Recombinant ribitol dehydrogenase strain and application thereof

Abstract

The invention discloses a recombinant ribitol dehydrogenase strain and application thereof, and belongs to the technical field of bioengineering. According to the recombinant ribitol dehydrogenase strain disclosed by the invention, pichia pastoris GS115 is taken as a starting strain, a recombinant ribitol dehydrogenase carrier is transformed, and the ribitol dehydrogenase gene sequence is shown as SEQ ID. NO 1. After fermentation in a 50L fermentation tank, the fermentation liquor is centrifuged, the supernate contains recombinase RDH with the purity of 95% or above, and the enzyme activity can reach 1059U / mL. The recombinase catalyzes a redox reaction between D-psicose and D-psilol to produce D-psilol, and the conversion rate can reach 56% after the reaction is balanced. The enzyme has good heat resistance, does not need metal ions to participate in a catalytic reaction, is low in product separation and purification difficulty, can keep the maximum enzyme activity of 80% or above in a wide pH (6-9) range, and can be applied to large-scale industrial production of rare sugar D-psilol.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically to a recombinant ribitol dehydrogenase strain and its application. Background Technology

[0002] Pichiapastoris is being developed as a widely used host organism for recombinant protein production, with advantages including rapid growth and high secretion capacity.

[0003] Alloyl alcohol is also known as allitol (C6H) 14 Allitol (O6), also known as Allitol, is a colorless crystalline solid with a sweet taste. It is easily soluble in water and is a non-hazardous rare sugar alcohol with industrial applications.

[0004] Allol's applications in rare sugar synthesis extend beyond its functional properties. It can also serve as a substrate for biotransformation, converting into a series of valuable rare sugar compounds, such as D-allulose, L-allulose, D-allose, and L-allose, through specific enzymatic or whole-cell catalytic biotransformation processes. These rare sugars hold broad application prospects in pharmaceuticals, food additives, health products, and fine chemicals.

[0005] The synthesis of allool using enzymatic catalysis dates back to 2000, when Takeshita et al. first synthesized allool from D-allulose using ribitol dehydrogenase (RDH) and formate dehydrogenase (FDH), obtaining 10 g / L allool after 48 h of reaction at 30 °C and pH 8.0. However, the ribitol dehydrogenase activity obtained in E. coli was <100 U / mL, resulting in an allool conversion rate of <50%, leading to high production costs.

[0006] Therefore, providing a recombinant ribitol dehydrogenase strain and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a recombinant ribitol dehydrogenase strain and its application.

[0008] This invention uses bioinformatics methods to compare and screen, selecting the Providencia ribitol dehydrogenase gene as the target gene, and successfully constructs the genetically engineered bacterium GS115 / pPIC9K-RDH, obtaining a soluble recombinant enzyme with good heat resistance, a wide pH range, and high enzyme activity, showing good potential for industrial application.

[0009] This invention provides a method for recombinant Pichia pastoris to improve the enzyme activity of ribitol dehydrogenase in the supernatant of Pichia pastoris fermentation broth, thereby solving the problem of low ribitol dehydrogenase activity.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] This invention provides a method for the soluble expression of ribitol 2-dehydrogenase (RDH, EC 1.1.1.56) from Providencia strain in Pichia pastoris GS115, as well as the isolation and purification of the recombinant enzyme expressed and its application in the production of the rare sugar D-allitol.

[0012] A recombinant ribitol dehydrogenase vector, using pPIC9K plasmid as the starting vector, is linked with a ribitol dehydrogenase gene sequence; the ribitol dehydrogenase gene sequence is shown in SEQ ID NO 1.

[0013] Furthermore, a recombinant ribitol dehydrogenase strain, using Pichia pastoris GS115 as the starting strain, is transformed into the recombinant ribitol dehydrogenase vector.

[0014] Furthermore, the application of the recombinant ribitol dehydrogenase vector or the recombinant ribitol dehydrogenase strain in improving the activity of ribitol dehydrogenase.

[0015] Furthermore, the application of the recombinant ribitol dehydrogenase vector or the recombinant ribitol dehydrogenase strain in improving the activity of ribitol dehydrogenase.

[0016] Furthermore, the recombinant ribitol dehydrogenase vector or the recombinant ribitol dehydrogenase strain is used in the production of heat- and pH-resistant ribitol dehydrogenase.

[0017] Furthermore, the recombinant ribitol dehydrogenase vector or the recombinant ribitol dehydrogenase strain is used in the production of D-alool.

[0018] Furthermore, the recombinant ribitol dehydrogenase vector or the recombinant ribitol dehydrogenase strain is used to improve the conversion rate of D-alool.

[0019] As can be seen from the above technical solution, compared with the prior art, this invention discloses a recombinant ribitol dehydrogenase strain and its application. The supernatant of the fermentation broth obtained from fermentation in a 50L fermenter has a protein content of 3.01 g / L and a ribitol dehydrogenase activity of 1059 U / ml, which is the highest reported to date. The conversion rate is as high as 56%. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 It is a recombinant plasmid containing the ribitol dehydrogenase gene sequence;

[0022] Figure 2 This is an SDS-PAGE image of the recombinant pPIC9K-RDH enzyme; the first lane represents the protein molecular weight standard; the second lane represents the recombinant RDH enzyme.

[0023] Figure 3 The effect of pH on the activity of recombinant RDH enzyme;

[0024] Figure 4 pH tolerance of recombinant RDH; where a is pH 6, b is pH 7, and c is pH 8;

[0025] Figure 5 The effect of temperature on the activity of recombinant RDH enzyme;

[0026] Figure 6 Temperature tolerance of recombinant RDH; where a is 55℃, b is 65℃, c is 75℃, and d is 80℃;

[0027] Figure 7 The results show the biotransformation of D-allosanol; the retention times are: 4.77 min: phosphate peak; 11.79 min: allose peak; 17.29 min: allulose peak. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The nucleotide sequence of ribitol dehydrogenase is shown in SEQ ID NO.1.

[0030] ATGGCTATTTCTCTGGAAAATAAAGTCGCGGCTATCACCGGTGCAGCTTCTGGCATCGGCCTGGAATGCGCACGTACCCTGCTGAAAGCAGGCGCTAAAGTGGTGCTGATCGACCGCGCTGAAGAGCGCCTGAACCAGCTGGTAGCAGAACTGGGTGACAACGCTATCCCGCTGGTAGTTGATCTGATGAAACCGGAACAGGTGGATGGCATGCTGGACGCTATCCTGGCAAAAGCTGGCCGTCTGGATATTTTCCACGCAAACGCAGGTGCGTACATCGGCGGTCCGGTTGCAGAAGGTGATCCGGATGTTTGGGACAAAGTGCTGAATCTGAACATCAACGCGGCTTTCCGTTCTGTACGTGCTGTGCTGCCGCACTTCATCGAACAGAAATCTGGCGACGTACTGTTCACCAGCTCTATCGCAGGTATGGTGCCGGTCATCTGGGAACCTATCTACACCGCCAGCAAATTCGCTGTCCAAGCTTTCGTTCATAGCACTCGTCGTCAGGTGTCTCAGTATGGCGTTCGTGTAGGCGCGGTACTGCCGGGTCCAGTTGTTACGGCACTGCTGGATGACTGGCCGAAAGAAAAGATGGAGGAAGCGCTGGCAAACGGTTCTCTGATGCAGCCAATCGAAGTTGCTGAGGCGGTGCTGTTCATGCTGACCCGTCCTAAGAACGTTACCATCCGCGACCTGGTTATC CTGCCAAACAGCGTAGATCTG ; SEQ ID NO.1.

[0031] The amino acid sequence of ribitol dehydrogenase is shown in SEQ ID NO.2.

[0032] MAISLENKVAAITGAASGIGLECARTLLKAGAKVVLIDRAEERLNQLVAELGDNAIPLVVDLMKPEQVDGMLDAILAKAGRLDIFHANAGAYIGGPVAEGDPDVWDKVLNLNINAAFRSVRAV SEQ ID NO.2.

[0033] This invention utilizes conventional techniques and methods from the fields of genetic engineering and molecular biology. Using the pPIC9K plasmid as a backbone, the ribitol dehydrogenase gene and the linearized pPIC9K vector were ligated using Gibson ligase at 50°C for 30 minutes. The ligation was then performed on *E. coli* DH5α competent cells to obtain the recombinant plasmid pPIC9K-RDH. The recombinant plasmid pPIC9K-RDH was electroporated into *Pichia pastoris* GS115 competent cells with electroporation parameters of 1.5 kV, 25 μF, 200 Ω, and an electroporation time of 4–10 ms. After incubation on ice for 30 min, the cells were evenly spread onto YPD plates containing 100 μg / ml genimycin and incubated upside down at 30°C for 48–72 h to obtain positive clones, named recombinant *Pichia pastoris* GS115 / pPIC9K-RDH.

[0034] Example 1: Construction of recombinant expression vector pPIC9K-RDH and genetically engineered strain

[0035] Obtaining the GS115 / pPIC9K-RDH

[0036] Culture medium formulation:

[0037] Escherichia coli culture medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0.

[0038] Yeast medium (YPD medium): 1% yeast extract, 2% peptone, 2% glucose.

[0039] Yeast selection medium:

[0040] LB-G418 medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 100 μg / mL genimycin, pH 7.0.

[0041] YPD-G418 medium: 1% yeast extract, 2% peptone, 2% glucose, 100 μg / mL genimycin, pH 7.0.

[0042] Fermentation medium formula: potassium dihydrogen phosphate 2.2g / L, magnesium sulfate 1.5g / L, diammonium hydrogen phosphate 4.5g / L, calcium sulfate 2.3g / L, citric acid 1.7g / L, with 30% methanol added as carbon source to maintain the methanol concentration in the tank at 1% until fermentation is complete.

[0043] The RDH sequence derived from Providencia was linked to the α signal peptide of the pPIC9K vector via homologous recombination. The pPIC9K vector itself carries the α signal peptide, resulting in the recombinant vector pPIC9K-RDH.

[0044] Using the fully synthesized RDH sequence (as shown in SEQ ID NO.1) as a template, the RDH fragment was amplified using primers RDH fragment-F / R; using the pPIC9K vector as a template, the vector sequence was amplified using vector-F / R. The PCR reaction system is shown in Table 1.

[0045] Table 1 PCR reaction system

[0046]

[0047] The PCR reaction program was 98℃ for 2 min; 98℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, for 30 cycles; 72℃ for 10 min.

[0048] Primer sequences are shown in Table 2.

[0049] Table 2 Primer sequence list

[0050]

[0051] The obtained RDH fragment and vector sequence were ligated using a homologous recombination kit (Wuhan Junuode Biotechnology Co., Ltd., one-step homologous recombination directional cloning kit, catalog number V6006). The homologous recombination reaction system is shown in Table 3.

[0052] Table 3 Homologous recombination reaction system

[0053]

[0054] The homologous recombination reaction procedure was 50℃ for 30 minutes.

[0055] The entire ligation system was added to DH5α competent cells, incubated on ice for 30 min, then at 42℃ for 90 s. 5 ml of LB liquid medium was added, and the mixture was incubated at 37℃ and 150 rpm for 90 min. The cells were then completely plated onto LB agar plates containing 100 μg / ml genimycin. After 16 h, single colonies were picked and expanded to obtain a large number of recombinant plasmids pPIC9K-RDH. Figure 1 ).

[0056] The recombinant plasmid pPIC9K-RDH was linearized using primer linearization-F / R (Table 4). The linearized fragment of the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The recombinant strain GS115 / pPIC9K-RDH of Pichia pastoris was obtained by screening on YPD-G418 plates, and positive transformants were screened.

[0057] Table 4

[0058]

[0059] Example 2: Obtaining Recombinant RDH Enzyme

[0060] A single GS115 / pPIC9K-RDH transformant was selected and cultured in a 1L YPD shake flask seed culture. OD 600 When the concentration was 0.8, all cells were transferred to a 50L fermenter (containing 35L of fermentation medium) and fermented at 27℃ and 300rpm for 120h to obtain the fermentation broth. The cells were removed by centrifugation, and the supernatant was obtained. The supernatant was then subjected to SDS-PAGE protein electrophoresis. Figure 2 ).

[0061] The protein content and ribitol dehydrogenase activity in the fermentation supernatant of transformants were determined using the following methods:

[0062] (1) Principle of protein concentration measurement

[0063] The Coomassie Brilliant Blue method for protein concentration determination is a rapid and sensitive method for quantitatively measuring trace amounts of protein, utilizing the principle of protein-dye binding. This protein assay method has significant advantages over other methods and is therefore widely used. It is currently the most sensitive protein assay method. Coomassie Brilliant Blue G-250 dye binds to proteins in acidic solution, causing the dye's maximum absorption peak (max) to shift from 465 nm to 595 nm, and the solution color to change from brownish-black to blue. The amount of protein bound can be determined by measuring the increase in light absorption at 595 nm. Studies have found that the dye primarily binds to basic amino acid residues (especially arginine) and aromatic amino acid residues in proteins.

[0064] (2) Methods for determining protein concentration

[0065] Coomassie Brilliant Blue G-250 100mg was dissolved in 50mL of 95% ethanol, 100mL of 85% phosphoric acid was added, and the solution was diluted with distilled water to 1000mL to obtain Coomassie Brilliant Blue reagent.

[0066] Crystallized bovine serum albumin was prepared by first determining the protein nitrogen content using the micro Kjeldahl method, and then using pure water to prepare a 1 mg / mL protein solution based on its purity.

[0067] Protein concentration standard curve:

[0068] Take 7 test tubes and perform the same procedure as in Table 5.

[0069] Table 5

[0070]

[0071] Shake well. Within 1 hour, use tube 0 as a blank control and measure the color at 595nm. Plot a standard curve on graph paper with A595nm as the ordinate and the standard protein content as the abscissa.

[0072] Determination of protein concentration in fermentation broth supernatant samples:

[0073] The determination method is the same as above. Take an appropriate volume of the unknown sample so that its measured value falls within the linear range of the standard curve. Based on the measured A595nm value, find the equivalent amount of standard protein on the standard curve, and then calculate the protein concentration (mg / mL) of the unknown sample.

[0074] (3) Principle of ribitol dehydrogenase activity assay

[0075] D-Allol Determination Method: Stock solutions of D-allulose and D-allol standards at concentrations of 10 g / L were prepared, and then successively diluted with deionized water to concentrations of 2, 4, 6, and 8 g. Finally, the prepared standards at different concentrations were filtered through a 0.22 mm aqueous filter into HPLC vials for HPLC detection and analysis. HPLC detection conditions are shown in Table 6.

[0076] Table 6

[0077] condition parameter Remark chromatographic column CarbomixPb-NP(10:8%,7.8x300mm,10μm) Saifen Technology Differential refractive index detector RID-20A Shimadzu mobile phase Double deionized water Filtration and ultrasonic degassing Flow rate 0.5 mL / min none Injection volume 10ul none Column temperature 78℃ none

[0078] HPLC analysis yielded peak areas corresponding to standard solutions of different concentrations. Therefore, a standard curve was plotted with concentration on the x-axis and the peak areas of each concentration standard solution on the y-axis. The resulting D-allulose standard curve is Y = 166647X - 9654(R). 2 -0.99997), the standard curve for D-alool is Y = 162414X - 19128.3 (R 2 -0.99995). When the peak area of ​​the substance being tested is known, its corresponding concentration can be obtained from the standard curve.

[0079] Ribitol dehydrogenase catalyzes the oxidation of ribitol, and the coenzyme NAD in the reaction... + (or NADP) +It is reduced to NADH (or NADPH), and its absorbance increases significantly at 340 nm (ε = 6.22 mM). -1 cm -1 Enzyme activity can be calculated by monitoring the rate of change of absorbance at 340 nm over time (ΔA / min).

[0080] (4) Assay of ribitol dehydrogenase activity

[0081] Take 1 mL of reaction solution (containing 50 mM Tris-HCl buffer, 1 mM coenzyme, and saturated ribitol) into a quartz cuvette and preheat to 30 °C;

[0082] Add 10 μL of enzyme solution (liquid samples diluted to an activity ≤15 U / mL);

[0083] Immediately scan at a wavelength of 340 nm for 1 minute, record the absorbance change curve, and calculate the slope (k, Abs / min);

[0084] Enzyme activity (U / mL) = (K·V1·D) / (ε·V2)

[0085] Note: In the formula, V1: substrate volume (μL); V2: enzyme solution volume (μL); D: dilution factor.

[0086] The results showed that the concentration of fermented protein in the transformant reached 3.01 g / L and the enzyme activity reached 1059 U / ml; the transformant in the experimental group was named Pichia pastoris RDH-FY1.

[0087] Example 3: Effect of pH on recombinant RDH enzyme activity

[0088] In a 100 ml system, the substrate D-allulose concentration was 50 mM. 1000 U of fermentation broth supernatant (crude enzyme solution) was added. The reaction temperature was 65℃, and the reaction time was 15 min. Three buffer systems were used: citrate buffer (50 mM, pH 5-5.5), phosphate buffer (50 mM, pH 6.0-7.5), and Tris hydrochloride buffer (50 mM, pH 8.0-9.0). The optimal pH for recombinant RDH enzyme is 7. Between pH 6 and 9, 80% of the maximum enzyme activity can be retained. See [link to relevant documentation]. Figure 3 .

[0089] The recombinant RDH enzyme was dissolved in solutions with pH values ​​of 6, 7, and 8 (corresponding to...). Figure 4 The enzyme activity of enzymes a, b, and c) was measured after incubation at room temperature for 48 hours. Figure 4 The results showed that the recombinant RDH enzyme had good stability at pH 7 and retained 60% of its activity after being placed at room temperature for 24 hours.

[0090] Example 4: Effect of temperature on recombinant RDH enzyme activity

[0091] In a 100 ml system, with a substrate concentration of 50 mM D-allulose, 1000 U of fermentation broth supernatant (crude enzyme solution) was added, and the reaction was carried out in pH 7 phosphate buffer at different temperatures for 15 min. The optimal temperature for recombinant RDH enzyme is 65℃. Figure 5 .

[0092] The recombinant RDH enzyme was placed in phosphate buffer at pH 8 and incubated at 55℃, 65℃, 75℃, and 80℃ (corresponding to...). Figure 6 Incubate at (a, b, c, d) for 15 min, then react at the optimum temperature (65℃) for 15 min and measure enzyme activity. Figure 6 The results showed that the recombinant RDH enzyme was relatively heat-resistant, with a half-life of 1 hour at 65°C.

[0093] Example 5: Biotransformation of D-allool

[0094] The enzyme reaction buffer was 50 mM pH 8.0 phosphate buffer. The substrate, D-allulose, was 100 mM, and the enzyme solution was added at a ratio of 20 U / g substrate. The reaction was carried out in an enzyme reactor at 55 °C for 12 h, and the reaction was terminated by adding concentrated HCl to a final concentration of 200 mM. The product composition was determined by high-performance liquid chromatography (HPLC). The chromatographic column was an HPLC-Waters Sugar-Pak1, the detector was a differential refractive index detector, the column temperature was 85 °C, the mobile phase was pure water, and the flow rate was 0.4 mL / min. The reaction solution contained 56% D-allol and 43% D-allulose, indicating that the enzyme catalyzed the ketaldehyde isomerization reaction between D-allose and D-allulose to reach a final equilibrium of 56:43. (See [link to relevant documentation]). Figure 7 .

[0095] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A recombinant ribitol dehydrogenase vector, characterized in that, Using pPIC9K plasmid as the starting vector, a ribitol dehydrogenase gene sequence was ligated; the ribitol dehydrogenase gene sequence is shown in SEQ ID NO 1.

2. A recombinant ribitol dehydrogenase strain, characterized in that, Using Pichia pastoris GS115 as the starting strain, the recombinant ribitol dehydrogenase vector described in claim 1 was transformed.

3. The application of the recombinant ribitol dehydrogenase vector of claim 1 or the recombinant ribitol dehydrogenase strain of claim 2 in improving the activity of ribitol dehydrogenase.

4. The application of the recombinant ribitol dehydrogenase vector of claim 1 or the recombinant ribitol dehydrogenase strain of claim 2 in improving the activity of ribitol dehydrogenase.

5. The application of the recombinant ribitol dehydrogenase vector of claim 1 or the recombinant ribitol dehydrogenase strain of claim 2 in the production of heat-resistant and pH-resistant ribitol dehydrogenase.

6. The use of the recombinant ribitol dehydrogenase vector of claim 1 or the recombinant ribitol dehydrogenase strain of claim 2 in the production of D-alool.

7. The application of the recombinant ribitol dehydrogenase vector of claim 1 or the recombinant ribitol dehydrogenase strain of claim 2 in improving the conversion rate of D-alool.

Images