Thiamine enzyme II as well as mutant and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses thiamine enzyme II as well as a mutant and application thereof, an amino acid sequence of the thiamine enzyme II is shown as SEQ ID NO.4, and a nucleotide sequence of a coding gene of the thiamine enzyme II is shown as SEQ ID NO.1. The invention further provides a thiamine enzyme II mutant. The thiamine enzyme II mutant is obtained by mutating one or more sites of tyrosine at the 48th site, lysine at the 49th site, glycine at the 78th site, tyrosine at the 113th site, tyrosine at the 140th site, tyrosine at the 164th site, tryptophan at the 168th site, phenylalanine at the 169th site and glutamic acid at the 206th site of the thiamine enzyme II. The thiamine enzyme II or each mutant provided by the invention has higher capability of catalytically synthesizing the 4-methyl-5-(beta-ethoxyl)-thiazole, and can be used for preparing the 4-methyl-5-(beta-ethoxyl)-thiazole, and the enzyme activity of the mutant is obviously higher than that of a wild type, so that the thiamine enzyme II or each mutant has a huge application prospect in the fields of health care products, medicines and food additives.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically discloses a thiamine enzyme II and a mutant and application thereof. Background Art

[0002] 4-Methyl-5-(β-hydroxyethyl)-thiazole, also known as thiothiazole, is a light yellow liquid with no volatility and an unpleasant odor of thiazole compounds, but it has a pleasant aroma at extremely dilute concentrations and can react with HCl to form hydrochloride that is soluble in water and alcohol. Thiothiazole is the basic structural ring of vitamin VB1 and an important intermediate in the synthesis of VB1. It also has aromas such as nuts, beans, milk, eggs, and meat, and can be used as a spice and flavor.

[0003] At present, 4-methyl-5-(β-hydroxyethyl)-thiazole, which is widely used as a food additive on the market, is a chemically synthesized product. Although the synthesis method has been optimized, the optimized method is still costly and accompanied by many side reactions, and the reagents used generate large pollution and high energy consumption. For example, the invention patent with publication number CN111635375A discloses a synthesis method of thiothiazole, using methyl acetoacetate and 3-bromo-3-chloropropyl acetate as raw materials, preparing 4-acetoxy-2-acetyl-2-chlorobutyric acid methyl ester under alkaline conditions, and obtaining thiothiazole through acid hydrolysis, condensation, oxidation, and alkaline hydrolysis. In the above synthesis process, the raw material cost of 3-bromo-3-chloropropyl acetate is high, and it needs to undergo a two-step hydrolysis reaction, which has many side reactions, affects the product yield, and increases the difficulty of product purification. Therefore, how to develop a green, environmentally friendly, safe, and mild biosynthetic method to achieve the large-scale synthesis of 4-methyl-5-(β-hydroxyethyl)thiazole is a technical problem that needs to be solved urgently. Summary of the invention

[0004] In view of these defects, the present invention provides a thiaminease II and its mutants and applications. By screening thiaminease II in different Bacillus strains, a thiaminease II from Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )Thiaminase II for efficient synthesis of 4-methyl-5-(β-hydroxyethyl)thiazole and its expression gene in HZ12 tenA At the same time, the enzyme was subjected to point mutation to obtain a series of thiaminease II mutants. The engineered strains capable of expressing the mutants were constructed to synthesize the food flavoring 4-methyl-5-(β-hydroxyethyl)thiazole through whole-cell catalysis, and the yield reached 1955.9 mg / L, which was 6.5 times higher than that of the original TenA thiaminease II derived from Bacillus amyloliquefaciens HZ12.

[0005] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a use of thiaminase II in producing 4-methyl-5-(β-hydroxyethyl)thiazole, wherein the amino acid sequence of the thiaminase II is shown in SEQ ID NO.4.

[0006] Amino acid sequence SEQ ID NO.4: MMTFSEECRQAAAEWWDGSFVHPFVTGIGDGSLPIDRFSYYVLQDSYLTHFAKVQAFGAAYAEDLFTTGRMAGHAQGTYEAEMALHREFTELLGITEEERAAFKPAPTAYSYTSHMY RSVMSGNFGEILAALLPCYWLYYEVGEHLKECKPEHPIYEKWIGTYGGDWFRQQVEEQISRFDEIAENSTEEIRAKMKENFVISSYEYQFWGMAYQKEGWSEKDGKEVESFGAARGN.

[0007] The inventors mined the key enzymes in the thiamine biosynthesis pathway based on the literature, and queried the metabolic pathways of thiamine through KEGG and compared the additives that can be used in food, found the crystal structure and active site analysis of thiamine enzyme, and analyzed that there is a key gene encoding thiaminease II in Bacillus. tenA , which encodes thiaminase II that can hydrolyze thiamine to produce 4-methyl-5-(β-hydroxyethyl)thiazole.

[0008] The present invention screened thiaminease II in a series of food safety grade Bacillus strains and found that compared with other Bacillus, the thiaminease II from Bacillus amyloliquefaciens HZ12 tenA The thiamine enzyme II encoded by the gene has excellent effect in synthesizing 4-methyl-5-(β-hydroxyethyl)thiazole. tenA Genetically constructed Bacillus amyloliquefaciens engineered strain BAX-10 / pT17-HZ12- capable of expressing thiaminease II tenA The yield of 4-methyl-5-(β-hydroxyethyl)thiazole synthesized by whole-cell catalysis can reach 113.08 mg / L.

[0009] Preferably, the nucleotide sequence encoding the thiaminease II is as shown in SEQ ID NO.1.

[0010] Nucleotide sequence SEQ ID NO.1: .

[0011] In a second aspect, the present invention also provides a thiaminease II mutant, which is obtained by mutating one or more of the 48th tyrosine, 49th lysine, 78th glycine, 113th tyrosine, 140th tyrosine, 164th tyrosine, 168th tryptophan, 169th phenylalanine and 206th glutamate of thiaminease II, and the amino acid sequence of the thiaminease II is shown in SEQ ID NO.4.

[0012] The thiamine enzyme II mutant provided by the present invention includes a single-point mutation, and the mutation position is one of the 48th tyrosine, the 49th lysine, the 78th glycine, the 113th tyrosine, the 140th tyrosine, the 164th tyrosine, the 168th tryptophan, the 169th phenylalanine and the 206th glutamic acid.

[0013] The thiaminease II mutant provided by the present invention also includes multi-point mutations, and the mutation positions are two or more of the 48th tyrosine, the 49th lysine, the 78th glycine, the 113th tyrosine, the 140th tyrosine, the 164th tyrosine, the 168th tryptophan, the 169th phenylalanine and the 206th glutamate.

[0014] On the basis of the thiaminease II to be protected by the present invention, homologous protein modeling and molecular docking are performed according to the data in the database established by the analysis of the structure and function of natural proteins, and the binding site between thiaminease II and the substrate thiamine is further analyzed and modified to find that the above site is the active site of thiaminease II. After the modification, the activity of wild-type thiaminease II can be improved.

[0015] Preferably, the thiaminease II mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to at least one of the following mutations: Mutating tyrosine at position 48 to alanine; and / or mutating lysine at position 49 to alanine; and / or Mutating glycine at position 78 to alanine; and / or Mutating tyrosine at position 113 to alanine; and / or mutating tyrosine at position 140 to alanine; and / or Mutating tyrosine at position 164 to alanine; and / or Mutating tryptophan at position 168 to alanine; and / or Mutating phenylalanine at position 169 to alanine; and / or The glutamic acid at position 206 was mutated to alanine.

[0016] The amino acid site mutation "the 48th tyrosine is mutated to alanine" of the present invention means that the 48th amino acid of the amino acid sequence shown in SEQ ID NO.4 is mutated from tyrosine to alanine; the expressions of the mutations at the remaining sites are similar.

[0017] Primers were designed to perform alanine single base mutation on the nucleotide sequence shown in SEQ ID NO.1, and vectors and engineering bacteria were constructed to obtain engineering bacteria with single-point alanine mutations at positions 48, 49, 78, 113, 140, 164, 168, 169 and 206. The successfully constructed engineering bacteria were fermented and HLPC tested for target products, and the yields of the target products obtained were better than those of the non-mutated engineering bacteria.

[0018] Preferably, the thiaminease II mutant is a single-site mutant or a double-site mutant; The single-site mutant is a single-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO. 4 to any one of Y113A, Y140A, F169A or E206A single-site mutations.

[0019] The double-site mutant is a double-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to any one of Y113A / F169A, Y140A / F169A or E206 / F169A double-site mutations.

[0020] The amino acid site mutation "Y113A" of the present invention means that the 113th amino acid in the amino acid sequence shown in SEQ ID NO.4 is mutated from tyrosine (Y) to alanine (A); the expressions of the other site mutations are similar.

[0021] The amino acid site mutation "Y113A / F169A" described in the present invention means that the 113th amino acid of the amino acid sequence shown in SEQ ID NO.4 is mutated from tyrosine (Y) to alanine, and the 169th amino acid is mutated from phenylalanine (F) to alanine (A); the expressions of the remaining site mutations are similar.

[0022] The production of 4-methyl-5-(β-hydroxyethyl)thiazole by engineering bacteria with single-point alanine mutations at Y113, Y140, F169 and E206 sites was significantly increased.

[0023] The Y113, Y140, F169 and E206 sites were subjected to superimposed mutations, and the amount of 4-methyl-5-(β-hydroxyethyl)thiazole produced by the Y113A / F169A engineering bacteria, Y140A / F169A engineering bacteria and E206 / F169A engineering bacteria expressing superimposed double mutations was significantly increased compared with that of the unmutated engineering strains.

[0024] More preferably, the thiaminease II mutant is a single-site mutant or a double-site mutant, and the single-site mutant is a single-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to a single-site mutation of F169A; The double-site mutant is a double-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to double-site mutations of Y113A / F169A.

[0025] The amount of 4-methyl-5-(β-hydroxyethyl)thiazole produced by the engineered bacteria expressing the single-site F169A mutation thiaminease II mutant can reach 1312.6 mg / L, which is 4.3 times that of the unmutated engineered bacteria. The amount of 4-methyl-5-(β-hydroxyethyl)thiazole produced by the engineered bacteria expressing the double-site Y113A / F169A mutation thiaminease II mutant can reach 1955.9 mg / L, which is 6.5 times that of the unmutated engineered bacteria.

[0026] In a third aspect, the present invention also provides a gene encoding the above-mentioned thiaminease II mutant.

[0027] The coding genes of the single-site mutants or multi-site mutants described in the present invention also fall within the protection scope of the present invention.

[0028] In a fourth aspect, the present invention also provides a recombinant expression vector containing a nucleotide sequence such as that shown in SEQ ID NO.1 or the above-mentioned encoding gene and a recombinant engineered bacterium containing the recombinant expression vector.

[0029] The recombinant expression vector or recombinant engineered bacteria containing the thiaminease II encoding gene or the thiaminease II mutant encoding gene described in the present invention also falls within the protection scope of the present invention.

[0030] The recombinant expression vector may be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector, including but not limited to pHY300PKL, pMA5 and T2.

[0031] The present invention illustratively provides a method for preparing a recombinant host cell containing the above-mentioned encoding gene, comprising: S1: Connecting the coding gene of thiaminease II or its mutant to the expression regulatory element to construct a recombinant expression vector; S2: The recombinant expression vector described in S1 is transformed into a host cell, and the recombinant engineered bacteria are obtained after expression verification.

[0032] In a fifth aspect, the present invention also provides an engineered Bacillus amyloliquefaciens strain that produces high 4-methyl-5-(β-hydroxyethyl)thiazole and can express the gene of the above-mentioned thiaminease II mutant; the host cell is Bacillus amyloliquefaciens BAX-10 and / or the overexpression vector is pT17.

[0033] Bacillus amyloliquefaciens BAX-10 is a strain of Bacillus amyloliquefaciens HZ12 in which seven important extracellular protease genes were knocked out. epr, nprE, aprE-a, mpr, pbpF, vpr, ykct1 ), 1 important intracellular protease gene ( apr ) and 2 redundant protein genes ( htrB,hag ) and the deletion of 10 genes had no negative impact on its growth. Bacillus amyloliquefaciens BAX-10, as a host cell, has the ability to efficiently express target extracellular proteins. Experiments have shown that alkaline protease (gene is expressed by BAX-10) aprE ) was 57% higher than that of the original strain without knockout.

[0034] The pT17 plasmid is based on the pHY300PLK original plasmid and incorporates the P43 promoter and Tamly terminator, which is beneficial to the expression of the target gene.

[0035] In a sixth aspect, the present invention further provides a water-in-water Pickering emulsion encapsulating the above-mentioned recombinant engineered bacteria, comprising a continuous phase and a dispersed phase; the continuous phase comprises a continuous phase polymer system and a solid particle stabilizer, and the dispersed phase comprises the above-mentioned recombinant engineered bacteria and the dispersed phase polymer system; the continuous phase polymer system and the dispersed phase polymer system contain one or more water-soluble polymers, and the absolute value of the difference between the solubility parameter of the water-soluble polymer contained in the continuous phase polymer system relative to water and the solubility parameter of the water-soluble polymer contained in the dispersed phase polymer system relative to water is greater than 0.5.

[0036] The water-in-water Pickering emulsion is particularly suitable for preparing, loading and transporting active ingredients and nutrients. As a microreactor, it can provide a good living environment for active substances such as probiotics. The water-in-water Pickering emulsion containing the above-mentioned recombinant engineered bacteria is fermented and dried to obtain a microcapsule preparation containing the recombinant engineered bacteria.

[0037] Preferably, the continuous phase comprises 3.0 wt% to 5.5 wt% of a water-soluble polymer and a solid particle stabilizer.

[0038] Preferably, the mass fraction of the solid particle stabilizer in the water-in-water Pickering emulsion is 0.025wt%-0.100wt%. Preferably, the solid particle stabilizer is water-insoluble micro-nanoparticles, which are one or more of inorganic nanoparticles, organic nanoparticles and inactivated microorganisms.

[0039] More preferably, the solid particle stabilizer is one or more combinations of SiO2, liposomes or cellulose nanocrystals.

[0040] Preferably, the dispersed phase comprises 2.5wt%-4.0wt% of a water-soluble polymer and a viable cell count of 10 7 -10 10 CFU / g of the above recombinant engineered bacteria.

[0041] Preferably, the water-soluble polymer in the dispersed phase polymer system and the water-soluble polymer in the continuous phase polymer system are independently selected from synthetic macromolecular polymers, water-soluble natural polysaccharides and water-soluble proteins; wherein the water-soluble synthetic macromolecular polymers include but are not limited to polyethylene glycol, polyvinyl alcohol or polyethylene oxide, etc.; the water-soluble natural polysaccharides include but are not limited to starch, pullulan, dextran, chitosan, konjac glucomannan, hydroxypropyl methylcellulose, methylcellulose, dextrin, carrageenan or guar gum, etc.; the water-soluble proteins include but are not limited to gelatin, collagen or casein, etc.

[0042] More preferably, the water-soluble polymer in the continuous phase is hydroxypropyl methylcellulose.

[0043] More preferably, the water-soluble polymer in the dispersed phase is maltodextrin.

[0044] Preferably, the dispersed phase includes nutrients that are beneficial to the growth and reproduction of the above-mentioned recombinant engineered bacteria, and the nutrients are selected from the nutritional components required for the growth of the embedded recombinant engineered bacteria.

[0045] In a seventh aspect, the present invention also provides the use of the above-mentioned thiaminease II mutant, the above-mentioned recombinant expression vector, the recombinant engineered bacteria containing the recombinant expression vector, the above-mentioned Bacillus amyloliquefaciens engineered bacteria or the above-mentioned water-in-water Pickering emulsion in the catalytic synthesis of 4-methyl-5-(β-hydroxyethyl)thiazole.

[0046] The thiamine enzyme II provided by the present invention has high activity in each single-site mutant or multi-site mutant, and can efficiently synthesize 4-methyl-5-(β-hydroxyethyl)thiazole. Therefore, the mutant and the recombinant expression vector, recombinant engineering bacteria or water-in-water Pickering emulsion containing the recombinant engineering bacteria capable of expressing the mutant have broad application prospects in the production of food flavor 4-methyl-5-(β-hydroxyethyl)thiazole.

[0047] A feasible industrial biosynthesis method can be selected to produce 4-methyl-5-(β-hydroxyethyl)thiazole using the thiaminease II mutant, recombinant engineered bacteria or water-in-water Pickering emulsion containing recombinant engineered bacteria provided by the present invention. The industrial biosynthesis method includes but is not limited to a fermentation method, an extraction enzyme catalysis method and a whole-cell catalysis method, or using a water-in-water Pickering emulsion to culture the recombinant engineered bacteria at a high density to obtain a reaction liquid, and then separating and purifying the obtained reaction liquid to obtain 4-methyl-5-(β-hydroxyethyl)thiazole.

[0048] In an eighth aspect, the present invention further provides a method for synthesizing 4-methyl-5-(β-hydroxyethyl)thiazole, comprising: A whole-cell catalytic reaction is performed on a genetically engineered bacterium capable of expressing thiaminase II having an amino acid sequence as shown in SEQ ID NO.4 or a thiaminase II mutant as described in any one of claims 3 to 6 to obtain a whole-cell catalytic reaction liquid, wherein the whole-cell catalytic reaction liquid contains the 4-methyl-5-(β-hydroxyethyl)thiazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 The free expression plasmid pT17-HZ12- tenA The construction process.

[0051] Figure 2 is pT17-HZ12- tenA Results of double restriction enzyme digestion of free expression plasmid; (lane M: DL5000 DNA Maker; lane 1: tenA Fragment; Lane 2: linear plasmid pT17) Figure 3 BAX10 / pT17-HZ12- constructed in Example 1 of the present invention tenA Colony PCR identification results of engineered bacteria; (lane M: DL5000 DNA Marker; lane 1: BAX10 / pT17-HZ12- tenA PCR results) Figure 4 is the concentration of 4-methyl-5-(β-hydroxyethyl)thiazole in the whole cell catalytic solution of the four strains in Example 2 of the present invention; Figure 5The predicted structure of thiaminase II protein from Bacillus amyloliquefaciens HZ12 and HZ12- tenA With BS168- tenA Amino acid correspondence diagram; Figure 6 This is a docking diagram of thiamine and the predicted active site of thiaminease II protein from Bacillus amyloliquefaciens HZ12; Figure 7 This is a diagram showing the interaction between thiamine and thiaminase II protein from Bacillus amyloliquefaciens HZ12; Figure 8 This is a graph showing the target product yield of the alanine mutant engineered bacteria constructed in Example 3 of the present invention; Fig. 9 This is a graph showing the target product yield of the alanine superposition mutant engineered bacteria constructed in Example 3 of the present invention; Fig.10 This is the microscopic image of the HPMC-MD W / W emulsion at 0 h in the verification example 1 of the present invention, in which different concentrations of LB medium were added to the emulsion system of HPMC:MD=4:3; Fig.11 This is a microscopic image of HPMC-MD W / W emulsions added with different concentrations of LB culture medium based on the emulsion system of HPMC:MD=4:3 in Verification Example 1 of the present invention at 24 h; Fig.12 The microscopic images of HPMC-MD W / W emulsions in which different concentrations of LB medium were added to the emulsion system of HPMC:MD=4:3.5 in Verification Example 1 of the present invention; Fig.13 The microscopic images of HPMC-MD W / W emulsions in LB medium with different concentrations after adding a single stable granule to the emulsion system of HPMC:MD=4:3.5 in Verification Example 1 of the present invention; Fig.14 These are microscopic images of HPMC-MD W / W emulsions in LB culture medium with different concentrations after adding mixed stabilizing granules to the emulsion system of HPMC:MD=4:3.5 in Verification Example 1 of the present invention. DETAILED DESCRIPTION

[0052] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Description of the source of biological materials: Bacillus amyloliquefaciens BAX-10, Bacillus amyloliquefaciens HZ12, Bacillus subtilis 168 and Bacillus licheniformis BL9 all come from Wei Xuetuan's research group at Huazhong Agricultural University.

[0054] Bacillus amyloliquefaciens BAX-10 has been disclosed in the article “Efficient production of extracellularalkaline protease in Bacillus amyloliquefaciens by host strain construction” (LWT-Food Science and Technology, 2022, 163, 113620.); Bacillus amyloliquefaciens HZ12 has been disclosed in the article "Multiple Microbial Engineering of Bacillus amyloliqucracicns for Production of the Plasma Chemical Putrcscinc from Sustainable Biomass llydrolysates" (ACS Sustainable Chemistry & Engineering, 2020, 8, 2147-2157.).

[0055] The culture medium and whole cell catalytic solution used in the embodiments of the present invention are: LB medium: 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and 15 g / L agar powder added to the solid medium, pH 7.2-7.4.

[0056] Whole cell catalytic solution: NaH2PO4·H2O 27.6 g / L, Na2HPO4·12H2O 71.6 g / L, 0.4% TritonX-100, thiamine 20 g / L, pH=6.6.

[0057] Detection method of 4-methyl-5-(β-hydroxyethyl)thiazole (THZ) and preparation method of standard curve: 4-Methyl-5-(β-hydroxyethyl)thiazole was detected by high performance liquid chromatography. High performance liquid chromatography, Agilent 1100; chromatographic column: ZORBAX Eclipse Plus XDB-C18 (4.6 mm × 250 mm, 5 μm); mobile phase: 0.01 mol / L PH 6.6 PB buffer (phase A) and methanol (phase B); detection conditions: flow rate 0.6 mL / min, detection wavelength 254 nm, column temperature 30 o C, injection volume 10 μL; gradient elution conditions, as shown in Table 1, 4-methyl-5-(β-hydroxyethyl)thiazole eluted at 17.2 min.

[0058] Table 1 Gradient elution conditions

[0059] Use a pipette to accurately draw the standard of 4-methyl-5-(β-hydroxyethyl)thiazole to prepare samples of 4-methyl-5-(β-hydroxyethyl)thiazole with concentrations of 2.0, 5.0, 8.0, 10, 20, 50, 80, 100, 150 and 200 mg / L. After filtering the prepared samples through a 0.22 μm aqueous filter membrane, the filtered sample solution is placed in a liquid phase bottle for standby use, and then placed in a high performance liquid chromatograph, with the solution concentration as the horizontal coordinate and the peak area as the vertical coordinate, to draw a standard curve. The linear equation of the standard curve used in the embodiment of the present invention is Y=21.40X+9.253, and the correlation coefficient r 2 =0.9998.

[0060] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

[0061] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0062] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

[0063] Example 1 Thiaminase II gene tenA A single colony of Bacillus amyloliquefaciens HZ12 was picked and inoculated into 5 mL LB liquid medium, cultured overnight at 37°C with shaking, collected by centrifugation and washed once with STE; 100 µL Solution I and 20 µL lysozyme (final concentration of 1 mg / mL) were added to the centrifuged cells to resuspend the cells, incubated at 37°C for 2-3 h or placed in a 4°C refrigerator overnight, 200 µL 2% SDS was added, 50-60°C water bath for 30 min, 100 µL 5 mol / L NaCl was added, mixed and centrifuged at 12000 r / min for 5 min, and the supernatant was taken; after extracting the supernatant twice with an equal volume of phenol / chloroform, the supernatant was taken and placed in a new centrifuge tube, 2 volumes of anhydrous ethanol were added, precipitated at -20°C for 2 h, centrifuged at 12000 r / min for 5 min, the supernatant was discarded, the precipitate was washed once with 70% ethanol, dried and dissolved in 50 µL RNase (20 The genomic DNA of Bacillus amyloliquefaciens HZ12 was obtained after being treated with 5% paraformaldehyde and 1% paraformaldehyde at 37°C for 30 min and stored at -20°C.

[0064] The genome of Bacillus amyloliquefaciens HZ12 was used as a template and specific primers (see Table 2) were used to amplify the HZ12- tenA The nucleotide sequence of the gene consists of 711 bases, and the sequence is shown in SEQ ID NO.1. The HZ12- tenA Gene fragment, please refer to the instruction manual for specific steps.

[0065] Table 2 Primer sequences

[0066] PCR system: 2×Rapid Taq Master Mix 25 µL; template DNA 1 µL; upstream primer 2 µL; downstream primer 2 µL; add pure water to make the total system 50 µL.

[0067] PCR cycles: (1) 95°C, 15 min; (2) 95°C, 30 s; (3) 55°C, 30 s; (4) 72°C, 1 min; (5) 30 cycles; (6) 72°C, 5 min; (7) 25°C, 5 min (annealing temperature varies according to the Tm value of the primer, and extension time varies according to the size of the target fragment to be amplified).

[0068] The same method was used to obtain the genomic DNA of Bacillus subtilis 168 and Bacillus licheniformis BL9, and the genomes of Bacillus subtilis 168 and Bacillus licheniformis BL9 were used as templates to amplify the 168- tenA Gene and BL9- tenA The nucleotide sequences of the genes are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

[0069] After the amplified DNA sequence was purified, restriction endonuclease XB I and B H After enzyme digestion, the purified DNA was recovered and cloned into the pT17 vector. The construction process is shown in Figure 1 The successfully connected pT17 vector was transformed into Escherichia coli DH5α and tested for tetracycline (Tet) resistance and Bm I and XB I double enzyme digestion identification (such as Figure 2 After that, the positive clones were selected and cultured, the plasmids were extracted and sequenced by the company, and the results were compared on NCBI to confirm that the inserted sequence was correct, indicating that the free expression plasmid pT17-HZ12- tenA, pT17-168- tenA and pT17-BL9- tenA .

[0070] The engineering strain BAX-10 / pT17-HZ12-tenA was constructed by OMEGA kit. The above-mentioned free expression plasmid pT17-HZ12- tenA A small amount of DNA was extracted and recombinant plasmid DNA was obtained. Then 10 μL of recombinant plasmid DNA (50 ng / μL) was added to the competent cells of Bacillus amyloliquefaciens BAX-10, gently mixed and transferred to a pre-cooled 2 mm electroporation cup. After ice bathing for 10 min, it was electroporated once with an electric pulse converter at 2.4 KV. After the electroporation, 800 μL of recovery medium was quickly added and cultured for 3 h at 37°C and 100 r / min to obtain transformants. The transformants were tested for tetracycline (Tet) resistance and verified by colony PCR using pT17-F / R as the detection primer (such as Figure 3 As shown), the engineered bacteria BAX-10 / pT17-HZ12- tenA , and stored in a -80°C ultra-low temperature freezer.

[0071] The same method can be used to obtain the engineered bacteria BAX-10 / pT17-168- tenA and engineered bacteria BAX-10 / pT17-BL9- tenA .

[0072] Example 2 Bacillus amyloliquefaciens BAX-10 / pT17-HZ12- tenA Whole-cell catalytic reactions Pick the engineered bacteria BAX-10 / pT17-HZ12- tenA A single colony was inoculated into LB liquid medium (50 mL in a 250 mL Erlenmeyer flask) and cultured at 180 rpm and 37°C for 8-12 h. 600 When the concentration reaches 3.5-4.0, inoculate 3% of the culture medium into LB liquid medium (50 mL in a 250 mL Erlenmeyer flask), culture at 37°C, 180 r / min for 24 h, centrifuge at 6000 r / min for 10 min, discard the supernatant, and collect the cells. Wash the cells twice with 0.01 mol / L PH 6.6 PBS buffer, then resuspend in 10 mL (10 mL in a 50 mL Erlenmeyer flask) of 4-methyl-5-(β-hydroxyethyl)thiazole whole cell catalytic solution to form a cell suspension until the OD reaches 0. 600 =10, 37°C, 180 r / min shaking culture for 48 h, and a whole-cell catalytic reaction solution containing 4-methyl-5-(β-hydroxyethyl)thiazole was obtained. tenA and engineered bacteria BAX-10 / pT17-BL9- tenA As a control.

[0073] Take 1 mL of the above four whole-cell catalytic solutions and centrifuge at 4°C, 10000 r / min for 10 min. After the supernatant is filtered through a 0.22 μm aqueous phase filter membrane, 4-methyl-5-(β-hydroxyethyl)thiazole in the whole-cell catalytic reaction solution is detected by high performance liquid chromatography. The content of 4-methyl-5-(β-hydroxyethyl)thiazole is determined based on the peak time and peak area. The results are as follows Figure 4 shown.

[0074] Depend on Figure 4 It can be seen that the engineered strain BAX-10 / pT17-HZ12- tenA The yield of 4-methyl-5-(β-hydroxyethyl)thiazole can reach 113.08 mg / L, which is significantly better than the control group, indicating that the tenA The gene-edited thiaminease II has excellent ability to catalyze the synthesis of 4-methyl-5-(β-hydroxyethyl)thiazole.

[0075] Example 3 Determination of mutation sites of thiaminase II and construction of mutants 1. HZ12- tenA Homology comparison of sequences Since there is no crystal structure analysis and report of TenA protein from Bacillus amyloliquefaciens HZ12, the SWISS-MODEL software was used to analyze the crystal structure of TenA protein from HZ12- tenA The homologous sequence prediction and alignment were performed on the sequences, and the sequence derived from Bacillus subtilis 168 (BS168- tenA ) and HZ12- tenA The closest sequence homology, two different sources tenA The amino acid correspondence of the sequence is as follows Figure 5 A. The three-dimensional structure of the protein TenA thiaminase II from Bacillus subtilis 168 has been reported, such as Figure 5 B. The subsequent molecular docking is carried out based on the three-dimensional structure of the protein. The resulting amino acids need to be Figure 5 A corresponds to Bacillus amyloliquefaciens HZ12- tenA on sequence.

[0076] 2. Preparation of receptor protein and treatment of small molecule ligands MOE 2019 (Chemical Computing Group, Canada) was used for this docking study. First, the BS168-TenA protein was optimized using the Quickprep module provided by MOE. The thiamine small molecule ligand was hydrogenated, charged, and energy optimized using the Wash, PartialCharges, and Energy minimize modules of MOE.

[0077] 3. Molecular docking of small molecule ligands and receptor proteins Use the Site Finder module of MOE to predict the active site of the protein and dock the small molecule with the predicted active site. Figure 6 As shown. In setting the docking parameters, select the triangle matcher to place the ligand in the binding site. Set 50 docking poses and sort them using the London dG scoring function. Select the 30 poses with the best scores for refinement and energy minimization using the rigid receptor method, and then re-score using the GBVI / WSA dG scoring function. Select the docking result with the highest score for visualization study, as shown in Figure 7 shown.

[0078] Then, the molecules predicted to have an effect were docked BS168-tenA The amino acids in the sequence (D71, Y74, L75, F78, Q82, H101, G104, T105, C162, Y163, Y166, Y190, W194, F195, and Q198) were identified by Figure 5 A corresponds to HZ12- tenAIn the sequence of , D45, Y48, L49, F52, Q56, H75, G78, T79, C136, Y137, Y140, Y164, W168, F169 and Q172 were determined to be predicted and mapped to HZ12- tenA The amino acid position of the sequence. The amino acid at position 108 is predicted to be alanine, so it is not set as a predicted mutation site.

[0079] Supplementary mutation sites According to the existing literature reports, the three active site residues D44, C135 and E205 of BS168-TenA enzyme may be involved in substrate binding and catalysis, and there is a hydrogen bond network near the Sγ atom of C135 (through the side chains of residues E205, Y112 and Y47), so these five sites are also added to the mutation plan. tenA Sequence and Figure 5 A sites were compared one by one, corresponding to HZ12- tenA D45, Y48, Y113, C136 and E206 amino acids of the sequence, among which D45, Y48, and C136 amino acids have appeared in the molecular docking results, so Y113 and E206 amino acids are added to the predicted mutation sites.

[0080] In summary, 17 mutation sites were identified, namely D45, Y48, L49, F52, Q56, H75, G78, T79, Y113, C136, Y137, Y140, Y164, W168, F169, Q172 and E206.

[0081] The construction of alanine mutants was based on the HZ12- tenA nucleotide sequences, and designed and synthesized site-directed mutagenesis primers for introducing alanine single mutations. tenA The gene was subjected to site-directed mutagenesis, and the primers were designed as shown in Table 3. The genes of the above-mentioned single-site mutants D45A, Y48A, L49A, F52A, Q56A, H75A, G78A, T79A, Y113A, C136A, Y137A, Y140A, Y164A, W168A, F169A, Q172A or E206A of thiaminease II were constructed into the pT17 vector respectively by the method used in Example 1. After the sequencing was correct, the corresponding mutant engineering bacteria were transformed into Bacillus amyloliquefaciens BAX-10 to construct the corresponding mutant engineering bacteria. The successfully constructed engineering bacteria and engineering strains BAX-10 / pT17-HZ12- tenAThe fermentation culture of 4-methyl-5-(β-hydroxyethyl)thiazole was carried out. The culture method was to inoculate the activated engineering strain into LB liquid culture medium at a 3% inoculum, culture it in a shaking table at 37°C for 12 h, then add 5 g / L thiamine, and collect 1 mL of fermentation liquid after 48 h for the detection of 4-methyl-5-(β-hydroxyethyl)thiazole production. The results Figure 8 shown.

[0082] Table 3 Primer sequences

[0083] Depend on Figure 8 It can be seen that under the same culture conditions, the 4-methyl-5-(β-hydroxyethyl)thiazole content in the reaction solution of the engineering bacteria expressing the thiaminease II single-point mutants of Y48A, L49A, G78A, Y113A, Y140A, Y164A, W168A, F169A and E206A was higher than that of the engineering strain BAX-10 / pT17-HZ12- tenA , indicating that Y48, L49, G78, Y113, Y140, Y164, W168, F169 and E206 of wild-type thiaminase II are active sites of the enzyme. Among them, the ability of thiaminase II single-point mutants expressing Y113A, Y140A, F169A and E206A to produce 4-methyl-5-(β-hydroxyethyl)thiazole was significantly improved, and the yields of the target product in the reaction solution were 559.2 mg / L, 1067.3 mg / L, 1312.6 mg / L and 762.0 mg / L, respectively. F169A had the greatest improvement in the catalytic activity of thiamine, and Y113, Y140, F169 and E206 were screened for further mutation improvement.

[0084] The four sites of Y113, Y140, F168 and E206 were subjected to superimposed mutation (double mutation, triple mutation and quadruple mutation). The genes of 10 mutants of thiaminease II, Y113A / Y140A, Y113A / F169A, Y113A / E206A, Y140A / F169A, Y140A / E206A, F169A / E206A, Y113A / Y140A / F169A, Y113A / Y140A / E206A, Y140A / F169A / E206A and Y113A / Y140A / F169A / E206A, were respectively constructed into the pT17 vector using the method used in Example 1. After correct sequencing, they were transformed into Bacillus amyloliquefaciens BAX-10 to construct the corresponding mutant engineering bacteria. The successfully constructed multi-site mutant engineering bacteria and the engineering bacteria expressing Y113A, Y140A, F169A and E206A, respectively, and BAX-10 / pT17-HZ12- tenA The engineered bacteria were used for fermentation to produce 4-methyl-5-(β-hydroxyethyl)thiazole. At the same time, 1 mL of the fermentation liquid was collected after 48 h of cultivation to detect the yield of 4-methyl-5-(β-hydroxyethyl)thiazole. The results Fig. 9 shown.

[0085] The results showed that the ability of three engineering bacteria expressing double-site mutants to produce 4-methyl-5-(β-hydroxyethyl)thiazole was significantly better than that of the engineering bacteria BAX-10 / pT17-HZ12- tenA The target product yields in the reaction solution of the engineering bacteria expressing Y113A / F169A, Y140A / F169A and F169A / E206A thiaminease II double-point mutants were 1955.9 mg / L, 785.9 mg / L and 1226.0 mg / L, respectively. Among them, the yield of the Y113A / F169A engineering bacteria with only double mutations exceeded that of the single mutation F169A engineering bacteria, and was higher than that of the engineering bacteria BAX-10 / pT17-HZ12- tenA The production increased by 6.5 times.

[0086] Example 4 This embodiment provides a method for encapsulating BAX-10 / pT17-HZ12- tenA Y113A / F169A Water-in-water Pickering emulsions of recombinant engineered bacteria.

[0087] Weigh maltodextrin (MD) and hydroxypropyl methylcellulose (HPMC) powders, use deionized water as solvent, prepare 20wt% MD aqueous solution and 8wt% HPMC aqueous solution respectively, and dissolve or swell them under magnetic stirring at room temperature overnight for use.

[0088] The above solutions were mixed in a mass ratio of MD aqueous solution to HPMC aqueous solution of 7:20, and after standing, the phases were separated to prepare an emulsion system of HPMC:MD=4:3.5, in which the dispersed phase was the MD phase and the continuous phase was the HPMC phase. SiO2 with a mass fraction of 0.05% (the total mass of the polymer mixed system consisting of the continuous phase and the dispersed phase was used as a reference), liposomes with a mass fraction of 0.1% or cellulose nanocrystals (CNCs) with a mass fraction of 0.05% were added and dispersed in the continuous phase. BAX-10 / pT17-HZ12- tenA Y113A / F169A and 2wt% LB liquid medium (engineered bacteria content 1.23×10 9 CFU / g) was added to the dispersed phase under mechanical stirring to generate a stable water-in-water Pickering emulsion containing engineered bacteria in one step (emulsification conditions: temperature 5°C, stirring speed 200 rpm).

[0089] Example 5 Example 5 provides a method for encapsulating BAX-10 / pT17-HZ12- tenA Y113A / F169A The water-in-water Pickering emulsion of the recombinant engineered bacteria is different from that of Example 4 in that the solid particle stabilizers used are SiO2 and liposomes, and the added mass fractions are 0.05% and 0.1%, respectively; or the solid particle stabilizers used are SiO2 and cellulose nanocrystals (CNCs), and the added mass fractions are both 0.05%; or the solid particle stabilizers used are liposomes and cellulose nanocrystals (CNCs), and the added mass fractions are 0.1% and 0.05%, respectively.

[0090] Verification Example 1 According to the preparation method provided in Example 4, LB medium of different concentrations (0wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%) was added to the emulsion system with HPMC:MD=4:3, and the emulsion stability and bacterial inclusion were observed. The results are as follows: Fig.10 , 11 shown.

[0091] Fig.10 The microscopic images of HPMC-MD W / W emulsions added with different concentrations of LB medium at 0 h. Fig.11 The microscopic images of HPMC-MD W / W emulsions with different concentrations of LB medium added at 24 h; a-f are LB medium concentrations of 0wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, and 2.5wt% in order. As can be seen from the figure, at 0 h, the droplet size in the same system is quite different, the overall droplet is small, and the droplet size will gradually decrease over time, and the encapsulation is not ideal. This shows that the emulsion system of HPMC:MD=4:3 is not suitable for the encapsulation of the engineered bacteria provided by the present invention.

[0092] According to the preparation method provided in Example 4, LB medium of different concentrations (0wt%, 1wt%, 2wt%) was added to the emulsion system with HPMC:MD=4:3.5, and the emulsion stability and bacterial inclusion were observed at 0 h. The results are as follows: Fig.12 As shown, the overall droplet size increases with the increase in the concentration of the culture medium, but the emulsion size varies greatly at the same concentration, indicating that the emulsion is unstable.

[0093] According to the preparation method provided in Example 4, different concentrations (1wt%, 2wt%) of LB culture medium and different single stable granules (0.05% SiO2, 0.1% liposomes, 0.05% CNCs) were added to the emulsion system with HPMC:MD=4:3.5, and the emulsion stability and bacterial encapsulation were observed at 0 h. The results are as follows: Fig.13 As shown in the figure, after adding a single stable granule, the emulsion is distributed more evenly and has better stability. Moreover, as the content of LB medium increases, the droplet diameter becomes larger and more engineered bacteria can be encapsulated.

[0094] According to the preparation method provided in Example 4, different concentrations (1wt%, 2wt%) of LB culture medium and different mixed stable granules (0.05% SiO2+0.1% liposomes, 0.05% SiO2+0.05% CNCs, 0.1% liposomes+0.05% CNCs) were added to the emulsion system with HPMC:MD=4:3.5, and the emulsion stability and bacterial encapsulation were observed at 0 h. The results are as follows. Fig.14 As shown in the results, the addition of mixed stabilizers will increase the stability of the emulsion. The emulsion containing 2wt% LB medium and mixed stabilizers has a large amount of bacteria and is evenly distributed, and has good stability.

[0095] In summary, the thiaminase II and mutants thereof provided by the present invention show significant enzyme activity in catalyzing the synthesis of 4-methyl-5-(β-hydroxyethyl)-thiazole. Compared with the wild-type thiaminase II, the catalytic efficiency and substrate conversion rate of the mutant are significantly enhanced. The mutant provided by the present invention and the engineered bacteria capable of expressing the mutant show excellent application potential in the preparation of 4-methyl-5-(β-hydroxyethyl)-thiazole, and can be widely used in the fields of health care products, medicines and food additives.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A use of thiaminase II in producing 4-methyl-5-(β-hydroxyethyl)thiazole, wherein the amino acid sequence of the thiaminase II is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that The nucleotide sequence encoding the thiaminease II is shown in SEQ ID NO.

1.

3. A thiaminease II mutant, characterized in that The thiaminease II mutant is obtained by mutating one or more of the 48th tyrosine, 49th lysine, 78th glycine, 113th tyrosine, 140th tyrosine, 164th tyrosine, 168th tryptophan, 169th phenylalanine and 206th glutamate of thiaminease II, and the amino acid sequence of the thiaminease II is shown in SEQ ID NO.

4.

4. The thiaminease II mutant according to claim 3, characterized in that The thiaminease II mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to at least one of the following mutations: Mutating tyrosine at position 48 to alanine; and / or mutating lysine at position 49 to alanine; and / or Mutating glycine at position 78 to alanine; and / or Mutating tyrosine at position 113 to alanine; and / or mutating tyrosine at position 140 to alanine; and / or Mutating tyrosine at position 164 to alanine; and / or Mutating tryptophan at position 168 to alanine; and / or Mutating phenylalanine at position 169 to alanine; and / or The glutamic acid at position 206 was mutated to alanine.

5. The thiaminease II mutant according to claim 4, characterized in that The thiaminease II mutant is a single-site mutant or a double-site mutant; The single-site mutant is a single-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to any one of Y113A, Y140A, F169A or E206A single-site mutations; The double-site mutant is a double-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to any one of Y113A / F169A, Y140A / F169A or E206 / F169A double-site mutations.

6. The thiaminease II mutant according to claim 5, characterized in that The thiaminease II mutant is a single-site mutant or a double-site mutant, characterized in that: The single-site mutant is a single-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to a single-site mutation of F169A; The double-site mutant is a double-site mutant obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to double-site mutations of Y113A / F169A.

7. A gene encoding the thiaminease II mutant according to any one of claims 3 to 6.

8. A recombinant expression vector containing the gene with a nucleotide sequence as shown in SEQ ID NO.1 or as described in claim 7, and a recombinant engineered bacterium containing the recombinant expression vector.

9. An engineered bacillus amyloliquefaciens strain that produces high yield of 4-methyl-5-(β-hydroxyethyl)thiazole, characterized in that: The gene of the thiaminease II mutant according to claim 7 can be expressed; the host cell is Bacillus amyloliquefaciens BAX-10 and / or the overexpression vector is pT17.

10. A water-in-water Pickering emulsion encapsulating the recombinant engineered bacteria according to claim 8, characterized in that: It comprises a continuous phase and a dispersed phase; the continuous phase comprises a continuous phase polymer system and a solid particle stabilizer, and the dispersed phase comprises the recombinant engineered bacteria and the dispersed phase polymer system according to claim 8; the continuous phase polymer system and the dispersed phase polymer system contain one or more water-soluble polymers, and the absolute value of the difference between the solubility parameter of the water-soluble polymer contained in the continuous phase polymer system relative to water and the solubility parameter of the water-soluble polymer contained in the dispersed phase polymer system relative to water is greater than 0.

5.

11. Use of the thiaminease II mutant according to any one of claims 3 to 6, the recombinant expression vector according to claim 8 and the recombinant host cell containing the recombinant expression vector, the engineered Bacillus amyloliquefaciens according to claim 9 or the water-in-water Pickering emulsion according to claim 10 in catalytic synthesis of 4-methyl-5-(β-hydroxyethyl)thiazole.

12. A method for synthesizing 4-methyl-5-(β-hydroxyethyl)thiazole, characterized in that: include: A whole-cell catalytic reaction is performed on a genetically engineered bacterium capable of expressing thiaminase II having an amino acid sequence as shown in SEQ ID NO.4 or a thiaminase II mutant as described in any one of claims 3 to 6 to obtain a whole-cell catalytic reaction liquid, wherein the whole-cell catalytic reaction liquid contains the 4-methyl-5-(β-hydroxyethyl)thiazole.

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