactobacillus acidophilus, lactobacillus casei, lactobacillus crispatus, lactobacillus fermentum, lactobacillus gasseri, lactobacillus helveticus, lactobacillus johnsonii, lactobacillus paracasei, lactobacillus plantarum, lactobacillus salivarius, lactobacillus delbrueckii, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus fermentum or Lactobacillus helveticus.
Specifically, the original strain is lactobacillus acidophilus, the lactobacillus acidophilus can be planted in the stomach and the small intestine, the stomach and the small intestine are main sites for digestion and absorption of protein in food, and the lactobacillus acidophilus engineering probiotics can effectively degrade branched-chain amino acids in the food. In one practical implementation mode, the starting strain is lactobacillus acidophilus NCFM, is the most abundant probiotic strain studied at present, mainly exists in the small intestine, can release lactic acid, acetic acid and some antibiotics acting on harmful bacteria, has antagonism on pathogenic microorganisms, is a common strain of the existing dairy fermentation, medicines or probiotic preparations, and has higher safety in application to human bodies.
Preferably, the leucine dehydrogenase (LeuDH) is derived from bacillus cereus, and the leucine dehydrogenase can degrade branched-chain amino acids into alpha-keto acids, so that the leucine dehydrogenase is beneficial to supplement an amino acid transaminase degradation pathway, and the degradation effect of branched-chain amino acids in intestinal tracts is improved. Specific examples are Bacillus cereus DSM 626, and the leucine dehydrogenase isolated from the strain has an optimal reaction temperature of 37 ℃, is close to the temperature of a human body, has a half-life of 330 hours in an environment of 30 ℃, and maintains more than 80% of the original enzyme activity after being stored in a buffer solution with pH of 7.0-8.0 for 24 hours. The characteristics determine that the leucine dehydrogenase used by the invention is hopefully well adapted to the intestinal environment of a human body. Furthermore, the invention carries out codon optimization on leucine dehydrogenase (LeuDH) in wild Bacillus cereus (DSM 626), and the optimized sequence is shown as SEQ ID NO. 1.
Preferably, the alpha-hydroxy acid dehydrogenase gene (MorA) is derived from Aspergillus oryzae, in particular Aspergillus oryzae (Aspergillus Oryzae) RIB40. The alpha-hydroxy acid dehydrogenase can continuously convert the alpha-keto acid into the alpha-hydroxy acid which is a nutrient substance beneficial to human bodies. The invention also carries out codon optimization on an alpha-hydroxy acid dehydrogenase gene (MorA) in the wild aspergillus oryzae (Aspergillus Oryzae) RIB40, and the optimized sequence is shown as SEQ ID NO. 2.
In one embodiment of the above preferred embodiment, the engineering bacterium has both leucine dehydrogenase and α -hydroxy acid dehydrogenase gene expression, and the modification includes introducing exogenous active gene, and the copy number of the exogenous active gene may be adjusted according to the expression effect of the recombinant strain. The modification means comprises ligating the gene of interest into a vector which is a construct comprising the nucleotide sequence of the gene of interest and which is capable of expression in lactobacillus, and integrating it into the host cell genome; further, the vector is one of natural or recombinant plasmid, cosmid, virus and phage. In one embodiment of the invention where validation is feasible, the vector is a recombinant plasmid.
In addition, the vector insertion into the chromosome may be performed by any method known in the art, for example, homologous recombination. In a specific embodiment, the construction method of the engineering bacterium for producing dehydrogenase is as follows:
(1) Placing the gene LeuDH and the gene MorA at the downstream of a lactobacillus acidophilus promoter P32, and synthesizing the promoter P32, the gene LeuDH and the gene MorA by adopting a chemical synthesis method;
(2) Ligating the above gene fragment to a pORI28 vector;
(3) The lactobacillus acidophilus NCFM is taken as an original strain, and the gene LeuDH and the gene MorA are integrated on a genome by a homologous recombination method to obtain an integrated strain of the gene LeuDH and the gene MorA.
The gene LeuDH and the gene MorA integrated strain are named as engineering probiotics MS312; the engineering bacteria have the following physiological characteristics: gram positive bacillus, the end of the thallus is round. Rough, raised, edge curled colonies formed on the solid plates. Metabolism produces lactic acid and acetic acid.
The optimal culture mode of the engineering bacteria is as follows: MRS liquid culture medium, anaerobic culture at 35-39 deg.C, shaking culture at 150-250 rpm; a further preferred mode of cultivation is: anaerobic, 200 rpm shaking culture at 37 ℃.
In a second aspect, the present invention provides a microbial inoculum, which is a culture or metabolite of the leucine dehydrogenase and alpha-hydroxy acid dehydrogenase producing engineering bacteria of the first aspect.
In a third aspect, the invention provides the use of the engineering bacterium of the first aspect and/or the microbial inoculum of the second aspect as a branched-chain amino acid degrading agent.
Proved by verification, the engineering bacteria can stably express leucine dehydrogenase and alpha-hydroxy acid dehydrogenase under the in vitro culture condition, realize the degradation of branched chain amino acids (leucine, isoleucine and valine), and can be used as a branched chain amino acid degradation agent.
In a specific embodiment, the leucine dehydrogenase and the alpha-hydroxy acid dehydrogenase produced by the engineering bacteria can be used for decomposing branched-chain amino acids of food sources in intestinal tracts, so that accumulation conditions of the branched-chain amino acids and keto acids in urine and blood of MSUD patients are improved, namely, the leucine dehydrogenase and the alpha-hydroxy acid dehydrogenase are used for treating and improving maple diabetes. Namely the application of the product as a branched chain amino acid degradation agent, and further, the product is used for preparing a product for treating maple diabetes, and the product comprises, but is not limited to, medicaments and health-care foods; the health food includes, but is not limited to, special medical foods, candies, drinks, etc.
In a fourth aspect, the present invention provides a pharmaceutical composition, wherein the engineering bacterium of the first aspect or the microbial inoculum of the second aspect is used as an active ingredient.
In a fifth aspect, the present invention provides a pharmaceutical composition for treating maple syrup urine disease, wherein the pharmaceutical composition comprises the engineering bacterium of the first aspect, the microbial inoculum of the second aspect and/or the pharmaceutical composition of the third aspect.
Preferably, the therapeutic drug further comprises pharmaceutically necessary auxiliary materials.
Preferably, the therapeutic agent is a solid formulation or a liquid formulation, the solid formulation is one of the formulations including, but not limited to, tablets, pills, capsules, powders, microcapsules; the liquid preparation is solution, suspension or emulsion; further preferably, the therapeutic agent is a solid preparation, which is a powder or a tablet.
The beneficial effects of the above technical scheme are:
(1) The invention adopts lactobacillus to construct engineering probiotics for treating maple diabetes for the first time, utilizes the characteristics of lactobacillus acidophilus which is mainly planted in the stomach and small intestine of protein digestion and amino acid absorption sites to effectively degrade and transform branched chain amino acids in food, and reduces the intake of the branched chain amino acids of patients.
(2) The engineering probiotics provided by the invention are beneficial to treating maple diabetes patients, improve the life quality of the patients, and have important clinical treatment value and social benefit.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a graph showing the effect of the engineered probiotic MS312 described in example 2 on the concentration of branched-chain amino acids in the blood of MSUD mice.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments. The starting strain lactobacillus acidophilus NCFM used in the examples below was purchased commercially.
Example 1
1. Engineering probiotic MS312 construction
Integrating leucine dehydrogenase gene LeuDH and alpha-hydroxy acid dehydrogenase gene MorA on genome by using lactobacillus acidophilus NCFM original strain, comprising the following steps:
in this example, total gene synthesis and sequencing were performed by Shanghai Biotechnology Inc. Molecular biology experiments include competent cell preparation, electrotransformation, etc. as described in the guidelines for molecular cloning experiments (third edition).
A gene fragment MSUD1 containing a promoter P32 (SEQ ID NO. 3), a leucine dehydrogenase gene LeuDH (SEQ ID NO. 1) and an alpha-hydroxy acid dehydrogenase gene MorA (SEQ ID NO. 2) is designed, a DNA fragment is obtained by a total gene synthesis method and constructed into a cloning vector pUC57, and a pUC57-MSUD1 vector is obtained.
The upstream homology arm L-arm is obtained by PCR amplification by taking the NCFM genome of lactobacillus acidophilus as a template and L-For and L-Rev as primers. The NCFM genome is used as a template, R-For and R-Rev are used as primers, and a downstream homology arm R-arm is obtained through PCR amplification. The pUC57-MSUD1 vector is used as a template, MSUD1-For and MSUD1-Rev are used as primers, and the MSUD1 fragment is obtained through PCR amplification. The L-arm, R-arm and MSUD1 fragments were cloned into the pORI28 vector using the recombinase method to obtain the pORI28-MSUD1 vector.
The pORI28-MSUD1 vector and the pTRK669 auxiliary plasmid are transferred into NCFM strain together through electric shock transformation, and the Val-For and Val-Rev primers are used For PCR screening, so as to obtain recombinant strain with LeuDH and MorA genes integrated on genome. Then the plasmid is eliminated by continuous passage at 42 ℃ and on a medium without antibiotics, and finally the engineering bacteria MS312 is obtained.
TABLE 1 primer list
2. Engineering probiotic MS312 degrading branched-chain amino acid activity
Inoculating NCFM original strain and MS312 engineering bacteria into MRS broth culture medium, respectively, culturing at 37deg.C and 250rpm to OD 600 Cells were collected by centrifugation and washed 2 times with PBS buffer =1.0. Finally, the mixture is suspended and diluted to OD by PBS buffer solution containing 0.5 percent of glucose, 40mg/L of leucine, isoleucine or valine 600 Culture at 37 ℃ at 250rpm, sampling at 0, 2, 4 and 8 hours, respectively, and centrifugation to collect supernatant samples, and concentration of leucine, isoleucine or valine was measured. Each treatment was repeated 3 times and the results averaged and the test results are shown in table 2. Statistical analysis shows that the decrease rate of the branched chain amino acid concentration in the MS312 engineering bacteria treated sample is obviously higher than that of NCFM control treatment,the MS312 engineering bacteria have better capability of degrading branched chain amino acid.
TABLE 2 average concentration of branched-chain amino acids under probiotic treatment conditions (mg/L)
EXAMPLE 2 Effect of engineering probiotic MS312 on maple syrup urine mice
A maple diabetes mouse model was purchased from biotechnology company and has a genotype of C57BL/6J-Bckdha em1 . Under normal feeding conditions, the model mice had typical symptoms of maple diabetes. The experiment is carried out by adopting C57BL/6J-Bckdha with the age of 6 weeks em1 Mice were fed with unbranched amino acid feed and were fed with 100 μl of purified water containing leucine, isoleucine and valine as branched amino acid feed at regular daily intervals. After the animal experiment is started, the mice are divided into 3 groups, and the mice are filled with 200 mu L of physiological saline, NCFM probiotics or MS312 engineering probiotics respectively at regular time and 2 times per day, and the dosage of each probiotic is about 1 x 10 10 cfu/mouse. The probiotics were fed for 14 days, sampled and urine collected, and the branched chain amino acid concentration in the blood was checked (fig. 1). The results show that after oral administration of MS312 engineered probiotics, C57BL/6J-Bckdha em1 Compared with the oral normal saline and NCFM probiotics, the concentration of the branched chain amino acid in the blood of the mice is obviously reduced, which proves that the MS312 engineering probiotics can degrade and convert the branched chain amino acid in food, and reduce the intake of the branched chain amino acid of the mice.
In conclusion, the lactobacillus acidophilus engineering probiotic MS312 constructed by the invention can degrade and transform branched chain amino acids in food, obviously reduce the concentration of the branched chain amino acids in blood of a maple diabetes gene mouse, and is expected to be applied to treating maple diabetes.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Shandong university of first medical science (Shandong province medical academy of sciences)
<120> an engineering bacterium for transforming branched-chain amino acid and application thereof in preparing products for treating maple diabetes
<130> 2010
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 1101
<212> DNA
<213> artificial sequence
<400> 1
atgacattag aaatcttcga atacttagaa aaatacgact acgagcaagt agtattctgc 60
caagacaaag aatctggctt aaaagcaatc atcgcaatcc atgacacaac acttggaccg 120
gcacttggcg gaacaagaat gtggacatac gactctgaag aagcagcaat cgaagacgca 180
ttgcgtcttg caaaagggat gacatacaaa aacgcagcag caggattaaa cttagggggc 240
gcaaaaacag taatcatcgg cgacccacgt aaagacaaga gcgaagcaat gttccgtgca 300
ctgggacgtt acatccaagg actgaacgga cgttacatca cagcagaaga cgtaggaaca 360
acagtagacg acatggacat catccatgaa gaaactgact tcgtaacagg catctcacca 420
tcattcggct cctcaggaaa cccatcgccg gtaactgcat acggggtata ccgtggcatg 480
aaagcagcag caaaagaagc attcggcact gacaacttag aaggaaaagt aatcgcagta 540
caaggcgtag gaaacgtagc ataccaccta tgcaaacatt tacacgcaga aggagcaaaa 600
ttaatcgtaa cagacatcaa caaagaagca gtacaacgtg cagtagaaga attcggcgca 660
tcagcagtag aaccaaacga aatctacggg gtagaatgcg acatctacgc accatgcgca 720
ctgggcgcaa cagtaaacga cgaaactatc ccacaactta aagcaaaagt aatcgcagga 780
tccgcaaaca accaattaaa agaagaccgt catggcgaca tcatccatga aatgggcatc 840
gtatacgcac cagactacgt aatcaacgca ggcggcgtaa tcaacgtagc agacgaatta 900
tacggataca acagagaacg tgcactgaaa cgtgtagagt caatctacga cacgatcgca 960
aaagtaatcg aaatctcaaa acgtgacggc atcgcaactt acgtagcagc agaccgtctt 1020
gcagaagagc gtatcgcaag cttgaagaac tcgcgtagca cttacttacg taacggccac 1080
gacatcatca gccgtcgtta g 1101
<210> 2
<211> 990
<212> DNA
<213> artificial sequence
<400> 2
atgccgagcg cactgctgat cggcgaaatc acccacgcac gtaaagaatg ggaagaactg 60
agcagcatcc tgaccctgac cgaattcccg agcggcaccc gtgaagactt catccgtaac 120
tgcaaagaag gccagtacga cgacgtactg gtaatctacc gtagcaacac cagcaccaaa 180
ttcaccggcc cgttcgacgc agaactgctg gcagtactgc cgaaaagcct gaaatacatc 240
tgccacaacg gcgcaggcta cgacaacatc gacgtaaaag gctgcaccga caaaggcatc 300
gcagtaagca gcaccccggt agcagtaaac cacgcaaccg cagacgtagg catcttcctg 360
atgatcggcg cactgcgtca ggcatacgta ccgctgagcg cactgcgtgc aggccagtgg 420
cagggccaga ccaccctggg ccgtgacccg cagggcaaag tactgggcat cctgggcatg 480
ggcggcatcg gccgtgaaat ggcaaaccgt gcaaaagcat tcggcatgaa aatccagtac 540
cacaaccgta gccgtctgag cccggaactg gaaggcgacg caacctacgt aagcttcgac 600
gaactgctgg caagcagcga cgtactgagc ctgaacctgg cactgaacgc aagcacccgt 660
cacatcatcg gcgaaaaaga attccagaaa atgaaagacg gcatcgtaat cgtaaacacc 720
gcacgtggcg cactgatcga cgaaaaagca ctggtagcag cactggacag cggcaaagta 780
ctgagcgcag gcctggacgt atacgaaaac gaaccggtag tagaacaggg cctggtaaac 840
aacccgaaag taatgctgct gccgcacatc ggcaccatga cctacgaaac ccagaaagac 900
atggaactgc tggtactgaa caacctgcgt agcgcagtag aaaaaggcaa aatgatcacc 960
ctggtaccgg aacagaaaaa cgtattctag 990
<210> 3
<211> 179
<212> DNA
<213> artificial sequence
<400> 3
tcgaattcgg tcctcgggat atgataagat taatagtttt agctattaat ctttttttat 60
ttttatttaa gaatggctta ataaagcggt tactttggat ttttgtgagc ttggactaga 120
aaaaaacttc acaaaatgct atactaggta ggtaaaaaaa tattcggagg aattttgaa 179
<210> 4
<211> 26
<212> DNA
<213> artificial sequence
<400> 4
tctagacttt gtttctaact ctccag 26
<210> 5
<211> 26
<212> DNA
<213> artificial sequence
<400> 5
ctggagagtt agaaacaaag tctaga 26
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> 6
tcaggcaaga ttattaaacc 20
<210> 7
<211> 26
<212> DNA
<213> artificial sequence
<400> 7
agatctgaaa tcaatttctt aattga 26
<210> 8
<211> 45
<212> DNA
<213> artificial sequence
<400> 8
tctagacttt gtttctaact ctccagtcga attcggtcct cggga 45
<210> 9
<211> 41
<212> DNA
<213> artificial sequence
<400> 9
gaacagaaaa acgtattcta gagatctgaa atcaatttct t 41
<210> 10
<211> 21
<212> DNA
<213> artificial sequence
<400> 10
gagtcaatct acgacacgat c 21
<210> 11
<211> 21
<212> DNA
<213> artificial sequence
<400> 11
tgcgctcagc ggtacgtatg c 21

Claims (11)

1. An engineered bacterium for converting branched-chain amino acids, wherein the engineered bacterium has an original strain of lactobacillus acidophilus NCFM modified to have enhanced leucine dehydrogenase and α -hydroxy acid dehydrogenase gene expression compared to a wild strain;
the leucine dehydrogenase is derived from bacillus cereusBacillus cereus）DSM 626；
The leucine dehydrogenase is subjected to codon optimization, and the optimized sequence is shown as SEQ ID NO. 1;
the alpha-hydroxy acid dehydrogenase gene is derived from Aspergillus oryzae;
is aspergillus oryzaeAspergillus Oryzae）RIB40；
The alpha-hydroxy acid dehydrogenase gene is subjected to codon optimization, and the optimized sequence is shown as SEQ ID NO. 2.
2. The engineered bacterium for converting branched-chain amino acids according to claim 1, wherein the engineered bacterium has expression of leucine dehydrogenase and α -hydroxy acid dehydrogenase genes simultaneously.
3. The engineering bacterium for converting branched-chain amino acids according to claim 2, wherein the construction mode of the engineering bacterium for converting branched-chain amino acids is as follows:
(1) Placing the gene LeuDH and the gene MorA at the downstream of a lactobacillus acidophilus promoter P32, and synthesizing the promoter P32, the gene LeuDH and the gene MorA by adopting a chemical synthesis method;
(2) Ligating the above gene fragment to a pORI28 vector;
(3) The lactobacillus acidophilus NCFM is taken as an original strain, and the gene LeuDH and the gene MorA are integrated on a genome by a homologous recombination method to obtain an integrated strain of the gene LeuDH and the gene MorA.
4. A bacterial agent, characterized in that the bacterial agent is a culture or metabolite of the branched-chain amino acid-converting engineering bacterium according to any one of claims 1 to 3.
5. Use of the engineering bacterium according to any one of claims 1 to 3 and/or the microbial inoculum according to claim 4 as branched-chain amino acid degradation agent.
6. The use according to claim 5, wherein the use as a branched-chain amino acid degradation agent is for the preparation of a product against maple syrup urine disease.
7. The use according to claim 6, wherein the product comprises a pharmaceutical, a health food; the health food comprises special medical food, candy or beverage.
8. A pharmaceutical composition, characterized in that the engineering bacterium according to any one of claims 1 to 3 or the microbial inoculum according to claim 4 is used as an active ingredient in the pharmaceutical composition.
9. A maple syrup urine treatment drug, which comprises the engineering bacterium according to any one of claims 1 to 3, the microbial inoculum according to claim 4 and/or the pharmaceutical composition according to claim 8.
10. The maple syrup urine disease treatment drug according to claim 9, wherein the treatment drug further comprises pharmaceutically necessary auxiliary materials;
or the therapeutic drug is a solid preparation or a liquid preparation, and the solid preparation is one of a tablet, a pill, a capsule, a powder and a microcapsule; the liquid preparation is solution, suspension or emulsion.
11. The maple syrup urine disease treatment drug according to claim 10, wherein the treatment drug is a solid preparation, and is a powder or a tablet.
CN202210170368.7A 2022-02-23 2022-02-23 Engineering bacterium for converting branched chain amino acid and application thereof in preparation of products for treating maple diabetes Active CN114561333B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210170368.7A CN114561333B (en) 2022-02-23 2022-02-23 Engineering bacterium for converting branched chain amino acid and application thereof in preparation of products for treating maple diabetes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210170368.7A CN114561333B (en) 2022-02-23 2022-02-23 Engineering bacterium for converting branched chain amino acid and application thereof in preparation of products for treating maple diabetes

Publications (2)

Publication Number Publication Date
CN114561333A CN114561333A (en) 2022-05-31
CN114561333B true CN114561333B (en) 2023-08-25

Family

ID=81714590

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210170368.7A Active CN114561333B (en) 2022-02-23 2022-02-23 Engineering bacterium for converting branched chain amino acid and application thereof in preparation of products for treating maple diabetes

Country Status (1)

Country Link
CN (1) CN114561333B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114836357B (en) * 2022-06-08 2023-07-21 华南农业大学 Lactobacillus reuteri strain LN0214 for reducing serum branched-chain amino acid level and derivative product and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008029920A1 (en) * 2006-09-08 2008-03-13 Toyama Prefecture Method for analysis of l-methionine in biological sample using function-modified phenylalanine dehydrogenase
WO2012169819A2 (en) * 2011-06-08 2012-12-13 한국과학기술원 Method for producing polyhydroxyalkanoate containing 2-hydroxybutyrate as the monomer
CN114008209A (en) * 2019-04-12 2022-02-01 马萨诸塞大学 AAV-mediated Maple Syrup Urine Disease (MSUD) gene therapy

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016201380A1 (en) * 2015-06-10 2016-12-15 Synlogic, Inc. Bacteria engineered to treat disorders involving the catabolism of a branched chain amino acid

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008029920A1 (en) * 2006-09-08 2008-03-13 Toyama Prefecture Method for analysis of l-methionine in biological sample using function-modified phenylalanine dehydrogenase
WO2012169819A2 (en) * 2011-06-08 2012-12-13 한국과학기술원 Method for producing polyhydroxyalkanoate containing 2-hydroxybutyrate as the monomer
CN114008209A (en) * 2019-04-12 2022-02-01 马萨诸塞大学 AAV-mediated Maple Syrup Urine Disease (MSUD) gene therapy

Also Published As

Publication number Publication date
CN114561333A (en) 2022-05-31

Similar Documents

Publication Publication Date Title
CN109415683B (en) Novel bacterial species
US11384123B2 (en) Mucoadhesive microorganism
JP6998892B2 (en) How to make a bacterial composition
CN110747157B (en) Engineering probiotics capable of degrading uric acid in intestinal tract and preparation method and application thereof
JP2020528264A (en) Methods for Producing Bacterial Compositions Containing Biofilms with Beneficial Bacteria
CN115181710B (en) Lactobacillus salivarius B12WU and application thereof
WO2022148362A1 (en) Engineering probiotic having surface display phenylalanine ammonia-lyase
CN114561333B (en) Engineering bacterium for converting branched chain amino acid and application thereof in preparation of products for treating maple diabetes
CN112662606A (en) Engineering probiotics for treating phenylketonuria
CN110747218A (en) Production method and application of special dietary protein
CA2493644C (en) Composition for promoting the proliferation of lactobacillus casei subsp. casei
US20240175039A1 (en) Engineering probiotic for degrading uric acid, and construction method therefor and use thereof
CN113151070A (en) Lactobacillus fermentum capable of improving relative abundance of Guttiferae in intestinal tract
CN113969292B (en) Engineering probiotics for treating phenylketonuria and construction method and application thereof
CN113473998A (en) Lactococcus lactis strains for the prevention and/or treatment of visceral pain
WO2023044479A1 (en) Methods for reducing hyperphenylalaninemia
CN115851542A (en) Bacillus coagulans strain taking D-psicose as carbon source and derivative product and application thereof
CN114774335A (en) Bifidobacterium longum 070103 with effects of targeting glucokinase and remarkably reducing blood sugar and blood fat and application thereof
KR101444216B1 (en) Probiotic Lactobacillus brevis JSB22 Having Extracellular Polysaccharide Producing Actibity
US20220047654A1 (en) Pharmabiotic treatments for metabolic disorders
US20230172997A1 (en) Recombinant bacteria for production of indole-3-acetic acid (iaa) and uses thereof
US20220031771A1 (en) Microencapsulated and chromosome integrated compositions for l-dopa microbiome therapy
WO2023165521A1 (en) Engineered microorganism for treating hyperphenylalaninemia and use thereof
US20240026310A1 (en) Microorganisms converting inositol to butyrate
US20240024376A1 (en) Microorganisms synthesizing anti-inflammatory molecules

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant