Hydroxyacetaldehyde synthetase mutant and application thereof in synthesis of D-xylulose

Abstract

The invention discloses a hydroxyacetaldehyde synthetase mutant and application of the hydroxyacetaldehyde synthetase mutant in synthesis of D-xylose. According to the invention, three GALS-M6 enzyme mutants are obtained through an enzyme directed evolution strategy, and cheap formaldehyde can be converted into D-xylulose only through two-step enzyme catalysis. The method is mild in reaction conditions, and the conversion rate reaches 50%. And the D-xylulose can also be utilized by microorganisms, so that a route from the carbon-I compound to various rare sugars, materials, biological energy sources and the like is opened, and a foundation is laid for resource utilization of the carbon-I compound.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and bioengineering, and specifically relates to a hydroxyacetaldehyde synthase mutant and an application thereof in synthesizing D-xylulose. Background Art

[0002] D-Xylulose is an important intermediate product of D-xylose metabolism that is widely present in nature. It is converted into D-xylulose-5-phosphate through the action of corresponding kinases, and then converted into various chemicals through the pentose phosphate metabolic pathway; D-Xylulose can be easily converted into furfural through dehydration reaction. Furfural is a renewable bioenergy and starting material for biochemical products, with great market development prospects; D-Xylulose can be used as a raw material to produce 5-carbon rare sugars such as L-arabinose, L-ribose, L-ribulose, L-xylulose, L-xylose and L-lyxose. These rare sugars have broad application prospects in the field of medical health; D-Xylulose can also be used as a raw material for xylitol, which is one of the important sweeteners in the food industry.

[0003] Conventional preparation methods for D-xylulose include extraction, chemical synthesis and biological methods. Extraction method: Since the abundance of D-xylulose in nature is not high, and it has high polarity, high water solubility and is mixed with many similar sugars, the separation, extraction and purification methods are costly, and this method only exists in the early research of D-xylulose. Chemical method: Xylulose can be synthesized by selective oxidation of cheap xylitol. However, due to the presence of multiple hydroxyl groups on xylitol, the selective protection, oxidation and deprotection of the substrate hydroxyl groups in this method make the entire synthesis process cumbersome and complicated, and the production cost remains high. Biological method: At present, the raw materials for the biological synthesis of D-xylulose are mainly D-xylose and D-glucose. At present, the industrial production of D-xylulose is mainly through the isomerization of D-xylose, but this method has the defects of low yield and high cost. The production of D-xylulose using glucose as raw material often converts glucose into D-arabitol first, and then into D-xylulose. For example, D-arabitol is obtained by fermenting glucose with Debaryomyces hansenii ATCC20121, and then converted into D-xylulose using Acetobacter oxydans (Appl Microbiol. 1969; 18 (6): 1031-5). D-arabitol is produced by fermenting with hyperosmotic yeast, and then converted into D-xylulose using bacteria of the genus Acetobacter, Gluconobacter or Klebsiella (EP403392A; EP421882A; CN1284564A). D-xylulose can be directly fermented with glucose using Gluconobacter, Acetobacter or Flatella (CN1263944A; CN1269421A). However, the final purification cost is high because the product is mixed with other similar monosaccharide impurities such as xylitol and xylose.

[0004] The cost of D-xylulose synthesis is determined by the price of raw materials. The high price on the market (￥16,000 / g, Carbosynth) has greatly restricted the further development of D-xylulose in the research field and industrial application field. Therefore, it is important to develop a method for preparing D-xylulose using cheap raw materials, simple and high conversion rate to reduce its market price. Summary of the invention

[0005] The purpose of the present invention is to provide a hydroxyacetaldehyde synthase mutant in view of the current status and shortcomings of the prior art. The present invention implements a directed evolution strategy on the GALS-M6 enzyme to obtain three mutants that can convert formaldehyde into hydroxyacetaldehyde and 1,3-dihydroxyacetone, and then catalyzes the synthesis of D-xylulose through the TalB mutant. Only two steps of enzyme catalysis are needed to convert cheap formaldehyde into D-xylulose; D-xylulose can also be used by microorganisms, opening up the route from carbon compounds to various rare sugars, materials, bioenergy, etc., laying the foundation for the resource utilization of carbon compounds.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a hydroxyacetaldehyde synthase mutant, wherein the hydroxyacetaldehyde synthase mutant is selected from any one of the following:

[0008] (a) the amino acid sequence of which is mutated from the sequence shown in SEQ ID NO: 1, with mutations at the following sites: C398M, F397Y and F464G, and mutation at the W463 site to E, H or K; the hydroxyacetaldehyde synthase mutant is capable of catalyzing the condensation of formaldehyde into hydroxyacetaldehyde and 1,3-dihydroxyacetone;

[0009] or

[0010] (b) the hydroxyacetaldehyde synthase variant has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence of (a), and has the function of the hydroxyacetaldehyde synthase mutant of (a);

[0011] or

[0012] (c) the hydroxyacetaldehyde synthase variant consists of 1-30, more preferably 1-10, even more preferably 1-6, most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence of (a), and has the function of the hydroxyacetaldehyde synthase mutant of (a);

[0013] It is to be understood that in the context of the present invention, glycolaldehyde synthase (GALS) has the amino acid sequence defined above.

[0014] In a second aspect, the present invention provides a polynucleotide encoding the hydroxyacetaldehyde synthase mutant described in the first aspect.

[0015] In a third aspect, the present invention provides an expression vector, wherein the expression vector comprises the polynucleotide described in the second aspect of the present invention.

[0016] In a fourth aspect, the present invention provides a host cell, wherein the host cell comprises the expression vector described in the third aspect of the present invention or a polynucleotide having the hydroxyacetaldehyde synthase mutant described in the second aspect of the present invention integrated into its genome.

[0017] According to an embodiment of the present invention, the host cell is a bacterium; more preferably, the host cell is Escherichia coli, Corynebacterium glutamicum, or Bacillus subtilis; most preferably, the host cell is Escherichia coli.

[0018] In the context of the present invention, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0019] In the context of the present invention, mutants are described according to their mutations at specific residues, the positions of which are determined by alignment with or reference to the wild-type enzyme sequence. In the context of the present invention, any variants carrying these same mutations at functionally equivalent residues are also contemplated.

[0020] In this article, the mutation site and its substitution are expressed by the position number of the mutation site and the type of amino acid at the site, for example, C398M indicates that the cysteine ​​at the 398th position of SEQ ID NO: 1 is mutated to methionine; F464G indicates that the phenylalanine at the 464th position of SEQ ID NO: 1 is mutated to glycine. In the present invention, "," is used to indicate the combination of mutation sites, for example, "C398M, F397Y and F464G, and the mutation at the W463 site is E" indicates that it contains four mutation sites, which is a quadruple mutant.

[0021] In a fourth aspect, the present invention provides a method for biosynthesizing D-xylulose, comprising the following steps:

[0022] 1) Formaldehyde is condensed into hydroxyacetaldehyde and 1,3-dihydroxyacetone under the catalysis of the hydroxyacetaldehyde synthase mutant or an enzyme having such catalytic function described in the first aspect of the present invention;

[0023] 2) Hydroxyacetaldehyde and 1,3-dihydroxyacetone are used to synthesize D-xylulose under the action of TalB mutant or enzyme with this catalytic function. Figure 1 (shown)

[0024] According to an embodiment of the present invention, the hydroxyacetaldehyde synthase mutant or the enzyme having the function of catalyzing the condensation of formaldehyde into hydroxyacetaldehyde and 1,3-dihydroxyacetone can be in the form of a purified enzyme or its mutant, enzyme lysis supernatant or whole cells.

[0025] According to an embodiment of the present invention, the TalB mutant used is derived from the sequence shown in SEQ ID NO: 8, and is mutated at the following sites: F178Y and R181E, ie, TalB-F178Y / R181E.

[0026] According to an embodiment of the present invention, step 1) of the method is carried out in a protein buffer, which comprises: potassium phosphate buffer, MgCl2, TPP, pH 7-8.

[0027] According to an embodiment of the present invention, the concentration of potassium phosphate buffer in the protein buffer in step 1) of the method is 10-100 mM;

[0028] According to an embodiment of the present invention, the concentration of MgCl2 in the protein buffer in step 1) of the method is 1-10 mM;

[0029] According to an embodiment of the present invention, the concentration of TPP in the protein buffer in step 1) of the method is 0.1-1 mM, exemplarily 0.5 mM;

[0030] According to an embodiment of the present invention, the concentration of the substrate formaldehyde in the protein buffer is 1-10 g / L, exemplarily 6 g / L; the concentration of the hydroxyacetaldehyde synthase mutant in the reaction system is 1-5 mg / ml, exemplarily 2 mg / ml.

[0031] According to an embodiment of the present invention, the reaction in step 1) may be carried out at 30-40°C, exemplarily at 37°C.

[0032] According to an embodiment of the present invention, the reaction in step 1) can be carried out for 0.5-3 hours, exemplarily for 1 hour.

[0033] According to an embodiment of the present invention, the reaction in step 1) can be carried out under centrifugal conditions, and the rotation speed can be 500-1000 rpm, and exemplarily 750 rpm.

[0034] According to an embodiment of the present invention, step 2) of the method is to add TalB-F178Y / R181E after the reaction in step 1) is completed, and its concentration in the reaction system is 2-10 mg / ml.

[0035] According to an embodiment of the present invention, the ratio of the amount of the hydroxyacetaldehyde synthase mutant to the amount of the TalB mutant is 1:1-1:5, and exemplarily 1:5.

[0036] According to an embodiment of the present invention, the reaction time of step 2) of the method is 1-24 hours.

[0037] In a fifth aspect, the present invention provides the use of the hydroxyacetaldehyde synthase mutant described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, or the combination of the host cell and the TalB mutant described in the fourth aspect in the biosynthesis of D-xylulose.

[0038] According to an embodiment of the present invention, the combination is used to synthesize D-xylulose using formaldehyde as a substrate.

[0039] Beneficial effects of the present invention:

[0040] The invention obtains three GALS-M6 enzyme mutants through the directed evolution strategy of enzymes, which can convert cheap formaldehyde into D-xylulose with only two steps of enzyme catalysis. The reaction conditions of the method are mild, and the conversion rate reaches 50%. In addition, D-xylulose can also be used by microorganisms, opening up the route from carbon compounds to various rare sugars, materials, bioenergy, etc., laying the foundation for the resource utilization of carbon compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 .Technical roadmap of the present invention;

[0042] Figure 2 . HPLC chart of GALS-M6 enzyme mutant catalyzing the condensation of formaldehyde into glycolaldehyde and 1,3-dihydroxyacetone;

[0043] Figure 3 .The yield and conversion rate of formaldehyde condensation to hydroxyacetaldehyde and 1,3-dihydroxyacetone catalyzed by GALS-M6 enzyme and its mutants; DHA represents 1,3-dihydroxyacetone and GALD represents hydroxyacetaldehyde;

[0044] Figure 4 .HPLC chart of TalB-F178Y / R181E catalyzing the production of D-xylulose from hydroxyacetaldehyde and 1,3-dihydroxyacetone;

[0045] Figure 5 .pET-28a-GALS-M6 plasmid map. DETAILED DESCRIPTION

[0046] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0047] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0048] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0049] Example 1: Construction of a vector expressing a GALS-M6 mutant

[0050] The coding gene of GALS-M6 enzyme comes from the literature Tianzhen Li, et al. Green Chem., 2020, 22, 6809. Based on GALS-M6 enzyme, three mutants were obtained through the directed evolution strategy of enzyme and named GALS-M6_C398M_F397Y_F464G_W463E, GALS-M6_C398M_F397Y_F464G_W463H, and GALS-M6_C398M_F397Y_F464G_W463K. The nucleotide sequence of the GALS-M6_C398M_F397Y_F464G_W463E gene is shown in SEQ ID No.2, and its amino acid sequence is shown in SEQ ID No.5; the nucleotide sequence of the GALS-M6_C398M_F397Y_F464G_W463H gene is shown in SEQ ID No.3, and its amino acid sequence is shown in SEQ ID No.6; the nucleotide sequence of the GALS-M6_C398M_F397Y_F464G_W463K gene is shown in SEQ ID No.4, and its amino acid sequence is shown in SEQ ID No.7. The vectors expressing GALS-M6, GALS-M6_C398M_F397Y_F464G_W463E, GALS-M6_C398M_F397Y_F464G_W463H, and GALS-M6_C398M_F397Y_F464G_W463K were prepared by inserting the above coding genes into the NdeI and XhoI restriction sites of the pET-28a vector. The obtained vectors were named pET-28a-GALS-M6, pET-28a-GALS-M6_C398M_F397Y_F464G_W463E, pET-28a-GALS-M6_C398M_F397Y_F464G_W463H, and pET-28a-GALS-M6_C398M_F397Y_F464G_W463K, respectively. Figure 5 shown.

[0051] Example 2. Expression of GALS-M6 mutant protein

[0052] In order to detect the function of the GALS-M6 mutant in vitro, the enzyme was exogenously expressed and purified in Escherichia coli.

[0053] (1) The prepared E. coli expression recombinant plasmids pET-28a-GALS_M6, pET-28a-GALS-M6_C398M_F397Y_F464G_W463E, pET-28a-GALS-M6_C398M_F397Y_F464G_W463H, and pET-28a-GALS-M6_C398M_F397Y_F464G_W463K were respectively transferred into E. coli BL21 (DE3) to obtain recombinant bacteria. Positive clones were screened using kanamycin resistance plates (Kan+, 100 mg / mL) and cultured overnight at 37°C;

[0054] (2) Pick a single clone and transfer it to 5 mL LB liquid medium (Kan+, 100 mg / mL) and culture at 37°C and 220 rpm until OD 600 Transfer 5 mL of the bacterial solution in LB medium to 800 mL of 2YT medium (Kan+, 100 mg / mL) and culture at 37°C and 220 rpm until the OD 600 When the pH value was 0.6-0.8, the temperature was lowered to 16°C, IPTG was added to a final concentration of 0.5 mM, and the expression was induced for 16 h;

[0055] (3) Collect the above culture solution into a collection bottle and centrifuge at 5500 rpm for 15 min;

[0056] (4) Discard the supernatant, suspend the obtained bacterial pellet with 35 mL of protein buffer (50 mM phosphate buffer, 5 mM MgSO4, pH 7.4), pour it into a 50 mL centrifuge tube, and store it in a -80°C refrigerator to obtain bacteria expressing GALS_M6, GALS-M6_C398M_F397Y_F464G_W463E, GALS-M6_C398M_F397Y_F464G_W463H, and GALS-M6_C398M_F397Y_F464G_W463K.

[0057] Example 3. Purification of GALS-M6 mutant protein

[0058] (1) Bacterial disruption: The expressed bacteria were disrupted twice using a high-pressure low-temperature disruptor at a pressure of 1200 bar and 4°C. Centrifuge at 4°C and 10,000 rpm for 45 min;

[0059] (2) Purification: The supernatant was filtered through a 0.45 μm microporous filter membrane and purified by nickel affinity chromatography. The specific steps are as follows:

[0060] a: Column balancing: Before loading the supernatant, wash the Ni affinity column with ddH2O for 2 column volumes, and then balance the Ni affinity column with protein buffer for 1 column volume;

[0061] b: Sample loading: slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 0.5 mL / min, repeat once;

[0062] c: Elution of impurities: Wash one column volume with protein buffer, and then use 50 mL of protein buffer containing 50 mM and 100 mM imidazole to elute the strongly bound impurities;

[0063] d: Elution of target protein: Elute the target protein with 20 mL of protein buffer (50 mM potassium phosphate, pH=7.4, 5 mM MgSO4) containing 200 mM imidazole, take the first few drops of flow sample, prepare the sample, and detect it with 12% SDS-PAGE.

[0064] (3) Liquid replacement: The collected target protein was concentrated by centrifugation (4°C, 3400 r / min) using a 50 mL Amicon ultrafiltration tube (30 kDa, Millipore) to 1 mL. 15 mL of protein buffer without imidazole was added and concentrated to 1 mL. This was repeated once to obtain the GALS mutant proteins.

[0065] (4) The concentration of the concentrated protein was detected using a Nondrop 2000 micro-spectrophotometer to obtain the GALS mutant proteins.

[0066] Example 4. Functional detection of GALS-M6 mutant protein

[0067] GALS-M6 mutant catalyzes the condensation of formaldehyde into glycolaldehyde and 1,3-dihydroxyacetone

[0068] The prepared GALS-M6 mutant protein was washed with the culture medium in protein buffer (50mM potassium phosphate buffer, 5mM MgCl2, 0.5mM TPP, pH7.5), the bacteria were collected, and then resuspended in 20mL of protein buffer with 0.5mM TPP. 500μL of the resuspended bacterial solution was added with 500μL of formaldehyde solution prepared by protein buffer with a final concentration of 6g / L containing 0.5mM TPP, and reacted at 37℃, 750rpm for 1h, and the supernatant was centrifuged for liquid phase detection.

[0069] Liquid phase detection uses AMINEX HPX-87H, 300X7.8MM column, 5mM sulfuric acid as mobile phase, 20μL injection each time, column oven at 35℃, 200nm UV conditions to detect hydroxyacetaldehyde and 1,3-dihydroxyacetone, and the content is determined by external standard method. Figure 2 shown.

[0070] The results are as follows Figure 3As shown, it can be seen that compared with the unmutated GALS-M6, the GALS-M6_C398M_F397Y_F464G_W463E, GALS-M6_C398M_F397Y_F464G_W463H, and GALS-M6_C398M_F397Y_F464G_W463K products in the four-mutated or five-mutated GALS mutants catalyzed the condensation of formaldehyde into hydroxyacetaldehyde and 1,3-dihydroxyacetone, and the final ratio was close to 1: 1. And starting from the one-carbon molecular formaldehyde, the conversion rate of the two-carbon molecular hydroxyacetaldehyde and the three-carbon molecular 1,3-dihydroxyacetone produced by enzyme catalysis, the GALS mutant proteins are all greater than 0.50, while the unmutated GALS-M6 is only 0.37. Figure 3 The conversion rate is calculated by dividing the amount of substrate decomposed from all products produced by the reaction (for example, 10 hydroxyacetaldehyde and 10 1,3-dihydroxyacetone are produced, and these 10 hydroxyacetaldehyde and 10 1,3-dihydroxyacetone are completely decomposed into 50 formaldehyde) by the amount of substrate actually added.

[0071] Example 5. Functional detection of GALS-M6 mutant protein

[0072] The GALS-M6 mutant protein prepared in Example 3 was added to 200 μl of a protein buffer reaction system (50 mM potassium phosphate buffer, 5 mM MgCl2, 0.5 mM TPP, pH 7.5, and a final concentration of 1.5 mg / ml of enzyme protein in the reaction system). A standard formaldehyde solution was then added to make the final concentration of formaldehyde in the reaction system 6 g / L. The reaction was carried out at 37°C and 750 rpm for 1 h. TalB-F178Y / R181E (Sarah Schneider, et al. Chembiochem. 2010 Mar 22; 11(5): 681-90) was added and the reaction was continued for 24 h. The supernatant was collected by centrifugation and liquid phase detection was performed. Derivatization method: The sample was derivatized with a benzyloxyamine hydrochloride derivatization reagent at 50°C for 1 h, and then the sample was diluted and liquid phase detection was performed. Liquid phase method: Mobile phase A: acetonitrile: water 4-1 (0.095% trifluoroacetic acid); Mobile phase B: 0.1% trifluoroacetic acid; phase A 10-70% gradient elution for 30 minutes, detection at 215nm. HPLC test results are as follows Figure 4 As shown, starting from formaldehyde, the conversion rate to D-xylulose is 50%.

[0073] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydroxyacetaldehyde synthase mutant, It is characterized in that The hydroxyacetaldehyde synthase mutant is selected from any one of the following: (a) Its amino acid sequence is mutated from the sequence shown in SEQ ID NO: 1, with mutations at the following sites: C398M, F397Y and F464G, and mutation at the W463 site to E, H or K; the hydroxyacetaldehyde synthase mutant is capable of catalyzing the condensation of formaldehyde into hydroxyacetaldehyde and 1,3-dihydroxyacetone; or (b) the hydroxyacetaldehyde synthase variant has 95%, preferably 98%, more preferably 99% sequence identity with the amino acid sequence of (a), and has the function of the hydroxyacetaldehyde synthase mutant of (a); or (c) The hydroxyacetaldehyde synthase variant consists of 1-30, more preferably 1-10, even more preferably 1-6, and most preferably 1-3 amino acid residues added or deleted at the C-terminus and / or N-terminus of the amino acid sequence of (a), and has the function of the hydroxyacetaldehyde synthase mutant of (a).

2. A polynucleotide encoding the hydroxyacetaldehyde synthase mutant according to claim 1.

3. An expression vector, It is characterized in that The expression vector comprises the polynucleotide according to claim 2.

4. A host cell, It is characterized in that The host cell comprises the expression vector of claim 3 or a polynucleotide having the hydroxyacetaldehyde synthase mutant of claim 2 integrated into its genome; Preferably, the host cell is a bacterium; more preferably, the host cell is Escherichia coli, Corynebacterium glutamicum, or Bacillus subtilis; most preferably, the host cell is Escherichia coli.

5. A method for biosynthesizing D-xylulose, It is characterized in that The following steps are involved: 1) Formaldehyde is condensed into hydroxyacetaldehyde and 1,3-dihydroxyacetone under the catalysis of the hydroxyacetaldehyde synthase mutant according to claim 1 or an enzyme having this catalytic function; 2) synthesizing D-xylulose from hydroxyacetaldehyde and 1,3-dihydroxyacetone under the action of a TalB mutant or an enzyme having this catalytic function.

6. The method according to claim 5, It is characterized in that The hydroxyacetaldehyde synthase mutant or the enzyme having the function of catalyzing the condensation of formaldehyde into hydroxyacetaldehyde and 1,3-dihydroxyacetone may be in the form of a purified enzyme or a mutant thereof, an enzyme lysis supernatant or whole cells.

7. The method according to claim 5, It is characterized in that The TalB mutant used was derived from the sequence shown in SEQ ID NO: 8, with mutations at the following sites: F178Y and R181E; According to an embodiment of the present invention, the ratio of the amount of the hydroxyacetaldehyde synthase mutant to the amount of the TalB mutant is 1:1-1:5, and exemplarily 1:

5.

8. The method according to claim 5, It is characterized in that Step 1) of the method is performed in a protein buffer, which comprises potassium phosphate buffer, MgCl 2 , TPP, pH 7-8; According to an embodiment of the present invention, the concentration of the potassium phosphate buffer in the protein buffer in step 1) of the method is 10-100 mM, exemplarily 50 mM; According to an embodiment of the present invention, the MgCl 2 The concentration is 1-10 mM, exemplarily 5 mM; According to an embodiment of the present invention, the concentration of TPP in the protein buffer in step 1) of the method is 0.1-1 mM, exemplarily 0.5 mM; According to an embodiment of the present invention, the concentration of the substrate formaldehyde in the protein buffer is 1-10 g / L, exemplarily 6 g / L; the concentration of the hydroxyacetaldehyde synthase mutant in the reaction system is 1-5 mg / ml, exemplarily 2 mg / ml; According to an embodiment of the present invention, the reaction in step 1) may be carried out at 30-40°C, exemplarily 37°C; According to an embodiment of the present invention, the reaction in step 1) can be carried out for 0.5-3 hours, exemplarily for 1 hour; According to an embodiment of the present invention, the reaction in step 1) may be carried out under centrifugal conditions, and the rotation speed may be 500-1000 rpm, exemplarily 750 rpm.

9. The method according to claim 5, It is characterized in that Step 2) of the method is to add TalB-F178Y / R181E after the reaction in step 1) is completed, and its concentration in the reaction system is 2-10 mg / ml; According to an embodiment of the present invention, the reaction time of step 2) of the method is 1-24 hours.

10. Use of the hydroxyacetaldehyde synthase mutant according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3 or the host cell according to claim 4 and the TalB mutant in the biosynthesis of D-xylulose; According to an embodiment of the present invention, the combination is used to synthesize D-xylulose using formaldehyde as a substrate.

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