A method for synthesizing D-xylitol based on NAD (H) cofactor circulation system and its special engineering bacteria

Abstract

The present invention discloses a method for synthesizing D-xylitol based on the NAD(H) cofactor circulation system and a special engineering bacterium thereof. The present invention provides a method for constructing an engineering strain for synthesizing D-xylitol, comprising the following steps: allowing a receptor bacterium to simultaneously express NAD(H)-dependent D-xylose reductase and NAD(H)-dependent formate dehydrogenase to obtain an engineering strain for synthesizing D-xylitol. The present invention will generate huge economic benefits for the production of D-xylitol and has great promotion and application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for synthesizing D-xylitol based on an NAD (H) cofactor circulation system and a special engineering bacterium thereof. Background Art

[0002] D-xylitol is a five-carbon sugar alcohol that is widely found in plants and microorganisms. D-xylitol is a natural sweetener. In 2004, it was identified by the U.S. Department of Energy as one of the twelve high-value-added chemicals synthesized from biomass. D-xylitol is widely used in many fields such as chemical industry, medicine, food, cosmetics, and agricultural production. (1) As a pharmaceutical intermediate, D-xylitol is used in the synthesis of various drugs, such as anti-tumor drugs, vitamins, and cardiovascular drugs; (2) D-xylitol has a wetting function and can be used as a wetting agent in the production of cosmetics; (3) D-xylitol can be used as a moisturizer and antifreeze agent in toothpaste, which can inhibit the growth of common oral bacteria (such as caries-causing bacteria) and maintain oral health; (4) D-xylitol produces a cooling sensation when consumed, so it is widely used in dairy products, soft drinks, chewing gum, and chocolate. In addition, spraying D-xylitol as foliar fertilizer on crops can enhance their resistance to stress, promote their absorption of nutrients, and improve their quality.

[0003] At present, the main method for industrial production of D-xylitol is chemical catalytic hydrogenation. However, this chemical synthesis method has problems such as harsh reaction conditions (high temperature and high pressure), expensive catalysts (heavy metals) and pollution, low yield, and many by-products that are not conducive to separation. Biotransformation (microbial fermentation and enzyme catalysis) is an environmentally friendly and rapidly developing method. Its key enzyme D-xylose reductase can catalyze D-xylose to D-xylitol. This enzyme usually requires reduced NADP (H) as a cofactor (Jin, LQ., Xu, W., Yang, B. et al. Efficient Biosynthesis of Xylitol from Xylose by Coexpression of Xylose Reductase and Glucose Dehydrogenase in Escherichia coli. Appl Biochem Biotechnol 187, 1143–1157, 2019). Due to the high price of the cofactor NADP(H), it is difficult to obtain large-scale production and application in industry by biotransformation. NAD(H) is cheaper than NADP(H). Some researchers have synthesized D-xylitol by co-utilizing cofactors NAD(H) and NADP(H), thereby reducing the cost of cofactors (Jo JH, Oh SY, Lee HS, Park YC, Seo JH. Dual utilization of NADPH and NADHcofactors enhances xylitol production in engineered Saccharomyces cerevisiae .Biotechnol J. 10(12), 1935-43, 2015).

[0004] In summary, there is an urgent need in the art to develop a method for synthesizing D-xylitol with lower cofactor costs. Summary of the invention

[0005] The purpose of the present invention is to provide a method for synthesizing D-xylitol based on the NAD (H) cofactor circulation system and a special engineering bacterium thereof.

[0006] In a first aspect, the present invention claims a method for constructing an engineered strain for synthesizing D-xylitol.

[0007] The method for constructing an engineered strain for synthesizing D-xylitol claimed in the present invention may include the following steps: allowing the recipient bacteria to simultaneously express NAD(H)-dependent D-xylose reductase and NAD(H)-dependent formate dehydrogenase to obtain an engineered strain for synthesizing D-xylitol.

[0008] Furthermore, the NAD(H)-dependent D-xylose reductase may be derived from Kluyveromyces marxianus ( Kluyveromyces marxianus The NAD(H)-dependent formate dehydrogenase may be derived from Thiobacillus ( Starkeya ).

[0009] Furthermore, the NAD(H)-dependent D-xylose reductase may be any of the following:

[0010] (A1) a protein having an amino acid sequence as shown in SEQ ID No. 1;

[0011] (A2) a protein having the same function as SEQ ID No. 1 with one or more amino acid residues substituted and / or deleted and / or added;

[0012] (A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with SEQ ID No. 1 and derived from Kluyveromyces marxianus and having the same function;

[0013] (A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 1.

[0014] Furthermore, the NAD(H)-dependent formate dehydrogenase may be any of the following:

[0015] (B1) a protein having an amino acid sequence as shown in SEQ ID No. 3;

[0016] (B2) a protein having the same function as SEQ ID No. 3 with one or more amino acid residues substituted and / or deleted and / or added;

[0017] (B3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with SEQ ID No. 3 and derived from Thiobacillus and having the same function;

[0018] (B4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 3.

[0019] In the above protein, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0020] In the above proteins, the identity of the amino acid sequence can be determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gapexistence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be calculated.

[0021] In the above proteins, the above 95% identity or more may be at least 96%, 97%, 98% identity. The above 90% identity or more may be at least 91%, 92%, 93%, 94% identity. The above 85% homology or more may be at least 86%, 87%, 88%, 89% identity. The above 80% identity or more may be at least 81%, 82%, 83%, 84% identity.

[0022] In the method, causing the recipient bacteria to express the NAD(H)-dependent D-xylose reductase and the NAD(H)-dependent formate dehydrogenase can be achieved as follows: introducing the coding gene of the NAD(H)-dependent D-xylose reductase and the coding gene of the NAD(H)-dependent formate dehydrogenase into the recipient bacteria.

[0023] Furthermore, the gene encoding the NAD(H)-dependent D-xylose reductase may be any of the following:

[0024] (C1) a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 2;

[0025] (C2) a DNA molecule that hybridizes with the DNA molecule shown in SEQ ID No. 2 under stringent conditions and encodes the protein shown in SEQ ID No. 1;

[0026] (C3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the DNA sequence defined by SEQ ID No. 2 and encodes the protein shown by SEQ ID No. 1.

[0027] Furthermore, the coding gene of the NAD(H)-dependent formate dehydrogenase may be any of the following:

[0028] (D1) a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 4;

[0029] (D2) a DNA molecule that hybridizes with the DNA molecule shown in SEQ ID No. 4 under stringent conditions and encodes the protein shown in SEQ ID No. 3;

[0030] (D3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the DNA sequence defined by SEQ ID No. 4 and encodes the protein shown by SEQ ID No. 3.

[0031] In the above coding genes, the identity of the nucleotide sequence can be determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gapexistence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of nucleotide sequences, the identity value (%) can be obtained.

[0032] In the above-mentioned coding genes, the above 95% identity may be at least 96%, 97%, 98% identity. The above 90% identity may be at least 91%, 92%, 93%, 94% identity. The above 85% identity may be at least 86%, 87%, 88%, 89% identity. The above 80% identity may be at least 81%, 82%, 83%, 84% identity.

[0033] In the method, the recipient bacteria may be Escherichia coli.

[0034] In one embodiment of the present invention, the Escherichia coli is Escherichia coli BW25113.

[0035] In the method, the gene encoding the NAD(H)-dependent D-xylose reductase and the gene encoding the NAD(H)-dependent formate dehydrogenase can be introduced into the recipient bacteria in the form of a recombinant vector.

[0036] In one embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by cloning the coding gene of the NAD(H)-dependent D-xylose reductase and the coding gene of the NAD(H)-dependent formate dehydrogenase into the pBAD / HisB vector. Specifically, the coding gene of the NAD(H)-dependent D-xylose reductase (SEQ ID No. 2) is cloned between the restriction sites XhoI and SpeI of the pBAD / HisB vector, and the coding gene of the NAD(H)-dependent formate dehydrogenase (SEQ ID No. 4) is cloned between the restriction sites SpeI and PstI of the pBAD / HisB vector.

[0037] In a second aspect, the present invention claims protection for the engineered strain constructed using the method described in the first aspect above.

[0038] In a third aspect, the present invention claims a complete set of products for synthesizing D-xylitol.

[0039] The complete set of products for synthesizing D-xylitol claimed in the present invention may be any of the following:

[0040] (E1) Complete product 1: comprising the engineered strain described in the second aspect above, D-xylose, formate and NAD(H).

[0041] Furthermore, the complete set of products 1 may also include L-arabinose.

[0042] Wherein, L-arabinose is used to induce the engineered strain to express the NAD(H)-dependent D-xylose reductase and the NAD(H)-dependent formate dehydrogenase.

[0043] (E2) Complete product 2: comprising the NAD(H)-dependent D-xylose reductase described in the first aspect above, the NAD(H)-dependent formate dehydrogenase described in the first aspect above, D-xylose, formate and NAD(H);

[0044] The NAD(H) is NAD + and / or NADH.

[0045] The synthesis of D-xylitol using the complete set of products uses D-xylose as a substrate. Figure 1 shown.

[0046] In one embodiment of the present invention, the formate is sodium formate.

[0047] In a fourth aspect, the present invention claims protection for any of the following applications:

[0048] (F1) Use of the engineered strain described in the second aspect above in the synthesis of D-xylitol;

[0049] (F2) Use of the complete set of products described in the third aspect above in the synthesis of D-xylitol.

[0050] In a fifth aspect, the present invention claims a method for synthesizing D-xylitol.

[0051] The method for synthesizing D-xylitol claimed in the present invention may include the following steps (G1) or (G2):

[0052] (G1) The engineered strain described in the second aspect above is cultured by induction with L-arabinose to obtain an induced strain, and the whole cells of the induced strain are used to catalyze the D-xylose reaction to obtain D-xylitol.

[0053] (G2) Using the NAD(H)-dependent D-xylose reductase and the NAD(H)-dependent formate dehydrogenase described in the first aspect to catalyze the reaction of D-xylose to obtain D-xylitol.

[0054] Among them, (G1) is the whole cell catalysis method; (G2) is the enzyme catalysis method.

[0055] Furthermore, in (G1), the engineered strain is induced to culture with L-arabinose in a culture medium containing L-arabinose at a final concentration of 2 g / L, the induction culture temperature is 30° C., and the induction culture time is 12-16 hours (e.g., 12 hours).

[0056] Further, in (G1), the final concentration of D-xylose in the reaction system using the induced strain whole cell to catalyze the D-xylose reaction (the induced strain is a whole cell catalyst) is 100-200 g / L (such as 100 g / L). The temperature of the reaction system is 30°C-50°C (such as 37°C), the reaction pH is 5.0-7.0 (such as pH 6.0), and the reaction time is 12-24 hours (such as 24 hours).

[0057] Furthermore, in (G1) and (G2), the reaction system for catalyzing the D-xylose reaction further contains formate (such as sodium formate) and NAD(H); the NAD(H) is NAD + and / or NADH.

[0058] In one embodiment of the present invention, in (G1), in the reaction system catalyzing the D-xylose reaction, the final concentration of formate (sodium formate) is 1.0 mol / L, and the NAD(H) is NAD + , NAD + The final concentration was 0.5 mmol / L.

[0059] The present invention constructs a special engineering bacterium coupling NAD(H)-dependent D-xylose reductase and formate dehydrogenase, and catalyzes the synthesis of D-xylitol from substrate D-xylose based on the NAD(H) cofactor circulation system. Experiments have shown that the maximum yield of D-xylitol obtained by the engineering bacterium constructed by the present invention is 140g / L. The present invention will generate huge economic benefits for the production of D-xylitol and has great promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram of the pathway for coupling D-xylose reductase (KMXR) and formate dehydrogenase (SFDH) to catalyze the synthesis of D-xylose from D-xylose based on the NAD(H) cofactor cycle system.

[0061] Figure 2 It is the liquid phase spectrum of D-xylose and D-xylitol standard. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0063] The experimental methods in the following examples are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The quantitative tests in the following examples are set up for three repeated experiments, and the results are averaged. For the chromatogram under the same condition parameters, the peak in the sample to be tested with the same retention time (±0.1min) as the standard can be identified as the target peak.

[0064] Escherichia coli BW25113: NBRP E. coli strain, catalog number: ME9062 (https: / / shigen.nig.ac.jp / ecoli / strain / resource / strainGeneMutant / list).

[0065] The Trans1-T1 competent cells in the following examples are products of Beijing Quanshijin Biotechnology Co., Ltd., with the product catalog number CD501.

[0066] Vector pBAD / HisB: Invitrogen, product catalog number V430-01.

[0067] D-Xylitol: Aladdin Company, product catalog number X100092.

[0068] D-Xylose: Aladdin Company, product catalog number X101009.

[0069] Example 1: Construction of D-xylitol-producing recombinant engineering bacteria X01

[0070] The NAD(H)-dependent D-xylose reductase involved in this embodiment is derived from Kluyveromyces marxianus ( Kluyveromyces marxianus ), whose amino acid sequence is shown in SEQ ID No.1, and the corresponding encoding nucleotide sequence is shown in SEQ ID No.2. The Starkeya ) NAD(H)-dependent formate dehydrogenase, the amino acid sequence of which is shown in SEQ ID No.3, and the corresponding encoding nucleotide sequence is shown in SEQ ID No.4.

[0071] 1. Acquisition of KMXR gene fragment

[0072] Using the artificial synthetic sequence containing SEQ ID No. 2 (synthesized by Nanjing GenScript Biotech Co., Ltd.) as a template, PCR amplification was performed using primers P1 and P2 to obtain a PCR product, namely the coding gene sequence of D-xylose reductase. The primer sequences are as follows:

[0073] P1: 5'-gtgccgcgcggcagc ctcgag ATGACCTACCTGGCTCCGAC-3′ (the underlined part is the XhoI recognition sequence);

[0074] P2: 5'-ctcaccgaattcacc actagt TTAGATGAAGGTCGGAAATTC-3' (the underlined part is the SpeI recognition sequence).

[0075] The PCR product was detected by 1% agarose gel electrophoresis, and the size of the PCR product was about 1000 bp, which was consistent with the target fragment and was named KMXR gene fragment. The KMXR gene fragment was recovered by gel electrophoresis.

[0076] 2. Construction of pBAD / HisB-KMXR recombinant vector

[0077] After the vector pBAD / HisB was double-digested with XhoI and SpeI, the vector fragment (about 3500bp) was recovered; the KMXR gene fragment recovered in step 1 was connected with the recovered vector fragment using the Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6:343-345.) to obtain a connection product. The connection product was transformed into Trans1-T1 competent cells and coated on a LB solid plate containing streptomycin. Incubate at 37℃ overnight, pick a single clone to extract the plasmid, and perform sequencing verification. The vector with the correct sequencing was named pBAD / HisB-KMXR. pBAD / HisB-KMXR structure description: The recombinant plasmid obtained by replacing the small fragment between the restriction sites XhoI and SpeI of the pBAD / HisB vector with the DNA fragment shown in SEQ ID No.2.

[0078] 3. Obtaining SFDH gene fragment

[0079] Using the artificially synthesized sequence containing SEQ ID No. 4 (synthesized by Nanjing GenScript Biotech Co., Ltd.) as a template, PCR amplification was performed using primers P3 and P4 to obtain a PCR product, namely, the coding gene sequence of performate dehydrogenase. The primer sequences are as follows:

[0080] P3: 5'-cgcggggtgctcgcgggctgaggagatatacc actagt ATGGCTAAAGTTCTGTGCGTTC-3′ (the underlined part is the SpeI recognition sequence);

[0081] P4: 5'-ctgccgcgcggcaccag ctgcag TTAAACAGCTTTTTTGAATTTAG-3' (the underlined part is the PstI recognition sequence).

[0082] The PCR product was detected by 1% agarose gel electrophoresis, and the size of the PCR product was about 1200 bp, which was consistent with the target fragment and was named SFDH gene fragment. The SFDH gene fragment was recovered by gel electrophoresis.

[0083] 4. Construction of pXRFDH recombinant vector

[0084] After the pBAD / HisB-KMXR vector obtained in step 2 was double-digested with SpeI and PstI, the vector fragment (about 4500bp) was recovered; the SFDH gene fragment recovered in step 3 was connected with the recovered vector fragment by the Gibson method to obtain a connection product. The connection product was transformed into Trans1-T1 competent cells and coated on a solid LB plate containing streptomycin. Overnight at 37°C, a single clone was picked to extract the plasmid, and sequencing verification was performed. The vector with the correct sequencing was named pXRFDH. pXRFDH structure description: The small fragment between the restriction sites XhoI and SpeI of the pBAD / HisB vector was replaced with the DNA fragment shown in SEQ ID No.2, and the small fragment between SpeI and PstI was replaced with the DNA fragment shown in SEQ ID No.4 to obtain a recombinant plasmid.

[0085] 5. Construction of recombinant engineered bacteria X01

[0086] The expression vector pXRFDH constructed in step 4 was transformed into Escherichia coli BW25113 by chemical transformation, and positive clones were screened on LB plates containing streptomycin (50 μg / mL) to obtain the corresponding recombinant engineered strain, which was named X01.

[0087] Example 2: Recombinant engineered bacteria X01 catalyzes the synthesis of xylitol from D-xylose

[0088] Figure 1The schematic diagram is a pathway for coupling D-xylose reductase (KMXR) and formate dehydrogenase (SFDH) and catalyzing D-xylose to synthesize D-xylitol based on the NAD(H) cofactor cycle system. In this example, the recombinant engineered bacteria X01 capable of expressing KMXR and SFDH prepared in Example 1 is used as a whole-cell catalyst to catalyze D-xylose to synthesize D-xylitol.

[0089] 1. Take a single clone of the test strain and inoculate it into liquid LB medium containing 50 μg / mL streptomycin, and culture at 37°C and 220 rpm until OD 600nm =0.6-0.8.

[0090] 2. After completing step 1, add L-arabinose to the culture system at a concentration of 2 g / L, and culture at 30°C and 200 rpm for 12 hours.

[0091] 3. After completing step 2, take the entire culture system, centrifuge it at 4°C and 6000 rpm for 15 min, and collect the bacterial precipitate.

[0092] 4. Recombinant engineered bacteria catalyze the synthesis of D-xylitol from substrate D-xylose

[0093] The reaction system comprises the bacterial precipitate obtained in step 3, D-xylose and formate buffer.

[0094] In the reaction system, the initial concentrations of the components were as follows: bacterial concentration 30 g (wet weight) / L, D-xylose 100 g / L, sodium formate solution 1.0 mol / L, cofactor NAD + The addition amount was 0.5 mmol / L (each concentration was the final concentration of the corresponding component in the system).

[0095] The pH of the reaction system was adjusted to 6.0, and the temperature of the reaction system was controlled to 37° C. The reaction was terminated after 24 hours of reaction, and the supernatant was collected by centrifugation for testing.

[0096] 5. Perform HPLC detection on the supernatant obtained in step 4 to determine the D-xylitol concentration in the system. Further calculate the conversion rate, conversion rate (%) = D-xylitol concentration (g / L) × final volume (L) / D-xylose concentration (g / L) × initial volume (L).

[0097] HPLC system: Agilent 1260; Chromatographic column: Aminex HPX-87C (300 mm × 7.8 mm);

[0098] Mobile phase: double distilled water.

[0099] Flow rate: 0.5 mL / min;

[0100] Temperature: 80℃;

[0101] Detector:RID.

[0102] Figure 2 The liquid phase spectrum of D-xylose and D-xylitol standards.

[0103] The results are shown in Table 1. When the initial raw material concentration was 150 g / L, the conversion rate was higher than 90%, the maximum concentration of D-xylitol was 142 g / L, and the conversion rate decreased with the increase of the substrate D-xylose concentration.

[0104] Table 1. Synthesis of D-xylitol from D-xylose catalyzed by engineered bacteria X01

[0105]

[0106] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for constructing an engineered strain for synthesizing D-xylitol, comprising the following steps: The recipient bacteria is made to express NAD(H)-dependent D-xylose reductase and NAD(H)-dependent formate dehydrogenase to obtain an engineered strain for synthesizing D-xylitol, wherein: The amino acid sequence of the NAD(H)-dependent D-xylose reductase is shown in SEQ ID No. 1, and the amino acid sequence of the NAD(H)-dependent formate dehydrogenase is shown in SEQ ID No. 3; The nucleotide sequence of the gene encoding the NAD(H)-dependent D-xylose reductase is shown in SEQ ID No. 2, and the nucleotide sequence of the gene encoding the NAD(H)-dependent formate dehydrogenase is shown in SEQ ID No. 4; The recipient bacteria is Escherichia coli BW25113; Using the vector pXRFDH, the coding gene of the NAD(H)-dependent D-xylose reductase and the coding gene of the NAD(H)-dependent formate dehydrogenase are introduced into the Escherichia coli BW25113; The structure of the vector pXRFDH is as follows: the small fragment between the restriction sites XhoI and SpeI of the pBAD / HisB vector is replaced with the DNA fragment shown in SEQ ID No.2, and the small fragment between SpeI and PstI is replaced with the DNA fragment shown in SEQ ID No.4 to obtain a recombinant plasmid.

2. An engineered strain constructed by the method of claim 1, wherein the engineered strain catalyzes D-xylose to synthesize D-xylitol in a reaction system with a pH of 5.0-7.

0.

3. Use of the engineered strain according to claim 2 in whole-cell catalysis of D-xylose to synthesize D-xylitol.

4. A complete set of products for synthesizing D-xylitol, comprising the engineered strain according to claim 2, D-xylose, formate, L-arabinose and NAD + .

5. Use of the complete set of products according to claim 4 in whole-cell catalysis of D-xylose to synthesize D-xylitol.

6. A method for synthesizing D-xylitol by whole-cell catalysis of D-xylose, comprising the following steps: The engineered strain of claim 2 is induced and cultured with L-arabinose to obtain an induced strain, and the whole cells of the induced strain are used to catalyze the D-xylose reaction to obtain D-xylitol; in, The engineered strain is cultured by L-arabinose induction in a medium containing L-arabinose at a final concentration of 2 g / L, the temperature of the induction culture is 30° C., and the time of the induction culture is 12-16 hours; In the reaction system in which the whole cell of the induced strain catalyzes D-xylose to synthesize D-xylitol, the reaction pH is 5.0-7.0, the initial concentration of formate is 1.0 mol / L, and NAD + The initial concentration was 0.5mmol / L.

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