Thiaminase ii and mutants and uses thereof

Abstract

The application belongs to the technical field of genetic engineering, and specifically discloses a thiaminase II, a mutant of the thiaminase II and application, wherein the amino acid sequence of the thiaminase II is shown as SEQ ID NO. 4, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1. The application further provides a mutant of the thiaminase II, wherein the mutant of the thiaminase II is obtained by mutating one 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 glutamic acid of the thiaminase II. The thiaminase II or each mutant provided by the application has higher ability in catalyzing synthesis of 4-methyl-5-(beta-hydroxyethyl)-thiazole, can be used for preparing 4-methyl-5-(beta-hydroxyethyl)-thiazole, and the activity of the mutant is obviously higher than that of the wild type, so that the application has great application prospect in the fields of health products, medicines and food additives.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and specifically discloses a thiaminase II and a mutant and application thereof. BACKGROUND

[0002] 4-methyl-5-(beta hydroxyethyl)-thiazole, also known as thiazole, is a light yellow liquid, has no volatility, has an unpleasant smell of thiazole compounds, but has a pleasant fragrance at a very dilute concentration, and can form a hydrochloride salt dissolved in water and alcohol with HCl. Thiazole is the basic structure ring of vitamin B1, is an important intermediate for synthesizing vitamin B1, has nutty, milk, egg smell, meat aroma, etc., and can be used as a spice and essence.

[0003] At present, 4-methyl-5-(beta-hydroxyethyl)-thiazole widely used as a food additive on the market is a chemical synthetic product. Although the synthesis method has been optimized, the optimized method is still high in cost and accompanied by many side reactions, and the reagents used produce large pollution and are high in energy consumption. For example, the invention patent with the publication number CN111635375A discloses a synthesis method of thiazole. Methyl acetoacetate and 3-bromo-3-chloropropyl acetate are used as raw materials to prepare 4-acetyloxy-2-acetyl-2-chlorobutyric acid methyl ester under alkaline conditions, and thiazole is obtained through acid hydrolysis, condensation, oxidation and alkaline hydrolysis. In the above synthesis process, the raw material 3-bromo-3-chloropropyl acetate is high in cost, and needs to be hydrolyzed in two steps, which is accompanied by many side reactions, affects the yield of the product, and increases the difficulty of product purification. Therefore, how to develop a green, environmentally friendly, safe and mild biological synthesis method to realize the large-scale synthesis of 4-methyl-5-(beta-hydroxyethyl) thiazole is a technical problem to be solved. SUMMARY

[0004] In view of the above defects, the application provides a thiaminase II and a mutant and application thereof. By screening thiaminase II in different bacillus strains, high-efficiency thiaminase II for synthesizing 4-methyl-5-(beta-hydroxyethyl) thiazole from Bacillus amyloliquefaciens (B.amyloliquefaciens) HZ12 and an expression gene thereof are obtained. Bacillus amyloliquefaciens The thiaminase II and the mutant and application thereof tenA ; a series of thiaminase II mutants are obtained by point mutation of the enzyme, and an engineering strain expressing the mutants is constructed. The yield of 4-methyl-5-(beta-hydroxyethyl) thiazole synthesized by whole-cell catalysis reaches 1955.9 mg / L, which is 6.5 times higher than that of the original TenA thiaminase II from Bacillus amyloliquefaciens HZ12.

[0005] To achieve the above object, the application provides the following technical solutions.

[0006] In a first aspect, the present application provides an application of thiaminase II in producing 4-methyl-5-(beta-hydroxyethyl)thiazole, wherein the amino acid sequence of the thiaminase II is shown as SEQ ID NO. 4.

[0007] The amino acid sequence of SEQ ID NO. 4:

[0008] MMTFSEECRQAAAEWWDGSFVHPFVTGIGDGSLPIDRFSYYVLQDSYYLTHFAKVQAFGAAYAEDLFTTGRMAGHAQGTYEAEMALHREFTELLGITEEERAAFKPAPTAYSYTSHMYRSVMSGNFGEILAALLPCYWLYYEVGEHLKECKPEHPIYEKWIGTYGGDWFRQQVEEQISRFDEIAENSTEEIRAKMKENFVISSYYEYQFWGMAYQKEGWSEKDGKEVESFGAARGN.

[0009] The inventors mined the key enzymes in the thiamin biosynthesis pathway according to the literature, and found the thiaminase crystal structure and active site analysis by querying the KEGG about the synthesis metabolic pathway of thiamin and comparing the additives available in food, and analyzed that Bacillus has a key gene encoding thiaminase II tenA , and the thiaminase II encoded by the gene can hydrolyze thiamin to generate 4-methyl-5-(beta-hydroxyethyl)thiazole.

[0010] The inventors screened thiaminase II in a series of food safety level Bacillus strains, and found that compared with other Bacillus, the thiaminase II encoded by the tenA gene from Bacillus amyloliquefaciens HZ12 has excellent effect of synthesizing 4-methyl-5-(beta-hydroxyethyl)thiazole. tenA The Bacillus amyloliquefaciens engineering strain BAX-10 / pT17-HZ12- tenA constructed by using the gene can produce 4-methyl-5-(beta-hydroxyethyl)thiazole through whole cell catalysis, and the yield can reach 113.08 mg / L.

[0011] Preferably, the nucleotide sequence encoding the thiaminase II is shown as SEQ ID NO. 1.

[0012] The nucleotide sequence of SEQ ID NO. 1:

[0013] ATGATGACATTTTCTGAAGAATGCAGACAGGCGGCTGCCGAATGGTGGGACGGAAGTTTTGTTCATCCGTTTGTAACGGGAATCGGTGACGGAAGCCTGCCGATTGACCGTTTCAGCTATTACGTGCTGCAAGATTCCTACTATTTGACGCATTTTGCAAAAGTTCAGGCGTTCGGCGCCGCATATGCCGAAGACTTGTTTACGACGGGCAGGATGGCGGGCCACGCTCAAGGGACATATGAAGCGGAAATGGCGCTGCACCGCGAGTTTACGGAGCTTTTAGGAATTACGGAAGAAGAGCGTGCGGCATTTAAACCGGCACCGACCGCCTATTCTTATACGTCTCATATGTACAGGTCGGTGATGAGCGGCAATTTTGGAGAAATATTAGCTGCGCTTCTGCCTTGTTATTGGCTTTATTATGAAGTCGGCGAACACCTAAAAGAGTGCAAACCGGAGCATCCGATTTATGAAAAATGGATCGGTACATACGGCGGAGACTGGTTCAGACAGCAGGTCGAAGAACAGATCAGCCGTTTTGACGAGATTGCTGAAAACAGCACAGAGGAAATCCGCGCGAAAATGAAAGAGAATTTTGTGATTTCAAGCTATTATGAATATCAATTTTGGGGAATGGCTTATCAAAAAGAAGGCTGGTCCGAGAAGGACGGAAAAGAGGTGGAATCCTTTGGAGCTGCACGCGGTAACTGA.

[0014] In a second aspect, the present application also provides a mutant of thiaminase II, which is obtained by mutating one 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 glutamic acid of thiaminase II, the amino acid sequence of which is shown as SEQ ID NO. 4.

[0015] The thiaminase II mutant provided by the present application includes 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.

[0016] The thiaminase II mutant provided by the present application also includes multiple point mutations, and the mutation position is 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 glutamic acid.

[0017] On the basis of the thiaminase II to be protected by the present application, homologous protein modeling and molecular docking are carried out according to the data in the database established according to the analysis of the structure and function of natural proteins, and the binding site of thiaminase II and substrate thiamin is further analyzed and modified, and it is found that the above-mentioned site is the active site of thiaminase II, and the activity of wild-type thiaminase II can be improved after modification.

[0018] Preferably, the thiaminase II mutant is obtained by at least one mutation of the amino acid sequence shown in SEQ ID NO. 4:

[0019] the 48th tyrosine is mutated into alanine; and / or

[0020] the 49th lysine is mutated into alanine; and / or

[0021] the 78th glycine is mutated into alanine; and / or

[0022] the 113th tyrosine is mutated into alanine; and / or

[0023] the 140th tyrosine is mutated into alanine; and / or

[0024] the 164th tyrosine is mutated into alanine; and / or

[0025] the 168th tryptophan is mutated into alanine; and / or

[0026] the 169th phenylalanine is mutated into alanine; and / or

[0027] the 206th glutamic acid is mutated into alanine.

[0028] The amino acid site mutation "the 48th tyrosine is mutated into alanine" of the present application means that the 48th amino acid of the amino acid sequence shown in SEQ ID NO. 4 is mutated from tyrosine to alanine; the rest of the site mutation is described in the same way.

[0029] The nucleotide sequence shown in SEQ ID NO. 1 is subjected to alanine single base mutation to construct a vector and an engineered bacterium, thereby obtaining an engineered bacterium with single-point alanine mutation at positions 48, 49, 78, 113, 140, 164, 168, 169 and 206. The successfully constructed engineered bacterium is subjected to fermentation and HPLC detection of the target product, and the yield of the target product is better than that of the unmutated engineered bacterium.

[0030] Preferably, the thiaminase II mutant is a single-site mutant or a double-site mutant.

[0031] The single-site mutant is a single-site mutant obtained by any one of Y113A, Y140A, F169A or E206A amino acid single-site mutation of the amino acid sequence shown in SEQ ID NO. 4.

[0032] The double-site mutant is a double-site mutant obtained by any one of Y113A / F169A, Y140A / F169A or E206 / F169A amino acid double-site mutation of the amino acid sequence shown in SEQ ID NO. 4.

[0033] The amino acid site mutation "Y113A" of the present application means that the 113th amino acid of the amino acid sequence shown in SEQ ID NO. 4 is mutated from tyrosine (Y) to alanine (A); the rest of the site mutation is described in the same way.

[0034] The amino acid site mutation "Y113A / F169A" of the present application 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 rest of the site mutation is described in the same way.

[0035] The yield of 4-methyl-5-(β-hydroxyethyl)thiazole of the engineered bacterium with single-point alanine mutation at Y113, Y140, F169 and E206 sites is significantly improved.

[0036] The Y113, Y140, F169 and E206 sites are subjected to superimposed mutation, and the yield of 4-methyl-5-(β-hydroxyethyl)thiazole of the engineered bacterium with superimposed double mutation of Y113A / F169A, Y140A / F169A and E206 / F169A is significantly improved compared with the unmutated engineered strain.

[0037] More preferably, the thiaminase II mutant is a single-site mutant or a double-site mutant, the single-site mutant is a single-site mutant obtained by mutating the amino acid sequence shown in SEQ ID NO. 4 at F169A;

[0038] the double-site mutant is a double-site mutant obtained by mutating the amino acid sequence shown in SEQ ID NO. 4 at Y113A / F169A.

[0039] The amount of 4-methyl-5-(β-hydroxyethyl)thiazole produced by the engineered bacteria expressing the thiaminase II mutant with the single-site F169A mutation 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 thiaminase II mutant with the double-site Y113A / F169A mutation can reach 1955.9 mg / L, which is 6.5 times that of the unmutated engineered bacteria.

[0040] In a third aspect, the present application also provides a gene encoding the thiaminase II mutant described above.

[0041] The gene encoding each single-site mutant or multi-site mutant described in the present application also falls within the protection scope of the present application.

[0042] In a fourth aspect, the present application also provides a recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO. 1 or the gene described above, and a recombinant engineered bacteria containing the recombinant expression vector.

[0043] The recombinant expression vector or the recombinant engineered bacteria containing the thiaminase II encoding gene or the gene encoding each mutant of thiaminase II described in the present application also falls within the protection scope of the present application.

[0044] The recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic vector, including but not limited to pHY300PKL, pMA5 and T2.

[0045] The present application exemplarily provides a preparation method of a recombinant host cell containing the gene described above, comprising:

[0046] S1: connecting the thiaminase II or mutant encoding gene with an expression regulatory element to construct a recombinant expression vector;

[0047] S2: transforming the recombinant expression vector described in S1 into a host cell, and obtaining a recombinant engineered bacteria after verification of expression.

[0048] In a fifth aspect, the present application further provides a high-yield 4-methyl-5-(β-hydroxyethyl)thiazole Bacillus amyloliquefaciens engineering bacteria, which can express the gene of the above-mentioned thiaminase II mutant; the host cell is Bacillus amyloliquefaciens BAX-10 and / or the overexpression vector is pT17.

[0049] Bacillus amyloliquefaciens BAX-10 is a modified engineering bacteria obtained by knocking out 7 important extracellular protease genes (aprE, aprX, nprE, nprX, nprS, nprM and nprL) of Bacillus amyloliquefaciens HZ12 original strain, epr, nprE, aprE-a, mpr, pbpF, vpr, ykct1 1 important intracellular protease gene (prc) and 2 redundant protein genes (yvdH and yvdI) on the basis of the original strain, and the deletion of the 10 genes has no negative impact on the growth of the bacteria. aprX htrB, hag Bacillus amyloliquefaciens BAX-10, as a host cell, has the ability to efficiently express target extracellular proteins, and experiments have proved that the activity of the alkaline protease (the gene is aprE) expressed by BAX-10 is increased by 57% compared with the original strain without knocking out. aprE

[0050] The pT17 plasmid is fused with P43 promoter and Tamly terminator on the basis of the original pHY300PLK plasmid, which is beneficial to the expression of the target gene.

[0051] In a sixth aspect, the present application further provides a water-in-water Pickering emulsion loaded with the above-mentioned recombinant engineering bacteria, which 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 above-mentioned recombinant engineering bacteria and a dispersed phase polymer system; the continuous phase polymer system and the dispersed phase polymer system comprise 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 with respect to water and the solubility parameter of the water-soluble polymer contained in the dispersed phase polymer system with respect to water is greater than 0.5.

[0052] The water-in-water Pickering emulsion is particularly suitable for preparing and delivering active ingredients and nutrients, and as a microreactor, it can provide a good living environment for active substances such as probiotics. After fermentation and drying of the water-in-water Pickering emulsion loaded with the above-mentioned recombinant engineering bacteria, a microcapsule preparation embedding the recombinant engineering bacteria can be obtained.

[0053] Preferably, the continuous phase comprises 3.0wt%-5.5wt% of water-soluble polymers and a solid particle stabilizer.

[0054] Preferably, the mass fraction of the solid particle stabilizer in the water-in-water Pickering emulsion is 0.025wt%-0.100wt%

[0055] ​​Preferably, the solid particulate stabilizer is a water-insoluble micro-nanoparticle, which is one or more of inorganic nanoparticles, organic nanoparticles and inactivated microorganisms.

[0056] More preferably, the solid particulate stabilizer is one or more of SiO2, liposome or cellulose nanocrystal in combination.

[0057] Preferably, the dispersed phase comprises 2.5wt%-4.0wt% of water-soluble polymer and the viable cell count of the above-mentioned recombinant engineering bacteria is 10 7 -10 10 CFU / g.

[0058] Preferably, the water-soluble polymer in the dispersed phase polymer system and the water-soluble polymer in the continuous phase polymer system are each 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 methyl cellulose, methyl cellulose, dextrin, carrageenan or guar gum, etc.; the water-soluble proteins include but are not limited to gelatin, collagen or casein, etc.

[0059] More preferably, the water-soluble polymer in the continuous phase is hydroxypropyl methyl cellulose.

[0060] More preferably, the water-soluble polymer in the dispersed phase is maltodextrin.

[0061] Preferably, the dispersed phase comprises nutrients that are beneficial to the growth and reproduction of the above-mentioned recombinant engineering bacteria, and the nutrients are selected from the nutritional components required for the growth of the recombinant engineering bacteria.

[0062] In a seventh aspect, the present application also provides the use of the thiaminase II mutant, the recombinant expression vector, the recombinant engineering bacteria containing the recombinant expression vector, the Bacillus amyloliquefaciens engineering bacteria or the water-in-water Pickering emulsion in the catalytic synthesis of 4-methyl-5-(β-hydroxyethyl)thiazole.

[0063] The thiaminase II mutant provided by the present application has very high activity and can efficiently synthesize 4-methyl-5-(β-hydroxyethyl)thiazole. Therefore, the mutant, the recombinant expression vector capable of expressing the mutant, the recombinant engineering bacteria or the water-in-water Pickering emulsion containing the recombinant engineering bacteria have wide application prospects in the production of food flavor 4-methyl-5-(β-hydroxyethyl)thiazole.

[0064] The 4-methyl-5-(β-hydroxyethyl)thiazole can be produced by using the thiaminase II mutant, the recombinant engineering bacteria or the water-in-water Pickering emulsion encapsulating the recombinant engineering bacteria provided by the present application through a feasible industrial biosynthesis method, including but not limited to fermentation method, enzyme catalysis extraction method and whole cell catalysis method, or using the water-in-water Pickering emulsion to culture the recombinant engineering bacteria at high density to obtain a reaction solution, and then separating and purifying the 4-methyl-5-(β-hydroxyethyl)thiazole from the obtained reaction solution.

[0065] In an eighth aspect, the present application further provides a method for synthesizing 4-methyl-5-(β-hydroxyethyl)thiazole, comprising:

[0066] The whole cell catalysis reaction is carried out on the genetic engineering bacteria capable of expressing the thiaminase II or the thiaminase II mutant as claimed in any one of claims 3-6, to obtain a whole cell catalysis reaction solution, wherein the whole cell catalysis reaction solution contains the 4-methyl-5-(β-hydroxyethyl)thiazole. BRIEF DESCRIPTION OF DRAWINGS

[0067] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0068] Figure 1 The construction process of the free expression plasmid pT17-HZ12- tenA in Example 1 of the present application.

[0069] Figure 2 The double enzyme digestion identification results of the pT17-HZ12- tenA free expression plasmid in Example 1 of the present application; (Lane M: DL5000 DNA Maker; Lane 1: tenA Fragment; Lane 2: Linear plasmid pT17)

[0070] Figure 3 The colony PCR identification results of the BAX10 / pT17-HZ12- tenA engineering bacteria constructed in Example 1 of the present application; (Lane M: DL5000 DNA Maker; Lane 1: PCR results of BAX10 / pT17-HZ12- tenA )

[0071] Figure 4The concentration of 4-methyl-5-(beta-hydroxyethyl)thiazole in the liquid catalyzed by the whole cells of the four strains in Example 2 of the present application;

[0072] Figure 5 The protein structure prediction diagram of thiaminase II from Bacillus amyloliquefaciens HZ12 and the amino acid corresponding diagram of HZ12- tenA and BS168- tenA ;

[0073] Figure 6 The docking diagram of thiamin with the predicted thiaminase II protein from Bacillus amyloliquefaciens HZ12;

[0074] Figure 7 The interaction diagram of thiamin with the thiaminase II protein from Bacillus amyloliquefaciens HZ12;

[0075] Figure 8 The target product yield diagram of the alanine mutant engineering bacteria constructed in Example 3 of the present application;

[0076] Figure 9 The target product yield diagram of the alanine superimposed mutant engineering bacteria constructed in Example 3 of the present application;

[0077] Figure 10 The micrograph of the HPMC-MD W / W emulsion at 0 h in Example 1 of the present application, which is based on the emulsion system of HPMC:MD=4:3 and added with different concentrations of LB medium;

[0078] Figure 11 The micrograph of the HPMC-MD W / W emulsion at 24 h in Example 1 of the present application, which is based on the emulsion system of HPMC:MD=4:3 and added with different concentrations of LB medium;

[0079] Figure 12 The micrograph of the HPMC-MD W / W emulsion in Example 1 of the present application, which is based on the emulsion system of HPMC:MD=4:3.5 and added with different concentrations of LB medium;

[0080] Figure 13 The micrograph of the HPMC-MD W / W emulsion in Example 1 of the present application, which is based on the emulsion system of HPMC:MD=4:3.5 and added with single stable granules;

[0081] Figure 14 The micrograph of the HPMC-MD W / W emulsion in Example 1 of the present application, which is based on the emulsion system of HPMC:MD=4:3.5 and added with mixed stable granules. DETAILED DESCRIPTION

[0082] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0083] Biological material source description:

[0084] Bacillus amyloliquefaciens BAX-10, Bacillus amyloliquefaciens HZ12, Bacillus subtilis 168 and Bacillus licheniformis BL9 are all from the Wei Xue team of Huazhong Agricultural University.

[0085] Bacillus amyloliquefaciens BAX-10 has been disclosed in the article "Efficient production of extracellular alkaline protease in Bacillus amyloliquefaciens by host strain construction" (LWT-Food Science and Technology, 2022, 163, 113620.);

[0086] Bacillus amyloliquefaciens HZ12 has been disclosed in the article "Multilcvel Mctabolic Engincering of Bacillus amyloliqucracicns for Production of the Platlorm Chemical Putrcscincfrom Sustainablc Biomass llydrolysates" (ACS Sustainablc Chemistry & Engineering, 2020, 8, 2147-2157.).

[0087] Culture medium and whole cell catalytic liquid used in the embodiments of the present application:

[0088] LB culture medium: 10 g / L of proteose peptone, 5 g / L of yeast extract powder, 10 g / L of sodium chloride, and 15 g / L of agar powder in solid culture medium, pH 7.2-7.4.

[0089] Whole cell catalytic liquid: 27.6 g / L of NaH2PO4·H2O, 71.6 g / L of Na2HPO4·12H2O, 0.4% Triton X-100, 20 g / L of thiamine, pH=6.6.

[0090] Method for detecting 4-methyl-5-(beta-hydroxyethyl)thiazole and method for making standard curve:

[0091] The detection of 4-methyl-5-(beta-hydroxyethyl)thiazole is carried out by high performance liquid chromatography. The high performance liquid chromatograph is Agilent 1100; the chromatographic column is ZORBAX Eclipse Plus XDB-C18 (4.6 mm x 250 mm, 5 μm); the mobile phase is 0.01 mol / L PH 6.6 PB buffer (A phase) and methanol (B phase); the detection condition is that the flow rate is 0.6 mL / min, the detection wavelength is 254 nm, and the column temperature is 30 o C, the injection amount is 10 μL; the gradient elution condition is shown in Table 1, and 4-methyl-5-(beta-hydroxyethyl)thiazole is eluted at 17.2 min.

[0092] Table 1 Gradient elution condition

[0093]

[0094] The standard sample of 4-methyl-5-(beta-hydroxyethyl)thiazole is accurately taken by a pipette to prepare 4-methyl-5-(beta-hydroxyethyl)thiazole samples with concentrations of 2.0, 5.0, 8.0, 10, 20, 50, 80, 100, 150 and 200 mg / L, the prepared samples are filtered through a water phase filter membrane with a pore size of 0.22 μm, then the filtered sample solution is loaded into a liquid phase bottle for standby, and then placed in a high performance liquid chromatograph, to draw a standard curve with the solution concentration as the abscissa and the peak area as the ordinate. The linear equation of the standard curve used in the embodiment of the application is Y=21.40X+9.253, and the correlation coefficient r 2 =0.9998.

[0095] Unless otherwise specified, the technical means used in the examples is the routine means familiar to those skilled in the art. The test methods in the following examples are routine methods unless otherwise specified. The reagents and materials used are commercially available unless otherwise specified.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are merely illustrative.

[0097] Unless otherwise indicated, the practice of the present invention will utilize conventional botanical techniques, microbiology, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics techniques readily apparent to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods employed in the present invention for DNA extraction, phylogenetic tree construction, gene editing methods, gene editing vector construction, and gene-edited plant production, in addition to the methods employed in the following examples, can all be accomplished using methods disclosed in the existing literature.

[0098] Example 1

[0099] Thiaminase II gene tenA

[0100] A single colony of Bacillus amyloliquefaciens HZ12 was inoculated into 5 mL of LB liquid medium and cultured with shaking at 37°C overnight. The cells were collected by centrifugation and washed once with STE. The centrifuged cells were resuspended in 100 μL of Solution I and 20 μL of lysozyme (final concentration of 1 mg / mL). The cells were incubated at 37°C for 2-3 h or placed in a refrigerator at 4°C overnight. 200 μL of 2% SDS was added and the cells were incubated in a 50-60°C water bath for 30 min. 100 μL of 5 mol / L NaCl was added and mixed. The cells were centrifuged at 12000 r / min for 5 min and the supernatant was collected. The supernatant was extracted twice with an equal volume of phenol / chloroform. The supernatant was collected and placed in a new centrifuge tube. 2 volumes of anhydrous ethanol were added and the cells were precipitated at -20°C for 2 h. The cells were centrifuged at 12000 r / min for 5 min and the supernatant was discarded. The pellet was washed once with 70% ethanol, dried and dissolved in 50 μL of RNase (20 The genomic DNA of Bacillus amyloliquefaciens HZ12 was obtained after incubation with 4% paraformaldehyde (P < 0.05) at 37°C for 30 min and stored at -20°C.

[0101] 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, as shown in SEQ ID NO.1. The HZ12- tenA Gene fragments, please refer to the instruction manual for specific steps.

[0102] Table 2 Primer sequences

[0103]

[0104] PCR system: 2× Rapid Taq Master Mix 25 µL; template DNA 1 µL; upstream primer 2 µL; downstream primer 2 µL; add purified water to make the total system 50 µL.

[0105] 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).

[0106] 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 and specific primers (see Table 2) were used 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.

[0107] After the amplified DNA sequence was purified, restriction enzyme Xba I and BamHI After enzyme digestion, the purified fragment 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 BamH I and Xba 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. The free expression plasmid pT17-HZ12- tenA, pT17-168- tenA and pT17-BL9- tenA .

[0108] The engineering strain BAX-10 / pT17-HZ12-tenA was constructed by OMEGA kit and the above-mentioned episomal expression plasmid pT17-HZ12- tenAA small amount of DNA extraction was performed, and recombinant plasmid DNA was obtained. Then, 10 μL of the recombinant plasmid DNA (50 ng / μL) was added to the B. amyloliquefaciens BAX-10 competent cells, which were gently mixed and then transferred to a pre-cooled 2 mm electrotransformation cup. After 10 min of ice bath, the cells were subjected to a single electric pulse using an electric pulse transformation instrument at 2.4 KV. After the electric pulse, 800 μL of recovery medium was quickly added, and the cells were recovered at 37°C and 100 r / min for 3 h to obtain the transformants. The transformants were subjected to tetracycline (Tet) resistance test and colony PCR verification using pT17-F / R as the detection primer (as shown in Figure 3 , and the engineering bacteria BAX-10 / pT17-HZ12- tenA were obtained.

[0109] The same method was used to obtain the engineering bacteria BAX-10 / pT17-168- tenA and the engineering bacteria BAX-10 / pT17-BL9- tenA .

[0110] Example 2: Whole-cell catalytic reaction of B. amyloliquefaciens BAX-10 / pT17-HZ12- tenA

[0111] The engineering bacteria BAX-10 / pT17-HZ12- tenA were inoculated into LB liquid medium (50 mL in a 250 mL flask) and cultured at 180 r / min and 37°C for 8-12 h. When the OD 600 reached 3.5-4.0, the bacteria were inoculated into LB liquid medium (50 mL in a 250 mL flask) at a 3% inoculation amount, and cultured at 37°C and 180 r / min for 24 h. Then, the bacteria were centrifuged at 6000 r / min for 10 min, and the supernatant was discarded. The bacteria were washed twice with 0.01 mol / L PBS buffer (pH 6.6), and then resuspended in 10 mL (10 mL in a 50 mL flask) of 4-methyl-5-(β-hydroxyethyl)thiazole whole-cell catalytic liquid to form a cell suspension with an OD 600 of 10. The cell suspension was cultured at 37°C and 180 r / min for 48 h to obtain a whole-cell catalytic reaction liquid containing 4-methyl-5-(β-hydroxyethyl)thiazole. B. amyloliquefaciens BAX-10, the engineering bacteria BAX-10 / pT17-168- tenA , and the engineering bacteria BAX-10 / pT17-BL9- tenA were used as controls.

[0112] ​The 1 mL of the four whole cell catalytic liquids above was centrifuged at 10,000 r / min for 10 min at 4°C to obtain the supernatant, which was filtered through a 0.22 μm water phase filter membrane and then detected for 4-methyl-5-(β-hydroxyethyl) thiazole by high performance liquid chromatography. The content of 4-methyl-5-(β-hydroxyethyl) thiazole was determined according to the peak time and peak area size, and the results are shown in Table 1. Figure 4

[0113] As can be seen from Table 1, the yield of 4-methyl-5-(β-hydroxyethyl) thiazole of the engineering strain BAX-10 / pT17-HZ12- Figure 4 may reach 113.08 mg / L, which is significantly better than the control group, indicating that the thiamine II edited by the TenA gene from Bacillus amyloliquefaciens HZ12 has excellent ability to catalyze the synthesis of 4-methyl-5-(β-hydroxyethyl) thiazole. tenA tenA

[0114] Example 3 Determination of mutation points of thiamine II and construction of mutant

[0115] 1. Homologous alignment of HZ12- tenA sequence

[0116] Since there is no crystal structure analysis and report of TenA protein from Bacillus amyloliquefaciens HZ12, homologous sequence prediction and alignment of HZ12- tenA sequence were performed by SWISS-MODEL software. Finally, it was predicted that the sequence from Bacillus subtilis 168 (BS168- tenA ) is the closest to the HZ12- tenA sequence. The amino acid correspondence of the two different source tenA sequences is shown in Figure 5 A. And the three-dimensional structure of TenA thiamine II from Bacillus subtilis 168 has been reported, as shown in Figure 5 B. The subsequent molecular docking was performed on this three-dimensional structure of protein. The amino acids of the docking results need to be corresponded to the HZ12- Figure 5 sequence by tenA A.

[0117] 2. Preparation of receptor protein and treatment of small molecule ligand

[0118] ​​​This docking study was conducted using MOE 2019 (Chemical Computing Group, Canada). The BS168-TenA protein was first optimized using MOE's Quickprep module. The thiamine small molecule ligand was hydrogenated, charged, and energetically optimized using MOE's Wash, PartialCharges, and Energy Minimization modules.

[0119] 3. Molecular docking of small molecule ligands and receptor proteins

[0120] 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, such as Figure 6 As shown. In setting the docking parameters, the triangle matcher is selected to place the ligand in the binding site. 50 docking poses are set and sorted using the London dG scoring function. The 30 poses with the best scores are selected for refinement and energy minimization using the rigid receptor method. They are then re-scored using the GBVI / WSA dG scoring function. The docking results with the highest scores are selected for visualization, as shown in the figure below. Figure 7 shown.

[0121] Then the molecules predicted to have an effect were 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- tenA In the sequence of , D45, Y48, L49, F52, Q56, H75, G78, T79, C136, Y137, Y140, Y164, W168, F169 and Q172 were predicted and mapped to HZ12- tenA The amino acid position of the sequence. The amino acid at position 108 is predicted to be alanine and is therefore not set as a predicted mutation site.

[0122] Supplementation of mutation sites According to the 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- tenAThe D45, Y48, Y113, C136 and E206 amino acids of the sequence, among which the D45, Y48, and C136 amino acids have appeared in the molecular docking results, so the Y113 and E206 amino acids are added to the predicted mutation sites.

[0123] 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.

[0124] The construction of alanine mutants was based on the HZ12- tenA The nucleotide sequence of HZ12- 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 using the method used in Example 1. After correct sequencing, 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 the engineering strain BAX-10 / pT17-HZ12- tenA The fermentation culture of 4-methyl-5-(β-hydroxyethyl)thiazole was carried out. The activated engineered strain was inoculated into LB liquid medium at a 3% inoculum, cultured on a shaking table at 37°C for 12 h, and then 5 g / L thiamine was added. After culture for 48 h, 1 mL of fermentation liquid was collected for detection of 4-methyl-5-(β-hydroxyethyl)thiazole production. The results showed that Figure 8 shown.

[0125] Table 3 Primer sequences

[0126]

[0127] 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 engineered bacteria expressing the thiaminease II single point mutants Y48A, L49A, G78A, Y113A, Y140A, Y164A, W168A, F169A and E206A was higher than that of the unmutated engineered strain BAX-10 / pT17-HZ12- tenA, Y48, L49, G78, Y113, Y140, Y164, W168, F169 and E206 of wild-type thiaminase II are active sites of the enzyme. Among them, the 4-methyl-5-(β-hydroxyethyl) thiazole production capacity of the thiaminase II single-point mutant engineering bacteria expressing Y113A, Y140A, F169A and E206A is significantly improved, and the yield of the target product in the reaction solution is 559.2 mg / L, 1067.3 mg / L, 1312.6 mg / L and 762.0 mg / L, respectively. F169A has the largest improvement in catalytic activity of thiamine, and Y113, Y140, F169 and E206 are selected for further mutation improvement.

[0128] The Y113, Y140, F168 and E206 four-point sites are subjected to superimposed mutation (double, triple, and quadruple). The genes of 10 thiaminase II mutants 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 are respectively constructed into pT17 vector using the method of Example 1. After sequencing, the corresponding mutant engineering bacteria are constructed by transforming the amylolytic Bacillus BAX-10. The above successfully constructed multi-point mutant engineering bacteria and the thiaminase II single-point mutant engineering bacteria expressing Y113A, Y140A, F169A and E206A, respectively, and the BAX-10 / pT17-HZ12- tenA The fermentation culture of the thiaminase II double-point mutant engineering bacteria is carried out for 48 h, and then 1 mL of the fermentation solution is collected for detection of the yield of 4-methyl-5-(β-hydroxyethyl) thiazole. The results are shown in Table 2. Figure 9

[0129] The results show that the 4-methyl-5-(β-hydroxyethyl) thiazole production capacity of three engineering bacteria expressing double-point mutant is obviously better than that of the engineering bacteria BAX-10 / pT17-HZ12- tenA The yield of the target product in the reaction solution of the thiaminase II double-point mutant engineering bacteria expressing Y113A / F169A, Y140A / F169A and F169A / E206A is 1955.9 mg / L, 785.9 mg / L and 1226.0 mg / L, respectively. The yield of the engineering bacteria Y113A / F169A with only double-point superposition is higher than that of the single-point F169A engineering bacteria, which is 6.5 times higher than that of the engineering bacteria BAX-10 / pT17-HZ12- tenA .​

[0130] Example 4

[0131] This example provides a BAX-10 / pT17-HZ12- tenA Y113A / F169A Water-in-water Pickering emulsion of recombinant engineering bacteria

[0132] Maltodextrin (MD) and hydroxypropyl methyl cellulose (HPMC) powder were weighed out, deionized water was used as the solvent, and 20wt% MD aqueous solution and 8wt% HPMC aqueous solution were respectively configured, and were dissolved or swelled at room temperature under magnetic stirring overnight for standby use.

[0133] The above solutions were mixed according to the mass ratio of MD aqueous solution to HPMC aqueous solution of 7:20, and after standing, the emulsion system of HPMC:MD = 4:3.5 was prepared, the dispersed phase was MD phase, and the continuous phase was HPMC phase. 0.05wt% SiO2 (based on the total mass of the polymer mixture system composed of the continuous phase and the dispersed phase), 0.1wt% liposome or 0.05wt% cellulose nanocrystal (CNCs) were added and dispersed in the continuous phase, and BAX-10 / pT17-HZ12- tenA Y113A / F169A 2wt% LB liquid medium (engineering bacteria content of 1.23 x 10 9 CFU / g) was added to the dispersed phase under mechanical stirring to generate a stable water-in-water Pickering emulsion containing engineering bacteria by one-step method (emulsification conditions: temperature 5℃, stirring rate 200 rpm).

[0134] Example 5

[0135] Example 5 provides a BAX-10 / pT17-HZ12- tenA Y113A / F169A Water-in-water Pickering emulsion of recombinant engineering bacteria, which is different from Example 4 in that the solid particle stabilizer used is SiO2 and liposome, and the mass fraction added is 0.05% and 0.1% respectively; or the solid particle stabilizer used is SiO2 and cellulose nanocrystal (CNCs), and the mass fraction added is 0.05% respectively; or the solid particle stabilizer used is liposome and cellulose nanocrystal (CNCs), and the mass fraction added is 0.1% and 0.05% respectively.

[0136] Verification Example 1

[0137] The preparation method provided in Example 4 is used, and different concentrations (0wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%) of LB medium are added to the emulsion system of HPMC:MD=4:3 to observe the emulsion stability and the bacteria-encapsulating condition, and the results are shown in Figure 10 、 11

[0138] Figure 10 Fig. 1 is a micrograph of the HPMC-MD W / W emulsion with different concentrations of LB medium at 0 h; Figure 11 Fig. 2 is a micrograph of the HPMC-MD W / W emulsion with different concentrations of LB medium at 24 h; a-f are in order of LB medium concentration of 0wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt% respectively. As can be seen from the figures, at 0 h, the droplet size difference is obvious in the same system, the overall droplet is small, the droplet size will gradually decrease over time, and the bacteria-encapsulating condition is not ideal. It is shown that the emulsion system of HPMC:MD=4:3 is not suitable for the embedding of the engineering bacteria provided in the application.

[0139] The preparation method provided in Example 4 is used, and different concentrations (0wt%, 1wt%, 2wt%) of LB medium are added to the emulsion system of HPMC:MD=4:3.5 to observe the emulsion stability and the bacteria-encapsulating condition at 0 h, and the results are shown in Figure 12 It can be seen that the overall droplet size will increase with the increase of the concentration of the medium, but the emulsion size varies greatly at the same concentration, indicating that the emulsion is unstable.

[0140] The preparation method provided in Example 4 is used, and different concentrations (1wt%, 2wt%) of LB medium and different single stable granules (0.05% SiO2, 0.1% liposome, 0.05% CNCs) are added to the emulsion system of HPMC:MD=4:3.5 to observe the emulsion stability and the bacteria-encapsulating condition at 0 h, and the results are shown in Figure 13 As can be seen from the figures, after adding the single stable granule, the emulsion is distributed more uniformly, the stability is better, and with the increase of the content of the LB medium, the droplet diameter becomes larger, and more engineering bacteria can be encapsulated.

[0141] The preparation method provided in Example 4 is used, and different concentrations (1wt%, 2wt%) of LB medium and different mixed stable granules (0.05% SiO2+0.1% liposome, 0.05% SiO2+0.05% CNCs, 0.1% liposome+0.05% CNCs) are added to the emulsion system of HPMC:MD=4:3.5 to observe the emulsion stability and the bacteria-encapsulating condition at 0 h, and the results are shown in​Figure 14 It can be seen from the results that the addition of the mixed stabilizer increases the stability of the emulsion, the emulsion containing 2wt% LB medium and the mixed stabilizer has a large amount of bacteria and a uniform distribution, and has good stability.

[0142] In summary, the thiaminase II and the mutant thereof provided by the present application exhibit 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 application and the engineered bacteria capable of expressing the mutant exhibit excellent application potential in preparing 4-methyl-5-(β-hydroxyethyl)-thiazole, and can be widely applied in the fields of health products, medicines and food additives.

[0143] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A thiaminease II mutant, characterized in that The thiaminease II mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to the following mutations: The phenylalanine at position 169 was mutated to alanine.

2. The thiaminease II mutant according to claim 1, wherein The thiaminease II 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.

3. The thiaminease II mutant according to claim 2, wherein The thiaminease II 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.

4. A gene encoding the thiaminease II mutant according to any one of claims 1 to 3.

5. A recombinant expression vector containing the gene according to claim 4 and a recombinant engineered bacterium containing the recombinant expression vector.

6. An engineered Bacillus amyloliquefaciens strain capable of producing high yields of 4-methyl-5-(β-hydroxyethyl)thiazole, characterized in that: The gene of the thiaminease II mutant according to claim 4 can be expressed; and the host cell is Bacillus amyloliquefaciens BAX-10.

7. The engineered Bacillus amyloliquefaciens strain according to claim 6, wherein The gene of the thiaminease II mutant according to claim 4 is cloned into the vector pT17.

8. A water-in-water Pickering emulsion containing the recombinant engineered bacteria according to claim 5, 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 5; 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.

9. Use of the thiaminease II mutant according to any one of claims 1 to 3, the recombinant expression vector according to claim 5 and the recombinant host cell containing the recombinant expression vector, the engineered Bacillus amyloliquefaciens strain according to claim 6, or the water-in-water Pickering emulsion according to claim 8 in catalytic synthesis of 4-methyl-5-(β-hydroxyethyl)thiazole.

10. 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 the thiaminease II mutant according to any one of claims 1 to 3 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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