Xylose reductase mutant and application thereof in redox synthesis of D-tagatose

Abstract

The invention provides a mutant of xylose reductase and application of the mutant in synthesis of D-tagatose. According to the present invention, the xylose reductase (XR) derived from Saccharomyces cerevisiae is subjected to molecular modification on the basis of the sequence and structure-oriented semi-rational modification strategy so as to obtain the high activity xylose reductase mutant, and the xylose reductase mutant has the following characteristics that: the xylose reductase (XR) derived from Saccharomyces cerevisiae is subjected to molecular modification so as to obtain the xylose reductase mutant, according to the invention, the mutant of the xylose reductase is integrated into a redox pathway for synthesizing the D-tagatose by virtue of a genetic engineering means, and is expressed in bacillus subtilis 168, so that a recombinant engineering bacterium for synthesizing the D-tagatose by fermenting D-galactose is successfully constructed, the efficient synthesis of the D-tagatose is realized, and the yield of the D-tagatose is improved. And the method has obvious practical application value and economic benefit.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a mutant of xylose reductase and an application thereof in the synthesis of D-tagatose. Background Art

[0002] D-tagatose is a rare ketohexose with a molecular formula of C6H 12 O6 is the ketose isomer of D-galactose and also the C-4 diastereomer of D-fructose. Pure D-tagatose is white anhydrous crystals with no odor, a melting point of 134-135°C, and is easily soluble in water at 21°C (solubility is 58% (w / w)). In addition, D-tagatose has good acid stability and is prone to Maillard and caramelization reactions under high temperature conditions. In a 10% aqueous solution, the sweetness of D-tagatose is equivalent to 92% of sucrose, but its calories are only 1 / 3 of sucrose (1.5kcal / g), and there is no bad aftertaste or bitterness. D-tagatose has the characteristics of high safety, high sweetness and low calories, and can be used as a low-calorie sweetener, which has attracted widespread attention from researchers.

[0003] The production methods of D-tagatose include natural extraction, chemical synthesis and biosynthesis. However, natural extraction faces many difficulties, such as resource shortage, rare content, low extraction efficiency, etc.; chemical synthesis has problems such as high energy consumption, large environmental pollution, difficulty in product purification, high catalyst cost, etc., which are not in line with the concept of sustainable green development. In contrast, the biological preparation of D-tagatose has the advantages of mild conditions, green environmental protection, and easy product purification, which has attracted much attention and has become the main direction of current research on D-tagatose synthesis. In recent years, due to the inherent thermodynamic equilibrium limitations of the isomerization pathway, researchers have begun to focus on the synthesis pathway that can produce D-tagatose by biofermentation, that is, the redox synthesis pathway of D-tagatose. Its metabolic pathway is the Galactose Metabolism pathway annotated by KEGG (https: / / www.genome.jp / kegg / ), that is, after lactose enters the cell, it is hydrolyzed by GAL to form D-galactose and D-glucose, and then D-galactose enters the redox pathway and is hydrogenated and reduced by NAD(P)H-dependent aldose reductase (AR) to form galactitol, which is then converted into D-galactose by NAD(P)H-dependent aldose reductase (AR). +The galactitol 2-dehydrogenase (GDH) dehydrogenates to generate D-tagatose; at the same time, the D-glucose generated by the hydrolysis of lactose can be used as the main carbon source for cell growth, and D-galactose can also be used by cells as a secondary carbon source. Studies have shown that the rate-limiting step of this pathway is the catalysis of D-galactose by the aldose reductase, which is mostly named xylose reductase (AR), resulting in insufficient supply of galactitol, thus affecting the effective synthesis of D-tagatose. The root cause of this bottleneck is that the xylose reductase used has insufficient substrate affinity for D-galactose, or its naturally low enzyme activity. Due to the influence of the rate-limiting step on the synthesis of D-tagatose, the enzyme activity of xylose reductase can be improved through the molecular modification strategy of the enzyme, and its ability to convert D-galactose to form galactitol can be enhanced, thereby breaking through the bottleneck that limits the synthesis of the D-tagatose redox pathway.

[0004] Semi-rational modification of enzymes overcomes the challenges of irrational modification screening difficulties and rational modification's high reliance on computers and enzyme structure analysis. It usually includes strategies such as site-directed mutagenesis, single-point saturation mutagenesis, and iterative mutagenesis, and has received widespread attention in recent years. Semi-rational modification of ScXR can be used to obtain positive mutants with significantly improved enzyme activity, replace the original wild-type ScXR, further optimize the redox pathway for synthesizing D-tagatose, and try to further remove the rate limit of D-galactose to D-tagatose synthesis through multiple copies of ScXR. Through enzyme engineering strategies, the high-version chassis cells for synthesizing D-tagatose are further optimized from the enzyme function level. Therefore, it is necessary to develop a mutant of xylose reductase that can efficiently synthesize D-tagatase. Summary of the invention

[0005] In view of some deficiencies in the prior art, the present invention provides a mutant of xylose reductase and its application in the synthesis of D-tagatose; the present invention molecularly transforms the xylose reductase (XR) from Saccharomyces cerevisiae based on a sequence- and structure-guided semi-rational transformation strategy, thereby obtaining a highly active mutant of xylose reductase; the present invention also integrates the above-mentioned mutant of xylose reductase into the redox pathway for synthesizing D-tagatose by genetic engineering means, and expresses it in Bacillus subtilis 168, successfully constructs a recombinant engineered bacterium for fermenting D-galactose to synthesize D-tagatose, realizes the efficient synthesis of D-tagatose, and has significant practical application value and economic benefits.

[0006] Since the wild-type xylose reductase is derived from Saccharomyces cerevisiae (Xylose reductase, XR), it is denoted as ScXR.

[0007] In order to achieve the above technical effects, the present invention adopts the following technical means:

[0008] The present invention first provides a mutant of xylose reductase, wherein the mutant of xylose reductase includes a mutant obtained by performing any one or more of the following mutations on the basis of the wild-type xylose reductase ScXR as shown in the amino acid sequence of SEQ ID NO: 1:

[0009] (a) mutating the amino acid L at position 18 of ScXR to F;

[0010] (b) mutating the amino acid I at position 227 of ScXR to F;

[0011] (c) mutating the amino acid F at position 312 of ScXR to Y. Preferably, the mutant of xylose reductase is a mutant obtained by mutating the amino acid I at position 227 of ScXR to F and the amino acid F at position 312 to Y, and the amino acid sequence thereof is shown in SEQ ID NO:2.

[0012] The present invention also provides a nucleic acid encoding the mutant of the xylose reductase.

[0013] The present invention also provides a recombinant expression vector, which comprises the above nucleic acid.

[0014] The present invention also provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the above nucleic acid or the above recombinant expression vector.

[0015] Preferably, the host bacteria of the recombinant engineered bacteria include Bacillus subtilis.

[0016] Preferably, the Bacillus subtilis is Bacillus subtilis 168.

[0017] The present invention also provides the use of the mutant of the xylose reductase, or the recombinant expression vector, or the recombinant engineering bacteria in the efficient synthesis of D-tagatose.

[0018] The present invention also provides a method for efficiently synthesizing D-tagatose, which comprises: fermenting D-galactose to synthesize D-tagatose using a recombinant engineering bacterium expressing the mutant of the xylose reductase.

[0019] Preferably, the method comprises:

[0020] (1) constructing a recombinant expression vector expressing the mutant of the xylose reductase, and expressing the recombinant expression vector in a host cell to obtain a recombinant engineered bacterium expressing the mutant of the xylose reductase;

[0021] (2) Using a recombinant engineered bacterium expressing a mutant of xylose reductase as a fermentation strain, D-tagatose is fermented and synthesized in a fermentation medium containing D-galactose.

[0022] Preferably, the fermentation medium comprises: 10 g / L peptone, 5 g / L yeast extract, 3 g / L K2HPO4, 0.27 g / L MgSO4·7H2O, 0.53 g / L CaCl2, 50 g / L D-glucose, and 20 g / L D-galactose.

[0023] Preferably, the fermentation conditions are: fermentation at 37-45°C and an initial pH value of 7.5-8.5;

[0024] When the D-galactose content was lower than 5 g / L, feeding was performed using a semi-continuous feeding strategy to maintain the D-galactose content at 20-30 g / L, with 4-6 feedings every 12 hours.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) Compared with the prior art, the present invention obtains a mutant of xylose reductase by molecular modification of the enzyme, and the enzyme activity of the mutant of xylose reductase reaches 1.20-1.35 U / mg, which is 30-60% higher than that of the prior art. This helps to improve the enzyme activity of xylose reductase, the rate-limiting enzyme in the redox pathway of synthesizing D-tagatose, and thus improve the production efficiency of the synthetic redox pathway of D-tagatose. After experimental verification, based on the modified xylose reductase mutant, the production efficiency of the synthetic redox pathway of D-tagatose is increased by 25%-35%. This is because the conversion efficiency of the rate-limiting enzyme xylose reductase for D-galactose is improved, so that the generation of the intermediate product galactitol is increased, and the catalysis of the substrate galactitol by galactitol dehydrogenase is improved, so that the yield of D-tagatose is increased.

[0027] (2) The present invention constructs a B. subtilis genetically engineered bacterium that synthesizes D-tagatose through a heterologous redox pathway based on a mutant of the constructed xylose reductase. Compared with the prior art, the B. subtilis genetically engineered bacterium improves the product synthesis efficiency while ensuring the fermentation advantages of the fermentation strain itself. Compared with the prior art, the B. subtilis genetically engineered bacterium constructed by the present invention increases the yield of D-tagatose synthesized by fermenting D-galactose by 20% to 30%, which can reach 30g / L to 40g / L, which can reduce production costs and enhance economic benefits.

[0028] (3) The biocatalytic pathway adopted by the present invention complies with the principles of green chemistry and reduces environmental impact. The mutant of xylose reductase described in the present invention shows significant advantages over the prior art in terms of enzyme activity, synthesis efficiency, production cost and environmental friendliness, and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 For the construction of pANY1-GRE3 plasmid and protein expression.

[0030] Figure 2 are the enzymatic property parameters of ScXR.

[0031] Figure 3 The changes in the enzyme activity of the ScXR single-point mutants.

[0032] Figure 4 The changes in enzyme activity of ScXR combination mutants.

[0033] Figure 5 Effects of ScXR combination mutants on the redox pathway for D-tagatose synthesis.

[0034] Figure 6 ScXR I227F / F312Y 5 L fermentation tank profile for redox synthesis of D-tagatose. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The sources, trade names, and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are not specifically stated, and are the same as the first indicated content.

[0036] Example 1. Expression, purification and enzymatic performance characterization of ScXR:

[0037] (1) Expression and purification of ScXR:

[0038] S11. Using the cDNA of strain S.cerevisiae W303-1A as a template (Genbank No. GCA_000292815.1), primers SEQ ID NO: 3 and SEQ ID NO: 4 were designed based on the characteristics of the multiple cloning site and homology arms on the vector pANY1. The GRE3 gene fragment encoding ScXR (960 bp, Figure 1b); The PCR reaction conditions are: pre-denaturation: 95°C, 2 min, denaturation: 94°C, 10 s, annealing: 58°C, 30 s, extension: 72°C, 12 s, final extension: 72°C, 5 min, 34 cycles.

[0039] SEQ ID NO:3: taccgagaatttgtattttcagATGTCTTCACTGGTTACTCTTAAT;

[0040] SEQ ID NO: 4: aactgcaggagctcccatggTCAGGCAAAAGTGGGGAATTTA.

[0041] S12. The vector pANY1 was linearized using primers SEQ ID NO: 5 and SEQ ID NO: 6, and purified and recovered to obtain a linearized vector. The obtained GRE3 gene fragment and the linearized vector were used to construct a recombinant plasmid using seamless cloning technology. The recombinant plasmid map is shown in FIG. Figure 1 As shown in a.

[0042] SEQ ID NO:5: CCATGGGAGCTCCTGC;

[0043] SEQ ID NO:6: CTGAAAATACAAATTCTCGG.

[0044] S13. The obtained recombinant plasmid was transformed into E. coli BL21 competent cells, and the positive clones with successful transformation were screened by PCR verification, plasmid extraction verification and sequencing verification, and named as E. coli BL21 / pANY1-GRE3.

[0045] S14. The strain E. coli BL21 / pANY1-GRE3 was inoculated into LB medium (10 g / L yeast powder, 20 g / L tryptone, 20 g / L sodium chloride), and cultured at 37°C and 200 rpm with shaking until the OD was between 0.4 and 0.6. IPTG inducer with a final concentration of 1 mM was added, and ScXR expression was induced at 25°C and 120 rpm overnight. The cells were collected, washed twice with PBS, and then ultrasonically disrupted. The crude enzyme solution was obtained after high-speed centrifugation, and the pure enzyme ScXR was purified by Ni column.

[0046] The expression and purification of ScXR were detected by SDS-PAGE. Figure 1 As shown in c. As can be seen from the figure, ScXR was successfully expressed and purified to obtain a single pure protein band of 35 kDa. The pure enzyme sample was preserved and named ScXR, and its amino acid sequence is shown in SEQ ID NO:1.

[0047] SEQ ID NO:1

[0048] MSSLVTLNNGLKMPLVGLGCWKIDKKVCANQIYEAIKLGYRLFDGACDYGNEKEVGEGIRKAISEGLVSRKDIFVVSKLWNNFHHPDHVKLALKKTLSDMGLDYLDLYYIHFPIAFKYVPFEEKYPPGFYTGADDEKKGHITEAHVPIIDTYRALEECVDEGLI KSIGVSNFQGSLIQDLLRGCRIKPVALQIEHHPYLTQEHLVEFCKLHDIQVVAYSSFGPQSFIEMDLQLAKTTPTLFENDVIKKVSQNHPGSTTSQVLLRWATQRGIAVIPKSSKKERLLGNLEIEKKFTLTEQELKDISALNANIRFNDPWTWLDGKFPTFA.

[0049] (2) Characterization of the enzymatic properties of ScXR:

[0050] Xylose reductase can convert aldose to the corresponding sugar alcohol under the action of cofactors ( Figure 2 a), in order to evaluate the properties of the obtained ScXR, this step characterizes the enzymatic properties of the pure enzyme ScXR, and the specific steps are as follows:

[0051] S21. In different coenzymes (type I coenzyme: NADH / NAD + Or type II coenzyme: NADPH / NADP + ) was used to determine the coenzyme preference of ScXR. Figure 2 shown.

[0052] As can be seen from the figure, the reduction and oxidation activities of ScXR are strictly dependent on type II coenzymes NADPH and NADP + , while type I coenzymes NADH and NAD + The enzyme cannot be activated.

[0053] S22. The effect of different temperatures (20℃~70℃) on the enzyme activity of ScXR was studied, and the effect of pH on the enzyme activity of ScXR was evaluated under different pH conditions (pH 4~6: 0.1M potassium acetate buffer, pH 7~8: 0.1M potassium phosphate buffer, pH 9~12: 0.1M glycine-NaOH buffer). The results are shown in 2b and 2c.

[0054] As can be seen from the figure, the optimum pH of ScXR is 7.0 and 7.5 respectively; in the pH range of 6.0-7.5, the reduction activity is maintained above 50%, and in the pH range of 7.0-8.0, the oxidation activity is maintained above 50%. This shows that the ScXR enzyme activity will decrease significantly under over-acid or over-alkaline conditions, and its optimal reaction pH tends to be neutral. In addition, the optimum temperatures for the reduction activity and oxidation activity of ScXR are 60℃ and 50℃ respectively; below the optimum temperature, the enzyme activity increases slowly with the increase of temperature, while above the optimum temperature, the enzyme activity drops sharply to inactivation.

[0055] S23. Using no metal ions as control, evaluate the effect of different metal ions (EDTA, Ni 2+ 、Zn 2+ , Mn 2+ 、Co 2+ , Ca 2+ Mg 2+ , Fe 2+ , Cu 2+ ) on the activation or inhibition of ScXR enzyme activity, and the evaluation results are as follows Figure 2 As shown in d.

[0056] As can be seen from the figure, Ni 2+ 、Co 2+ , Ca 2+ and Mg 2+ It can significantly enhance its reducing ability, among which Ni 2+ The relative activity of ScXR was enhanced by 3.7 times; while Fe 2+ and Cu 2+ It will completely inhibit its enzyme activity.

[0057] S24. In this step, various aldoses, such as D-xylose, L-arabinose, D-galactose, D-glucose and D-mannose, were used as substrates to analyze the substrate spectrum of ScXR and explore its substrate affinity for different aldoses. Figure 2 As shown in e.

[0058] As can be seen from the figure, ScXR has the highest substrate affinity for D-xylose, followed by L-arabinose, while the relative enzyme activity for D-galactose is 78% of the optimal substrate.

[0059] In summary, ScXR only depends on type II coenzyme, and the optimum temperature and pH for its reduction activity are 60℃ and 7.0, respectively. 2+ It has a prominent activation effect on ScXR, D-xylose is the most suitable substrate for ScXR, and the relative activity is 78% when D-galactose is the substrate. Based on the characterized enzymatic properties of ScXR, it provides a basis for further modification of the enzyme and the redox synthesis of D-tagatose.

[0060] Example 2. Screening of single-point mutations of ScXR and single-point xylose reductase mutants:

[0061] In this example, the mutation target of ScXR and the target amino acid of site-directed mutation were determined by online website prediction and rational analysis based on sequence and structure, so as to perform molecular modification of ScXR enzyme and improve the xylose reductase activity of synthesizing D-tagatose. The modification steps are as follows:

[0062] (1) HotSpot Wizard 3.1 (https: / / loschmidt.chemi.muni.cz / hotspotwizard / ) was used to predict ScXR mutation hotspots, and site-directed mutagenesis was performed on potential sites to obtain eight single-point mutants, including D48V, K22E, F112W, F226R, M229V, S220P, F221L, and F312Y.

[0063] Combining the multiple sequence alignment of ScXR and key amino acid information such as substrate / co-substrate binding sites, the potential mutation sites and target amino acids of ScXR were determined according to the rational analysis of sequence homology, structural characteristics and amino acid properties, so as to obtain the single-point mutants L18F, I227F, M229L, T258P, K279N, E281G, L283D, G285Q, as well as C47A, C47D, C47E, C47F, C47M, C47R, C47W, C47Y, F112G, F112S, F112W, I110G, I110V, Q224L, Y109D, Y109F, Y109G and Y109I.

[0064] (2) Using primers SEQ ID NO: 7 and SEQ ID NO: 8, reverse PCR was used to introduce the L18F mutation, SEQ ID NO: 9 and SEQ ID NO: 10, reverse PCR was used to introduce the I227F mutation, and SEQ ID NO: 11 and SEQ ID NO: 12, reverse PCR was used to introduce the F312Y mutation, and linearized fragments were obtained and then circularized to form recombinant plasmids pANY1-GRE3 containing different mutants. L18F 、pANY1-GRE3 I227F 、pANY1-GRE3 F312Y .

[0065] Similarly, the recombinant plasmids of D48V, K22E, F112W, F226R, M229V, S220P, F221L, M229L, T258P, K279N, E281G, L283D, G285Q, C47A, C47D, C47E, C47F, C47M, C47R, C47W, C47Y, F112G, F112S, F112W, I110G, I110V, Q224L, Y109D, Y109F, Y109G and Y109I were obtained and recorded as pANY1-GRE3. D48V , pANY1-GRE3 K22E , pANY1-GRE3 F112W , pANY1-GRE3 F226R , pANY1-GRE3 M229V , pANY1-GRE3 S220P , pANY1-GRE3 F221L 、pANY1-GRE3 M229L , pANY1-GRE3 T258P , pANY1-GRE3 K279N , pANY1-GRE3 E281G , pANY1-GRE3 L283D , pANY1-GRE3 G285Q , pANY1-GRE3 C47A 、pANY1-GRE3 C47D , pANY1-GRE3 C47E , pANY1-GRE3 C47F , pANY1-GRE3 C47M 、pANY1-GRE3 C47R , pANY1-GRE3 C47Y 、pANY1-GRE3 F112G , pANY1-GRE3 F112S , pANY1-GRE3 F112W 、pANY1-GRE3 I110G , pANY1-GRE3 I110V , pANY1-GRE3 Q224L 、pANY1-GRE3 Y109D , pANY1-GRE3 Y109F 、pANY1-GRE3 Y109G , pANY1-GRE3 Y109I .

[0066] SEQ ID NO:7: CttcGGGTGCTGGAAAATTGACAAA;

[0067] SEQ ID NO:8:TTTTCCAGCACCCgaaGCCGACTAGGGGCATTTTCA;

[0068] SEQ ID NO:9:CATTCttcGAGATGGACTTACAGTTGGCAAAA;

[0069] SEQ ID NO:10: GTCCATCTCgaaGAATGATTGAGGACCGAAGGAG;

[0070] SEQ ID NO: 11: CATTCttcGAGATGGACTTACAGTTGGCAAAA;

[0071] SEQ ID NO: 12: GTCCATCTCgaaGAATGATTGAGGACCGAAGGAG.

[0072] (3) The obtained recombinant plasmids containing different mutants were transformed into E. coli BL21, the strains were cultured to induce the expression of the enzyme, and the pure enzymes of each mutant were further purified and recorded as mutant ScXR F312Y ScXR L18F and ScXR I227F ,ScXR D48V ScXR K22E ScXR F112W ScXR F226R ScXR M229V ScXR S220P ScXR F221L ScXR M229L ScXR T258P ScXR K279N ScXR E281G ScXR L283D ScXR G285Q ScXR C47A ScXR C47D ScXR C47E ScXR C47F ScXR C47M ScXR C47R ScXR C47W ScXR C47Y ScXR F112G ScXR F112S ScXR F112W ScXR I110G ScXR I110V ScXR Q224L ScXR Y109D ScXRY109F ScXR Y109G and ScXR Y109I .

[0073] Using D-galactose as substrate, the enzyme activity of each mutant was measured, and the wild-type ScXR was used as control to evaluate the changes in the enzyme activity of each mutant obtained above. The evaluation results are as follows: Figure 3 As shown in the figure, compared with the wild-type ScXR, the mutant ScXR F312Y ScXR L18F and ScXR I227F The enzyme activities of the mutants were increased by 30.74%, 19.41% and 36.62%, respectively, while the enzyme activities of the other mutants were reduced or completely inactivated.

[0074] Therefore, in the single-point mutants obtained by molecular modification of ScXR, ScXR F312Y ScXR L18F and ScXR I227F The enzyme activity was significantly improved, and these three mutations were selected for further experiments.

[0075] Example 3. Combined mutation based on forward mutation points and screening of combined xylose reductase mutants

[0076] In order to further improve the enzyme activity, based on the results of single-point mutation, multi-point combined mutation was performed on the forward mutation points F312Y, L18F and I227F in Example 2. The specific steps are as follows:

[0077] pANY1-GRE3 F312Y As a template, reverse PCR was performed using primers SEQ ID NO:7 and SEQ ID NO:8 to introduce the L18F mutation and obtain the double-point mutant pANY1-GRE3 F312Y / L18F ; pANY1-GRE3 F312Y As a template, I227F mutation was introduced by reverse PCR using SEQ ID NO:9 and SEQ ID NO:10 to obtain the double point mutant pANY1-GRE3 I227F / F312Y ; pANY1-GRE3 L18F As a template, the I227F mutation was introduced by reverse PCR using SEQ ID NO:9 and SEQ ID NO:10 to obtain the double point mutant pANY1-GRE3 L18F / I227F ; pANY1-GRE3 L18F / I227F Using SEQ ID NO:11 and SEQ ID NO:12 as templates, reverse PCR was performed to introduce the F312Y mutation, and the triple-point mutant pANY1-GRE3 was obtained. F312Y / L18F / I227F . Four recombinant plasmids pANY1-GRE3 were respectivelyF312Y / L18F 、pANY1-GRE3 I227F / F312Y 、pANY1-GRE3 L18F / I227F 、pANY1-GRE3 F312Y / L18F / I227F According to the method described in Example 2, the four recombinant plasmids were transformed into E. coli BL21, cultured, induced for expression, and purified to obtain the mutant enzyme ScXR L18F / I227F ScXR L18F / F312Y ScXR I227F / F312Y and ScXR L18F / I227F / F312Y .

[0078] Purified mutant enzyme ScXR L18F / I227F ScXR L18F / F312Y ScXR I227F / F312Y and ScXR L18F / I227F / F312Y Enzyme activity assays were performed to evaluate the changes in D-galactosidase activity of the multiple point combination mutations based on the substrate, and the changes in the enzyme activity of these mutants based on D-xylose as the substrate were also evaluated for in vitro enzyme activity analysis. Figure 4 shown.

[0079] from Figure 4 As can be seen, compared with wild-type ScXR, the combined mutant ScXR L18F / I227F ScXR L18F / F312Y ScXR I227F / F312Y and ScXR L18F / I227F / F312Y The enzyme activities of ScXR I227F / F312Y It showed the best enzyme activity and could be applied to further redox synthesis of D-tagatose.

[0080] Example 4. Application of combined xylose reductase mutants in the synthesis of D-tagatose:

[0081] This example uses the four combined mutants obtained in Example 3 to replace the wild-type ScXR in the heterologous redox pathway, so as to use the xylose reductase mutants for the biosynthesis of D-tagatose. The specific steps are as follows:

[0082] (1) Using SEQ ID NO: 13 and SEQ ID NO: 14 as primers, pP43NMK.1-P laps -ScXR-P srfA -RoGDH-RBS-pdhABC was used as template, and reverse PCR was performed to form a linearized vector. laps -ScXR-P srfA-The construction method of RoGDH-RBS-pdhABC refers to the paper Zhang, GY, An, YF, Zabed, HM, Yun, JH, Parvez, A., Zhao, M., Zhang, CS, Yuvaraj, R., Li, J., Qi, XH*. Rewiring Bacillus subtilis and bioprocess optimization for oxidoreductive reaction-mediated biosynthesis of D-tagatose. Bioresource Technology, 2023, 389: 129843.

[0083] Using SEQ ID NO:3 and SEQ ID NO:4 as primers, pANY1-GRE3 obtained in Example 3 was used to generate the PCR product. L18F / I227F 、pANY1-GRE3 L18F / F312Y 、pANY1-GRE3 I227F / F312Y and pANY1-GRE3 L18F / I227F / F312Y As a template, PCR amplification was performed to obtain the mutant encoding gene GRE3 L18F / I227F 、GRE3 L18F / F312Y 、GRE3 I227F / F312Y and GRE3 L18F / I227F / F312Y The obtained genes encoding different mutants were combined with pP43NMK.1-P laps -ScXR-P srfA -RoGDH-RBS-pdhABC linearized vector was purified, and then seamless cloning, transformation and positive clone verification were performed, and the recombinant vector was extracted from E. coli with successful plasmid verification to obtain pP43NMK.1-P laps -Mut-ScXR-P srfA -RoGDH-RBS-pdhABC(Mut_ScXR represents ScXR L18F / I227F ScXR L18F / F312Y ScXR I227F / F312Y ScXR L18F / I227F / F312Y )

[0084] SEQ ID NO: 13: AAAATAACCAAAAGCAAGGACT;

[0085] SEQ ID NO: 14: CGTTCATGTCTCCTTTTTTA.

[0086] (2) The four recombinant vectors pP43NMK.1-P laps-Mut-ScXR-P srfA -RoGDH-RBS-pdhABC(Mut_ScXR represents ScXR L18F / I227F ScXR L18F / F312Y ScXR I227F / F312Y ScXR L18F / I227F / F312Y ) was transferred into B. subtilis 168 (purchased from Beina Biotechnology) to obtain a recombinant strain, which was recorded as B. subtilis / pP43NMK.1-P laps -Mut_ScXR-P srfA -RoGDH-RBS-pdhABC(Mut_ScXR represents ScXR L18F / I227F ScXR L18F / F312Y ScXR I227F / F312Y ScXR L18F / I227F / F312Y ).

[0087] (3) The four recombinant strains obtained above were respectively subjected to fermentation verification to evaluate the effect of the combined mutants of ScXR on the fermentation of D-galactose to synthesize D-tagatose. The results are as follows Figure 5 shown.

[0088] As can be seen from the figure, compared with the role of wild-type ScXR in the pathway, mutant ScXR L18F / I227F ScXR L18F / F312Y ScXR I227F / F312Y ScXR L18F / I227F / F312Y , replacing the redox pathway formed by the wild-type ScXR in the host, the yield of D-tagatose synthesized in 24h increased by 14.75%, 21.27%, 43.97% and 8.68%, reaching 2.69g / L, 3.07g / L, 4.39g / L and 2.34g / L, respectively. The in vivo fermentation results of these mutants were consistent with the in vitro enzyme activity change trend, highlighting the positive effect of mutant ScXR on promoting D-tagatose synthesis, and all showed that ScXR I227F / F312Y The best mutant.

[0089] The ScXR I227F / F312Y The amino acid sequence is shown in SEQ ID NO:2.

[0090] SEQ ID NO:2

[0091] MSSLVTLNNGLKMPLVGLGCWKIDKKVCANQIYEAIKLGYRLFDGACDYGNEKEVGEGIRKAISEGLVSRKDIFVVSKLWNNFHHPDHVKLALKKTLSDMGLDYLDLYYIHFPIAFKYVPFEEKYPPGFYTGADDEKKGHITEAHVPIIDTYRALEECVDEGLI KSIGVSNFQGSLIQDLLRGCRIKPVALQIEHHPYLTQEHLVEFCKLHDIQVVAYSSFGPQSFFEMDLQLAKTTPTLFENDVIKKVSQNHPGSTTSQVLLRWATQRGIAVIPKSSKKERLLGNLEIEKKFTLTEQELKDISALNANIRYNDPWTWLDGKFPTFA.

[0092] Example 5. Amplification verification

[0093] This example uses the B. subtilis / pP43NMK.1-P constructed in Example 4. laps -GRE3 I227F / F312Y -P srfA -rogdh-pdhABC strain was used as a fermentation strain. A semi-continuous feeding strategy was used to ferment D-galactose in a 5L fermenter to synthesize D-tagatose. The scale-up verification was carried out. The specific steps were as follows:

[0094] The genetically engineered bacteria B. subtilis / pP43NMK.1-P laps -GRE3 I227F / F312Y -P srfA -rogdh-pdhABC were streaked in LB and cultured in an inverted plate at 37°C for about 24 h. Subsequently, a single colony with good growth was picked and inoculated into LB liquid culture medium and cultured in a shaking incubator at 240 rpm overnight until the logarithmic growth phase to prepare a seed solution. Then, 200 mL of the seed solution was inoculated into a 5 L fermentation tank containing 1.8 L fermentation medium at an inoculum volume fraction of 10%, and the airflow rate and stirring speed were set to 500 rpm and 1 vvm, respectively.

[0095] In order to ensure good growth of the strain in the early stage of the fermentation tank, the glucose as the carbon source for cell growth was increased to 50g / L, and the other culture medium components remained unchanged. Sampling was taken every 12h to monitor the production of D-tagatose and the remaining amount of substrate galactose and the synthesis of intermediates, and the biomass of the engineered bacteria in the fermentation tank was characterized. During the fermentation process, a batch feeding strategy was adopted. When the D-galactose content was about 5g / L, it was supplemented to 20g / L to achieve continuous synthesis of the product. The fermentation results are shown in Figure 2. Figure 6 shown.

[0096] from Figure 6 It can be seen that 30-40 g / L D-tagatose was generated and about 80-90 g / L D-galactose was consumed in 120 hours, which was 20%-30% higher than the prior art.

[0097] In summary, the present invention has molecularly modified the xylose reductase from Saccharomyces cerevisiae based on a sequence- and structure-guided semi-rational modification strategy, thereby obtaining a highly active xylose reductase mutant; the present invention has also integrated the above-mentioned xylose reductase mutant into the redox pathway for synthesizing D-tagatose by genetic engineering means, and expressed it in Bacillus subtilis, successfully constructed a recombinant engineered bacterium for fermenting D-galactose to synthesize D-tagatose, achieved efficient synthesis of D-tagatose, and has significant practical application value and economic benefits.

[0098] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A mutant of xylose reductase, characterized in that: The xylose reductase mutant includes a mutant obtained by performing any one or more of the following mutations on the basis of the wild-type xylose reductase ScXR as shown in the amino acid sequence of SEQ ID NO: 1: (a) mutating the amino acid L at position 18 of ScXR to F; (b) mutating the amino acid I at position 227 of ScXR to F; (c) The amino acid F at position 312 of ScXR was mutated to Y.

2. The mutant of xylose reductase according to claim 1, characterized in that The mutant of xylose reductase is a mutant obtained by mutating the 227th amino acid I of ScXR to F and the 312th amino acid F to Y, and its amino acid sequence is shown in SEQ ID NO:

2.

3. A nucleic acid encoding the mutant of xylose reductase according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid of claim 3.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria comprises the nucleic acid according to claim 3 or the recombinant expression vector according to claim 4.

6. The recombinant engineered bacterium according to claim 5, characterized in that The host bacteria of the recombinant engineering bacteria include Bacillus subtilis.

7. Use of the mutant of xylose reductase according to claim 1 or 2, or the recombinant expression vector according to claim 4, or the recombinant engineered bacteria according to any one of claims 5 to 6 in the efficient synthesis of D-tagatose.

8. A method for efficiently synthesizing D-tagatose, characterized in that: The method comprises: using a recombinant engineering bacterium expressing the mutant of the xylose reductase according to claim 1 or 2 to ferment D-galactose to synthesize D-tagatose.

9. The method for efficiently synthesizing D-tagatose according to claim 8, characterized in that: The method comprises: (1) constructing a recombinant expression vector expressing the mutant of xylose reductase according to claim 1 or 2, and expressing the recombinant expression vector in a host cell to obtain a recombinant engineered bacterium expressing the mutant of xylose reductase; (2) Using the obtained recombinant engineered bacteria expressing the mutant of xylose reductase as a fermentation strain, D-tagatose is fermented and synthesized in a fermentation medium containing D-galactose.

10. The method for efficiently synthesizing D-tagatose according to claim 9, characterized in that: The fermentation medium comprises: 10 g / L peptone, 5 g / L yeast extract, 3 g / L K2HPO4, 0.27 g / L MgSO4·7H2O, 0.53 g / L CaCl2, 50 g / L D-glucose, and 20 g / L D-galactose; The fermentation conditions are: fermentation at 37-45°C and an initial pH of 7.5-8.5; When the D-galactose content was lower than 5 g / L, feeding was performed using a semi-continuous feeding strategy to maintain the D-galactose content at 20-30 g / L, with 4-6 feedings every 12 hours.

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