Preparation method of probiotics based on synthetic biology

Abstract

The invention belongs to the technical field of synthetic biology, and particularly relates to a preparation method of probiotics based on synthetic biology. By further expressing threonine inner carrier proteins of different sources and types in the isoleucine production strain, the threonine absorption capability of cells is improved, so that the yield and content of L-isoleucine in a fermentation product are improved, and the relative yield and content of a by-product heteroacid (valine) are reduced. And further deleting an exporter for excreting threonine from the cells so as to obtain a genetic engineering high-yield strain.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a preparation method of probiotics based on synthetic biology. Background Art

[0002] L-isoleucine, also known as "L-isoleucine", is one of the three essential branched-chain amino acids (BCAA) for the human body. It has multiple physiological functions, participates in the synthesis of hormones and enzymes in the human body, promotes protein production and inhibits its decomposition, and plays an important regulatory role in human and animal health. Therefore, it has a wide range of applications and commercial value in the fields of food, medicine, and feed.

[0003] In the prior art, probiotics are widely used in the fields of chemical industry, food, medicine, animal feed and cosmetics. After searching, the patent with publication number CN113968758A discloses the application of probiotics in making fertilizers, the patent with publication number CN113908251A discloses a yogurt containing probiotics, the patents with publication numbers CN114053310A, CN114129707A, CN114107122A and CN113999805A disclose the application of probiotics in lipid reduction, anti-allergy, relieving wind and treating hyperuricemia, the patent with publication number CN114027397A discloses a feed additive containing probiotics, and the patent CN113827521A discloses the application of probiotics in the field of cosmetics.

[0004] At present, many domestic companies use fermentation to produce L-isoleucine, but there are still problems such as low acid production level, low conversion rate, many by-products and poor fermentation product extraction technology. There is a lack of research on improving the metabolic pathway of L-isoleucine in recombinant strains as probiotics to treat diseases. There is no existing technology about modified probiotics for improving the symptoms of ulcerative colitis, obesity and fatty liver. Summary of the invention

[0005] In order to solve the problems pointed out in the above background technology, the purpose of the present invention is to propose a preparation method of probiotics based on synthetic biology, wherein the probiotics knock out ldhA, pta, poxB, adhE, pflB, mgsA, frdA, dadA, avtA and ilvE genes;

[0006] Overexpression of ilvAIH, ilvE and ygaZH genes.

[0007] Preferably, the probiotics are used in the preparation of a medicine for treating ulcerative colitis or improving obesity and fatty liver.

[0008] Preferably, the probiotics may be microorganisms or other organisms including but not limited to wild or genetically engineered Escherichia coli, Bacillus, Yeast, Corynebacterium, Brevibacterium or Streptomyces.

[0009] Preferably, the probiotics are modified to maximize the concentration of carbon metabolic flow to pyruvate and direct it to isoleucine, and the production pathway of miscellaneous acid and alcohol metabolic byproducts represented by ethanol, acetic acid and lactic acid in the engineered strain is weakened or blocked.

[0010] Preferably, the method for constructing the probiotics includes plasmid expression, genome integration such as homologous recombination mediated by CRISPR, lambda-red, phage, etc.

[0011] Preferably, each gene in the overexpressed genes has a single copy or more than two copies.

[0012] Preferably, the overexpressed gene is expressed via a plasmid.

[0013] Preferably, the treatment of colorectal inflammation includes reducing mucosal damage, alleviating shortening of colon length, or reducing the content of pro-inflammatory factors including TNFα, IL 1β, and IL 6 in the colon.

[0014] Preferably, the improvement of obesity and fatty liver is to improve the symptom phenotype of obesity and fatty liver, and the improvement of the symptom phenotype of obesity and fatty liver includes: (1) reducing body weight; (2) reducing fat mass; (3) improving the fatty liver phenotype under the pathological condition of obesity; (4) reducing the level of inflammation.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention further expresses threonine endonuclease carriers of different sources and types in isoleucine production strains to improve the ability of cells to absorb threonine, thereby increasing the yield and content of L-isoleucine in fermentation products and reducing the relative yield and content of byproduct heteroacids (valine); furthermore, the extrinsic carriers for threonine excretion from cells are deleted to obtain genetically engineered high-yield strains. The genetically engineered strains are fermented and cultured, threonine is added during the culture process, and L-isoleucine is separated from the culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The probiotic eEcN is shown to alleviate the intestinal inflammation symptoms of mice. A is a graph showing the effect of eEcN on the colon length of mice with colorectal inflammation; B is a graph showing the effect of eEcN on the content of proinflammatory factors in the colon of mice with colorectal inflammation; C is a graph showing the effect of eEcN on the pathological score of the colon tissue of mice with colorectal inflammation.

[0018] Figure 2 It is shown that the probiotic eEcN improves the symptoms of obesity and fatty liver in mice. A is a picture of liver tissue HE staining; B is the effect of eEcN on the fat mass (left picture) or lean mass (right picture) of obese mice, C is the effect of eEcN on the body weight of obese mice; D is the effect of eEcN on the blood white blood cell (WBC) or neutrophil (Gran) level of obese mice (involving a range of 0.1-1.8). DETAILED DESCRIPTION

[0019] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0020] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0021] The articles "a" and "the" are used herein to refer to one or more than one of the grammatical objects of the article. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof in the alternatives. The term "and / or" should be understood to mean one or two of the alternatives. As used herein and unless otherwise specified, the term "about" refers to a measurable value such as an amount, a time period, etc., indicating a variation of ±10% from a given value, more preferably ±5%, even better ±1%, and still more preferably ±0.1%, as long as such variation is suitable for implementing the disclosed method.

[0022] As used herein, the term "gene synthesis" refers to the production of recombinant DNA technology or the acquisition of synthetic DNA or amino acid sequence technology available and known in the art. "Encoding" refers to the inherent properties of other polymers and macromolecules synthesized in biological processes as templates by specific sequences of nucleotides in polynucleotides such as genes, cDNAs or mRNAs, which have any of the defined sequences of nucleotides (i.e., rRNA, tRNA and mRNA) or defined sequences of amino acids and the biological properties produced therefrom. Therefore, if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system, the gene encodes a protein. The coding strands that are equivalent to the mRNA sequence and are usually provided in the sequence table, and the non-coding strands used as templates for transcribed genes or cDNAs, can both be referred to as encoding proteins or other products of that gene or cDNA.

[0023] As used herein, the term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0024] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside of an organism, cell, tissue or system.

[0025] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0026] As used herein, the term "threo / serine endorporter" includes a threo / serine-hydrogen ion symporter, a serine-hydrogen ion symporter, a threo / serine transporter, a threonine transporter, and a threo / serine permease.

[0027] Unless otherwise specified, "a polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding the protein may contain intron(s) in certain versions.

[0028] As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from a recombinant library or a cell genome using cloning techniques, or using synthetic means.

[0029] Any of the aforementioned polynucleotide molecules may also include one or more of a threonine dehydratase gene (e.g., tdcB gene), a threonine transporter gene (e.g., tdcC gene), an export protein gene (e.g., Escherichia coli export protein ygaZH gene, Corynebacterium glutamicum export protein brnEF gene), an acetohydroxyacid isomerase gene (e.g., ilvC gene), a dihydroxyacid dehydratase (e.g., ilvD gene), a branched-chain amino acid aminotransferase (e.g., ilvE gene), and a branched-chain amino acid dehydrogenase (e.g., leuDH gene). The gene may be a wild-type or mutant type. For example, in one embodiment, the ilvC gene may be a wild-type ilvC or a mutant ilvGm. The mutant ilvGm refers to a mutant gene of ilvC that uses NADH as a coenzyme. The mutant ilvC is different from the wild-type ilvC that uses NADPH as a coenzyme. The mutant ilvC carries mutations of L67E, R68F, and K75E.

[0030] In one embodiment, the threonine dehydratase (tdcB), dihydroxyacid dehydratase (ilvD), branched-chain amino acid aminotransferase (ilvE) and branched-chain amino acid export protein ygaZH can be derived from the Escherichia coli genome.

[0031] In one embodiment, the branched-chain amino acid dehydrogenase (leuDH) can be derived from Bacillus subtilis. In one embodiment, the branched-chain amino acid export protein (brnEF) can be derived from Corynebacterium glutamicum.

[0032] As known to those skilled in the art, the above-mentioned gene can be obtained by PCR reaction using DNA primers. Wherein, the gene (tdcC) gene of the threonine transporter directly affects the flux of threonine entering the cell, and its expression intensity can be carefully regulated to achieve the optimal expression of isoleucine. And the control of the expression intensity of the threonine transporter gene can be achieved by using promoters of different strengths (such as constitutive promoters or inducible promoters), using inducers of different concentrations (for inducible promoters) or using different types of plasmids (free outside the chromosome), and integrating in different positions in the genome. Therefore, in one embodiment of the present invention, it is related to a vector, comprising any of the aforementioned polynucleotide molecules, and the vector also comprises common elements, including but not limited to one or more of the origin of replication (ORI), antibiotic resistance gene, multiple cloning site (MCS), promoter, enhancer, primer binding site, and selection marker.

[0033] In various illustrative embodiments, the polynucleotides herein include, but are not limited to, polynucleotides comprising expression vectors, viral vectors, transfer plasmids, expression cassettes, and polynucleotides encoding polypeptides of cytokine antibodies or antibody fragments or antigen binding fragments.

[0034] As disclosed herein or as known in the art, regardless of the length of the coding sequence itself, the polynucleotide can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites, stop codons, transcription termination signals, post-transcriptional response elements, and polynucleotides encoding self-cleaving polypeptides, epitope tags, so that its overall length can vary significantly. Therefore, polynucleotide fragments of almost any length can be used, and the total length is preferably limited by ease of preparation and use in the intended recombinant DNA protocol.

[0035] As used herein, the term "vector" is a material composition that includes an isolated nucleic acid, and it can be used to transfer an isolated nucleic acid to the interior of a cell. The nucleic acid transferred is usually connected to, for example, inserted into a carrier nucleic acid molecule. The vector may include a sequence that guides the autonomous replication in the cell or may include a sequence that is sufficient to allow integration into the host cell DNA. Many vectors are known in the art, including but not limited to plasmids, phagemids, artificial chromosomes, bacteriophages, and animal viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses.

[0036] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are those non-translated regions of the vector—origins of replication, selection cassettes, promoters, enhancers, translation start signal introns, post-transcriptional regulatory elements, polyadenylation sequences, 5' and 3' untranslated regions—which interact with host cell proteins for transcription and translation. The length and specificity of such elements can vary. Depending on the vector system and host utilized, any number of suitable transcription and translation elements can be used, including ubiquitous promoters and inducible promoters. In a specific embodiment, the polynucleotide is a vector including, but not limited to, expression vectors and viral vectors and comprises exogenous, endogenous or heterologous control sequences, such as promoters and / or enhancers. "Endogenous" control sequences are sequences naturally linked to a given gene in a genome. "Exogenous" control sequences are sequences that are placed in juxtaposition with a gene by genetic manipulation (i.e., molecular biotechnology) so that transcription of that gene is directed by the connected enhancer / promoter. "Heterologous" control sequences are exogenous sequences from species different from the genetically manipulated cell. "Synthetic" control sequences can include one or more elements of endogenous and / or exogenous sequences and / or sequences determined in vitro or in silico to provide optimal promoter and / or enhancer activity for a particular gene therapy. As used herein, the term "promoter" refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to a promoter.

[0037] The term "promoter" refers to a fragment of DNA containing sequences capable of providing promoter functions.

[0038] The term "conditional expression" may refer to any type of conditional expression, including but not limited to: inducible expression; repressible expression; expression in cells or tissues having a specific physiological, biological or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, expression is controlled by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes the polynucleotide to be expressed or that increases or decreases the expression of a polynucleotide encoded by the polynucleotide of interest.

[0039] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid or estrogen receptors, metallothionein promoters; MX-1 promoter, the "gene switch" mifepristone-regulatable system, tetracycline-dependent regulatory systems, etc.

[0040] In certain embodiments, the genetically modified cell comprises a polynucleotide further comprising a positive marker for the selection of a cell belonging to an external negative selective phenotype. The positive selective marker can be a gene that, when introduced into a host cell, expresses a dominant phenotype that allows positive selection of cells carrying the gene. This type of gene is known in the art.

[0041] In one embodiment, the positive selectable marker and the negative selectable marker are linked so that loss of the negative selectable element is also accompanied by loss of the positive selectable marker. In a specific embodiment, the positive and negative selectable markers are fused so that loss of one necessarily results in loss of the other.

[0042] As used herein, the term "transfection" or "conversion" or "transduction" refers to a process by which exogenous nucleic acids are transferred or introduced into a recipient strain. A "transfected" or "converted" or "transduced" strain is a strain that has been transfected, transformed or transduced with exogenous nucleic acids. The strain includes a primary generation and its progeny. The recipient strain can be Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Vibrionatriegens, Bacillus amyloliquefaciens, or Saccharomyces cerevisiae, etc.

[0043] In the present specification, the ilvA gene encoding the threonine dehydratase that is substantially relieved of the inhibition by L-isoleucine is referred to as a "relieving type ilvA gene".

[0044] Materials and Methods

[0045] LB medium: 1% peptone, 0.5% yeast extract and 1% NaCl, adjusted to pH 7.2 with 30% NaOH, sterilized at 1×105Pa for 20 min, and 1.5% agar was added when making plates. When screening for resistance, a final concentration of 100 mg / L ampicillin and / or 50 mg / L kanamycin was added according to the type of resistance gene.

[0046] Shake flask fermentation medium: glucose 40g / L, yeast powder 5g / L, ammonium sulfate 10g / L, magnesium sulfate 2mM, calcium chloride 0.1mM, potassium dihydrogen phosphate 0.8g / L, manganese sulfate pentahydrate 0.02g / L, threonine 25g / L, betaine 1g / L, nicotinic acid 0.01g / L, VB1 0.005g / L, IPTG 0.3mM and calcium carbonate 10g / L. The pH of the culture medium was adjusted to 7.3-7.4 with concentrated ammonia water and sterilized at 11°C for 15min.

[0047] Shake flask fermentation method: Pick a single clone of the activated bacteria from the LB plate (containing an appropriate concentration of antibiotics) at 37°C and inoculate it into LB liquid culture medium (containing an appropriate concentration of antibiotics), culture it at 37°C for 12-16h, at a rotation speed of 220rpm, inoculate the LB overnight culture at a 1% inoculum amount into a 250ml shake flask (containing 10g / L CaCO3 as a pH stabilizer) containing 25ml fermentation medium containing an appropriate concentration of ampicillin and kanamycin, seal it with a breathable membrane, and culture it at 30°C and 220rpm. When the OD600 reaches 0.6, add IPTG (final concentration of 0.2mM) to induce plasmid expression. Stop the fermentation after 40 hours of culture and take samples.

[0048] Determination of amino acid concentration: Amino acid standards were purchased from Sigma-Aldrich (www.sigmaaldrich.cn). Take 1 mL of fermentation broth, centrifuge at 10,000 r / min for 5 min to remove the bacteria, filter the filtrate through a filter membrane with a pore size of 0.22 μm, dilute it to an appropriate multiple, and use high-performance liquid chromatography HPLC (https: / / www.agilent.com / library / applications / 5990-4547EN.pdf) to determine the concentration of branched-chain amino acids in the sample. Among them, the high-performance liquid chromatograph is Shimadzu Nexera LC-40, the chromatographic column is Agilent ZORBAX Eclipse Plus C18, 4.6×250mm 5μm; the detector is a DAD diode array detector, the detection wavelength is 338nm, and the reference wavelength is 390nm; the mobile phase composition, ratio change, flow rate and column temperature are all set according to the above method.

[0049] The enzyme types involved in the present invention are shown in Table 1.

[0050] Table 1

[0051]

[0052] The biomaterials constructed by the present invention are shown in Table 2.

[0053] Table 2

[0054]

[0055] Example 1 Construction of isoleucine high-yielding strain and plasmid

[0056] Using λ-Red recombination technology, the pflB gene encoding pyruvate-formate lyase in the genome of Escherichia coli K bacteria background strain BW25113 was knocked out and replaced with the kanamycin resistance gene Kan to obtain the recombinant strain BW25113ΔpflB::Kan. The plasmid pCP20 was transformed into the above-mentioned Kan-resistant transformant, spread on an LB plate containing 100 mg / L ampicillin, and cultured at 30°C for 24 hours. The correct transformant in which the kanamycin resistance gene Kan gene was removed in the target gene site was identified by PCR to obtain the non-resistant Escherichia coli BW25113ΔpflB. By analogy, the same method and strategy were used to obtain strain SP7 (BW25113ΔldhAΔptaΔpoxBΔadhEΔpflBΔmgsAΔdadAΔavtAΔilvE), in which most of the genes of mixed acid fermentation of Escherichia coli were knocked out, which can prevent the cells from producing by-products such as miscellaneous acids during the fermentation process that interfere with the substrate conversion rate. The absence of pflB can reduce the decomposition of the precursor 2-ketobutyrate, which is beneficial to direct the isoleucine anabolic flow starting from threonine to the synthesis of isoleucine.

[0057] The ygaZH and ilvAIH fragments (encoded deregulated IlvA protein with L447F and L451A mutations; ilvH gene with G14D and S17F mutations) were amplified from plasmids pZE-ygaZH and pZA-ilvAIH (plasmids derived from Chinese patent CN114410701A) by PCR, and the gene fragments were seamlessly connected to the pZElac vector PCR fragment in one step using a recombinant cloning kit to obtain the recombinant plasmid pZE-ilvAIHygaZH (pZE-SP), and the above recombinant plasmid combination was transformed into the SP strain. For details of specific plasmids and strains, please refer to the Materials and Methods section.

[0058] Example 2 Shake flask fermentation of high-yield L-isoleucine probiotics

[0059] The plasmid pZE-SP was transferred into the starting strain BW25113, and the obtained recombinant strain ILE-SP and the starting strain ILE-BW were fermented according to the shake flask fermentation method, with the starting strain ILE-BW as the control, and the results are shown in Table 3. It can be seen that compared with the starting strain ILE-BW, the isoleucine expression level of the recombinant strain ILE-SP was significantly increased.

[0060] Table 3

[0061] Strain name ILE(g / L) ILE relative yield VAL(g / L) ILE / VAL ILE-BW 13.91±0.56 1 5.65±0.23 2.50 ILE-SP 16.70±0.35 1.21 2.74±0.05 6.08

[0062] Example 3 Application of probiotics in alleviating disease symptoms in mice with colorectal inflammation induced by dextran sulfate sodium (DSS)

[0063] Twenty-four healthy male C57BL / 6J mice weighing 18-22g were randomly divided into four groups with 6 mice in each group. The bacterial solution 20h before the end of the shake flask fermentation of the above-mentioned recombinant strain was used as the source of probiotics: blank group (conventional drinking water + intragastric administration of normal saline), model group (2% DSS drinking water + intragastric administration of normal saline), probiotic intervention group EcN (2% DSS drinking water + intragastric administration of ILE-BW bacterial solution), probiotic control group eECN (2% DSS drinking water + intragastric administration of ILE-SP bacterial solution). A cycle of 2% DSS drinking water for 5 days + regular drinking water for 7 days was repeated three times in total. Mice in the model group, probiotic intervention group and probiotic control group developed symptoms of chronic enteritis. The mice were gavaged once every two days, and the gavage dose of the probiotic intervention group and the probiotic control group was 5×10 9 CFU / mouse, and the other groups were gavaged with an equal volume of normal saline.

[0064] During the modeling period (i.e., during the treatment with DSS), the disease activity index (DAI) of the mice was calculated every two days based on the weight, stool characteristics and blood in the stool of the mice. Specifically, the disease activity index is combined with the percentage of weight loss of the animals (unchanged weight is 0, 1-5 is 1 point, 5-10 is 2 points, 10-15 is 3 points, and more than 15 is 4 points), stool viscosity (normal is 0, loose stool is 2 points, and diarrhea is 4 points) and stool bleeding (normal is 0, occult blood is 2 points, and overt bleeding is 4 points) to make a comprehensive score. The total score of the three results is divided by 3 to obtain the DAI value. That is, DAI = (body weight index + stool shape + bleeding) / 3.

[0065] After the mice were killed on day 36, the length of the colon was measured and recorded. The colorectal tissues were taken and the content of pro-inflammatory factors was detected by RT-PCR.

[0066] The experimental results are as follows Figure 1 As shown, probiotics can alleviate the symptoms of enteritis in mice, including reducing colon shortening ( Figure 1 A) Reducing pro-inflammatory cytokines ( Figure 1 B) and reduction in DAI disease scores ( Figure 1 C).

[0067] Example 4 Application of eEcN in improving obesity and fatty liver phenotype in mice

[0068] Twenty-two healthy male C57BL / 6J mice (18-22 g in weight) aged 4-5 weeks were randomly assigned to a control group of 6 mice, and the remaining 16 mice were fed a 60% (kcal) high-fat purified diet (HFD) for obesity modeling. The process lasted for 8-12 weeks until the mouse obesity model was successfully induced, and the mice with successful modeling were randomly and evenly assigned to three groups. At this point, the mice were divided into four groups: control group (normal diet group, gavaged with normal saline at the same time as the model group, referred to as ND); model group (HFD modeling, gavaged with normal saline after successful modeling, referred to as HFD), eEcN intervention group (HFD modeling, gavaged with eEcN after successful modeling, referred to as HFD+eEcN), EcN control group (HFD modeling, gavaged with EcN after successful modeling, referred to as HFD+EcN. Gavage was performed every four days, and the gavage dose of eEcN and EcN was 1×109 CFU / mouse. The control group and model group were gavaged with equal volumes of normal saline.

[0069] During the intragastric administration, the mice were weighed and recorded every four days. After 32 days of intragastric administration, all mice were placed in the EchoMRI small animal body composition analyzer for body fat composition detection, and the lean mass and fat mass of the mice were recorded. The mice were then euthanized and blood was collected. The whole blood samples were used to test the levels of white blood cells and neutrophils, and the liver tissue was taken for HE staining to observe the fatty liver phenotype of the mice.

[0070] The experimental results are as follows Figure 2 As shown, eEcN can reduce the body weight and fat mass of obese mice, improve the fatty liver phenotype under the pathological condition of obesity in mice, and reduce the level of systemic inflammation in obese mice.

[0071] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A preparation method of a synthetic biology probiotic, wherein the probiotic knocks out ldhA, pta, poxB, adhE, pflB, mgsA, frdA, dadA, avtA and ilvE genes; Overexpression of ilvAIH, ilvE and ygaZH genes.

2. The preparation method of synthetic biology-based probiotics according to claim 1, wherein the probiotics are used in the preparation of drugs for treating ulcerative colitis or improving obesity and fatty liver.

3. The preparation method based on synthetic biology probiotics according to claim 1 or 2, wherein the probiotics can be microorganisms or other organisms including but not limited to wild or genetically engineered Escherichia coli (E. coli), Bacillus (Bacillus), yeast (Yeast), Corynebacterium (Corynebacterium), Brevibacterium (Brevibacterium) or Streptomyces (Streptomyces).

4. According to the preparation method of synthetic biology probiotics according to claim 1, the probiotics are modified to maximize the carbon metabolic flow to pyruvate and direct it to isoleucine, and the production pathway of miscellaneous acid and alcohol metabolic byproducts represented by ethanol, acetic acid, and lactic acid in the engineered strain is weakened or blocked.

5. The method for preparing a synthetic biology probiotic according to any one of claims 1 to 4, wherein the probiotic construction method comprises plasmid expression, genome integration such as homologous recombination mediated by CRISPR, lambda-red, phage, etc.

6. According to the preparation method of synthetic biology probiotics according to any one of claims 1 to 5, each of the overexpressed genes has a single copy or more than two copies.

7. According to the preparation method based on synthetic biology probiotics according to any one of claims 1 to 6, the overexpressed gene is expressed by a plasmid.

8. According to any one of claims 2-7, the preparation method based on synthetic biology probiotics, the treatment of colorectal inflammation includes reducing mucosal damage, alleviating shortening of colon length, or reducing the content of pro-inflammatory factors including TNFα, IL 1β, IL 6 in the colon.

9. According to any one of claims 2-7, the preparation method based on synthetic biology probiotics, wherein the improvement of obesity and fatty liver is to improve the symptom phenotype of obesity and fatty liver, and the improvement of the symptom phenotype of obesity and fatty liver comprises: (1) Lose weight; (2) Reduce fat mass; (3) Improve the fatty liver phenotype in obesity pathological conditions; (4) Reduce inflammation levels.

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