CN114774338B - Probiotics for producing butyric acid, construction method and application thereof - Google Patents

Probiotics for producing butyric acid, construction method and application thereof Download PDF

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CN114774338B
CN114774338B CN202210323936.2A CN202210323936A CN114774338B CN 114774338 B CN114774338 B CN 114774338B CN 202210323936 A CN202210323936 A CN 202210323936A CN 114774338 B CN114774338 B CN 114774338B
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use according
gene
butyric acid
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artificial sequence
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CN114774338A (en
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杨昀
陈国强
巩旭
耿洪伟
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Tsinghua University
Beihang University
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Tsinghua University
Beihang University
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Priority to PCT/CN2023/084163 priority patent/WO2023185760A1/en
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Abstract

The invention provides a probiotics for producing butyric acid, a construction method and application thereof. The probiotics can improve ulcerative colitis, depression, obesity and fatty liver symptoms. Has potential application in the fields of chemical industry, food, medicine, animal feed or cosmetics.

Description

Probiotics for producing butyric acid, construction method and application thereof
Technical Field
The invention relates to the technical field of genetic engineering, in particular to probiotics for producing butyric acid, a construction method thereof and application thereof in the fields of chemical industry, food, medicine, animal feed or cosmetics.
Background
Probiotics are a class of active microorganisms beneficial to a host by colonizing the human body and altering the flora composition of a part of the host. By regulating the immune function of host mucous membrane and system or regulating the balance of flora in intestinal tract, the effect of promoting nutrient absorption and maintaining intestinal health is achieved, so that single microorganism or mixed microorganism with definite composition beneficial to health is produced.
At present, probiotics are widely used in the fields of chemical industry, food, medicine, animal feed or cosmetics. For example, patent CN113968758A discloses the use of probiotics in the manufacture of fertilizers. Patent CN113908251a discloses a yoghurt comprising probiotics. Patent CN114053310A, CN114129707A, CN114107122A, CN113999805a discloses the use of probiotics for reducing blood lipid, anti-allergy, alleviating ventilation and treating hyperuricemia, respectively. Patent CN114027397a discloses a feed additive comprising probiotics. Patent CN113827521a discloses the use of probiotics in the cosmetic field.
Further, diseases are treated by modification of probiotics. For example, patent CN113604493a discloses that a probiotic bacteria highly expressing LDH can effectively inhibit cell death, improve cerebral infarction volume, enhance and improve mucosa integrity, promote tissue morphology improvement, and have therapeutic effects on peptic ulcer, inflammatory bowel disease and cardiovascular and cerebrovascular diseases.
However, the prior art does not disclose the probiotic bacteria engineered in the present application, nor their use in ameliorating ulcerative colitis, depression, obesity and fatty liver symptoms.
Disclosure of Invention
The present inventors have made a careful study of the synthesis pathway of butyric acid (see fig. 1) to prepare a probiotic bacterium capable of producing butyric acid, and in particular, introduced a gene related to the synthesis of butyric acid into a probiotic bacterium, which can be applied to the fields of chemical industry, food, medicine, animal feed or cosmetics. In particular in the treatment or prevention of colitis or depression, and in the amelioration of obesity or fatty liver. Compared with the existing method for administering butyrate by oral and rectal routes, the targeting positioning is accurate, and the limitation of unpleasant taste released by butyric acid is solved.
In a first aspect of the present invention there is provided a butyric acid producing probiotic comprising an inserted gene in the genome of said probiotic, said inserted gene comprising a butyric acid synthesis associated gene.
Preferably, the butyrate synthesis-related gene comprises a 3-hydroxybutyryl-coa dehydrogenase gene (preferably hbd and/or phaB), a 3-hydroxybutyryl-coa dehydratase gene (preferably crt and/or phaJ) and/or an enoyl-coa reductase gene (preferably ter).
Further preferably, the gene involved in the synthesis of butyric acid further comprises an acetoacetyl-CoA thiolase gene (preferably atoB) or an acetyl-CoA acetyltransferase gene (preferably phaA and/or thl).
Still more preferably, the butyrate synthesis-related gene further comprises an acyl-coa thioesterase gene (preferably tesB and/or yciA), and/or a coa transferase gene (preferably cat 1).
In one embodiment of the present invention, the gene involved in the synthesis of butyric acid comprises:
a) An acetoacetyl-CoA thiolase gene (preferably atoB) or an acetyl-CoA acetyltransferase gene (preferably phaA and/or thl);
b) 3-hydroxybutyryl-coa dehydrogenase genes (preferably hbd and/or phaB);
c) 3-hydroxybutyryl-coa dehydratase gene (preferably crt and/or phaJ);
d) An enoyl-coa reductase gene (preferably ter); and, a step of, in the first embodiment,
e) Acyl-coa thioesterase genes (preferably tesB and/or yciA), and/or coa transferase genes (preferably cat 1).
Preferably, the atoB, tesB and yciA are derived from escherichia coli.
Preferably, phaA is derived from cupravidusneccator.
Preferably, thl and cat1 are derived from Clostridium tyrobutyricum.
Preferably, hbd and crt are derived from Clostridium acetobutylicum.
Preferably, ter is derived from Treponema denticola.
Preferably, phaB is derived from chromatium rnvinosum.
Preferably, phaJ is derived from Aeromonas cavae.
Preferably, each gene in the inserted gene is single copy or multiple copies.
Preferably, the inserted gene is expressed in a plasmid or on a chromosome.
In one embodiment of the invention, the insertion gene is inserted into the malEK, adhE or ldhA site of the probiotic genome.
The inserted genes may all be on one plasmid, or several inserted genes may be on one plasmid and others on another plasmid. Of course, the inserted genes may all be inserted into the same site, or different inserted genes may be inserted into different sites, or the same inserted gene may be inserted into different sites (two or more copy numbers).
Preferably, the probiotics do not express or reduce expression of genes related to the synthesis competition pathway of butyric acid. The gene related to the butyrate synthesis competition pathway comprises one or more than two of lactate dehydrogenase A gene (preferably ldhA), acetaldehyde ethanol dehydrogenase gene (preferably adhE) or/and fumaric acid reductase gene (preferably frdA).
In one embodiment of the present invention, the gene involved in the competing pathway of the synthesis of butyric acid is a lactate dehydrogenase A gene (preferably ldhA) and/or an acetaldehyde alcohol dehydrogenase gene (preferably adhE).
Preferably, the non-expression or reduced expression includes, but is not limited to, knockout, knockdown or silencing.
Preferably, the expression of the gene involved in the synthesis of butyrate competition pathway may be prevented or reduced in the probiotic bacteria by knocking out (e.g., homologous recombination, CRISPR, etc.) or knocking out (e.g., adding siRNA, etc.) the gene involved in the synthesis of butyrate competition pathway.
Preferably, the probiotics are intestinal probiotics.
Preferably, the probiotics include, but are not limited to, yeasts, probiotics, clostridium butyricum, lactobacillus, bifidobacterium, actinomycetes or escherichia coli.
In one embodiment of the invention, the probiotic is E.coli.
In a second aspect of the present invention, there is provided a method of constructing the probiotic described above, the method comprising introducing an insertion gene into the probiotic.
Preferably, the inserted gene comprises a gene related to butyrate synthesis.
Preferably, the butyrate synthesis-related gene comprises a 3-hydroxybutyryl-coa dehydrogenase gene (preferably hbd and/or phaB), a 3-hydroxybutyryl-coa dehydratase gene (preferably crt and/or phaJ) and/or an enoyl-coa reductase gene (preferably ter).
Further preferably, the gene involved in the synthesis of butyric acid further comprises an acetoacetyl-CoA thiolase gene (preferably atoB) or an acetyl-CoA acetyltransferase gene (preferably phaA and/or thl).
Still more preferably, the gene involved in the synthesis of butyric acid further comprises an acyl-CoA thioesterase gene (preferably tesB and/or yciA) or a CoA transferase gene (preferably cat 1).
In one embodiment of the present invention, the gene involved in the synthesis of butyric acid comprises:
a) An acetoacetyl-CoA thiolase gene (preferably atoB) or an acetyl-CoA acetyltransferase gene (preferably phaA and/or thl);
b) 3-hydroxybutyryl-coa dehydrogenase genes (preferably hbd and/or phaB);
c) 3-hydroxybutyryl-coa dehydratase gene (preferably crt and/or phaJ);
d) An enoyl-coa reductase gene (preferably ter); and, a step of, in the first embodiment,
e) Acyl-coa thioesterase genes (preferably tesB and/or yciA), and/or coa transferase genes (preferably cat 1).
Preferably, the atoB, tesB and yciA are derived from escherichia coli.
Preferably, phaA is derived from cupravidusneccator.
Preferably, thl and cat1 are derived from Clostridium tyrobutyricum.
Preferably, hbd and crt are derived from Clostridium acetobutylicum.
Preferably, ter is derived from Treponema denticola.
Preferably, phaB is derived from chromatium vinosum.
Preferably, phaJ is derived from Aeromonas cavae.
Preferably, each gene in the inserted gene is single copy or multiple copies.
Preferably, the inserted gene is expressed in a plasmid or on a chromosome.
Preferably, the introduction is by plasmid transformation of the inserted gene into the probiotic.
In one embodiment of the invention, the introducing is by inserting an insertion gene into the malEK, adhE or ldhA site of the probiotic chromosome genome.
The inserted genes may all be on one plasmid, or several inserted genes may be on one plasmid and others on another plasmid. Of course, the inserted genes may all be inserted into the same site, or different inserted genes may be inserted into different sites, or the same inserted gene may be inserted into different sites (two or more copy numbers).
Preferably, the construction method further comprises knocking out (e.g., homologous recombination, CRISPR, etc.) or knocking down (e.g., adding siRNA, etc.) or silencing the gene involved in the synthesis of butyrate, such that the probiotic does not express or reduces expression of the gene involved in the synthesis of butyrate. The gene related to the butyrate synthesis competition pathway comprises one or more than two of lactate dehydrogenase A gene (preferably ldhA), acetaldehyde ethanol dehydrogenase gene (preferably adhE) or/and fumaric acid reductase gene (preferably frdA).
In one embodiment of the present invention, the gene involved in the competing pathway of the synthesis of butyric acid is a lactate dehydrogenase A gene (preferably ldhA) and/or an acetaldehyde alcohol dehydrogenase gene (preferably adhE).
Preferably, the probiotics are intestinal probiotics.
Preferably, the probiotic is selected from the group consisting of yeasts, probiotics, clostridium butyricum, lactobacillus, bifidobacterium, actinomycetes and escherichia coli.
In one embodiment of the invention, the probiotic is E.coli.
In one specific embodiment of the invention, pRE112 suicide plasmid (Addgene: 43828) homologous recombination system is adopted to carry out genome editing on chassis cells, gene related to competitive pathway is knocked out, and simultaneously single copy or multiple copy butyric acid synthesis related genes atoB, phaA or thl, hbd or phaB, crt or phaJ, ter and tesB, yciA or cat1 are inserted to obtain the butyric acid-producing escherichia coli.
The pRE112 suicide plasmid homologous recombination system is characterized in that an upstream fragment and a downstream fragment of a gene to be knocked out or a gene to be inserted and the upstream fragment and the downstream fragment to be inserted are cloned to a pRE112 plasmid vector, and the pRE112 suicide plasmid vector enters host bacteria EcN through conjugation and the like, and as no replication protein (Pi protein) required for the initiation of a replication gene exists in EcN, the replication protein cannot be replicated, and under the action of external selective pressure, a mutant gene carried by the suicide plasmid vector and a wild type gene on a EcN chromosome undergo secondary homologous recombination, so that a target mutant strain is obtained.
In one embodiment of the invention, the atoB (or phaA or thl) and hbd (or phaB) expression cassettes, the crt (or phaJ) and ter expression cassettes, the tesB (or yciA or cat 1) expression cassettes, the genomic adhE site and/or ldhA site are inserted in sequence at the malEK site. The promoter used in the expression cassette comprises P fnrs A promoter.
In a third aspect of the present invention, there is provided a plasmid comprising an inserted gene comprising a gene related to butyrate synthesis.
Preferably, the plasmid further comprises a plasmid backbone.
Preferably, the plasmid contains regulatory elements required for the expression of the inserted gene, such as promoters and the like.
In a specific embodiment of the invention, the plasmid backbone includes, but is not limited to, a plasmid containing a pBBR1 replicon or a pRE112 plasmid.
In a fourth aspect of the invention there is provided a medicament or functional bacterial agent or pharmaceutical composition or pharmaceutical adjuvant, the medicament or functional bacterial agent or pharmaceutical composition comprising a probiotic as described above. Wherein the probiotic may be a combination of one or more. Preferably also comprises pharmaceutically acceptable auxiliary materials. Preferably, other active ingredients than probiotics may also be included.
In a fifth aspect of the invention, there is provided a process for the preparation of a medicament or functional bacterial agent or pharmaceutical composition or pharmaceutical adjuvant, said process comprising culturing the probiotic bacteria described above in a medium to log phase, followed by washing with physiological saline and resuspension.
In one embodiment of the present invention, the preparation method packageComprises culturing the above probiotics in LB culture medium at 37deg.C to logarithmic phase, washing with physiological saline, and re-suspending until the bacterial concentration reaches 2.5X10 10 CFU/mL。
In a sixth aspect of the invention there is provided a feed or feed additive comprising a probiotic as described above. Wherein the probiotic may be a combination of one or more. Preferably also contains the ingredients of conventional animal feeds.
When the probiotics are used as feed additives, the probiotics can replace antibiotics or be used together with antibiotics, can balance animal ecological systems, maintain intestinal canal homeostasis, for example, form normal microbial flora in the body of the probiotics, synthesize main vitamins for hosts, provide nutrition, prevent invasion of pathogenic bacteria and the like.
In a seventh aspect of the invention, there is provided a food product comprising the probiotic bacteria described above. Wherein the probiotic may be a combination of one or more.
Preferably, the food product may be yogurt, bread, or the like.
The food can be eaten by human or non-human animals.
In an eighth aspect, the present invention provides the use of a probiotic or a medicament or a pharmaceutical composition or a pharmaceutical adjuvant or a functional microbial agent as described above in the fields of chemical industry, food, medicine, animal feed or cosmetics.
In a ninth aspect, the invention provides the use of a probiotic or a medicament or a pharmaceutical composition or a pharmaceutical adjuvant or a functional bacterial agent as described above for the treatment of proctitis, depression or improvement of obesity or fatty liver.
In a tenth aspect of the invention there is provided the use of a probiotic or a medicament or a pharmaceutical composition or a pharmaceutical adjuvant or a functional bacterial agent as described above for ameliorating the symptoms of proctitis, including but not limited to reducing mucosal damage, alleviating a shortened colon length or reducing the content of pro-inflammatory factors including TNF- α, IL-1 β, IL-6 in the colon.
In an eleventh aspect of the invention there is provided the use of a probiotic or a medicament or a pharmaceutical composition or a pharmaceutical adjuvant or a functional bacterial agent for improving depression-like behaviour, including but not limited to the following: (1) reducing behavior that gives up resistance, relieving depressed mood; (2) Reduces the content of perforin-degerming D (GSDMD) related to inflammatory cytokines IL-1 beta and cell apoptosis in Hippocampus.
In a twelfth aspect of the invention there is provided the use of a probiotic or a medicament or a pharmaceutical composition or a pharmaceutical adjuvant or a functional bacterial agent for improving the phenotype of obesity and fatty liver symptoms, including but not limited to (1) weight loss; (2) reducing fat mass; (3) improving fatty liver phenotype in obese pathological conditions; (4) reducing the level of inflammation.
In a thirteenth aspect of the invention there is provided a method of treating proctitis, depression, obesity or fatty liver, said method comprising administering to a subject a probiotic or a medicament or a pharmaceutical composition or a pharmaceutical adjuvant or a functional bacterial agent as described above.
According to the technical scheme, the probiotics which do not generate butyric acid originally can be used for stably generating butyric acid, the yield of the probiotics which can generate butyric acid originally is increased, and more importantly, the probiotics obtained by the method can be used for effectively relieving ulcerative proctitis, improving depression, obesity and fatty liver symptoms, and can be used for preparing foods, medicines, feed products and the like with the functions of relieving ulcerative proctitis, improving depression, obesity and fatty liver symptoms, so that the probiotics have wide application prospects.
The "inserted gene" according to the present invention may be an exogenous gene or an endogenous gene. Wherein the exogenous gene is a gene not contained in the organism before modification, namely the organism contains the gene through insertion of the exogenous gene; an endogenous gene is a gene that the organism has already contained prior to modification, i.e., the organism is over-expressed by insertion of the endogenous gene or contains the gene in excess of its original copy number. Of course, where the insertion gene is one or more, and where the insertion gene is a plurality, the genes may be part exogenous and part endogenous or both endogenous and exogenous.
The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.
The terms "comprising" or "includes" are used in this specification to be open-ended, having the specified components or steps described, and other specified components or steps not materially affected.
An "individual" as described herein may be a human or animal (or non-human animal) which may be a wild animal, zoo animal, economic animal, pet, laboratory animal, or the like. Preferably, the non-human animals include, but are not limited to, pigs, cows, sheep, horses, donkeys, foxes, raccoons, martens, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, dragon cats, squirrels), fish or monkeys, etc.
All combinations of items to which the term "and/or" is attached "in this description shall be considered as being individually listed in this document. For example, "a and/or B" includes "a", "a and B", and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".
By "pharmaceutically acceptable" is meant that the biological activity and properties of the active substance of the administered product are neither significantly stimulated nor inhibited by the organism.
The "pharmaceutically acceptable auxiliary materials" of the present invention include, but are not limited to, one or more of carriers, excipients, diluents, wetting agents, fillers, binders, lubricants, disintegrants, antioxidants, buffers, suspending agents, solubilizers, thickeners, stabilizers, flavoring agents, preservatives, etc.
The agents of the present invention may be administered by any suitable route, such as by the gastrointestinal (e.g., oral) or parenteral (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.) route.
The medicament according to the present invention may be in any suitable dosage form, for example parenterally or parenterally, preferably including, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder mists, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, drop pills, gels and the like.
The various dosage forms of the medicament can be prepared according to the conventional production method in the pharmaceutical field.
The medicament of the invention may contain 0.01-100% (e.g. 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%) by weight of the probiotic.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1: the butyric acid synthesis pathway.
Fig. 2: butyric acid production of the probiotic after modification.
Fig. 3: eEcN alleviates the symptoms of enteritis in mice. A is a graph of the effect of eEcN on mucosal injury in mice with proctitis; b is an effect diagram of eEcN on colon length of a proctitis mouse, the left diagram is a bar chart, and the right diagram is a diagram for actually measuring colon length of the mouse; c is a graph of the effect of eEcN on the pro-inflammatory factor content in the colon of proctitis mice; d is a graph of the effect of secn on colon histopathological scores of proctitis mice.
Fig. 4: the eEcN relieves the behavior of chronically unpredictable Wen Heying shock (CUMS) -induced depression in mice. Wherein con is a control group, eEcN is an eEcN intervention group, ecN is a EcN control group, sal is a model group; a is an effect diagram of eEcN on depressed mice in a forced swimming test; b is a graph of the effect of eEcN on IL-1B in the hippocampus of depressed mice; c is a graph of the effect of secn on mesothelin D (GSDMD) in the hippocampus of depressed mice.
Fig. 5: eEcN ameliorates obesity and fatty liver symptoms. A is a liver tissue HE staining pattern; b is the effect of secn on fat mass (left panel) or lean mass (right panel) of obese mice, C is the effect of secn on body weight of obese mice; d is the effect of eEcN on blood leukocyte (WBC) or neutrophil (Gran) levels in obese mice (ranging from 0.1 to 1.8 is involved).
Detailed Description
The embodiments of the present invention will be described in detail below, but the present examples are illustrative and not restrictive, and are not intended to limit the scope of the present invention.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The method for constructing the escherichia coli by utilizing suicide plasmid homologous recombination in the embodiment is as follows:
construction of suicide plasmid (suicide plasmid) containing pRE112 as backbone and containing "homologous arm upstream of target insertion site-insert-homologous arm downstream of target insertion site" (knock-in), or "homologous arm upstream of target knockout site-homologous arm downstream of target knockout site" (knock-out), or "homologous arm upstream of target replacement site-homologous arm downstream of target replacement site" (replacement) in E.coli S17-1 by taking knock-in, or knock-out, or replacement of genome site upstream-1000 bp as homology arm. The strains containing the suicide plasmid are subjected to joint transformation to Escherichia coli Nissle 1917 (EcN for short) or EcN of deletion of the stealth plasmids pMUT1 and/or pMUT2, NCBI Taxonomy ID is 316435, then a primary single exchange monoclonal screening is carried out by using a basic culture medium plate containing chloramphenicol, and a primary exchange positive strain is obtained after PCR verification. The positive strain is cultivated in LB culture medium containing 10g/L sucrose overnight to promote the second round of double exchange, and then the bacterial liquid is coated on LB plate containing 10g/L sucrose to obtain monoclonal, and the final genome editing successful strain is obtained by PCR verification and DNA sequencing.
Conditions for fermentation culture of E.coli in examples:
single colony with good growth is selected and inoculated in a shake flask containing LB liquid medium, and cultured for 10 hours at 37 ℃ and 200 r/min. And then the seed solution is subjected to static culture for 48 hours in a 14mL sealed shaking tube filled with culture medium (M9 basic culture medium+15 g/L glucose+5 g/L yeast powder), and the butyric acid content in the culture medium supernatant is detected.
Method for measuring butyric acid yield by HPLC in examples:
HPLC conditions: aminex HPX-87H (Bio-Rad, USA) chromatographic column, 5mmol/L dilute sulfuric acid mobile phase, ultraviolet detector, 210nm detection wavelength, column temperature 50 ℃, flow rate 0.6mL/min, 20. Mu.L sample injection.
Example 1 preparation of probiotics
EcN (reference: CRISPR-based curing and analysis of metabolic burden of cryptic plasmids in Escherichia coliNissle 1917,Eng Life Sci.2019Jun 3;19 (6): 478-485.) deleted of the cryptic plasmids pMUT1 and pMUT2 was used as the starting strain. Constructing escherichia coli by suicide plasmid homologous recombination, which comprises the following steps:
the adhE and ldhA genes of strain EcN were first knocked out and P was inserted at the genomic malEK (malE: sugar ABC transporter substrate binding protein, malK: sugar transporter ATP binding protein, see Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria, nat Biotechnol.2018Oct;36 (9): 857-864) site fnrs -phaA-hbd-P fnrs -crt-ter-P fnrs tesB (see SEQ ID NO:1, wherein 93 to 1274 are phaA, 1301 to 2149 are hbd, 2177 to 2962 are crt, 2989 to 4182 are ter, 4280 to 5140 are tesB), and the butyric acid-producing probiotic M3 is obtained after proper sequencing. And replacing the malEK locus hbd-crt of the M3 strain with phaB-phaJ gene to obtain butyric acid producing probiotics M2. M3 is taken as an original strain, P is respectively inserted into two positions of adhE and ldhA fnrs Ter (see SEQ ID NO: 2) to give probiotic M3A8L8 butyric acid comprising three copies of ter; replacing MalEK locus phaA of the M3A8L8 strain as thl gene to obtain butyric acid-producing probiotics M3A8L8T; replacing the malEK locus phaA of the M3A8L8 strain as an atoB gene to obtain butyric acid-producing probiotics eEcN. Replacing a malEK locus tesB of the eEcN strain as a yciA gene to obtain butyric acid-producing probiotics M3A8L8Y; substitution of the malEK locus tesB of the eEcN strain as the cat1 gene to obtain butyric acid-producing probioticsM3A8L8C; the adhE gene of EcN strain is knocked out, and the malEK site is inserted into P fnrs -atoB-hbd-P fnrs -crt-ter-P fnrs Insertion of the adhE site into P in tesB fnrs Ter, obtaining butyric acid-producing probiotics M3A8; taking eEcN as an original strain, knocking out frdA to obtain butyric acid-producing probiotics M3A9. Table 1 summarizes the strains constructed in example 1.
Table 1: the strain constructed in this example
Wherein, the atoB (see SEQ ID NO: 4), the tesB and the yciA (see SEQ ID NO: 6) are EcN (GenBank: CP 007799.1) source sequences, the phaA (Uniprot Entry: P14611) source sequences optimized by E.coli codons, the thl (see SEQ ID NO: 3) VJQ0 and the cat1 (see SEQ ID NO: 3) are Clostridium tyrobutyricum source sequences optimized by E.coli codons, the hbd (Uniprot Entry: P52041) and the crt (Uniprot Entry: P52046) are Clostridium acetobutylicum source sequences optimized by E.coli codons, the phaB (see SEQ 48 (3-hydroxybutyric acid) 24, the Eur J. 1992Oct 1;209 (see SEQ ID NO: 3-50) is SEQ ID NO: 42) is a SEQ ID NO: 24) source sequence optimized by E.coli codons, the hbd (Uniprot Entry: P52041) and the crt (Uniprot Entry: P52046) is Clostridium acetobutylicum source sequences optimized by E.coli codons, and the phaB (see SEQ ID NO: 3-hydroxybutyric acid) is SEQ ID NO: 24, the SEu 2) is a SEQ ID NO: 24, the Uniprot (Uniprot) is a sequence optimized by E.coli codons, and the great strain of E.coli codons is SEQ ID NO: 24. The primers of Table 2 were used to construct suicide plasmids containing the gene of interest.
As shown in FIG. 2, wild type EcN does not produce butyric acid, the transformed probiotics are fermented for 48 hours, the butyric acid yield is 0.3-1.6 g/L, and the intermediate yield strain eEcN (1.4 g/L) is selected for subsequent study.
Table 2: primers used in example 1
Example 2 use of eEcN for alleviating symptoms of sodium dextran sulfate (DSS) induced proctitis in mice
24 healthy male C57BL/6J mice weighing 18-22g were taken, and 6 mice in each group were randomly divided into 4 groups: blank (regular drinking water + lavage saline), model (2% dss drinking water + lavage saline), eEcN intervention (2% dss drinking water + lavage eEcN), ecN control (2% dss drinking water + lavage EcN). Mice in the model group, the eEcN intervention group and the EcN control group developed chronic enteritis symptoms with 5 days of 2% dss plus 7 days of normal water as one cycle, three times in total. The eEcN and EcN lavage doses were 5X 10 every two days 9 CFU/patient, other groups perfuse the stomach with equal volumes of saline.
During the modeling period (i.e., during DSS treatment), disease activity index (Disease activity index, DAI) was calculated from the mice body weight, fecal trait and hematochezia condition every two days. The method comprises the following steps: the disease activity index was combined with the percentage of weight loss (body weight not changed to 0,1-5 for 1 minute, 5-10 for 2 minutes, 10-15 for 3 minutes, and more than 15 for 4 minutes), stool consistency (normal 0, loose stool for 2 minutes, diarrhea for 4 minutes) and stool bleeding (normal 0 minutes, occult blood positive for 2 minutes, dominant bleeding for 4 minutes) for the animals, and the total score of 3 results was divided by 3 to obtain the DAI value. I.e. dai= (body mass index + stool shape + bleeding situation)/3.
After the mice were sacrificed on day 36, the colon length of the mice was measured and recorded. Colorectal tissues were taken, proinflammatory factor content was detected by RT-PCR, and paraffin sections of colon were prepared and HE stained.
Experimental results as shown in fig. 3, secn can reduce enteritis symptoms in mice, including reduced mucosal injury (fig. 3A), reduced colonic shortening (fig. 3B), reduced pro-inflammatory cytokines (fig. 3C), and reduced DAI disease scores (fig. 3D).
Example 3 application of eEcN in the alleviation of Chronic unpredictable Wen Heying shock (CUMS) induced depression-like behavior mice
60 healthy male C57BL/6J mice of eight weeks old were taken, 10 were randomly allocated as a control group, and the remaining 50 were subjected to CUMS modeling for 4 weeks. Behavioural tests were performed 4 weeks after moulding, and the mice that were successfully moulded were randomly and evenly distributed into 3 groups. Furthermore, mice are currently divided into 4 groups: control group (8 weeks of normal feeding); model group (CUMS modeling for 8 weeks, 5 th week starting to perfuse stomach saline), eEcN intervention group (CUMS modeling for 8 weeks, 5 th week starting to perfuse stomach eEcN), ecN control group (CUMS modeling for 8 weeks, 5 th week starting to perfuse stomach EcN). The eEcN and EcN lavage doses were 1X 10 once daily 9 CFU/patient, control and model groups perfuse the same volume of physiological saline.
The following day after the end of the 8 th week of gavage was performed with the stomach still being gavaged. When the behavioural test is finished, the mice are sacrificed, the whole brains of the mice are taken after the heart is perfused with 4% paraformaldehyde, the hippocampus is stripped off, and the content of the proinflammatory factors is detected by using RT-PCR.
Experimental results as shown in fig. 4, eEcN can increase the struggle duration of depressed mice forced swimming (panel a), alleviate their depressed phenotype, and reduce IL-1β (panel B) and mesothelin D (GSDMD, panel C) content in the hippocampus of depressed mice.
Example 4 use of eEcN for improving obesity and fatty liver phenotype in mice
22 healthy male C57BL/6J mice (weight 18-22 g) of 4-5 weeks old were taken, 6 were randomly allocated as control group, and the remaining 16 were subjected to obesity molding with 60% (kcal) high fat purified feed (HFD). The period of 8-12 weeks was continued until the mice obesity model was induced to be successful, and the mice with successful modeling were randomly and evenly distributed into 3 groups. To this end, mice were divided into 4 groups: control group (normal diet group, and model group synchronously lavage physiological saline, abbreviated as ND); model group (HFD molding, after successful molding, gastric normal saline, HFD for short), eEcN intervention group (HFD molding, after successful molding, gastric eEcN, HFD for short+eEcN), ecN control group (HFD molding, after successful molding, gastric EcN, HFD for short+EcN. Gastric lavage is performed once every four days, and the gastric dosage of eEcN and EcN is 1×10) 9 CFU/alone, controlThe group and model group perfuse the stomach with equal volumes of physiological saline.
Body weight was weighed and recorded every four days during gavage, and after 32 days of gavage, all mice were placed in an echo mri small animal body composition analyzer for body Fat composition detection, and Lean mass (Lean mass) and Fat mass (Fat mass) of the mice were recorded. Mice were then euthanized and bled, whole blood samples were tested for leukocyte and neutrophil levels, and liver tissue was HE stained to observe fatty liver phenotype.
As shown in the experimental results in FIG. 5, eEcN can reduce the weight and fat mass of obese mice, improve the fatty liver phenotype of the mice under the morbid condition of obesity, and reduce the systemic inflammation level of the obese mice.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Beijing aviation aerospace
TSINGHUA University
<120> a butyric acid-producing probiotic, construction method and application thereof
<130> P0102022030224W
<160> 43
<170> PatentIn version 3.5
<210> 1
<211> 5140
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
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aagcgttgaa gatattgata aagcgatgaa actgggtgcc aatcatccga tgggcccgct 1980
ggaactgggt gattttattg gcctggatat ttgcctggca attatggatg tgctgtattc 2040
agaaaccggt gatagcaaat atcgcccgca taccctgctg aaaaaatatg ttcgtgcggg 2100
ttggctgggc cgcaaaagtg gtaaaggctt ttatgattat tcaaaataat actagagtca 2160
cacaggaaag actagaatgg aactgaataa cgtgattctg gaaaaagaag gcaaagttgc 2220
ggtggttacc attaatcgtc cgaaagccct gaatgcactg aactcagata ccctgaaaga 2280
aatggattac gtgatcggtg aaatcgaaaa cgatagcgaa gtgctggccg ttattctgac 2340
cggcgcaggt gaaaaatcat ttgttgcggg cgccgatatc agcgaaatga aagaaatgaa 2400
caccattgaa ggtcgcaaat ttggcattct gggtaacaaa gtgtttcgtc gcctggaact 2460
gctggaaaaa ccggtgattg ccgcagttaa tggctttgca ctgggtggcg gttgtgaaat 2520
tgcgatgagt tgcgatattc gtattgcgag cagtaatgca cgttttggtc agccggaagt 2580
gggtctgggt attaccccgg gttttggcgg tacccagcgt ctgtcacgcc tggttggcat 2640
gggtatggcg aaacagctga tttttaccgc ccagaatatt aaagcggatg aagccctgcg 2700
tattggtctg gtgaataaag tggttgaacc gagcgaactg atgaacaccg caaaagaaat 2760
cgcgaacaaa atcgttagca acgcaccggt ggcggttaaa ctgagtaaac aggccattaa 2820
tcgcggcatg cagtgtgata ttgataccgc actggcgttt gaaagtgaag catttggtga 2880
atgcttttca accgaagatc agaaagatgc catgaccgca ttcatcgaaa aacgtaaaat 2940
cgaaggcttt aaaaaccgct aatactagag aaagaggaga aatctagtat gattgtgaaa 3000
ccgatggttc gtaataacat ttgtctgaat gcgcatccgc agggttgcaa gaaaggtgtg 3060
gaagatcaga tcgaatacac caaaaaacgc atcaccgccg aagttaaagc cggtgcaaaa 3120
gcgccgaaaa atgtgctggt tctgggctgt agcaacggtt atggcctggc gagtcgtatt 3180
accgccgcat ttggttatgg cgcggccacc attggtgtgt catttgaaaa agcaggcagc 3240
gaaaccaaat atggtacccc gggctggtat aataacctgg cgtttgatga agcagcgaaa 3300
cgcgaaggtc tgtattcagt gaccattgat ggcgatgcct ttagcgatga aattaaagca 3360
caggttatcg aagaagccaa aaagaaaggt atcaaattcg atctgatcgt gtacagcctg 3420
gcaagtccgg ttcgtaccga tccggatacc ggcattatgc ataaaagtgt gctgaaaccg 3480
tttggtaaaa cctttaccgg caaaaccgtt gatccgttta ccggtgaact gaaagaaatt 3540
tcagcagaac cggcgaacga tgaagaagcc gcagcgaccg tgaaagttat gggtggcgaa 3600
gattgggaac gttggattaa acagctgtca aaagaaggtc tgctggaaga aggctgtatt 3660
accctggcat atagctatat tggtccggaa gccacccagg cactgtatcg caaaggtacc 3720
attggcaaag ccaaagaaca tctggaagcc accgcacatc gtctgaataa agaaaacccg 3780
agtattcgcg catttgtgtc agttaataaa ggcctggtga cccgtgcgag tgccgtgatt 3840
ccggttattc cgctgtatct ggcatcactg ttcaaagtta tgaaagaaaa aggtaaccat 3900
gaaggctgca ttgaacagat tacccgcctg tatgcggaac gtctgtatcg caaagatggc 3960
accattccgg tggatgaaga aaatcgtatt cgcattgatg attgggaact ggaagaagat 4020
gtgcagaaag cggttagcgc cctgatggaa aaagttaccg gtgaaaacgc cgaaagtctg 4080
accgatctgg caggctatcg ccatgatttt ctggcgagca atggttttga tgtggaaggc 4140
attaactatg aagccgaagt tgaacgtttt gatcgcattt aagaattcaa aaacgccgca 4200
aagtttgagc gaagtcaata aactctctac ccattcaggg caatatctct ctttactaga 4260
gaaagaggag aaactgcata tgagtcaggc cctgaaaaat ctgctgaccc tgctgaacct 4320
ggaaaaaatt gaagaaggtc tgtttcgtgg ccagtcagaa gatctgggtc tgcgccaggt 4380
gtttggtggc caggtggttg gccaggcact gtatgccgca aaagaaaccg tgccggaaga 4440
acgtctggtt catagctttc atagttattt tctgcgcccg ggtgatagca aaaaaccgat 4500
catctacgat gtggaaaccc tgcgtgatgg taattcattt agcgcacgtc gcgttgcggc 4560
cattcagaac ggcaaaccga tcttttatat gaccgcgagt tttcaggccc cggaagcagg 4620
ttttgaacat cagaaaacca tgccatcagc accggcacca gatggcctgc cgtcagaaac 4680
ccagattgcg cagagtctgg cacatctgct gccgccggtg ctgaaagata aatttatttg 4740
tgatcgtccg ctggaagtgc gcccggttga atttcataat ccgctgaaag gtcatgtggc 4800
ggaaccgcat cgtcaggttt ggattcgcgc caacggtagc gtgccggatg atctgcgtgt 4860
tcatcagtat ctgctgggct atgccagtga tctgaatttt ctgccggttg cactgcaacc 4920
gcatggtatt ggctttctgg aaccgggcat tcagattgcg accattgatc attcaatgtg 4980
gtttcatcgc ccgtttaatc tgaacgaatg gctgctgtat agcgtggaaa gtacctcagc 5040
gagcagtgcc cgtggttttg ttcgcggcga attttatacc caggatggtg tgctggttgc 5100
aagtaccgtg caggaaggcg ttatgcgcaa tcataactaa 5140
<210> 2
<211> 1285
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
aaaaacgccg caaagtttga gcgaagtcaa taaactctct acccattcag ggcaatatct 60
ctctttacta gagaaagagg agaaatctag tatgattgtg aaaccgatgg ttcgtaataa 120
catttgtctg aatgcgcatc cgcagggttg caagaaaggt gtggaagatc agatcgaata 180
caccaaaaaa cgcatcaccg ccgaagttaa agccggtgca aaagcgccga aaaatgtgct 240
ggttctgggc tgtagcaacg gttatggcct ggcgagtcgt attaccgccg catttggtta 300
tggcgcggcc accattggtg tgtcatttga aaaagcaggc agcgaaacca aatatggtac 360
cccgggctgg tataataacc tggcgtttga tgaagcagcg aaacgcgaag gtctgtattc 420
agtgaccatt gatggcgatg cctttagcga tgaaattaaa gcacaggtta tcgaagaagc 480
caaaaagaaa ggtatcaaat tcgatctgat cgtgtacagc ctggcaagtc cggttcgtac 540
cgatccggat accggcatta tgcataaaag tgtgctgaaa ccgtttggta aaacctttac 600
cggcaaaacc gttgatccgt ttaccggtga actgaaagaa atttcagcag aaccggcgaa 660
cgatgaagaa gccgcagcga ccgtgaaagt tatgggtggc gaagattggg aacgttggat 720
taaacagctg tcaaaagaag gtctgctgga agaaggctgt attaccctgg catatagcta 780
tattggtccg gaagccaccc aggcactgta tcgcaaaggt accattggca aagccaaaga 840
acatctggaa gccaccgcac atcgtctgaa taaagaaaac ccgagtattc gcgcatttgt 900
gtcagttaat aaaggcctgg tgacccgtgc gagtgccgtg attccggtta ttccgctgta 960
tctggcatca ctgttcaaag ttatgaaaga aaaaggtaac catgaaggct gcattgaaca 1020
gattacccgc ctgtatgcgg aacgtctgta tcgcaaagat ggcaccattc cggtggatga 1080
agaaaatcgt attcgcattg atgattggga actggaagaa gatgtgcaga aagcggttag 1140
cgccctgatg gaaaaagtta ccggtgaaaa cgccgaaagt ctgaccgatc tggcaggcta 1200
tcgccatgat tttctggcga gcaatggttt tgatgtggaa ggcattaact atgaagccga 1260
agttgaacgt tttgatcgca tttaa 1285
<210> 3
<211> 1182
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgagtgaag tggttattgc gtcagccgtg cgtaccgcca ttggcaaatt tggtggcagc 60
ctgaaaagtg ttccggcgac cgatctgggt gccctggtga ttaaagaagc actgaaacgc 120
gcgaatgtga aaccggaact ggttgatgaa gtggttatgg gcaatgttct gcaagcaggt 180
ctgggtcaga acccagcacg tcaggccctg attaaaagcg gcattccgaa taccgtgccg 240
ggttttacca ttaacaaagt ttgtggtagt ggtctgcgtg cagtgagtct ggcagcacag 300
atgattaaag caggtgatga tgatattgtg gttgcgggtg gcatggaaaa catgtcagcg 360
gccccgtatg ttatgccaag tgcacgttgg ggtcagcgta tgttcgatgg taaaatcatc 420
gatgaaatgg ttaaagatgg cctgtgggat gcgttcaaca actaccatat gggtatcacc 480
gccgaaaata ttgcagaaaa atggaacatt acccgtcaga tgcaggatga atttgcagcg 540
gccagtcagc agaaagcggt tgcagcgatc aaatcaggca aattcaaaga tgaaatcgtg 600
ccggtggtta tcaaagatcg taagaaaggt gaaatcgttt tcgataccga tgaatttccg 660
cgcgatggcg tgaccgttga aggtattagc aaactgaaac cggcgtttaa gaaagatggt 720
ggcaccgtga ccgccgcaaa tgcctcaggc attaacgatg cggccgcagc gctggttatt 780
atgagcgcag ataaagcgaa agaactgggt attaaaccgc tggcaaaaat tacctcatat 840
ggcagcaaag gtctggatcc gagtattatg ggctatggtc cgtttcatgc gaccaaactg 900
gccctgaaga aagccaatct gaccgtggat gatctggatc tgattgaagc caacgaagca 960
tttgccgcac agagcctggc agttgcgaaa gatctgaaat tcgatatgag taaagtgaac 1020
gttaacggtg gcgccattgc actgggtcat ccaattggtg caagtggtgc acgtattctg 1080
accaccctgc tgtatgaaat gcagaaacgt gattcaaaac gcggcctggc aaccctgtgc 1140
attggtggcg gtatgggtac cgcaattatt gtggaacgct aa 1182
<210> 4
<211> 1185
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atgaaaaatt gtgtcatcgt cagtgcggta cgtactgcta tcggtagttt taacggttca 60
ctcgcttcca ccagcaccat cgacctgggg gcgacagtaa ttaaagccgc cattgaacgt 120
gcaaaaatcg attcactaca cgttgatgaa gtgattatgg gtaacgtgct acaagccggg 180
ctggggcaaa atccggcgcg tcaggcactg ttaaaaagcg gactggccga aacagtgtgc 240
ggattcacgg tcaataaagt gtgtggttcg ggtcttaaaa gtgtggcgct tgccgcccag 300
gcaattcagg caggtcaggc gcagagcatt gtggcggggg gtatggaaaa tatgagttta 360
gcgccctact tactcgatgc aaaagcacgc tctggttatc gtcttggaga cggacaggtt 420
tatgacgtaa tcctgcgcga tggtctgatg tgcgccaccc atggttatca tatggggatt 480
accgccgaaa acgtggctaa agagtacgga attacccgtg aaatgcagga tgaactggcg 540
ctacattcac agcgtaaagc ggcagccgca attgagtccg gtgcttttac agccgaaatc 600
gtcccggtaa atgttgtcac tcggaagaaa accttcgtct tcagtcaaga cgaattcccg 660
aaagcggatt ctacggctga agcgttaggc gcattgcgcc cggccttcga taaagcagga 720
acagtcaccg ccgggaacgc gtctggcatt aacgacggtg ctgccgctct ggtgattatg 780
gaagaatctt cggcgctggc agcaggcctt acccccctgg ctcgcattaa aagttatgcc 840
agcggtggcg tgccccccgc attgatgggt atggggccag tacctgccac gcaaaaagcg 900
ttacaactgg cggggctgca actggcggat attgatctca ttgaggctaa tgaagcattt 960
gctgcacagt tccttgccgt tgggaaaacc ctgggctttg agcctgagaa agtgaatgtc 1020
aacggcgggg ccatcgcgct ggggcatcct atcggtgcca gtggtgctcg tattctggtc 1080
acactattac atgcaatgca ggcacgcgat aaaacgctgg ggctggcaac actgtgcatt 1140
ggtggcggcc agggaattgc gatggtgatt gaacgattga attaa 1185
<210> 5
<211> 1302
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atgagcttcg aagaactgta taaaagtaaa gtggtttcag cggatgaagc cgtgagccat 60
atcaaaagtg gtgataaaat cgttttcggc catgcatgcg cggaaccgca ggtgctgctg 120
aatgcactgg ttaaaaacaa agaaaaatac aaaaatgtgg aactgtatca tatgattccg 180
aaaggtgaaa ccaactatgc ggaaccgggc gttgaaaaat acatccatga taacagcttt 240
ttcaccggta aaggcacctc agaagcaatt caggaaggtc gtgcggattt tattccggtg 300
tttttcagcg aaattccgcg cctgtttaag aaaggttata ttccggtgga tgttgcgctg 360
attgaagtta gtaaaccgga tgaacatggt tattgctcat ttggcgttag caacgattat 420
attcatgccg gtgcagaatg tgcgaaactg gtgattgccg aagttaatga aaacatgccg 480
cgtgtgtttg gcaataactt catccatgtt agtgatatcg attacatcgt tgaaagcagt 540
cgcccggtgg ttgaaatgca gccgtcaaaa attggtgatg tggaaaaagc cattggcaaa 600
tattgtgcaa gcctggttga agatggtagt accctgcaac tgggtattgg cgccattccg 660
gatgcagttc tgctgtttct gaaagataag aaagatctgg gcattcatag tgaaatgatt 720
tcagatggtg tggttgaact ggtgaaagca ggcgttatta ccaataaaaa gaaaaccctg 780
catccgggta aaattgtggt tacctttctg atgggcacca aaaaactgta cgatttcatc 840
gataacaacc cggaagtgga atcacatccg gtggattatg ttaacaaccc gaccgttatc 900
atgaaaaact gtaaaatggt gtgcatcaac agttgtgttg aaattgatct gatgggtcag 960
atttgctcag aaagcattgg tctgaaacag attagcggcg ttggtggcca gattgatttt 1020
acccgtggtg caagtatggc ggaagatggc aaatcaatta ttgccattcc gagcaccgcc 1080
gcacatggta aagtgtcacg tattgttccg tttctgaatc agggcgcggc cattaccacc 1140
agccgcaacg atgtgcatta tgttattacc gaatatggta ttgcgcagct gcgcggcaaa 1200
accctgaaac agcgtgcccg cgaactgatt aaaattagcc atccgaaatt tcgtgaaggt 1260
ctgatggcag aatttgaaaa acgctttggc tgtaaatatt aa 1302
<210> 6
<211> 399
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atgtctacaa cacataacgt ccctcagggc gatcttgttt tacgtacttt agccatgccc 60
gccgatacca atgccaatgg tgacatcttt ggtggttggt taatgtcaca aatggatatt 120
ggcggcgcta ttctggcgaa agaaattgcc catggtcgtg tagtgaccgt gcgggttgaa 180
ggaatgactt tcttacggcc ggttgcggtc ggcgatgtgg tgtgctgcta tgcacgctgt 240
gtccagaaag ggacgacatc agtcagcatt aatattgaag tgtgggtgaa aaaagtggcg 300
tctgaaccaa tcgggcaacg ctataaagca acagaagcat tatttaagta tgtcgcggtt 360
gatcccgaag gaaaacctcg cgccttaccc gttgagtaa 399
<210> 7
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gagctcgata tcgcatgcgg ccataattca ctttactggt gtcgatgttg 50
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atcatggcga ttttatgtgc g 21
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
ccgcatgcga tatcgagctc 20
<210> 10
<211> 55
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
gcacataaaa tcgccatgat tactagagaa agaggagaaa tctagtatga ttgtg 55
<210> 11
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
accgaggtca tgtaaggtta cgctgacttg acgggac 37
<210> 12
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
taaccttaca tgacctcggt ttag 24
<210> 13
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ccaagcttct tctagaggta cgtcagcgat atcactcggc ag 42
<210> 14
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
tacctctaga agaagcttgg gatcgg 26
<210> 15
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gcacataaaa tcgccatgat aaaaacgccg caaagtttga g 41
<210> 16
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ccaagcttct tctagaggta ccggaatggt gccatctttg 40
<210> 17
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
tacctctaga agaagcttgg gatcgg 26
<210> 18
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
cccgatccca agcttatgca atagccacaa agaaac 36
<210> 19
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
cggcgttttt cataatgctc tcctgataat gttaaac 37
<210> 20
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
cagcgtaatg aaagcgaaaa aatccgctta at 32
<210> 21
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
gagctcgata tcgcatgcgg gcgtccgtca tcgacgtc 38
<210> 22
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gagcattatg aaaaacgccg caaagtttga g 31
<210> 23
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ttttcgcttt cattacgctg acttgacggg ac 32
<210> 24
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tcccaagctt tgacgagtaa tttgccacg 29
<210> 25
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
cgttcgggca cataagactt tctccagtga tgttgaat 38
<210> 26
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
aagtcttatg tgcccgaacg aactggtt 28
<210> 27
<211> 52
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
gagctcgata tcgcatgcgg gcaaaaaaat agccataaaa aagtaaaaat gc 52
<210> 28
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
tcccaagctt ttgctcaaat ccagtttcgc 30
<210> 29
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
ccttctcctt cacgacattc ctccagattg 30
<210> 30
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gaatgtcgtg aaggagaagg cgaatggc 28
<210> 31
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
gagctcgata tcgcatgcgg gttgcactcc ttagcgtg 38
<210> 32
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
ttagcgttcc acaataattg c 21
<210> 33
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
caattattgt ggaacgctaa tactagagaa agaggagaaa ctgca 45
<210> 34
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
ttctctagta ttaattcaat cgttcaatca ccatcg 36
<210> 35
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
gaacgattga attaatacta gagaaagagg agaaactgca 40
<210> 36
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gaaagaggag aaactgcata tgtctacaac acataacgtc c 41
<210> 37
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
tgtgattacg catcacctta ctcaacgggt aaggcg 36
<210> 38
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gaaagaggag aaactgcata tgagcttcga agaactgta 39
<210> 39
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
tgtgattacg catcacctta atatttacag ccaaagcgtt tttcaaattc 50
<210> 40
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
taaggtgatg cgtaatcaca attaaactag t 31
<210> 41
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
atgcagtttc tcctctttct c 21
<210> 42
<211> 741
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
atggcacgta ttgcgctggt taccggtggc attggtggca ttggtacctc aatttgtacc 60
cgcctggcaa aagatggctg taccgtggtt gcgaattgtc atccaagtga agccgcagcg 120
gccgaagaat ggaaacaggc acgtgcagcg gaaggttttg atattgcggt gtttaccgcc 180
gatgttagca gttttgatga tagcgcccgt atggtgcgcg aaattaccga acaggttggt 240
ccgattgata ttctggtgaa ctgcgcaggc attacccgcg ataaaacctt taagaaaatg 300
gaacaggcac attgggaagc ggtgatcaac gttaacctga atagtgtgtt taacgttacc 360
cgtcaggttt gggatggcat gctggaacgt ggttttggcc gcattattaa catttcaagc 420
gtgaatggtc agcgcggcca gtttggtcag gcgaactatt cagccgcaaa agccggcatg 480
catggtttta caatggcact ggcacaggaa ggcgcaagca aaggtgtgac cgttaatacc 540
attagtccgg gttatgttga aaccgccatg accctggcaa tgaacgatga tgtgcgtaat 600
agtattattt caggcattcc gatgcgtcgc atggcgcagc cggatgaaat tgcggccgca 660
attgcctttc tggcaggcga tgaaagtggt tatatgacag gtgcaaacct gccggtgaat 720
ggtggcctgt ttatgcatta a 741
<210> 43
<211> 405
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
atgtcagcgc agagcctgga agtgggtcag aaagcccgtc tgagcaaacg ctttggcgcc 60
gcagaagttg cggcctttgc agcgctgagt gaagatttta atccgctgca tctggatccg 120
gcatttgccg caaccaccgc gtttgaacgt ccgattgtgc atggtatgct gctggccagt 180
ctgttttcag gtctgctggg ccagcagctg ccgggtaaag gcagcattta tctgggtcag 240
agcctgagtt ttaaactgcc ggtgtttgtt ggcgatgaag tgaccgccga agtggaagtt 300
accgcactgc gtgaagataa accgattgca accctgacca cccgcatttt tacacagggt 360
ggcgcgctgg cagttacagg tgaagcggtg gttaaactgc cgtaa 405

Claims (32)

1. The application of the probiotics for producing butyric acid in preparing feed or medicine is characterized in that the probiotics are escherichia coli @Escherichia coli) Nissle 1917, which maintains intestinal homeostasis, or treats proctitis or depression, or improves obesity or fatty liver, wherein the genome of the probiotic comprises an inserted gene comprising a gene related to butyrate synthesis, which comprises:
a) atoB, hbd, crt, ter and cat1;
b) atoB, hbd, crt, ter and yciA;
c) atoB, hbd, crt, ter and tesB;
d) phaA, hbd, crt, ter and tesB;
e) thl, hbd, crt, ter and tesB; or alternatively, the first and second heat exchangers may be,
f) phaA, phaB, phaJ, ter and tesB;
the probiotics do not express or reduce the expression of genes related to the synthesis competition pathway of the butyric acid, and the genes related to the synthesis competition pathway of the butyric acid are selected from one or more than two of lactate dehydrogenase A gene ldhA, acetaldehyde ethanol dehydrogenase gene adhE or fumaric acid reductase gene frdA.
2. The use according to claim 1, wherein atoB, tesB and yciA are derived from escherichia coli @Escherichia coli）。
3. The use according to claim 1, wherein phaA is derived fromCupriavidus necator。
4. The use according to claim 1, wherein thl and cat1 are derived fromClostridium tyrobutyricum。
5. The use according to claim 1, wherein hbd and crt are derived fromClostridium acetobutylicum。
6. The use according to claim 1, wherein ter is derived fromTreponema denticola。
7. The use according to claim 1, wherein phaB is derived fromChromatium vinosum。
8. The use according to claim 1, wherein phaJ is derived fromAeromonas caviae。
9. The use according to claim 1, wherein the nucleotide sequence of phaA is SEQ ID NO: 93 to 1274 of 1.
10. The use according to claim 1, wherein the nucleotide sequence of hbd is SEQ ID NO: bits 1301 to 2149 of 1.
11. The use according to claim 1, wherein the nucleotide sequence of crt is SEQ ID NO: 2177 to 2962 bits of 1.
12. The use according to claim 1, wherein the nucleotide sequence of ter is SEQ ID NO: positions 2989 to 4182 of 1.
13. The use according to claim 1, wherein the nucleotide sequence of tesB is SEQ ID NO: 4280 th to 5140 th bits of 1.
14. The use according to claim 1, wherein the nucleotide sequence of atoB is SEQ ID NO:4.
15. the use according to claim 1, wherein the nucleotide sequence of yciA is SEQ ID NO:6.
16. the use according to claim 1, wherein thl has the nucleotide sequence of SEQ ID NO:3.
17. the use according to claim 1, wherein the nucleotide sequence of cat1 is SEQ ID NO:5.
18. the use according to claim 1, wherein the nucleotide sequence of phaB is SEQ ID NO:42.
19. the use according to claim 1, wherein the nucleotide sequence of phaJ is SEQ ID NO:43.
20. the use according to any one of claims 1 to 19, wherein said treatment of proctitis comprises reducing mucosal lesions, alleviating a shortening of the length of the colon or reducing the content of pro-inflammatory factors including TNF- α, IL-1 β, IL-6 in the colon.
21. The use according to any one of claims 1-19, wherein said treating depression is improving depression-like behavior, said improving depression-like behavior comprising: (1) reducing behavior that gives up resistance, relieving depressed mood; (2) Reduce the content of perforin-vanishing factor D related to inflammatory cytokines IL-1 beta and cell apoptosis in Hippocampus.
22. The use according to any one of claims 1 to 19, wherein said improvement of obesity or fatty liver is an improvement of obesity and fatty liver symptom phenotypes comprising: (1) weight loss; (2) reducing fat mass; (3) improving fatty liver phenotype in obese pathological conditions; (4) reducing the level of inflammation.
23. The use according to any one of claims 1 to 19, wherein the feed is an animal feed.
24. The use according to any one of claims 1 to 19, wherein each gene in the inserted gene is a single copy or two or more copy numbers.
25. The use according to any one of claims 1 to 19, wherein the inserted gene is expressed in a plasmid or on a chromosome.
26. The use of claim 1 wherein ter is a single copy or more than two copy numbers.
27. The use according to claim 1, wherein the gene involved in the pathway of competing butyrate synthesis is the gene adhE for acetaldehyde alcohol dehydrogenase.
28. The use according to claim 1, wherein the genes involved in the competing pathways of butyrate synthesis are lactate dehydrogenase a gene ldhA and acetaldehyde ethanol dehydrogenase gene adhE.
29. The use according to claim 1, wherein the genes involved in the competing pathways of the synthesis of butyric acid are the lactate dehydrogenase a gene ldhA, the acetaldehyde ethanol dehydrogenase gene adhE and the fumaric acid reductase gene frdA.
30. The use according to any one of claims 1 to 19, wherein the method of constructing a butyric acid producing probiotic comprises introducing an insertion gene into the probiotic.
31. The use of claim 30, wherein said introducing is by inserting an insertion gene into the malEK, adhE or ldhA site of the genome of the probiotic chromosome.
32. The use according to claim 30, wherein the method of constructing a butyric acid producing probiotic comprises knocking down or knocking out genes involved in the competing pathways of butyric acid synthesis.
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