Engineering bacterium for degrading methylmalonic acid as well as construction method and application of engineering bacterium

Abstract

The invention discloses an engineering bacterium for degrading methylmalonic acid as well as a construction method and application of the engineering bacterium, and belongs to the technical field of biological medicines. Escherichia coli BL21 (DE3) is used as a protein expression host, and an expression vector containing exogenous genes (ACSF3 and MUT) is introduced through a genetic engineering technology, so that the enzyme-linked preparation for degrading methylmalonic acid is produced, and the enzyme-linked preparation has excellent degradation capacity; the enzyme-linked preparation can degrade methylmalonic acid of a patient in the digestive tract, reduce methylmalonic acid in the intestinal tract and relieve metabolic acidosis and nutrient absorption disorder of the patient. Meanwhile, the enzyme-linked preparation converts methylmalonic acid into succinyl coenzyme A which plays an important role in a human body, has relatively high safety and application value, and can be well applied to methylmalonic acid reducing medicines or health care products and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to an engineered bacterium for degrading methylmalonic acid, and a construction method and application thereof. Background Art

[0002] Methylmalonic acid (MMA) is a byproduct of the propionate metabolic pathway and is usually used as a clinical test marker for vitamin B12 deficiency. An increase in MMA levels in the human body is caused by a variety of factors, including but not limited to gene mutations related to the propionate metabolic pathway, gut microbiota dysbiosis, and cancer.

[0003] Methylmalonic acidemia belongs to a rare disease of amino acid metabolism disorders. This disease is related to mutations in genes encoding enzymes in the propionate metabolic pathway, including the MUT gene. Patients show excessive accumulation of MMA, methylmalonyl coenzyme A, 2-methylcitric acid, 2-hydroxypropionic acid, propionylcarnitine, and a lack of succinyl coenzyme A. At the same time, this disease is accompanied by mitochondrial dysfunction and is toxic to organs such as the brain, liver, and kidneys.

[0004] Although patients with methylmalonic acidemia can receive optimal diet and cofactor therapy, they still have a risk of metabolic instability or even life-threatening conditions and suffer from long-term complications.

[0005] Conventional treatment methods for methylmalonic acidemia include diet management, vitamin B12 supplementation, conventional drug therapy, dialysis, and liver transplantation. Diet management requires patients to restrict protein intake and consume nutritional supplements, which may lead to malnutrition and affect the quality of life of patients; the method of vitamin B12 supplementation is only effective for patients with absorption or utilization disorders of B12, and the supplementation effect varies among individuals; drug therapy, such as taking L-carnitine, can help promote fat metabolism and remove excess toxic components, but the dosage is limited and side effects may occur; severe patients can undergo dialysis and liver transplantation. The former needs to be carried out regularly, causing an economic burden on patients and their families. The latter has risks such as infection and rejection, and long-term intervention management is required after surgery.

[0006] Currently, emerging treatment strategies include two gene therapy strategies - adeno-associated virus (AAV) therapy and mRNA therapy. Gene therapy uses viruses or non-viruses as vector tools to deliver genes into cells and express specific proteins, thereby replacing defective genes. Viral vector delivery has high transfer efficiency, but high immunogenicity and high cost; non-viral vector delivery (such as exosomes, lipid carriers, nanoparticles, etc.) has lower cost and simple operation methods, but low transfection efficiency, short expression time of foreign genes in host cells, and high non-specific targeting. In short, it is impossible to have both high delivery efficiency and low immunogenicity in gene therapy, and the cost is high. In addition, this treatment requires long-term intravenous injection for patients, increasing the pain of patients, which limits the clinical application of gene therapy. Therefore, it is of great significance to develop new methods for treating methylmalonic acidemia.

[0007] Probiotics and engineered bacteria have shown great potential in the treatment of metabolic diseases. Currently, patients in clinical practice regulate blood sugar and treat type 2 diabetes by orally taking probiotics such as Lactobacillus and Bifidobacterium. More and more studies have constructed Escherichia coli Nissle 1917 engineered bacteria expressing urate oxidase gene to degrade uric acid in the mouse intestine, and at the same time reduce the uric acid content in the mouse blood, which is expected to be used to maintain the blood uric acid concentration of patients. At the same time, there are products that construct engineered bacteria expressing purine-degrading enzymes to degrade purines in food and the digestive tract to control the uric acid content in the blood. This indicates that constructing engineered bacteria to express enzyme preparations is expected to reduce the concentration of methylmalonic acid in the blood and urine of patients, but there is currently a lack of relevant research and applications. Summary of the Invention

[0008] In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide an engineered bacterium for degrading methylmalonic acid, its construction method and application.

[0009] The engineered bacterium heterologously expresses malonate-CoA ligase (ACSF3) and methylmalonyl-CoA mutase (MUT), and uses this combined enzyme preparation to degrade methylmalonic acid to generate succinyl-CoA, which plays an important role in the human body. And it has been experimentally proven that it has excellent degradation ability in vitro and can be well applied as a drug or health product for reducing methylmalonic acid.

[0010] The purpose of the present invention is achieved by the following technical solutions:

[0011] The first aspect of the present invention provides an application of methylmalonyl-CoA ligase ACSF3 in the preparation of a product for degrading methylmalonic acid or in the preparation of a product for treating methylmalonic academia.

[0012] The second aspect of the present invention provides an application of the combination of methylmalonyl-CoA ligase ACSF3 and methylmalonyl-CoA mutase MUT in the preparation of a product for degrading methylmalonic acid or in the preparation of a product for treating methylmalonic academia.

[0013] The methylmalonyl-CoA ligase ACSF3 includes at least one of ACSF3 bj ACSF3 hk ACSF3 nnj and ACSF3 human

[0014] The ACSF3 bj is derived from the beluga whale (Delphinapterus leucas), and its amino acid sequence is as shown in UniProt Accessions: A0A2Y9NUE1, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO: 4.

[0015] The ACSF3 hk is derived from the sea anemone (Actinia tenebrosa), and its amino acid sequence is as shown in SEQ ID NO: 1, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO: 5.

[0016] The ACSF3 nnj is derived from Arabidopsis thaliana, and its amino acid sequence is as shown in UniProt Accessions: Q8H151, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO: 6.

[0017] The ACSF3 human is derived from Homo sapiens, and its amino acid sequence is as shown in SEQ ID NO: 2, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO: 7.

[0018] The methylmalonyl-CoA mutase MUT includes at least one of MUT dxb MUT mxc MUT k12 and MUT human ; further includes at least one of MUT dxb and MUT human

[0019] The MUT​​dxb derived from Stygiella incarcerata, with its amino acid sequence shown as UniProt Accessions: A0A192ZIA6, and the nucleotide sequence of its encoding gene shown as SEQ ID NO: 8.

[0020] The MUT mxc derived from Trichinella nativa, with its amino acid sequence shown as UniProt Accessions: A0A0V1LU09, and the nucleotide sequence of its encoding gene shown as SEQ ID NO: 9.

[0021] The MUT k12 derived from Escherichia coli (strain K12), with its amino acid sequence shown as UniProt Accessions: P27253, and the nucleotide sequence of its encoding gene shown as positions 822881 - 820737 bp in CP097882.1.

[0022] The MUT human derived from Homo sapiens, with its amino acid sequence shown as SEQ ID NO: 3, and the nucleotide sequence of its encoding gene shown as SEQ ID NO: 10.

[0023] Preferably, the co - use of methylmalonyl - CoA ligase ACSF3 and methylmalonyl - CoA mutase MUT includes the co - use of ACSF3 and MUT in series through an RBS or a linker peptide.

[0024] Preferably, the nucleotide sequence of the RBS is AAGGAG; the amino acid sequence of the linker peptide is GGGGS, and its nucleotide sequence is GGTGGAGGCGGTTCA.

[0025] In the third aspect of the present invention, there is provided an engineered bacterium for degrading methylmalonic acid, which is obtained by performing any one of the following operations on an Escherichia coli starting strain:

[0026] (1) Heterologous expression of methylmalonyl - CoA ligase ACSF3; or,

[0027] (2) Heterologous expression of methylmalonyl - CoA ligase ACSF3 and methylmalonyl - CoA mutase MUT; wherein, ACSF3 and MUT are heterologously expressed in series through an RBS or a linker peptide;

[0028] The Escherichia coli starting strain includes Escherichia coli BL21(DE3).

[0029] The fourth aspect of the present invention provides a method for constructing the engineered bacterium for degrading methylmalonic acid, comprising the following steps:

[0030] S1. Based on the Escherichia coli starting strain, heterologously express the above-mentioned methylmalonyl-CoA ligase ACSF3 to construct a single-enzyme engineered bacterium;

[0031] S2. Based on the Escherichia coli starting strain, tandemly express the methylmalonyl-CoA ligase ACSF3 gene and the methylmalonyl-CoA mutase MUT gene through RBS or a linker peptide, and then heterologously express them to construct a tandem-enzyme engineered bacterium;

[0032] Wherein, the tandem is a ribosome (RBS) tandem or a linker peptide tandem.

[0033] The fifth aspect of the present invention provides the above-mentioned method for verifying heterologous expression of the enzyme to verify the existence of the above-mentioned enzyme and identify its function.

[0034] The verification method includes: S1. Provide a liquid fermentation culture of the engineered strain, add an inducer, and then induce the translation and expression of the heterologous protein at a low temperature; S2. Perform SDS-PAGE experiments to verify the protein expression level and molecular weight.

[0035] Among them, ACSF3 in the above-mentioned enzyme gene bj 、ACSF3 hk 、MUT dxb 、MUT mxc The existence and function of these translated proteins / enzymes have not been strictly proven. The method for confirming the existence of the enzyme is only to infer the existence based on orthologous species or based on gene transcripts. The present invention provides a more accurate experimental basis for the existence of the above-mentioned enzyme.

[0036] The sixth aspect of the present invention provides the application of the above-mentioned engineered bacterium for degrading methylmalonic acid in the preparation of products for degrading methylmalonic acid or in the preparation of products for treating methylmalonic acidemia.

[0037] The products include at least one of drugs, health foods, food additives, etc.

[0038] The present invention has the following advantages and effects compared with the prior art:

[0039] The present invention uses Escherichia coli BL21(DE3) as the protein expression host, and introduces an expression vector containing foreign genes (ACSF3 and MUT) through genetic engineering technology, thereby producing an enzyme preparation for degrading methylmalonic acid, and having excellent degradation ability; the enzyme preparation can degrade methylmalonic acid of patients in the digestive tract, reduce methylmalonic acid in the intestine, and relieve metabolic acidosis and nutritional absorption disorders of patients. At the same time, the enzyme preparation converts methylmalonic acid into succinyl coenzyme A that plays an important role in the human body, and has high safety and application value. Brief Description of the Drawings

[0040] Figure 1 It is the similarity analysis of the amino acid sequences of enzymes from different species sources in the present invention.

[0041] Figure 2 It is the structural schematic diagram of the expression vector of the enzyme gene from different species sources in the present invention.

[0042] Figure 3 It is the SDS-PAGE result diagram of the single enzyme obtained by constructing the engineering bacteria in the present invention; among them, M is Marker; (a): 2, 4, 6, 8 are the supernatant of the broken cells of the uninduced fermentation bacteria; 1, 3, 5, 7 are the supernatant of the broken cells of the induced fermentation bacteria; 1-2: ACSF3 hk Crude enzyme solution; 3-4: ACSF3 nnj Crude enzyme solution; 5-6: ACSF3 bj Crude enzyme solution; 7-8: ACSF3 human Crude enzyme solution; (b): Supernatant of the broken cells of the induced fermentation bacteria, 1: Crude enzyme solution of the control group; 2: MUT dxb Crude enzyme solution; 3: MUT mxc Crude enzyme solution; 4: MUT k12 Crude enzyme solution; 5: MUT human Crude enzyme solution; (c): 1: ACSF3 bj Purified enzyme solution; 2: ACSF3 bj Crude enzyme solution. Note: The crude enzyme solution of the control group is the supernatant of the broken cells of the induced fermentation BL21(DE3)-pET-28a(+).

[0043] Figure 4 It is the effect of methylmalonic acid degradation and succinyl coenzyme A production of the single enzyme obtained by constructing the engineering bacteria in the present invention in vitro.

[0044] Figure 5 It is the schematic diagram of different tandem ways of the two enzymes in the present invention.

[0045] Figure 6It is the SDS-PAGE result diagram of the tandem enzyme obtained by constructing the engineering bacteria in the present invention; wherein, M: protein Marker; (a): 1: empty pET-28a(+); 2: l-bd; 3: l-bm; 4: l-hd; 5: l-hm; 6: l-nd; 7: l-nm; 8: l-ad; 9: l-am; (b): 1: empty pET-28a(+); 2: r-bd; 3: r-bm; 4: r-hd; 5: r-hm; 6: r-nd; 7: r-nm; 8: r-ad; 9: r-am.

[0046] Figure 7 It is the effect of methylmalonic acid degradation by the tandem enzyme obtained by constructing the engineering bacteria in the present invention in vitro.

[0047] Figure 8 It is the effect of the tandem enzyme obtained by constructing the engineering bacteria in the present invention in generating succinyl coenzyme A in an in vitro reaction.

[0048] Figure 9 It is the schematic flow diagram of the degradation of methylmalonic acid by ACSF3 enzyme and MUT enzyme in the present invention. Detailed implementation manners

[0049] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the implementation manners of the present invention are not limited thereto.

[0050] For the test methods without specific experimental conditions noted in the following embodiments, they are usually in accordance with conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0051] In a specific implementation manner of the present invention, an engineering bacterium for degrading methylmalonic acid is provided, which is Escherichia coli BL21(DE3). A free expression vector is introduced into its cells, and any one or more of the methylmalonyl-CoA synthase gene ACSF3 and the methylmalonyl-CoA mutase gene MUT are inserted into the vector, and the tandem mode is RBS tandem or linker tandem. Among them, the nucleotide sequence of RBS is AAGGAG, and the amino acid sequence of the linker is GGGGS, and its nucleotide sequence is GGTGGAGGCGGTTCA.

[0052] The methylmalonyl-CoA ligase provided by the present invention specifically includes the enzyme ACSF3 bj , ACSF3 hk , ACSF3 nnj , ACSF3 human One or more of them, and its amino acid sequence and nucleotide sequence are shown in Table 2.

[0053] In the present invention, the methylmalonyl-CoA ligase is derived from Delphinapterus leucas, Actinia tenebrosa, Arabidopsis thaliana, and Homo sapiens respectively. By predicting the enzyme activity using its amino acid sequence, it is found that it has excellent ability to catalyze the degradation of methylmalonic acid.

[0054] More specifically, the enzyme ACSF3 nnj has a homology lower than 39.21% with other amino acid sequence fragments shown in Table 2, such as 39.21%, 39.05%, 36.6%. The enzyme ACSF3 hk has a homology lower than 49.21% with other amino acid sequence fragments shown in Table 2, such as 49.21%, 44.11%, 39.21%. The enzyme ACSF3 bj has a homology lower than 74.85% with other amino acid sequence fragments shown in Table 2, such as 74.85%, 44.11%, 36.60%. The enzyme ACSF3 human has a homology lower than 74.85% with other amino acid sequence fragments shown in Table 2, such as 74.85%, 49.21%, 39.05%. The schematic diagram of similarity analysis is as Figure 1 shown.

[0055] In some specific embodiments, the methylmalonyl-CoA ligase ACSF3 is preferably ACSF3 bj . At this time, the degradation rate of methylmalonic acid in vitro for 1 h reaches 2.45 μmol / h / μg.

[0056] The methylmalonyl-CoA mutase provided by the present invention specifically includes one or more of the enzymes MUT dxb , enzyme MUT mxc , enzyme MUT k12 , enzyme MUT human , and their amino acid sequences and nucleotide sequences are shown in Table 2.

[0057] In the present invention, the methylmalonyl-CoA mutase is derived from Stygiella incarcerata, Trichinella nativa, Escherichia coli (strain K12), and Homo sapiens respectively. By predicting the enzyme activity using their amino acid sequences, it is found that they have excellent ability to catalyze the conversion of methylmalonyl-CoA into succinyl-CoA. More specifically, among the amino acid sequences of the methylmalonyl-CoA mutase, any two have a homology lower than 70.62%, such as 70.62%, 66.39%, 61.89%, 59.66%, 59.49% or any value between them. The schematic diagram of similarity analysis is as shown in Figure 1 shown.

[0058] In some specific embodiments, the methylmalonyl-CoA mutase MUT is preferably MUT dxb . At this time, the in vitro succinyl-CoA production rate of the methylmalonyl-CoA mutase MUT reaches 19.87 μmol / h / μg in 1 h.

[0059] In the present invention, two tandem methods are used to tandem the methylmalonyl-CoA synthetase ACSF3 and the methylmalonyl-CoA mutase MUT to form a cascade reaction for further degrading methylmalonic acid in vitro. More specifically, the tandem methods are RBS tandem and linker tandem respectively. The tandem enzyme ACSF3 is any one of the 4 ACSF3s in Table 2, and the tandem enzyme MUT is the MUT in Table 2 dxb or MUT human .

[0060] Preferably, in some specific embodiments, the tandem method is RBS tandem, where the tandem enzyme ACSF3 is ACSF3 bj , and the tandem enzyme MUT is MUT dxb . The in vitro methylmalonic acid degradation rate of its crude enzyme solution in the cascade reaction reaches 0.835 μmol / h / mg in 1 h, and its rate of degrading methylmalonic acid in vitro is increased by 7.27 times compared with other tandem methods and / or different enzyme combinations.

[0061] In the present invention, the strain is constructed by using the gene engineering technology commonly used in the art. The construction method specifically includes: ligating the gene of the heterologous expressed protein to an expression vector, and transferring the recombinant expression vector into Escherichia coli BL21(DE3) for expression;

[0062] Among them, the recombinant expression vector is obtained by effectively connecting any one or more of the above gene sequences to an expression vector, and the expression vector is pET-28a(+); the transformation method is heat shock transformation. In the present invention, the gene of the heterologous expressed protein is obtained by specifically amplifying the genomes of Homo sapiens and Escherichia coli BW25113 (BW25113 is a K12-derived strain), or can be synthesized based on the relevant nucleotide sequences and by methods such as codon optimization, chemical synthesis, and PAGE purification. The above methods are all conventional technical means in the art, and no specific steps and conditions are particularly limited.

[0063] The present invention also provides the application of the above-mentioned engineered bacteria for degrading methylmalonic acid in the preparation of products for degrading methylmalonic acid, and the product for degrading methylmalonic acid can achieve the degradation of methylmalonic acid in the intestine. More specifically, specific examples of the product for reducing uric acid can be, but are not limited to, one or more of pharmaceuticals, health foods, and / or food additives.

[0064] The sources of the reagents, enzymes, and related reagent kits involved in the following examples are:

[0065] The bacterial genomic DNA extraction kit was purchased from Guangzhou Meiji Biotechnology Co., Ltd.;

[0066] The human genomic DNA extraction kit was purchased from Guangzhou Meiji Biotechnology Co., Ltd.;

[0067] The DNA polymerase for amplifying the target gene was purchased from Nanjing Novoprotein Scientific Inc., Takara Biotechnology (Beijing) Co., Ltd., and Beijing TransGen Biotech Co., Ltd.;

[0068] The DNA gel rapid purification kit was purchased from Guangzhou Meiji Biotechnology Co., Ltd.;

[0069] The DNA cloning reagent was purchased from Nanjing Novoprotein Scientific Inc.;

[0070] The homologous recombination reagent was purchased from Nanjing Novoprotein Scientific Inc.;

[0071] The bacterial lysate was purchased from Changzhou Boyi Biotechnology Co., Ltd.;

[0072] The enzyme purification kit was purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.;

[0073] The BCA protein content determination kit was purchased from Thermo Fisher Scientific;

[0074] The primers were purchased from Shanghai Sangon Biotech Co., Ltd.;

[0075] All chemical reagents are of analytical grade and are purchased from Sigma, Shanghai Macklin Biochemical Co., Ltd. and Beijing Solarbio Science & Technology Co., Ltd.

[0076] The schematic diagram of the process of ACSF3 enzyme and MUT enzyme degrading methylmalonic acid in the present invention is as Figure 9 shown.

[0077] Information on strains and plasmids involved in the specific implementation method:

[0078] Table 1 Information on plasmids or strains

[0079]

[0080]

[0081] Table 2 Gene information

[0082]

[0083] Note: SEQ ID NO: 4-6, 8-9 are all nucleotide sequences optimized by Escherichia coli codons.

[0084] Example 1

[0085] This example is used to illustrate the preparation of ACSF3 / MUT- strain and methylmalonic acid degrading enzymes ACSF3 and succinyl-CoA synthetase MUT, specifically including:

[0086] 1. Obtaining of heterologous expression genes

[0087] Enzyme gene ACSF3 bj 、ACSF3 hk 、ACSF3 nnj 、MUT dxb 、MUT mxc were codon-optimized and synthesized by Shanghai Bioengineering Co., Ltd., and the gene information is shown in Table 2.

[0088] Enzyme gene ACSF3 human 、MUT human were obtained by PCR amplification from human (homo sapiens) genomic DNA; enzyme gene MUT k12 was obtained by PCR amplification from Escherichia coli BW25113 genomic DNA. Among them, the extraction methods of human genome and Escherichia coli genome refer to the instruction manual of the extraction kit, and the gene information is shown in Table 2.

[0089] 2. Construction of expression vector

[0090] The pET-28a(+) plasmid was selected as the expression vector, and the T7 inducible promoter was used to express the target gene. A 6*His tag was ligated to the N-terminus of the target gene to facilitate the separation and purification of the translated target protein.

[0091] Using the method of homologous recombination commonly used in the field of molecular cloning, the target genes ACSF3 bj 、ACSF3 hk 、ACSF3 nnj 、MUT dxb 、MUT mxc were inserted between the sequences GGATCCGAATTC(5′-3′) and CGTCGACAAGCTTGCGGC(5′-3′) of the pET-28a(+) plasmid. The target genes ACSF3 human 、MUT human 、MUT k12 were inserted between the sequences GGATCCGAATTCGAGCTC(5′-3′) and GGCCGCACTCGAGCACCA(5′-3′) of the pET-28a(+) plasmid to obtain 8 single-gene expression plasmids, as shown in Table 1 for details. The schematic diagram of plasmid construction is shown in Figure 2 .

[0092] 3. Construction of engineering strains

[0093] Using E. coli BL21(DE3) as the host bacterium, the above expression vector was transferred into the host bacterium by heat shock. After overnight culture on an LB solid plate containing 50 μg / mL kanamycin sulfate, single colonies were picked and verified by colony PCR to screen for positive clones, namely, the engineering strains BL21(DE3)-pET28a-ACSF3 bj 、BL21(DE3)-pET28a-ACSF3 hk 、BL21(DE3)-pET28a-ACSF3 nnj 、BL21(DE3)-pET28a-ACSF3 human and BL21(DE3)-pET28a-MUT dxb 、BL21(DE3)-pET28a-MUT mxc 、BL21(DE3)-pET28a-MUT k12 、BL21(DE3)-pET28a-MUT human .

[0094] 4. Determination of the growth of engineering bacteria

[0095] The growth of the expression strains was detected using an automatic growth curve analyzer for 48 h (OD 600 ).

[0096] 5. Cultivation and induction of engineered bacteria

[0097] Streak plate the engineered bacteria, and take monoclonal colonies the next day to inoculate them into 5 mL of fresh liquid LB medium containing 50 μg / mL kanamycin sulfate, and culture them with shaking in an incubator at 37 °C and 200 rpm. The next day, measure the OD of the bacterial solution 600 And take 1 mL of the bacterial solution and inoculate it into a 250 mL conical flask containing 100 mL of fresh liquid LB medium and 50 μg / mL kanamycin sulfate. Culture it with shaking in an incubator at 37 °C and 200 rpm until the OD of the culture 600 reaches between 0.6 and 0.8, add isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, change the culture conditions to 16 °C and 150 rpm, and culture with shaking for 17 - 20 h.

[0098] 6. Preparation of crude enzyme and pure enzyme

[0099] Measure the OD of the induced culture 600 , and centrifuge the bacterial solution at 6000 rpm for 10 min at 4 °C to collect the bacterial cells. Keep the bacterial cell pellet, add 8 - 12 mL of 100 mM Tris-HCl buffer and vortex the bacterial cell pellet evenly, add 1 - 3 mL of Boyi Biotech bacterial lysis solution, and wait at room temperature for 10 - 30 min. Centrifuge the mixture at 8000 rpm for 20 min at 4 °C, and collect the supernatant to obtain the crude enzyme solution of the target enzyme.

[0100] For the purification of the enzyme, use the Ni NTA Beads nickel column from Tiandi Renhe Biotechnology Co., Ltd. Prepare imidazole solutions with concentrations of 20 mM, 40 - 50 mM, and 300 mM respectively according to its instruction manual, and perform operations such as column washing, elution of impurity proteins, and elution and collection of the target protein with a 6×His tag, and store it at 4 °C to obtain the purified enzyme solution of the target enzyme.

[0101] 7. Verification of heterologous protein expression effect by SDS-PAGE

[0102] Mix the crude enzyme solution or purified enzyme solution of the target protein evenly with Loading Buffer, and heat and denature it at 100 °C in a metal bath. Use MOPS-SDS Running Buffer (Boyi Biotech) electrophoresis solution and precast gel FuturePAGEM (Boyi Biotech) to perform protein electrophoresis experiments. Electrophoresis conditions: voltage is 100 V, and time is 1 hour. After electrophoresis, use a rapid staining solution (Nanjing Aisiyi Biotech) to stain the protein gel, and then use deionized water to decolorize it multiple times until the background is transparent. Finally, place the gel on a Bio-Rad gel imager for photography and analysis.

[0103] 8. Quantitative determination of enzyme protein by BCA method

[0104] According to the instructions of the Pierce kit from Thermo Fisher Scientific TM According to the method in the instructions of the BCA Protein Assay Kits, a standard solution with a concentration gradient of 0 - 2000 μM of bovine serum albumin (BSA) at 2 mg / mL of standard protein was prepared by gradient dilution. The BCA working solution was prepared according to the instructions. 25 μL of the above-mentioned standard solutions with different concentrations were respectively taken and mixed evenly with 200 μL of the BCA working solution into a 96-well microplate reader, with 3 replicates for each concentration. This microplate was incubated in a constant temperature incubator at 37 °C for 30 min, and then the absorbance value at 562 nm was measured using a grating microplate reader. With the protein concentration (0 - 2000 μg / mL) as the abscissa and the average absorbance as the ordinate, a protein concentration standard curve was obtained.

[0105] For subsequent sample determination, the same reaction system and reaction conditions were used, and the protein content of the sample was calculated based on the protein concentration standard curve.

[0106] 9. Chemical reaction

[0107] To determine the degradation of methylmalonic acid by the single enzyme ACSF3 reaction: Take 2 mL of the purified enzyme solution in a 5 mL brown light-proof glass test tube, and add 40 mM methylmalonic acid, 100 mM Tris-HCl, 50 μ M sodium coenzyme A hydrate (Maclean, C873372), 2 - 3.5 mM ATP, and 6 mM magnesium chloride hexahydrate (the above are all final concentrations). Invert and mix well, incubate in a metal bath at 25 °C for 50 min, and add perchloric acid with a final concentration of 0.4 M to terminate the reaction. Centrifuge the reaction solution at 8000 rpm for 20 min, take the supernatant, filter it through a 0.22 μm microporous filter membrane, and then perform liquid phase detection.

[0108] To determine the formation of succinyl coenzyme A by the single enzyme MUT reaction: Take 100 μL of the purified enzyme solution in a 2 mL brown light-proof centrifuge tube, and add 0.53 mM (R)-methylmalonyl coenzyme A (CAS No.: 1264 - 45 - 5), 100 mM Tris-HCl, and 0.65 μg / mL adenosylcobalamin (the above are all final concentrations). Invert and mix well, incubate in a metal bath at 25 °C for 10 min, and add perchloric acid with a final concentration of 0.4 M to terminate the reaction. Centrifuge the reaction solution at 8000 rpm for 20 min, take the supernatant, filter it through a 0.22 μm microporous filter membrane, and then perform liquid phase - mass spectrometry detection.

[0109] 10. Detection

[0110] Detection of methylmalonic acid by high performance liquid chromatography: The chromatographic column used was MicroPulite Perfect T3 (5μm 4.6×250mm), the mobile phase ratio was 10mM potassium dihydrogen phosphate buffer (added with 1mL - 1.5mL phosphoric acid to make the phosphoric acid concentration 0.1% - 0.15%): methanol = 99:1, the flow rate was 1.0mL / min, the detection wavelength was 210nm, the injection volume was 10μL, and the column temperature was 30°C.

[0111] Detection of succinyl coenzyme A by high performance liquid chromatography tandem mass spectrometry:

[0112] Liquid phase conditions: The chromatographic column was Waters BEH C18 1.7μm 2.1*100 column, the column length was 100mm, the inner diameter was 2mm, the particle size was 1.8μm, or an equivalent column. The flow rate was 0.3mL / min, the column temperature was 40°C, and the injection volume was 10μL. Mobile phase and gradient elution conditions: Mobile phase A was a solution containing 0.0075% ammonia water solution, and mobile phase B was 0.1% acetonitrile. The mobile phase and gradient elution conditions refer to Table 3.

[0113] Mass spectrometry conditions: The instrument was a Waters-TQS liquid chromatography-mass spectrometry instrument, the capillary voltage was 3kV; the desolvation gas temperature was 500°C, the sheath gas flow rate was 800L / h, and the auxiliary gas flow rate was 150L / h. The specific reference for the reaction detection precursor ion, product ion, and collision energy is Table 4.

[0114] Table 3 Mobile phase and gradient elution conditions

[0115]

[0116] Table 4 Selected reaction monitoring precursor ion, product ion, and collision energy

[0117]

[0118] Note: *Quantitative ion

[0119] 11. Calculation of degradation rate / generation rate:

[0120] Methylmalonic acid degradation rate = Methylmalonic acid degradation amount / (reaction time * enzyme protein content);

[0121] Succinyl coenzyme A generation rate = Succinyl coenzyme A generation amount / (reaction time * enzyme protein content).

[0122] The heterologous expression of single enzyme ACSF3 and single enzyme MUT is as Figure 3 shown. The results show that 4 kinds of ACSF3 and 4 kinds of MUT proteins are correctly expressed. Among them, ACSF3 hk 、ACSF3 nnj 、ACSF3 bj, ACSF3 human The molecular weights of the proteins are approximately 71 kDa, 73 kDa, 75 kDa, and 70 kDa respectively; MUT dxb , MUT mxc , MUT k12 , MUT human The molecular weights of the proteins are approximately 90 kDa, 90 kDa, 85 kDa, and 100 kDa respectively.

[0123] The in vitro degradation of methylmalonic acid by the single enzyme ACSF3 is shown in Table 5 and Figure 4 as shown, and the in vitro production of succinyl coenzyme A by the single enzyme MUT is shown in Table 6 and Figure 4 as shown. The results show that the group with the highest degradation rate of methylmalonic acid is ACSF3 bj , and its degradation rate reaches 2.446 μmol / h / μg, which is 1.521 times that of the group with the lowest degradation rate (ACSF3 nnj ); the group with the highest production rate of succinyl coenzyme A is MUT dxb , and its production rate reaches 19.869 μmol / h / μg, which is 1.98 times that of the group with the lowest production rate (MUT mxc ).

[0124] Table 5 In vitro methylmalonic acid degradation ability of the purified enzyme solution of the single enzyme ACSF3

[0125]

[0126] Table 6 In vitro succinyl coenzyme A production ability of the purified enzyme solution of the single enzyme MUT

[0127]

[0128] Example 2

[0129] This example is used to illustrate the preparation of the ACSF3-MUT- strain and the cascade reaction enzyme preparation.

[0130] For the construction of its engineering strain, determination of growth conditions, cultivation and induction of the engineering bacteria, and preparation of the crude enzyme solution, refer to the methods provided in Example 1. The difference lies in the construction of the expression vector. Any one of the ACSF3 genes is selected as the target gene and is tandemly connected with any one of the MUT dxb , MUT human with high enzyme activity. The tandem connection method is RBS (AAGGAG) or the linker GGGGS (GGTGGAGGCGGTTCA). Finally, 16 different expression vectors are obtained, and their names are shown in Table 1. The construction schematic diagrams of different tandem connection methods are as shown in Figure 5 .

[0131] The detection and calculation of the degradation rate of methylmalonic acid and the production rate of succinyl-CoA by the tandem enzyme were carried out according to the method of Example 1. Among them, for the determination of the tandem enzyme reaction: 300 μL of crude enzyme solution was taken into a 2 mL brown light-proof centrifuge tube, and 10 mM methylmalonic acid, 100 mM Tris-HCl, 200 μM sodium salt hydrate of coenzyme A, 2 mM ATP, 6 mM magnesium chloride hexahydrate, and 0.033 μg / mL adenosylcobalamin (the above are all final concentrations) were added. After inverting and mixing evenly, it was incubated in a metal bath at 25 °C for 30 min, and the reaction was terminated by adding perchloric acid with a final concentration of 0.4 M. The reaction solution was centrifuged at 8000 rpm for 20 min, and the supernatant was filtered through a 0.22 μm microporous membrane and then subjected to liquid phase or liquid chromatography-mass spectrometry detection.

[0132] The heterologous expression of the tandem enzyme was as Figure 6 shown. The results showed that all tandem proteins were correctly co-expressed or fusion-expressed. Among them, as shown in (a) of Figure 6 , the molecular weight of the fusion-expressed protein was between 140 and 180 kDa, corresponding to the sum of the molecular weights of the two related proteins; as shown in (b) of Figure 6 , the sizes of the co-expressed proteins were consistent with the molecular weights of the single enzyme proteins.

[0133] The situation of the tandem enzyme degrading methylmalonic acid and generating succinyl-CoA in vitro is shown in Table 7 and Figures 7-8 shown. The results showed that the top three combinations for the degradation rate of methylmalonic acid were r-bm, r-bd, and r-hd, and their degradation rates reached 0.847, 0.835, and 0.744 μmol / h / mg respectively, which were 7.370, 7.267, and 6.469 times that of the lowest degradation rate combination (l-nm); the top three combinations for the production rate of succinyl-CoA were l-nm, r-bd, and r-am, and their production rates reached 4.059, 4.018, and 3.950 μmol / h / mg respectively, which were 3.00, 2.970, and 2.919 times that of the lowest production rate combination (l-hd).

[0134] Table 7 In vitro methylmalonic acid degradation ability and succinyl-CoA production ability of crude enzyme solution of tandem ACSF3-MUT gene

[0135]

[0136] In summary, it can be seen that both the pure enzyme and the crude enzyme provided by the present invention can effectively degrade methylmalonic acid, and the degradation product can be further decomposed into succinyl-CoA that can be utilized by the human body. The methylmalonic acid-lowering product prepared by using the above-mentioned crude enzyme and / or pure enzyme can be administered to the human body to play a role in the intestine, reduce the concentration of methylmalonic acid in the intestine of patients, and further alleviate the clinical symptoms of metabolic acidosis caused by methylmalonic acidemia in patients.

[0137] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of methylmalonyl-CoA ligase ACSF3, or the combination of methylmalonyl-CoA ligase ACSF3 and methylmalonyl-CoA mutase MUT in the preparation of a product for degrading methylmalonic acid or a product for treating methylmalonic academia, characterized in that: The methylmalonyl-CoA ligase ACSF3 includes ACSF3 bj , ACSF3 hk , ACSF3 nnj and ACSF3 human and at least one of them; The ACSF3 bj is derived from the beluga whale (Delphinapterus leucas); The ACSF3 hk is derived from the sea anemone (Actinia tenebrosa); The ACSF3 nnj is derived from Arabidopsis thaliana; The ACSF3 human is derived from Homo sapiens; The methylmalonyl-CoA mutase MUT includes at least one of MUT dxb , MUT mxc , MUT k12 and MUT human ; The MUT dxb is derived from Stygiella incarcerata; The MUT mxc is derived from Trichinella nativa; The MUT k12 is derived from Escherichia coli (strain K12); The MUT human is derived from Homo sapiens.

2. The use according to claim 1, characterized in that: The ACSF3 bj has an amino acid sequence as shown in UniProt Accessions: A0A2Y9NUE1; The ACSF3 hk has the amino acid sequence shown in SEQ ID NO: 1; The ACSF3 nnj has an amino acid sequence as shown in UniProt Accessions: Q8H151; The ACSF3 human has the amino acid sequence shown in SEQ ID NO: 2; The MUT dxb has an amino acid sequence as shown in UniProt Accessions: A0A192ZIA6; The MUT mxc has an amino acid sequence as shown in UniProt Accessions: A0A0V1LU09; The MUT k12 has an amino acid sequence as shown in UniProt Accessions: P27253; The MUT human has the amino acid sequence shown in SEQ ID NO:

3.

3. The use according to claim 2, characterized in that: The ACSF3 bj encoding gene has a nucleotide sequence as shown in SEQ ID NO: 4; The ACSF3 hk encoding gene has a nucleotide sequence as shown in SEQ ID NO: 5; The ACSF3 nnj encoding gene has a nucleotide sequence as shown in SEQ ID NO: 6; The ACSF3 human encoding gene has a nucleotide sequence as shown in SEQ ID NO: 7; The MUT dxb has a nucleotide sequence of the coding gene as shown in SEQ ID NO: 8; The MUT mxc encoding gene has a nucleotide sequence as shown in SEQ ID NO: 9; The MUT k12 encoding gene has a nucleotide sequence as shown by positions 822881 to 820737 bp in CP097882.1; The MUT human encoding gene has a nucleotide sequence as shown in SEQ ID NO:

10.

4. The use according to any one of claims 1 to 3, characterized in that: The combination of methylmalonyl-CoA ligase ACSF3 and methylmalonyl-CoA mutase MUT includes the combination of ACSF3 and MUT in series through RBS or a linker peptide.

5. The use according to claim 4, characterized in that: The nucleotide sequence of the RBS is AAGGAG; the amino acid sequence of the linker peptide is GGGGS, and further, the nucleotide sequence of the linker peptide is GGTGGAGGCGGTTCA.

6. An engineered bacterium for degrading methylmalonic acid, characterized in that: is obtained by performing any one of the following operations on an Escherichia coli starting strain: (1) Heterologous expression of the methylmalonyl-CoA ligase ACSF3 described in any one of claims 1 to 5; or, (2) Heterologous expression of the methylmalonyl-CoA ligase ACSF3 and methylmalonyl-CoA mutase MUT described in any one of claims 1 to 5; wherein, ACSF3 and MUT are heterologously expressed in series through RBS or a linker peptide.

7. The engineered bacterium for degrading methylmalonic acid according to claim 6, characterized in that: The Escherichia coli starting strain includes Escherichia coli BL21(DE3).

8. The method for constructing an engineered bacterium for degrading methylmalonic acid according to claim 6 or 7, characterized in that: Comprises the following steps: S1. Based on the Escherichia coli starting strain, heterologously express the methylmalonyl-CoA ligase ACSF3 described in any one of claims 1 to 5 to construct a single-enzyme engineered bacterium; S2. Based on the Escherichia coli starting strain, heterologously express the methylmalonyl-CoA ligase ACSF3 gene and methylmalonyl-CoA mutase MUT gene described in any one of claims 1 to 5 in series through RBS or a linker peptide to construct a tandem-enzyme engineered bacterium.

9. Use of the engineered bacterium for degrading methylmalonic acid according to claim 6 or 7 in the preparation of a product for degrading methylmalonic acid or a product for treating methylmalonic academia.

10. The use according to any one of claims 1 to 5 or 9, characterized in that: The product includes at least one of drugs, health foods, and food additives.

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