A ribitol dehydrogenase mutant with high substrate selectivity and its application

Abstract

The present invention discloses a ribitol dehydrogenase mutant with high substrate selectivity and its application in the catalytic reduction of D-psicose. The ribitol dehydrogenase mutant is obtained by directed evolution of wild-type ribitol dehydrogenase and comprises any one of V152L, E157N, and V193L mutations or a combination thereof. The ribitol dehydrogenase mutant disclosed in the present invention can selectively recognize D-psicose as a substrate in a mixed substrate system of D-psicose and D-fructose, thereby significantly improving substrate selectivity and conversion rate. The present invention discloses a product, preparation method, and application of a ribitol dehydrogenase mutant with improved substrate selectivity. The temperature and pH required for the catalytic reaction of the enzyme mutant are easily achieved. The present invention discloses a nucleic acid molecule encoding the ribitol dehydrogenase mutant, an expression vector comprising the nucleic acid molecule encoding the ribitol dehydrogenase mutant, and a recombinant cell comprising the expression vector.

Description

Technical Field

[0001] The present invention relates to a ribitol dehydrogenase mutant, in particular to a ribitol dehydrogenase mutant with high substrate selectivity and application thereof in catalytic reduction of D-psicose, belonging to the technical fields of enzyme engineering and genetic engineering. Background Art

[0002] D-Psicose, a rare, naturally occurring ketohexose, boasts 70% of the sweetness of sucrose and only 10% of its calories. It is a new generation of natural, low-intensity sweetener with a wide range of applications. Studies have shown that D-Psicose has significant effects in lowering blood sugar, regulating glucose and lipid metabolism, and treating atherosclerosis, thus possessing promising application prospects and development value.

[0003] Because D-psicose is a rare natural sugar with extremely low concentrations in nature, purification of natural products is difficult, and therefore it is often synthesized using artificial synthesis methods, which are mainly divided into chemical synthesis and biosynthesis. Chemical synthesis is cumbersome, highly polluting, and produces an impure product. Biosynthesis, however, has advantages such as mild reaction conditions and good environmental compatibility, and has gradually become a hot topic in the research of psicose production. The mainstream biosynthesis method mainly uses D-psicose-3-epimerase to diastereoisomerize D-fructose at the 3-position. However, due to the existence of thermodynamic equilibrium in the reaction, the overall conversion rate is only 30%-35%. To address this issue, ribitol dehydrogenase (RDH) can be used to first reduce D-psicose to D-psicol (allitol) to promote the continuous forward isomerization reaction. Allitol can then be oxidized to D-psicose to increase the yield of the target product, D-psicose. Existing ribitol dehydrogenases have problems such as low substrate selectivity and poor activity. There is a side reaction of producing sorbitol using D-fructose as a substrate, and the cost of separating sorbitol from the target product allitol is high, which greatly limits the application of this reaction pathway in industry. Summary of the Invention

[0004] Object of the invention: The object of the present invention is to provide a ribitol dehydrogenase mutant with high substrate selectivity for D-psicose and its application.

[0005] Technical solution: In the first aspect, the present invention provides a ribitol dehydrogenase mutant with high substrate selectivity, wherein the amino acid sequence of the ribitol dehydrogenase mutant is obtained by subjecting the sequence shown in SEQ ID NO: 1 to at least one mutation among V152L, E157N, and V193L.

[0006] In a second aspect, the present invention provides a nucleic acid molecule encoding the ribitol dehydrogenase mutant according to the first aspect. The nucleotide sequence of the nucleic acid molecule is obtained by base mutation of the sequence shown in SEQ ID NO: 2.

[0007] In a third aspect, the present invention provides an expression vector comprising the nucleotide sequence described in the second aspect.

[0008] In a fourth aspect, the present invention provides a recombinant cell and a method for constructing the same, wherein the recombinant cell comprises the expression vector described in the third aspect. The method for constructing the recombinant cell comprises the following steps: (1) constructing the expression vector: ligating the nucleic acid molecule described in the second aspect with the enzyme-cleaved plasmid to obtain a recombinant expression vector; and (2) constructing the recombinant cell: transforming the constructed recombinant expression vector into competent Escherichia coli cells, culturing, and screening to obtain the recombinant cell.

[0009] In a fifth aspect, the present invention provides a product comprising the ribitol dehydrogenase mutant as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the expression vector as described in the third aspect, or the recombinant cell as described in the fourth aspect.

[0010] In a sixth aspect, the present invention provides use of the ribitol dehydrogenase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in catalyzing the production of D-psicose. The catalytic reaction conditions include a reaction temperature of 25-45°C, a pH of 6-9, and a reaction time of 12 hours.

[0011] In a seventh aspect, the present invention provides a method for preparing a ribitol dehydrogenase mutant, wherein the preparation method obtains the ribitol dehydrogenase mutant by culturing the recombinant cell as described in the fourth aspect.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0013] 1. The ribitol dehydrogenase mutant obtained in the present invention can selectively recognize D-psicose as a substrate in a mixed substrate system of D-psicose and D-fructose to produce D-psicol, thereby significantly improving substrate selectivity and conversion rate.

[0014] 2. The present invention provides a product comprising a ribitol dehydrogenase mutant with improved substrate selectivity. The temperature and pH conditions of the catalytic reaction are easy to achieve, and the product has a basis for industrial production and application.

[0015] 3. The present invention provides a nucleic acid molecule encoding the above-mentioned ribitol dehydrogenase mutant, an expression vector comprising a nucleic acid molecule encoding the above-mentioned ribitol dehydrogenase mutant, and a recombinant cell comprising the expression vector; the present invention provides a preparation method and application of the above-mentioned ribitol dehydrogenase mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram showing the conversion rates of wild-type ribitol dehydrogenase and enzyme mutants on D-psicose and D-fructose.

[0017] Figure 2 The reaction process of D-psicose to D-psicol catalyzed by ribitol dehydrogenase.

[0018] Figure 3 This is a diagram showing the conversion effect of the ribitol dehydrogenase mutant (V152L / E157N / V193L) on D-psicose and D-fructose at different pH values.

[0019] Figure 4 This is a diagram showing the conversion effect of ribitol dehydrogenase mutant (V152L / E157N / V193L) on D-psicose and D-fructose at different temperatures. DETAILED DESCRIPTION

[0020] The features and exemplary embodiments of various aspects of the present invention will be described in detail below. The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below.

[0021] For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should all be encompassed within the scope of the present application. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main idea of ​​the present application.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in this invention should be understood to include the inevitable systematic errors in industrial production.

[0023] In all discussions herein, the standard single-letter codes for amino acids are used. Standard substitution notation is also used, i.e., V152L means that the valine (V) at position 152 at the N-terminus is replaced by a leucine (L). V152L / E157N means that the valine (V) at position 152 at the N-terminus is replaced by a leucine (L), and the glutamic acid (E) at position 157 at the N-terminus is replaced by an asparagine (N).

[0024] The term "wild-type" refers to a gene or gene product that has been isolated from a naturally occurring source. A wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, truncations, or insertions), post-translational modifications, and / or functional properties (e.g., altered properties) compared to the wild-type gene or gene product. Note that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered properties compared to the wild-type gene or gene product. Methods for introducing or substituting natural, non-naturally occurring amino acids are well known in the art.

[0025] Wild-type ribitol dehydrogenase

[0026] After mining the enzyme genome, the present invention ultimately selected the wild-type ribitol dehydrogenase EcRDH (Primary accession: Q9F4L7) from Escherichia coli for plasmid construction. Polymerase chain reaction (PCR) technology was used for gene amplification and heterologous vector construction, and the gene was functionally expressed in an E. coli expression system. The amino acid sequence of EcRDH is shown in SEQ ID NO:1, and the nucleotide sequence encoding EcRDH is shown in SEQ ID NO:2.

[0027] Ribitol dehydrogenase mutants

[0028] By combining rational design with random mutation technology, the structure and function of ribitol dehydrogenase were modified and optimized. The obtained enzyme mutants were tested using two similar substrates, D-fructose and D-psicose, and finally a mutant strain with high substrate selectivity for D-psicose was obtained. The wild type and mutants in the present invention have the ability to catalyze D-psicose to produce D-psicol. Figure 2 .

[0029] Example

[0030] Example 1 Sequences of ribitol dehydrogenase mutants

[0031] Based on the amino acid sequence of wild-type ribitol dehydrogenase (WT) SEQ ID NO: 1, seven mutant sequences including V152L, E157N, V193L, V152L / E157N, V152L / V193L, E157N / V193L, and 152L / E157N / V193L were selected.

[0032] Example 2 Construction of Ribitol Dehydrogenase Mutant Expression Vector

[0033] 1. Obtain target mutant gene by whole plasmid PCR

[0034] Gene mutation was performed using whole-plasmid PCR to obtain the target mutant gene. Using V193L as an example, primers were specifically designed. Other mutants were designed using this principle and subjected to single-point iterative mutagenesis.

[0035] V193L upstream primer: CCTGGTCCG CTG GTCACCGCTC

[0036] V193L downstream primer: GAGCGGTGAC CAG CGGACCAGG

[0037] PCR system is shown in Table 1:

[0038] Table 1 PCR reaction system

[0039] Ingredients volume 10×BufferforKOD-Plus- 2.5 μL 2mM dNTP 2.5 μL 25mMMgSO4 1.5 μL DMSO 1 μL 10 pmol / μL Forward Primer 0.75μL 10 pmol / μL Reverse Primer 0.75μL DNA template <100ng KOD-Plus- 1 μL ddH2O up to 25 μL

[0040] PCR reaction conditions are shown in Table 2:

[0041] Table 2 PCR reaction conditions

[0042]

[0043]

[0044] After the PCR amplification was completed, the amplified product was detected by 0.9% agarose gel electrophoresis, and the results showed that the amplified product was a single band with a size of about 6000 bp. The amplified product was purified and recovered using a DNA recovery and purification kit.

[0045] 2. Construction of recombinant E. coli BL21 (DE3) / pET22b-EcRDH mutant

[0046] The purified gene fragment was digested with DpnI to remove the template and recombined with a recombinase. The recombinant plasmid was transformed into E. coli DH5α competent cells using the heat shock method. The recombinant E. coli DH5α / pET22b-EcRDH was spread on the surface of LB solid medium containing ampicillin and cultured overnight. A single clone was selected and cultured with shaking in LB liquid medium containing ampicillin until the OD value reached 0.8. A single clone with a correct sequencing result was selected and the plasmid was extracted using a bacterial plasmid extraction kit. The recombinant plasmid was stored in a -20°C refrigerator. The recombinant expression plasmid pET22b was heat-shocked into E. coli BL21(DE3) to construct the recombinant mutant expression strain E. coli BL21(DE3) / pET22b-EcRDH.

[0047] Example 3 Cultivation of ribitol dehydrogenase mutants and preparation of pure enzyme solution

[0048] The recombinant mutant expression strain E. coli BL21 (DE3) / pET22b-EcRDH was successfully constructed and spread on the surface of a plate containing 100 μg / mL ampicillin and cultured overnight. A single colony was picked and inoculated into 5 mL of LB medium containing resistance and cultured at 37°C and 200 rpm / min overnight. 1% of the inoculum was transferred to 500 mL of LB medium containing resistance and cultured until the OD 600 IPTG was added to a final concentration of 0.5 mM and induced at 20°C for 18 h.

[0049] After centrifugation, the cells were resuspended in K₂HPO₄-KH₂PO₄ (KPi) buffer, placed on ice, and disrupted by sonication (2 seconds on, 5 seconds off, for a total of 15 minutes). The cells were then centrifuged at 4°C, 12,000 rpm / min, for 20 minutes. The supernatant was collected and filtered through a 0.22 μm aqueous filter for use as a sample. The sample was then purified on a nickel column to obtain the crude enzyme solution.

[0050] Example 4 Ribitol dehydrogenase and its mutants selectively catalyze the reduction of D-psicose to D-psicol in a mixed system of D-psicose and D-fructose

[0051] The corresponding engineered bacteria expressing ribitol dehydrogenase and their mutants were cultured according to the construction of Examples 1-3, and the obtained crude enzyme solution was used as a catalyst.

[0052] The reaction system is: OD 600 =40 crude enzyme solution, 150mM D-psicose or D-fructose, 0.5mM NAD +The reaction system was prepared by mixing 500 mM sodium formate, 1 U / mL commercial formate dehydrogenase (FDH), and 200 mM KPi buffer (pH 7.5). The reaction temperature was controlled at 30°C in a water bath with magnetic stirring for 12 h.

[0053] After the reaction is complete, add an equal volume of methanol-water solution (methanol:water = 1:1) to the reaction mixture until the product concentration is approximately 5 mM. Shake thoroughly at 400 rpm. Centrifuge at 4000 rpm for 10 minutes. Discard the precipitate, and dilute the supernatant with water. High-performance liquid chromatography (HPLC) is used to determine the concentrations of the two reduction products, and the conversion rate is calculated. The results are shown in Table 3.

[0054] The HPLC detection conditions are as follows: chromatographic analysis column Carbomix-Pb-NP10: 8% (7.8×300 mm), mobile phase ultrapure water, flow rate 0.5 mL / min, column temperature 78° C.

[0055] Table 3. Conversion efficiency of D-psicose and D-fructose by ribitol dehydrogenase

[0056] strains Cell concentration D-psicose conversion rate D-fructose conversion rate EcRDH(WT) <![CDATA[OD 600 =40]]> 44％ 36％ E157N <![CDATA[OD 600 =40]]> 60％ 27％ V152L <![CDATA[OD 600 =40]]> 52％ 20％ V193L <![CDATA[OD 600 =40]]> 49％ 23％ V152L / V193L <![CDATA[OD 600 =40]]> 58％ 15％ E157N / V193L <![CDATA[OD 600 =40]]> 64％ 12％ V152L / E157N <![CDATA[OD 600 =40]]> 67％ 6％ V152L / E157N / V193L <![CDATA[OD 600 =40]]> 70％ 1％

[0057] Example 5 Ribitol dehydrogenase mutant (V152L / E157N / V193L) selectively identifies the optimal pH for catalyzing D-psicose to D-psicol in a mixed system of D-fructose and D-psicose

[0058] According to the method of Examples 1-3, an engineered bacterium capable of expressing ribitol dehydrogenase and its mutants were constructed and cultured, and the obtained crude enzyme solution was used as a catalyst.

[0059] The reaction system is: OD 600 =40 crude enzyme solution, 150mM D-psicose or D-fructose, 0.5mM NAD + , 500mM sodium formate, commercial formate dehydrogenase (FDH) concentration 1U / mL. The reaction temperature was controlled at 30℃ in a water bath, with magnetic stirring. After the reaction time was the same, the conversion rate of EcRDH to substrate at different pH was detected. Among them, the reaction buffer was a buffer of different pH, and the pH was controlled at 6-9. The buffers were: Kpi buffer at pH 6, 7, and 8, and Tris-HCl buffer at pH 9. The results are shown in Figure 3 .

[0060] Experiments have shown that different pH levels affect enzyme activity. Its activity exhibits a slightly normal trend with changes in pH. The best catalytic effect is observed at pH 9. However, there is little difference in enzyme activity at pH 6, 7, and 8, possibly due to changes in ions in the buffer at pH 9.

[0061] Example 6 Ribitol dehydrogenase mutant (V152L / E157N / V193L) selectively identifies the optimal temperature for catalyzing D-psicose to D-psicol in a mixed system of D-fructose and D-psicose

[0062] According to the method of Examples 1-3, an engineered bacterium capable of expressing ribitol dehydrogenase and its mutants were constructed and cultured, and the obtained crude enzyme solution was used as a catalyst.

[0063] The reaction system is: OD 600 =40 crude enzyme solution, 150mM D-psicose or D-fructose, 0.5mM NAD + , 500mM sodium formate, commercial formate dehydrogenase (FDH) concentration 1U / mL. Use 200mM KPi buffer with pH=7.5 as the reaction system. Magnetic stirring, after the same reaction time, the conversion rate of EcRDH to substrate at different temperatures was detected. The reaction system temperature was controlled to be constant at 25℃, 30℃, 35℃, and 40℃ by water bath, and the reaction time was the same. The product content was detected by HPLC and the conversion rate was calculated. The results are shown in the table. Figure 4 .

[0064] Experiments have shown that different temperatures affect the catalytic activity of EcRDH mutants. Their activity exhibits a skewed normal trend with temperature. Optimal catalytic activity is observed at 35°C. Enzyme activity decreases at temperatures above and below this temperature, with lower temperatures having a more pronounced effect.

Claims

1. A ribitol dehydrogenase mutant with high substrate selectivity, characterized in that: The amino acid sequence is obtained by subjecting the sequence shown in SEQ ID NO: 1 to at least one mutation among V152L, E157N, and V193L.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the ribitol dehydrogenase mutant according to claim 1.

3. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that The recombinant cell comprises the expression vector according to claim 3.

5. A method for constructing a recombinant cell, characterized in that: The steps include: (1) Constructing an expression vector: ligating the nucleic acid molecule of claim 2 with the enzyme-digested plasmid to obtain a recombinant expression vector; (2) Construction of recombinant cells: The constructed recombinant expression vector is transferred into competent Escherichia coli cells, cultured and screened to obtain recombinant cells.

6. A product, characterized in that The product comprises the ribitol dehydrogenase mutant according to claim 1 or the nucleic acid molecule according to claim 2 or the expression vector according to claim 3 or the recombinant cell according to claim 4.

7. Use of the ribitol dehydrogenase mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3, or the recombinant cell according to claim 4 in catalyzing the production of D-psicose.

8. Use according to claim 7, characterized in that The catalytic reaction conditions include a reaction temperature of 25 to 45° C., a pH of 6 to 9, and a reaction time of 12 h.

9. A method for preparing a ribitol dehydrogenase mutant, characterized in that: The preparation method obtains the ribitol dehydrogenase mutant by culturing the recombinant cell according to claim 4.

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