A mutant of xylose dehydrogenase, a nucleotide encoding the mutant and use thereof

Abstract

The present application provides a xylitol dehydrogenase mutant, a nucleotide for coding the mutant and application thereof, and belongs to the technical field of bioengineering; in the present application, tertiary structure prediction is performed on a natural xylitol dehydrogenase (XDH) by using protein engineering and enzyme engineering technology, rational analysis reveals the catalytic active center thereof, and a xylitol dehydrogenase mutant with significantly improved catalytic activity is obtained by performing rational design on the amino acid residues in the active center; the xylitol dehydrogenase mutant can be used for producing D-arabitol in cooperation with D-arabitol dehydrogenase.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a mutant of xyritol dehydrogenase, a nucleotide coding the mutant and application thereof. BACKGROUND

[0002] D-arabitol is a natural five-carbon polyol, and its molecular formula is C5H 12 O5, and its molecular weight is 152.15. D-arabitol is one of the twelve platform compounds with the highest added value. In the food field, D-arabitol is an important functional food additive. Compared with the heat value of sucrose (4kcal / g) and xylitol (2.4kcal / g), the heat value of D-arabitol is only 0.2kcal / g. Moreover, D-arabitol can inhibit the growth of cariogenic bacteria, and after entering the mouth, it is endothermically dissolved, so that the mouth produces a cool feeling. Therefore, D-arabitol has great application potential as an anti-caries polyol in oral care products. At the same time, D-arabitol can be used as an intermediate for synthesizing drugs or high-value-added chemicals, such as xylitol, propylene glycol, and anti-pathogenic drugs. Therefore, due to these characteristics, D-arabitol has a very broad development prospect in many fields such as food, medicine, and chemical industry, and is paid great attention by the world.

[0003] The production methods of D-arabitol include natural extraction method, chemical synthesis method and biological synthesis method. A small amount of D-arabitol can be directly extracted from lichens and mushrooms in nature, but the substrate is expensive, and the extraction process is difficult, so it cannot meet the demand in economy and yield. Therefore, chemical method and biological method are the main research directions of D-arabitol production. At present, the industrial production of D-arabitol mainly uses chemical synthesis method, which produces D-arabitol by hydrogenation reduction of arabinose, arabinose lactone or lyxose. However, the process of hydrogenation reduction needs expensive catalyst, high energy consumption, strict experimental conditions and high-cost production equipment, and at the same time, the chemical method cannot avoid environmental pollution, which makes it difficult for D-arabitol production to meet the market demand in various fields. Therefore, it is necessary to develop a method which can meet the large-scale production and reduce the economic cost.

[0004] In recent years, researchers gradually turn their attention to the more inexpensive and readily available agricultural by-products straw, hemicellulose hydrolysate xylose as starting cheap substrate, the whole metabolic pathway as follows: that is, from the orange pole, hydrolysis to obtain xylose, xylose after xylose reductase reaction, the generated D-xylitol is catalyzed by xylitol dehydrogenase (XDH) to generate D-xylulose, and the generated D-xylulose is catalyzed by arabinitol dehydrogenase (Ardh) to generate arabinitol. At present, the research on the production of D-arabinol by biological method mostly concentrates on the natural yeast fermentation to produce D-arabinol with glucose as raw material, and there is little research report on the construction of genetically engineered bacteria to produce D-arabinol from xylose. On the other hand, in the metabolic pathway of producing D-arabinol from xylose, the activity of xylitol dehydrogenase (XDH) is insufficient, which is the main bottleneck of the biosynthesis of D-arabinol from xylose. Therefore, it is necessary to develop a high-activity xylitol dehydrogenase. SUMMARY

[0005] In view of some deficiencies in the prior art, the present application provides a xylitol dehydrogenase mutant, a nucleotide sequence encoding the mutant and the application thereof. In the present application, by using protein engineering and enzyme engineering technology, the tertiary structure of a natural xylitol dehydrogenase (XDH) is predicted, and the catalytic active center is revealed by rational analysis. By rationally designing the amino acid residues in the active center, a xylitol dehydrogenase mutant with significantly improved catalytic activity is obtained, which is denoted as XDH-G161A-S163A. The xylitol dehydrogenase mutant has the potential for D-arabinol production.

[0006] The present application first provides a xylitol dehydrogenase mutant, and the amino acid sequence of the xylitol dehydrogenase mutant is shown in SEQ ID No: 2.

[0007] The present application also provides a nucleotide sequence encoding the xylitol dehydrogenase mutant, and the nucleotide sequence is shown in SEQ ID No: 1.

[0008] The present application also provides a construction method of the xylitol dehydrogenase mutant. The xylitol dehydrogenase mutant is obtained by mutating the glycine at the 161st position of the xylitol dehydrogenase amino acid sequence from GGC to alanine encoded by GCA, and mutating the serine at the 163rd position of the amino acid sequence from TCA to alanine encoded by GCA on the basis of the wild-type xylitol dehydrogenase (XDH), and the mutant is named G161A-S163A. The enzyme activity is verified by measuring the enzyme activity of the enzyme converting NAD to NADH by 340 nm ultraviolet spectrophotometer.

[0009] Further, the amino acid sequence of the wild-type xylitol dehydrogenase is shown as SEQ ID No: 4, and the nucleotide sequence encoding the wild-type xylitol dehydrogenase is shown as SEQ ID No: 3.

[0010] The application further provides a recombinant vector comprising the above-mentioned nucleotide sequence encoding the xylitol dehydrogenase mutant.

[0011] The application further provides a recombinant bacterium comprising the above-mentioned nucleotide sequence encoding the xylitol dehydrogenase mutant, or the above-mentioned recombinant vector, or expressing the above-mentioned xylitol dehydrogenase mutant.

[0012] Further, the recombinant bacterium is Escherichia coli.

[0013] The application further provides the above-mentioned xylitol dehydrogenase mutant, recombinant vector or recombinant bacterium for use in the production of D-arabitol in cooperation with D-arabitol dehydrogenase.

[0014] Further, the use is for the metabolism of xylitol to generate D-arabitol.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] In the application, through homologous multiple sequence alignment and online protein information analysis platform, tertiary structure prediction, molecular docking of substrate and enzyme, and conservation analysis of key catalytic residues in the active center, a xylitol dehydrogenase with significantly improved enzyme activity is obtained by semi-rational design, which has higher catalytic activity and can be used for the production of D-arabitol.

[0017] The application designs a natural xylitol dehydrogenase through enzyme engineering and synthetic biology, and obtains a high-activity unnatural enzyme through rational structure and evolutionary analysis, which is beneficial to promote the metabolism of xylitol to generate D-arabitol, and thus improve the yield of D-arabitol.

[0018] In the application, the traditional irrational design idea is abandoned, and a high-activity xylitol dehydrogenase (XDH-G161A-S163A) is designed by semi-rational design, which has the advantages of rapidity and convenience compared with the traditional irrational method, greatly reduces the mutant screening library, and has important theoretical and practical significance for the modification of other dehydrogenases. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1Figure 1 is an agarose gel electrophoresis map of XDH gene.

[0020] Figure 2 Figure 2 is an SDS-PAGE map of XDH mutant.

[0021] Figure 3 Figure 3 is a map of recombinant plasmid with XDH and Ardh genes.

[0022] Figure 4 Figure 4 is a distribution map of amino acid residues in the catalytic active center of XDH.

[0023] Figure 5 Figure 5 is a result map of amino acid conservation analysis near the active center of XDH.

[0024] Figure 6 Figure 6 is a comparison map of enzymatic properties of wild-type xylitol dehydrogenase and mutant, wherein a is reaction temperature, b is pH, c is metal ion, and d is enzyme activity.

[0025] Figure 7 Figure 7 is a result map of XDH mutant for D-arabitol production. DETAILED DESCRIPTION

[0026] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the scope of the present application is not limited thereto.

[0027] In the following examples, unless specific conditions are specified, the procedures are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless the manufacturer is specified, are conventional products that can be purchased on the market. Unless specifically specified, the present application adopts the existing technology in the field. In the examples, E. coli BL21 (DE3) is purchased from Yobo Biotech Co., Ltd., and pETDuet-1 plasmid is purchased from Moli Biotech Co., Ltd.

[0028] In the following examples, the definition of enzyme activity unit is as follows: XDH consumed to oxidize or reduce 1 μmol of NAD(P) + / NAD(P)H per minute; specific activity unit (U / mg) = activity unit (U / mL) / protein concentration (mg / mL)

[0029] Example 1: Construction of Xylitol Dehydrogenase (XDH) Recombinant Plasmid

[0030] (1) The primers were designed according to the sequence of xylitol dehydrogenase gene of Gluconobacter oxydans in Thailand using Oligo 7.0 software, wherein the nucleotide sequence of XDH is shown in SEQ ID No: 3, the amino acid sequence is shown in SEQ ID No: 4, and the primer sequences are shown in SEQ ID No: 7 and SEQ ID No: 8:

[0031] XDH F: tcatcaccacagccaggatccgATGGCATACGTGGTAAGTTCCT (SEQ ID No: 7);

[0032] XDH R: cgacttaagcattatgcggccgcTCAGCCTCCGGAGATTTCCAGAT (SEQ ID No: 8).

[0033] SEQ ID No: 3

[0034] atggcatacgtggtaagttcctttagcggtaaatcctgtctcgtgacgggtgcaggaggcaatatcggcctcgcaaccgcgctacgtctcgcagaaatggggacggacatcgctcttctggacatgaatccggaagcgttggcgaaagcagaagccgctgtccgcgagaaaggggtgaaggcagaatcgtatgtctgtgacgtgacgtctgaaacatccgtgaatgatgtcgtcagtcaggttgtcgcagattttgggaaaatcgatttcctgttcaataatgccggctatcagggcgcatttgctccagtccaggattatccggctgaagattttccgaaggttctgaacatcaacgtcacgggtgctttccatgtcctgaaagcagtctcccggcacatgattgcaaacggctttgggcggatcgtgaatacggccagcatggcaggcgtaaaaggcccgccaaacatggccgcttatggcgcttcaaaaggcgcaatcattgcgctgactgaaacggcagctctcgaccttgcgccttacaacatccgcgttaatgcaatcagccctggatatatggggccgggcttcatgtgggaccggcaggttgaactgcaggccaaggccaacacgcagtatttctcaaccaacccggaagaagtctccaagcagatgatcggcagtgttccgatgcgtcgttatggagatatcaatgaaattccgggcgtcgtagccttccttctcggagacgattccagcttcatgacaggtgtgaatctggaaatctccggaggctga

[0035] SEQ ID No:4

[0036] MAYVVSSFSGKSCLVTGAGGNIGLATALRLAEMGTDIALLDMNPEALAKAEAAVREKGVKAESYVCDVTSETSVNDVVSQVVADFGKIDFLFNNAGYQGAFAPVQDYPAEDFPKVLNINVTGAFHVLKAVSRHMIANGFGRIVNTASMAGVKGPPNMAAYGASKGAIIALTETAALDLAPYNIRVNAISPGYMGPGFMWDRQVELQAKANTQYFSTNPEEVSKQMIGSVPMRRYGDINEIPGVVAFLLGDDSSFMTGVNLEISGG.

[0037] (2) PCR amplification of XDH gene using XDH F and XDH R primers. The reaction parameters for PCR amplification are as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s; annealing at 55°C for 10 s; extension at 72°C for 30 s; final extension at 72°C for 5 min; after 33 cycles, the target gene is obtained by purifying the PCR product. The obtained target gene is subjected to agarose gel electrophoresis, and the electrophoresis result is shown in Figure 1 From Figure 1 it can be seen that the size of the gene is 798 bp, which is consistent with the theoretical size of XDH gene, i.e., the target gene is XDH gene.

[0038] (3) The primers are designed according to the sequence of pETDuet-1 plasmid by using Oligo7.0 software, and the sequences of the primers are shown in SEQ ID No: 9 and SEQ ID No: 10:

[0039] PET F: gcggccgcataatgcttaagtcg (SEQ ID No: 9);

[0040] PET R: cggatcctggctgtggtgatgat (SEQ ID No: 10).

[0041] Then, the pETDuet-1 plasmid is PCR amplified using the primers PET F and PET R, and the reaction parameters for PCR are as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s; annealing at 55°C for 10 s; extension at 72°C for 30 s; final extension at 72°C for 5 min; after 33 cycles, the pETDuet-1 plasmid skeleton is obtained.

[0042] (4) Gibson assembly of the XDH gene and pETDuet-1 plasmid backbone using 2x MultiF Seamless Assembly Mix at 50°C for 30 min to obtain a recombinant plasmid; then the recombinant plasmid is transformed into E. coli BL21 competent cells by a standard heat shock method. The specific steps of the standard heat shock method are as follows: the E. coli BL21 competent cells are ice-bathed for 5 min, then the recombinant plasmid is added, followed by ice-bathing for 30 min, then heat shock at 42°C for 45 s, followed by continued ice-bathing for 2 min, then 1 mL of antibiotic-free LB medium is added, and the culture is incubated at 37°C for 1 h, then plated on LB plates containing 100 μg / mL ampicillin. After a period of culture, a suitable amount of single colony is picked for culture and the recombinant plasmid is extracted, and sent to Suzhou Genewiz for sequencing verification. The verified recombinant plasmid is named pET-xdh, wherein the XDH gene expression is driven by the T7 promoter.

[0043] Example 2: Xylitol and xylitol molecular docking find the active center of intermolecular binding

[0044] (1) Analyze the amino acid sequence of XDH, predict the tertiary structure of the enzyme through the SWISS Model online website, and then evaluate the modeling results through online software such as Procheck and Verify 3D (https: / / saves.mbi.ucla.edu). Use Chemoffice 19.0 to draw a 3D structure model of the substrate D-Xylitol, and perform minimum energy calculation on the 3D structure model of D-Xylitol.

[0045] Subsequently, through the docking software AutoDock 1.5.6, the small molecule substrate D-Xylitol is subjected to molecular docking analysis with the macromolecular enzyme XDH, and the amino acid residues related to the enzyme activity of XDH are predicted through the online protein analysis platform Hotspot Wizard 3.0, including: SER (serine) at position 147, ALa (alanine) at position 149, Pro (proline) at position 154, Met (methionine) at position 157, TYR (tyrosine) at position 159, and TYR (tyrosine) at position 162. The amino acid residues near the docking pocket are screened as mutation hotspots, and through the analysis of the amino acid residues near the catalytic pocket and internal ion channel after molecular docking, the molecular docking results are as follows: Figure 4The key amino acid residues near the binding pocket of the molecular docking were GLY at position 161, SER at position 163 and GLN at position 202, which were determined by combining the xylose amino acid sequence and the results of protein homology modeling, substrate-protein docking and analysis and mapping using PyMOL software. The catalytic pocket volume and surface area were calculated using Proteins Plus online software, and the internal ion channel simulation was performed using the Caver3.0.3 plug-in in PyMOL software.

[0046] (2) According to the XDH amino acid homologous multi-sequence alignment, combined with the multi-sequence alignment results and the online protein design analysis platform Hotspot Wizard 3.0, GLY at position 161, SER at position 163 and GLN at position 202 were selected for saturation mutation, and the amino acid residues were mutated: the glycine (G) at position 165 was mutated to alanine (A), histidine (H) and tryptophan (W), respectively, denoted as G165A, G165H, G165W; the arginine (R) at position 201 was mutated to proline (P), denoted as R201P; the alanine (A) at position 158 was mutated to serine (S), denoted as A158S; the alanine (A) at position 159 was mutated to glycine (G), denoted as A159G; the alanine (A) at position 162 was mutated to threonine (T), denoted as A162. Then the primers were designed using Oligo7.0 software:

[0047] G161 F: CGCTTATnnnGCTTCAAAAGGCGCAATCATTG (SEQ ID No: 11);

[0048] G161 R: TTGAAGCnnnATAAGCGGCCATGTTTGGCGGG (SEQ ID No: 12);

[0049] S163 F: TATGGCGCTnnnAAAGGCGCAATCATTGCGCT (SEQ ID No: 13);

[0050] S163 R: CCTTTnnnAGCGCCATAAGCGGCCATGTTTGG (SEQ ID No: 14);

[0051] G202 F: GACCGGnnnGTTGAACTGCAGGCCAAGGCCAA (SEQ ID No: 15);

[0052] G202 R: AGTTCAACnnnCGGGTCCCACATGAAGCCCGG (SEQ ID No: 16);

[0053] G165A F: GCGCTTCAAAAgcaGCAATCATTGCGCTGACTGA (SEQ ID No: 17);

[0054] G165A R: TCGCtgcCTGCCGGGGATGGCGGCCGGAGAGT (SEQ ID No: 18);

[0055] G165H F: GCGCTTCAAAAcatGCAATCATTGCGCTGACTGA (SEQ ID No: 19);

[0056] G165H R: TGCatgTTTTGAAGCGCCATAAGCGGCCATGT (SEQ ID No: 20);

[0057] R201P F: GACccgCAGGTTGAACTGCAGGCCAAGGCCAA (SEQ ID No: 21);

[0058] R201P R: AGTTCAACCTGcggGTCCCACATGAAGCCCGG (SEQ ID No: 22);

[0059] A158S F: AACATGagcGCTTATGGCGCTTCAAAAGGCGC (SEQ ID No: 23);

[0060] A158S R: CCATAAGCgctCATGTTTGGCGGGCCTTTTAC (SEQ ID No: 24);

[0061] A162T F: GGCGCTTCAAAAtttGCAATCATTGCGCTGACTGA (SEQ ID No: 25);

[0062] A162T R: CCTTTTGAggtGCCATAAGCGGCCATGTTTGG (SEQ ID No: 26);

[0063] (3) PCR amplification of pET-xdh plasmid using G161 F and G161 R primer pairs, S163 F and S163 R primer pairs, G202 F and G202 R primer pairs, G165AF and G165A R primer pairs, G165H F and G165H R primer pairs, R201P F and R201P R primer pairs, A158S F and A158S R primer pairs, and A162T F and A162T R primer pairs, respectively, to obtain eight mutant plasmid backbones, namely G161A, S163A, G165A, G165H, R201P, A158S, A162T and G202A mutant plasmid backbones.

[0064] The above eight mutant plasmid backbones were Gibson assembled at 50°C for 30 min using 2x MultiF Seamless Assembly Mix to obtain recombinant plasmids; and then the recombinant plasmids were transformed into E. coli BL21 competent cells by a standard heat shock method. The specific steps of the standard heat shock method are as follows: the E. coli BL21 competent cells were ice-bathed for 5 min, then the recombinant plasmids were added, followed by ice-bathing for 30 min, then heat shock at 42°C for 45 s, followed by ice-bathing for 2 min, then 1 mL of antibiotic-free LB medium was added, and the culture was incubated at 37°C for 1 h, and then plated on LB plates containing 100 μg / mL ampicillin. After a period of culture, a suitable amount of single colony was picked and cultured to extract the recombinant plasmid, which was sent to Suzhou Genewiz for sequencing verification. The verified correct recombinant strains were named G161A, S163A, G165A, G165H, R201P, A158S, A162T and G202A, respectively.

[0065] The PCR reaction parameters are as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s; annealing at 55°C for 10 s; extension at 72°C for 1.5 min; final extension at 72°C for 5 min; and 33 cycles.

[0066] (4) The recombinant strains G161A, S163A, G165A, G165H, R201P, A158S, A162T and G202A obtained in step (3) were inoculated in LB medium (yeast extract 5 g / L, tryptone 10 g / L and sodium chloride 10 g / L), and cultured at 37°C and 200 rpm until the OD600 was between 0.6 and 0.8. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) inducer was added at a final concentration of 1 mM, and the XDH mutant protein expression was induced at 28°C and 180 rpm overnight. The expression of XDH protein was detected by SDS-PAGE (12% separation gel), and the results are shown in Figure 2 . Figure 2The SDS-PAGE diagram of the XDH mutant is shown in the figure. The size of the wild XDH protein and the mutant XDH protein is about 30 kDa, which is consistent with the expected size, indicating that the protein is a normal purifiable protein, and the protein size is consistent with the theoretical value.

[0067] (5) The wild XDH protein and the mutant XDH protein obtained in step (4) were respectively subjected to enzyme activity test under the following conditions:

[0068] The test system comprises 1 mM DTT, 2 mM NAD + and 2 mM xylitol and an appropriate amount of enzyme solution, and is supplemented with 100 mmol / L potassium phosphate buffer (pH 8.0) to 1 mL, and then incubated at 37°C. The change of absorbance value at 340 nm was determined by an enzyme marker. Through enzyme activity detection, it is found that the mutant G161A-S163A has an enzyme activity 50% higher than that of the wild type XDH.

[0069] Example 3: Analysis of amino acid conservation near the active center of XDH

[0070] (1) According to the amino acid sequence of XDH, the tertiary structure of the enzyme was predicted through the SWISS Model online website, and the molecular xylitol was docked with XDH through Auto Dock software.

[0071] (2) The above molecular docking model was visualized using PYMOL software, and the amino acid residues within 5A range of xylitol molecule were selected.

[0072] (3) The amino acids within 5A range shown in Figure 4 were analyzed for conservation using amino acid conservation analysis software to find the active center of XDH enzyme. The analysis results are shown in Figure 5 . Figure 5 The amino acid conservation analysis results near the active center of XDH are shown in the figure. The marked positions are conservative sites: G 17 -X-G 19 -G 20 -X2-G 23 , Asn 144 -Ser 147 -Tyr 160 -Lys 164 , and the corresponding interval of the conservative site near the binding pocket is found. Among them, Asn 144 -Ser 147 -Tyr 160 -Lys 164This interval is near the molecular docking binding pocket, and the site near it is selected as the mutation reference site. Except for the amino acids at positions 161 and 163, the amino acids near the pocket are highly conserved, so G161 and S163 have the potential for rational design.

[0073] Example 4: Rational design of XDH mutants

[0074] (1) According to the amino acid conservation results obtained in Example 3, it is shown that the amino acid G at position 161 of the wild XDH is mutated to A to obtain the mutant G161A, and the amino acid S at position 163 is mutated to A to obtain the mutant S163A. In combination with the enzyme activity determination of the two mutants, iterative mutation of the two key amino acid residues is performed to obtain the mutant G161A-S163A, which may help to improve the XDH enzyme activity.

[0075] (2) According to the XDH gene sequence, the primers are designed by using Oligo7.0 software, and the mutated bases represented by lowercase letters in the primers are as follows:

[0076] G161A F: CGCTTATgcaGCTTCAAAAGGCGCAATCATTG (SEQ ID No: 27);

[0077] G161A R: TTGAAGCtgcATAAGCGGCCATGTTTGGCGGG (SEQ ID No: 28);

[0078] S163A F: TATGGCGCTgcaAAAGGCGCAATCATTGCGCT (SEQ ID No: 29);

[0079] S163A R: CCTTTtgcAGCGCCATAAGCGGCCATGTTTGG (SEQ ID No: 30);

[0080] G161A-S163A F: CGCTTATGCAGCTGCAAAAGGCGCAATCATTGC (SEQ ID No: 31);

[0081] G161A-S163A R: TTTGCAGCTGCATAAGCGGCCATGTTTGGCGGG (SEQ ID No: 32);

[0082] (3) PCR amplification of pET-xdh plasmid using G161A F and G161A R primer pairs, S163A F and S163A R primer pairs, G161A-S163A F and G161A-S163A R primer pairs, respectively, to obtain G161A, S163A and G161A-S163A mutant plasmid backbones. Gibson assembly of the above mutant plasmid backbones using 2x MultiF Seamless Assembly Mix at 50°C for 30 min to obtain recombinant plasmids, the map of which is shown in Figure 3 Then, the recombinant plasmids are transformed into E. coli BL21 competent cells by standard heat shock method. The specific steps of the standard heat shock method are as follows: after ice bath for 5 min, the recombinant plasmids are added to the E. coli BL21 competent cells, then ice bath for 30 min, after ice bath, heat shock at 42°C for 45 s, then continue ice bath for 2 min, add 1 mL of LB medium without antibiotics, culture at 37°C for 1 h, then spread on LB plate containing 100 μg / mL ampicillin. After a period of culture, a suitable amount of single colony is picked and cultured to extract the recombinant plasmid, which is sent to Suzhou Genewiz for sequencing verification. The recombinant strains verified correctly are named as G161A, S163A and G161A-S163A, respectively. The nucleotide sequence of the G161A-S163A xylitol dehydrogenase mutant is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID No: 2

[0083] SEQ ID No: 1

[0084] atggcatacgtggtaagttcctttagcggtaaatcctgtctcgtgacgggtgcaggaggcaatatcggcctcgcaaccgcgctacgtctcgcagaaatggggacggacatcgctcttctggacatgaatccggaagcgttggcgaaagcagaagccgctgtccgcgagaaaggggtgaaggcagaatcgtatgtctgtgacgtgacgtctgaaacatccgtgaatgatgtcgtcagtcaggttgtcgcagattttgggaaaatcgatttcctgttcaataatgccggctatcagggcgcatttgctccagtccaggattatccggctgaagattttccgaaggttctgaacatcaacgtcacgggtgctttccatgtcctgaaagcagtctcccggcacatgattgcaaacggctttgggcggatcgtgaatacggccagcatggcaggcgtaaaaggcccgccaaacatggccgcttatgcagctgcaaaaggcgcaatcattgcgctgactgaaacggcagctctcgaccttgcgccttacaacatccgcgttaatgcaatcagccctggatatatggggccgggcttcatgtgggaccggcaggttgaactgcaggccaaggccaacacgcagtatttctcaaccaacccggaagaagtctccaagcagatgatcggcagtgttccgatgcgtcgttatggagatatcaatgaaattccgggcgtcgtagccttccttctcggagacgattccagcttcatgacaggtgtgaatctggaaatctccggaggctga SEQ ID No: 2

[0085] MAYVVSSFSGKSCLVTGAGGNIGLATALRLAEMGTDIALLDMNPEALAKAEAAVREKGVKAESYVCDVTSETSVNDVVSQVVADFGKIDFLFNNAGYQGAFAPVQDYPAEDFPKVLNINVTGAFHVLKAVSRHMIANGFGRIVNTASMAGVKGPPNMAAYAAAKGAIIALTETAALDLAPYNIRVNAISPGYMGPGFMWDRQVELQAKANTQYFSTNPEEVSKQMIGSVPMRRYGDINEIPGVVAFLLGDDSSFMTGVNLEISGG.

[0086] PCR reaction parameters: pre-denaturation, 98°C for 3 min; denaturation, 98°C for 10 s; annealing, 55°C for 10 s; extension, 72°C for 1.5 min; final extension, 72°C for 5 min; 33 cycles.

[0087] (4) The recombinant bacteria G161A, S163A and G161A-S163A obtained in step (3) were inoculated into LB medium (yeast powder 5 g / L, tryptone 10 g / L and sodium chloride 10 g / L) and cultured at 37°C, 200 rpm for 0.6-0.8 of OD600, and then 1 mM isopropyl-β-D-thiogalactoside (IPTG) inducer was added, and the XDH mutant protein expression was induced at 28°C, 180 rpm overnight.

[0088] (5) The wild type XDH protein and mutant XDH protein obtained in step (4) were respectively tested for enzyme activity under the following conditions:

[0089] The test system includes 1 mM DTT, 2 mM NAD + and 2 mM xylitol and an appropriate amount of enzyme solution, and 100 mmol / L potassium phosphate buffer (pH 8.0) was added to 1 mL, then incubated at 37°C, and the absorbance value change was determined at 340 nm by an enzyme marker, and the determination results are shown in Figure 6 .

[0090] Figure 6 Comparison of wild type xylitol dehydrogenase and mutant enzyme properties, wherein Figure 6 (a) The optimal reaction temperature of the enzyme was explored to be 35°C; Figure 6 (b) The effect of pH on enzyme activity was explored, and the optimal pH was 11; Figure 6 (c) The effect of metal ions on enzyme catalytic activity was explored, and manganese ions had the best catalytic activity; Figure 6(d)It can be seen that compared with wild type XDH, G161A and S163A are increased by 20% and 43% respectively compared with the wild strain enzyme activity, and the enzyme activity of mutant G161A-S163A is 50% higher than that of wild type XDH, indicating that it has better catalytic activity.

[0091] Example 5: Construction of recombinant plasmid of xylitol dehydrogenase mutant and D-arabitol dehydrogenase synergistic expression

[0092] (1) The primers were designed according to the sequence of D-arabitol dehydrogenase (ARDH) gene of Oxyglococcus thailandicus, wherein the nucleotide sequence of ARDH is shown as SEQ ID No: 5, and the amino acid sequence is shown as SEQ ID No: 6, and the primer sequence is as follows:

[0093] ARDHF: tcttagtatattagttaagtataagaaggagATATACATATGATGTACATGGAAAAACTTCGTC TCGA (SEQ ID No: 33);

[0094] ARDH R: agcggtggcagcagcctaGGTTACCAGACGGTGAAACCAGCA (SEQ ID No: 34)

[0095] SEQ ID No: 5:

[0096] atgtacatggaaaaacttcgtctcgatggccgcaccgcagttgtcactggcggcgcacagaacatcggtctggcctgcgtgacggcactggccgaagccggggcgcgtgttgtgattgcggatctggatgaggccatggccgcacaatctgcggaggaactctgcgcagagggcctggacgtcagaagcatccgcatggatgtcacgagcatggaaaatgttcaggcagccatcaagaccctgcacgagcaggaaggccatctggatattctggtggcctgtgcggggatctgcatttccgaagtcaaagctgaggacatgacggaaggtcagtggctcaagcaggtcgatatcaacctgaacggcatgttccgttgctgtcaggccgtgggtcgcatcatgcttgagcagaagaaaggcgcgattgtcgccatcggatccatgtccgggcaaatcgtcaaccgcccacagcagcaggccgcctataatgcctccaaggcgggtgtgcaccagtatatccgctcacttgcggcggaatgggcgccttatggtatccgtgccaatgcagttgctccgacctacatcgaaacaacactgacacgcttcggtatggaaaagccggaactgtatgatgcgtggattgccggaacaccgatggggcgcgtggggcagcccgacgaagtcgcctccgtcgtgcactttctggcctcggatgccgcaagcctgatgacgggttccatcgtcaacgtggatgctggtttcaccgtctggtaa

[0097] SEQ ID No:6:

[0098] MYMEKLRLDGRTAVVTGGAQNIGLACVTALAEAGARVVIADLDEAMAAQSAEELCAEGLDVRSIRMDVTSMENVQAAIKTLHEQEGHLDILVACAGICISEVKAEDMTEGQWLKQVDINLNGMFRCCQAVGRIMLEQKKGAIVAIGSMSGQIVNRPQQQAAYNASKAGVHQYIRSLAAEWAPYGIRANAVAPTYIETTLTRFGMEKPELYDAWIAGTPMGRVGQPDEVASVVHFLASDAASLMTGSIVNVDAGFTVW.

[0099] (2) PCR amplification of XDH gene using ARDH F and ARDH R primers. The reaction parameters for PCR amplification are as follows: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 30 s, final extension at 72°C for 5 min; after 33 cycles, the target gene is obtained by purifying the PCR product. The obtained target gene is subjected to agarose gel electrophoresis to verify that the obtained target gene is the ARDH gene.

[0100] (3) The primers for the xylitol dehydrogenase mutant recombinant plasmid G161A-S163A obtained in Example 4 are designed using Oligo7.0 software, and the primer sequences are shown in SEQ ID No: 35 and SEQ ID No: 36:

[0101] G161A-S163A F: catatgtatatctccttcttatacttaactaatatactaa (SEQ ID No: 35);

[0102] G161A-S163A R: cctaggctgctgccaccgctgagcaataactagcataacc (SEQ ID No: 36).

[0103] Then, the G161A-S163A recombinant plasmid is PCR amplified using the G161A-S163A F and G161A-S163A R primer pairs, and the PCR reaction parameters are as follows: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 30 s, final extension at 72°C for 5 min; after 33 cycles, the G161A-S163A plasmid backbone is obtained.

[0104] (4) Gibson assembly of the above ARDH gene and G161A-S163A plasmid backbone using 2x MultiF Seamless Assembly Mix at 50°C for 30 min to obtain a recombined plasmid; then transform the recombined plasmid into E. coli BL21 competent cells by a standard heat shock method. The specific steps of the standard heat shock method are as follows: add the recombined plasmid into the E. coli BL21 competent cells after ice-bath for 5 min, then ice-bath for 30 min, heat shock at 42°C for 45 s after ice-bath, then continue ice-bath for 2 min, add 1 mL LB medium without antibiotics, culture at 37°C for 1 h, then coat LB plate containing 100 μg / mL ampicillin. After a period of culture, take a proper amount of single colony for culture and extract the recombined plasmid, send to Suzhou Genewiz for sequencing verification, and name the recombined plasmid verified correctly as pET-XDH-G161A-S163A, which expresses the xyxitol dehydrogenase mutant and D-arabitol dehydrogenase described in Example 4 in coordination.

[0105] Example 6: Production of D-arabitol by xyxitol dehydrogenase mutant and D-arabitol dehydrogenase in coordination

[0106] (1) Transform the recombined plasmid pET-XDH-G161A-S163A obtained in Example 5 into E. coli BL21 competent cells by a standard heat shock method. The specific steps of the standard heat shock method are as follows: add the recombined plasmid into the E. coli BL21 competent cells after ice-bath for 5 min, then ice-bath for 30 min, heat shock at 42°C for 45 s after ice-bath, then continue ice-bath for 2 min, add 1 mL LB medium without antibiotics, culture at 37°C for 1 h, then coat LB plate containing 100 μg / mL ampicillin. After a period of culture, obtain a strain of D-arabitol-producing engineered bacteria, named GC S1;

[0107] (2) The genetically engineered bacteria GCS1 were activated in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride) at 37°C, 220 rpm overnight, and then inoculated into fermentation medium (xylitol 60 g / L, MgSO4·7H2O 1.2 g / L, (NH4)2SO4 4.0 g / L, KH2PO4 3 g / L, K2HPO4 0.8 g / L, yeast powder 11.5 g / L, peptone 19.5 g / L, pH 7.0) at 5% v / v inoculation amount, and then cultured at 37°C, 220 rpm for 2.5 hours until OD600 reached about 0.8, and then 0.05 mM IPTG was added, and then the culture was continued at 28°C, 220 rpm for 72 hours, and then the yield of D-arabitol was measured by high performance liquid chromatography, and the liquid chromatography conditions were as follows: Aminex HPX-Ga column, 0.4 mL / min, 65°C, RID detector 35°C, UV detector 210 nm, and the detection results were as shown in FIG. 2. Figure 7

[0108] Figure 7 The liquid chromatography analysis results of the engineered bacteria GCS1 for D-arabitol production are shown in the figure. As can be seen from the figure, the peak time of xylitol is near 23.84, and the peak time of D-arabitol is near 20.7, indicating that the engineered bacteria can be used to ferment D-arabitol from xylitol as a substrate.

[0109] In summary, the high-activity enzyme XDH-G161A-S163A designed in the present application has higher enzyme activity and has potential for D-arabitol production.

[0110] The above embodiments are preferred embodiments of the present application, but the present application is not limited to the above embodiments, and any obvious improvements, replacements or modifications made by those skilled in the art without departing from the essential content of the present application shall fall within the protection scope of the present application.​

Claims

1. A xylitol dehydrogenase mutant, characterized in that, The amino acid sequence of the xylitol dehydrogenase mutant is shown in SEQ ID No:

2.

2. The nucleic acid encoding the xylitol dehydrogenase mutant of claim 1, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID No:

1.

3. A recombinant vector comprising the nucleic acid encoding the xylitol dehydrogenase mutant of claim 2.

4. A recombinant bacterium, said recombinant bacterium comprising the nucleic acid encoding the xylitol dehydrogenase mutant of claim 2, or the recombinant vector of claim 3, or expressing the xylitol dehydrogenase mutant of claim 1.

5. The recombinant bacteria according to claim 4, characterized in that, The recombinant bacteria is Escherichia coli.

6. The method for constructing the xylitol dehydrogenase mutant according to claim 1, characterized in that, include: Based on wild-type xylitol dehydrogenase, glycine at position 161 and serine at position 163 of the wild-type xylitol dehydrogenase amino acid sequence were mutated to alanine to obtain xylitol dehydrogenase mutant.

7. The method for constructing the xylitol dehydrogenase mutant according to claim 6, characterized in that, The amino acid sequence of the wild-type xylitol dehydrogenase is shown in SEQ ID No:4, and the nucleotide sequence encoding the wild-type xylitol dehydrogenase is shown in SEQ ID No:

3.

8. The use of the xylitol dehydrogenase mutant of claim 1, the recombinant vector of claim 3, or the recombinant bacteria of any one of claims 4 to 5 in the co-production of D-arabitol with D-arabitol dehydrogenase.

9. The application according to claim 8, characterized in that, The application is to metabolize xylitol to produce D-arabinitol.

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