Enzyme for synthesizing hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone by catalyzing formaldehyde, and applications thereof

Abstract

An enzyme synthesizes hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone by catalyzing formaldehyde. Site-directed mutation of benzoylformate decarboxylase (BFD) creates a mutant of the enzyme, which can polymerize the formaldehyde, A phosphoketalose (F/XPK) generates acetyl phosphoric acid from the hydroxyl acetaldehyde or 1,3-dihydroxyacetone (DHA). Combination with phosphotransacetylase (Pta) provides a route from the formaldehyde to acetyl coenzyme A in three steps.

Claims

1. A method for preparing hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone, comprising: catalyzing formaldehyde to be condensed to hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone with a benzoylformate decarboxylase (BFD) mutant protein using formaldehyde as a substrate, wherein the BFD mutant protein comprises the amino acid sequence of SEQ ID NO: 1 with the exception of the mutations W86R and N87T, and optionally one or more mutations selected from the group consisting of L109G, L110E, H281V, Q282F, T377M, T380C, T380Y, and A460M, wherein the amino acid number corresponds to the amino acid sequence of SEQ ID NO: 1.

2. A method for producing acetyl phosphoric acid using formaldehyde, comprising: (i) catalyzing formaldehyde to be condensed to hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone with a BFD mutant protein, using formaldehyde as a substrate; (ii) using the hydroxyl acetaldehyde and/or 1, 3-dihydroxyacetone obtained in (i) as a substrate to prepare acetyl phosphoric acid, wherein the BFD mutant protein comprises the amino acid sequence of SEQ ID NO: 1 with the exception of the mutations W86R and N87T, and optionally one or more mutations selected from the group consisting of L109G, L110E, H281V, Q282F, T377M, T380C, T380Y, and A460M, wherein the amino acid number corresponds to the amino acid sequence of SEQ ID NO:1.

3. A method for producing acetyl coenzyme A using formaldehyde, the method comprising: (i) catalyzing formaldehyde to be condensed to hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone with a BFD mutant protein, using formaldehyde as a substrate; (ii) using hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone obtained in (i) as a substrate to prepare acetyl phosphoric acid; and (iii) using the acetyl phosphoric acid as a substrate to prepare acetyl coenzyme A, wherein the BFD mutant protein comprises the amino acid sequence of SEQ ID NO: 1 with the exception of the mutations W86R and N87T, and optionally one or more mutations selected from the group consisting of L109G, L110E, H281V, Q282F, T377M, T380C, T380Y, and A460M, wherein the amino acid number corresponds to the amino acid sequence of SEQ ID NO:1.

4. The method according to claim 2, wherein the step of preparing acetyl phosphoric acid comprises using hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone as a substrate and catalyzed with a phosphoketolase (F/XPK) protein to generate acetyl phosphoric acid.

5. The method according to claim 3, wherein the step of preparing acetyl phosphoric acid comprises using hydroxyl acetaldehyde and/or 1,3-dihydroxyacetone as a substrate and catalyzed with a F/XPK protein to generate acetyl phosphoric acid.

6. The method according to claim 3, wherein the step of preparing acetyl coenzyme A comprises using acetyl phosphoric acid as a substrate and catalyzed with phosphotransacetylase to generate acetyl coenzyme A.

7. The method according to claim 1, wherein the BFD mutant protein performs its catalyzing function in the form of a crude enzyme liquid, lyophilized powders of a crude enzyme liquid, a pure enzyme or whole cells.

8. The method according to claim 4, wherein the F/XPK protein performs its catalyzing function in the form of a crude enzyme liquid, lyophilized powders of a crude enzyme liquid, a pure enzyme or whole cells.

9. The method according to claim 6, wherein the phosphotransacetylase performs its catalyzing function in the form of a crude enzyme liquid, lyophilized powders of a crude enzyme liquid, a pure enzyme or whole cells.

10. The method according to claim 7, wherein the crude enzyme liquid, the lyophilized powders of a crude enzyme liquid and the pure enzyme are all prepared and obtained in accordance wherein the method comprising: obtaining a recombinant cell by expressing the BFD mutant protein in a host cell; the crude enzyme liquid, the lyophilized powders of a crude enzyme liquid or the pure enzyme are obtained by lysing the recombinant cell; the whole cells are all prepared and obtained in accordance with a method comprising: the BFD mutant protein is expressed in a host cell, and the obtained recombinant cell is the whole cell.

11. The method according to claim 10, wherein the recombinant cell is prepared and obtained in accordance with a method comprising: the recombinant cell expressing the BFD mutant protein is obtained by introducing a nucleic acid molecule capable of expressing the BFD mutant protein into the host cell, followed by induction culture.

12. The method according to claim 11, wherein the said nucleic acid molecule capable of expressing the BFD mutant protein is introduced into the host cell in the form of a recombinant vector; wherein the recombinant vector is a bacterial plasmid, phage, a yeast plasmid or a retrovirus packaging plasmid carrying the coding sequence of the BFD mutant protein; and/or the host cell is a prokaryotic cell or a lower eukaryotic cell.

13. The method according to claim 12, wherein the prokaryotic cell is bacteria; the lower eukaryotic cell is a yeast cell.

14. The method according to claim 13, wherein the bacteria are Escherichia coli.

15. The method according to claim 1, wherein the mutations are selected from the group consisting of: W86R-N87T-L109G-L110E, W86R-N87T-L109G-L110E-T377M, W86R-N87T-L109G-L110E-A460M, W86R-N87T-L109G-L110E-H281V-Q282F-A460M, W86R-N87T-T377M-T380C, and W86R-N87T-T377M-T380Y.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a plasmid map of pET-28a-BFD and pET-28a-F/XPK.

(2) FIG. 2 is the GC-MS detection of hydroxyl acetaldehyde.

(3) FIG. 3 is the GC-MS detection of 1,3-dihydroxyacetone.

(4) FIG. 4 is the catalyzing of hydroxyl acetaldehyde or 1,3-dihydroxyacetone by the F/XPK protein to obtain acetyl phosphoric acid.

(5) FIG. 5 is the double-enzyme catalyzing of formaldehyde by the BFD mutant and the F/XPK to obtain acetyl phosphoric acid.

(6) FIG. 6 is the LC-MS detection of acetyl coenzyme A.

DETAILED DESCRIPTION

(7) The following examples facilitate a better understanding of the present invention, but without limitation of the present invention. The experimental methods in the following examples are all conventional methods, unless specifically indicated. The experimental materials in the following examples are all available from a shop selling conventional biochemical reagents, unless specifically indicated. The quantitative tests in the following examples are all repeated in triplicate, the results of which are averaged.

Example 1. Construction of an Expression Vector

(8) 1. Construction of a Vector Expressing a BFD Mutant

(9) The genus of the coding gene of a BFD enzyme is sourced from Pseudomonas putida. The amino acids of the BFD enzyme are shown as SEQ ID No.1, and the nucleotide sequence of the coding gene of the BFD enzyme is shown as SEQ ID No.2. A vector expressing the BFD enzyme is one obtained by inserting the coding gene (SEQ ID No.2) of the above BFD enzyme between the enzymatic cleavage sites of NdeI and XhoI in a pET-28a vector, and is named as pET-28a-BFD (as shown by A in FIG. 1)

(10) Each of vectors expressing a BFD mutant is one obtained by inserting a coding gene (see Table 1) of different BFD mutants between the enzymatic cleavage sites of NdeI and XhoI in a pET-28a vector.

(11) 2. Construction of a Vector Expressing F/XPK

(12) The genus of a F/XPK gene is sourced from Bifidobacterium adolescentis. The amino acids of the F/XPK protein are shown as SEQ ID No.3, and the nucleotide sequence of the coding gene of the F/XPK protein is SEQ ID No.4.

(13) A vector expressing F/XPK is one obtained by inserting the coding gene (SEQ ID No.4) of the above F/XPK protein between the enzymatic cleavage sites of NdeI and XhoI in a pET-28a vector, and is named as pET-28a-F/XPK (as shown by B in FIG. 1).

(14) 3. Construction of a Vector Expressing PTA

(15) The genus of a PTA gene is sourced from Escherichia coli. The amino acids of the PTA protein are shown as SEQ ID No.5, and the nucleotide sequence of the coding gene of the PTA protein is SEQ ID No.6.

(16) A vector expressing PTA is one obtained by inserting the coding gene (SEQ ID No.6) of the above PTA protein between the enzymatic cleavage sites of NdeI and XhoI in a pET-28a vector, and is named as pET-28a-PTA(as shown by C in FIG. 1).

Example 2. Expression of a Protein

(17) In order to in vitro detect the activity of a BFD wild-type and a mutant, F/XPK, and a PTA enzymes, an exogenous expression and a purification of the enzymes are performed in E. coli.

(18) (1) The E. coli expressing recombinant plasmid pET-28a-BFD, each of vectors expressing the BFD mutant, the pET-28a-F/XPK, and the pET-28a-PTA prepared in the above II are respectively transformed into E. coli BL21 (DE3) to obtain recombinant bacteria expressing the BFD enzyme, each of recombinant bacteria expressing the BFD mutant, recombinant bacteria expressing F/XPK and recombinant bacteria expressing PTA. Positive clones are screened by using a kanamycin-resistant plate (Kan+, 100 mg/mL), followed by overnight culture at 37° C.;

(19) (2) A monoclone is streaked into a 5 mL LB liquid medium (Kan+, 100 mg/mL), followed by culture at 37° C., 220 r/min until OD.sub.600 reaches 0.6-0.8. The bacteria liquid in the 5 mL LB medium is transferred to a 800 mL 2YT medium (Kan+, 100 mg/mL), followed by culture at 37° C., 220 rpm until OD.sub.600 reaches 0.6-0.8, then the temperature is cooled to 16° C. and IPTG is added to a final concentration of 0.5 mM, followed by induction expression for 16 h;

(20) (3) The above cultured bacteria liquid is collected in a bacteria collecting bottle, followed by centrifugation for 15 min at 5500 rpm;

(21) (4) Supernatants are abandoned. The obtained bacteria precipitates are suspended in a 35 mL protein buffer (50 mM phosphate buffer, pH7.4) and poured into a 50 mL centrifuge tube, and then kept in a refrigerator at −80° C. Bacteria expressing the BFD enzyme, each of bacteria expressing the BFD mutant, bacteria expressing F/XPK and bacteria expressing PTA are obtained.

Example 3. Purification of a Protein

(22) (1) Disruption of bacteria: bacteria expressing the BFD enzyme, each of bacteria expressing the BFD mutant, bacteria expressing F/XPK and bacteria expressing PTA are respectively disrupted twice using a high pressure and low temperature disruptor at the pressure of 1200 bar, at 4° C. Centrifugation for 45 min at 4° C., 10000 rpm is performed;

(23) (2) Purification: suction filtration of supernatants through a 0.45 μm micropore filter membrane is performed, followed by Ni affinity chromatography purification, and the steps are detailed as follows.

(24) a: Column balance: the Ni affinity chromatography column is first washed by ddH.sub.2O with 2 column volumes, and then is balanced by a protein buffer with 1 column volume, before supernatants are loaded;

(25) b: Loading: the supernatants are slowly passed through the Ni affinity chromatography column in a flow rate of 0.5 mL/min, which is repeated once;

(26) c: Elution of impure proteins: a protein buffer is used for washing with 1 column volume, and then an elution of strongly binded impure proteins with 50 mL of a protein buffer containing 50 mM, 100 mM imidazole is performed respectively;

(27) d: Elution of a target protein: an elution of a target protein with 20 mL of a protein buffer (50 mM potassium phosphate, PH=7.4, 5 mM MgSO.sub.4) containing 200 mM imidazole is performed. The first several drops of flow-through samples are taken for making a sample, followed by a detection with 12% SDS-PAGE.

(28) (3) Liquid change: the collected target protein is concentrated to 1 mL in a 50 mL Amicon ultrafiltration tube (30 kDa, Millipore company) by centrifugation (4° C., 3400 r/min). 15 mL of a protein buffer free of imidazole is added and then concentrated to 1 mL, which is repeated once. BFD, F/XPK, and PTA proteins are obtained, respectively.

(29) (4) the concentration of the protein after concentration is detected by an Nondrop 2000 micro-spectrophotometer and diluted to 10 mg/mL, that is, a BFD protein, each of BFD mutant proteins, a F/XPK protein and a PTA protein are obtained.

Example 4. Detection of the Function of a BFD Wild-Type and a Mutant Protein

(30) 1. A BFD Wild-Type Protein Catalyzes Formaldehyde to be Condensed to Hydroxyl Acetaldehyde

(31) Sample: 1 mg/mL BFD, 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM thiamine pyrophosphate (TPP), at pH 7.5, and 2 g/L formaldehyde.

(32) Control: 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH 7.5, and 2 g/L formaldehyde.

(33) Standard: 0.1 g/L hydroxyl acetaldehyde.

(34) The reaction system is placed at 37° C. for reaction for 1 h after being uniformly mixed. The reaction system is lyophilized after the reaction is finished. Then 60 μL of pentafluorobenzene hydroxylamine hydrochloride (PFBOA, 200 mM) is added and whirled, followed by incubation for 1 h at room temperature. 300 μL of hexane is added and placed at room temperature for 5 min. 100 μL of the sample in the organic layer is taken, into which 30 μL of trimethylsilicyl trifluoroacetamide containing 1% trimethylchlorosilane and 20 μL of proline are added to silylanize the PFBOA derivative. The sample is detected by GC-MS.

(35) GC-MS detection: the detection system is an Agilent Gas Chromatography 7890A; detection conditions are: Agilent chromatographic column 19091S-433, 30 m×250 μm×0.25 μm; starting temperature is set to be 50° C., with retention time of 1 min, the temperature is increased to 150° C. in a linear increasing rate of 15° C./min, and then to 300° C. in 30° C./min, with retention time of 1 min; the injection port temperature is 250° C., the GC-MS interface temperature is 280° C. Helium gas is for a carrying gas with a flow rate of 1.2 mL/min. Injection volume is 1 μL, and the solvent delay for 5 min is detected.

(36) As can be known from the GC-MS analysis, BFD can catalyze formaldehyde to generate hydroxyl acetaldehyde, and the results are shown in FIG. 2.

(37) 2. A BFD Wild-Type Protein Catalyzes Formaldehyde to be Condensed to 1,3-Dihydroxyacetone

(38) Sample: 1 mg/mL BFD, 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH 7.5, and 2 g/L formaldehyde.

(39) Control: 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH 7.5, and 2 g/L formaldehyde.

(40) Standard: 0.1 g/L 1,3-dihydroxyacetone.

(41) The reaction system is placed at 37° C. for reaction for 1 h after being uniformly mixed. The reaction system is lyophilized after the reaction is finished. Then trimethylsilicyl trifluoroacetamide (60 μL) containing 1% trimethylchlorosilane and pyridine (200 μL) are added and whirled at 60° C. for 10 min. The sample is detected by GC-MS.

(42) GC-MS detection:

(43) Injection volume: 1 μL; injecting without splitting stream;

(44) Injection port temperature: 250° C.

(45) Chromatographic column: J&W HP-5 (30 m×250 μm×0.25 μm)

(46) Column box temperature: keeping at 80° C. for 1 min, and increasing to 280° C. in 20° C./min; increasing to 310° C. in 10° C./min and keeping for 6 min

(47) GC/MS interface temperature: 280° C.

(48) EI ion source temperature: 230° C.

(49) Ionization energy: 70 eV

(50) Solvent delay: 2.5 min

(51) Scanning range: 50-500 amu

(52) Collecting rate: 5 spectra/s

(53) As can be known from the GC-MS analysis, BFD can catalyze formaldehyde to generate 1,3-dihydroxyacetone, and the results are shown in FIG. 3.

(54) 3. Whole Cells of a BFD Wild-Type and Mutant Catalyze Formaldehyde to be Condensed to Hydroxyl Acetaldehyde and 1,3-Dihydroxyacetone

(55) The recombinant bacteria expressing the BFD wild-type and each of those expressing BFD mutants prepared in Example 2 are respectively cultured in 200 mL 2YT at 37° C. until OD.sub.600=0.6, followed by induction with 0.5 mM IPTG at 16° C. for 18 h, and centrifugation for 15 min at 3500 rpm. A protein buffer (50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH 7.5) is used for washing off the medium, and the bacteria are collected. Then, 20 mL of a protein buffer added with 0.5 mM TPP is used for respective resuspension. 500 μL of the resuspended bacteria liquid is taken, into which 500 μL of a formaldehyde solution with a final concentration of 5 g/L containing 0.5 mM TPP formulated by a protein buffer is then added, followed by reacting for 2 h at 37° C., 750 rpm, and centrifugation such that supernatants are taken for detection by liquid chromatography.

(56) The detection by liquid chromatography uses an AMINEX HPX-87H, 300×7.8MM column. The mobile phase is 5 mM sulfuric acid. 20 μL is injected each time. The column box temperature is 35° C. Hydroxyl acetaldehyde and 1,3-dihydroxyacetone are detected at 200 nm under ultraviolet conditions, and an external standard method is used to determine the content.

(57) The results are shown in Table 1. As can be seen, compared with a BFD without mutation, all the yields of 1,3-dihydroxyacetone condensed by catalyzing formaldehyde with BFD mutant proteins are increased;

(58) Compared with a BFD without mutation, the yield of hydroxyl acetaldehyde from W86R-N87T is the highest among BFD mutants having double mutations; the yield of hydroxyl acetaldehyde from W86R-N87T-L109G-L110E is the highest among BFD mutants having four mutations; the yield of hydroxyl acetaldehyde from W86R-N87T-L109G-L110E-A460M is the highest among BFD mutants having five mutations; the yield of hydroxyl acetaldehyde from W86R-N87T-L109G-L110E-H281V-Q282F-A460M is the highest among BFD mutants having seven mutations; and they are higher than those from other mutation sites.

(59) TABLE-US-00001 TABLE 1 shows detection of the activity of a BFD mutant hydroxyl 1,3- acetaldehyde dihydroxyacetone BFD mutant substitution g/L g/L BFD protein SEQ ID No. 1 0.00972 0.00882 wild-type gene SEQ ID No. 2 M1 protein N374D-S376V 0.00972 0.01242 gene AAC 1120-1122 GAT/TCT 1126-1128 GTT M2 protein S430A 0.01116 0.00246 gene TCT 1288-1290 GCA M3 protein T379R 0.01116 0.0117 gene ACC 1135-1137 CGT M4 protein S236M 0.01224 0.00192 gene TCT 706-708 ATG M5 protein W86R-N87T-R184H 0.0126 0.00954 gene TGG 256-258 CGT/AAC 259-261 ACC/CGT 550-552 CAT M6 protein G25H 0.01368 0.0096 gene GGT 73-75 CAT M7 protein N374D-T377G 0.01584 0.0048 gene AAC 1120-1122 GAT/ACC 1129-1131 GGT M8 protein T457C 0.018 0.0024 gene ACC 1369-1371 TGT M9 protein S376V 0.01944 0.00822 gene TCT 1126-1128 GTT M10 protein S26T-G401N 0.02304 0.04404 gene TCT 76-78 ACC/GGT 1201-1203 AAT M11 protein N87T-T377G 0.02592 0.00816 gene AAC 259-261 ACC/ACC 1129-1131 GGT M12 protein S26H 0.02736 0.00942 gene TCT 76-78 CAT M13 protein S261-N87T 0.03024 0.00852 gene TCT 76-78 ATT/AAC 259-261 ACC M14 protein F397A 0.0306 0.009 gene TTC 1189-1191 GCA M15 protein N374E 0.03096 0.01266 gene AAC 1120-1122 GAA M16 protein N87T-G401N 0.0378 0.00798 gene AAC 259-261 ACC/GGT 1201-1203 AAT M17 protein N87T-R184H 0.0378 0.00882 gene AAC 259-261 ACC/CGT 550-552 CAT M18 protein L109H-G459A 0.06084 0.01062 gene CTG 325-327 CAT/GGT 1375-1377 GCA M19 protein W86R-N87T 0.08064 0.01086 gene TGG 256-258 CGT/AAC 259-261 ACC M20 protein W86R-N87T-T377M-T380C 0.56052 0.09468 gene TGG 256-258 CGT/AAC 259-261 ACC/ACC 1129-1131 ATG/ACC 1138-1140 TGT M21 protein W86R-N87T-T377M-T380Y 0.56268 0.05058 gene TGG 256-258 CGT/AAC 259-261 ACC/ACC 1129-1131 ATG/ACC 1138-1140 TAT M22 protein W86R-N87T-L109G-L110E 0.57312 0.0189 gene TGG 256-258 CGT/AAC 259-261 ACC/CTG 325-327 GGT/CTG 328-330 GAA M23 protein W86R-N87T-L109G-L110E-T377M 0.57564 0.08562 gene TGG 256-258 CGT/AAC 259-261 ACC/CTG 325-327 GGT/CTG 328-330 GAA/ACC 1129-1131 ATG M24 protein W86R-N87T-L109G-L110E-A460M 0.756 0.27 gene TGG 256-258 CGT/AAC 259-261 ACC/CTG 325-327 GGT/CTG 328-330 GAA/GCT 1378-1380 ATG M25 protein W86R-N87T-L109G-L110E-H281V-Q282F-A460M 1.4357 0.221 gene TGG 256-258 CGT/AAC 259-261 ACC/CTG 325-327 GGT/CTG 328-330 GAA/CAC 841-843 GTT/CAG 844-846 TTT/GCT 1378-1380 ATG

(60) Notes: the numbering of a protein substitution is started from the N-terminal of the amino acid sequence shown by SEQ ID No.1; the numbering of a gene substitution is started from the 5′end of the nucleotide sequence shown by SEQ ID No.2. In the table, a following nomenclature is used for substitution of an amino acid: an original amino acid, a position (that is, the position in SEQ ID No.1), a substituted amino acid. Correspondingly, substitution of the original tryptophan at position 86 of SEQ ID No.1 with arginine is named as “W86R”. A following nomenclature is used for substitution of a base substitution: an original base, a position (that is, the positions in SEQ ID No.2), a substituted base. Correspondingly, substitution of the original TGG at positions 256-258 of SEQ ID No.2 with CGT is named as “TGG256-258CGT”. A variant in a protein comprising multiple variations is separated by symbol “-”; a variant in a gene comprising multiple variations are separated by symbol “/”.

Example 5. Catalyzing of Hydroxyl Acetaldehyde or 1,3-Dihydroxyacetone by a F/XPK Protein to Obtain Acetyl Phosphoric Acid

(61) 1. Formulation of Detection Reagents:

(62) 2M hydroxylamine hydrochloride (100 mL) at pH 7.5: 13.5 g of solid hydroxylamine hydrochloride is weighed and dissolved in 190 mL of ddH.sub.2O, the pH of which is adjusted to 6.5 with solid sodium hydroxide, followed by performing a constant volume to 200 mL with ddH.sub.2O.

(63) Developer 1: 15% trichloroacetic acid (10 mL); 150 mg of trichloroacetic acid is weighed and dissolved in 10 mL of ddH.sub.2O.

(64) Developer 2: 4M HCl (10 mL); 3.333 mL of concentrated hydrochloric acid is taken and dissolved in ddH.sub.2O, followed by performing a constant volume to 10 mL.

(65) Developer 3: 5% ferric trichloride (10 mL); 50 mg of ferric trichloride is weighed and dissolved in 0.1M HCl, followed by performing a constant volume to 10 mL.

(66) 2. Reaction

(67) 1) Catalyzing of hydroxyl acetaldehyde or 1,3-dihydroxyacetone with F/XPK:

(68) Experimental group: 10 mM 1,3-dihydroxyacetone or hydroxyl acetaldehyde, 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH 7.5, 2 mg/mL F/XPK; after shaking reaction for 2 h at 37° C., a reaction liquid is obtained.

(69) Enzyme-free control: 10 mM 1,3-dihydroxyacetone or hydroxyl acetaldehyde, 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH 7.5; after shaking reaction for 2 h at 37° C., a reaction liquid is obtained.

(70) Substrate-free control: 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH 7.5, 2 mg/mL F/XPK; after shaking reaction for 2 h at 37° C., a reaction liquid is obtained.

(71) Detection method: 75 μL of the above reaction liquid is taken to react with 75 μL of 2M hydroxylamine hydrochloride solution at pH 7.5 for 10 min at 30° C. Then 50 μL of each of developer 1, developer 2, and developer 3 are added. The reaction liquid after development is centrifuged for 2 min at 12000 rpm, and 130 μL of supernatant is taken to measure its (acetyl phosphoric acid) absorbance at 505 nm.

(72) The results are seen in FIG. 4. As can be seen, hydroxyl acetaldehyde or 1,3-dihydroxyacetone can generate acetyl phosphoric acid, which is catalyzed by a F/XPK protein.

Example 6. Synthesis of Acetyl Coenzyme A with Catalyzing of Acetyl Phosphoric Acid by a PTA Enzyme

(73) 1. Generation of Acetyl Coenzyme A with Catalyzing of Acetyl Phosphoric Acid by PTA

(74) 1 mg/ml PTA, 3 mM acetyl phosphoric acid, 2 mM CoA, 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH 7.5; after shaking reaction for 0 min and 10 min at 37° C., reaction liquids are obtained.

(75) Detection method: 1 mL of the reaction liquid is taken to measure its absorbance at 233 nm, and the results are seen in Table 2.

Table 2 Shows Detection Data for the Synthesis of Acetyl Coenzyme A by Acetyl Phosphoric Acid

(76) TABLE-US-00002 Reaction time OD.sub.233  0 min 0.640 10 min 1.077

(77) Calculation Method:

(78) OD.sub.233 for the reaction for 0 min is taken as E.sub.1, and OD.sub.233 for the reaction for 10 min is taken as E.sub.2. The difference of the molar extinction coefficients between acetyl coenzyme A and CoA is Δε.

(79) C.sub.acetyl coenzyme A is the concentration of acetyl coenzyme A
C.sub.acetyl coenzyme A=10*(E.sub.2−E.sub.1)/Δε

(80) The concentration of acetyl coenzyme A is 0.98 mM.

Example 7. Double-Enzyme Catalyzing of Formaldehyde with a BFD Mutant and a F/XPK to Obtain Acetyl Phosphoric Acid

(81) Experimental group: 2 g/L formaldehyde, 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH7.5, 1 mg/mL BFD or its mutant, 2 mg/mL F/XPK; after shaking reaction for 2 h at 37° C., a reaction liquid is obtained.

(82) Enzyme-free control: 2 g/L formaldehyde, 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH7.5; after shaking reaction for 2 h at 37° C., a reaction liquid is obtained.

(83) Substrate-free control: 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2, 0.5 mM TPP, at pH7.5, 1 mg/mL BFD or its mutant, 2 mg/mL F/XPK; after shaking reaction for 2 h at 37° C., a reaction liquid is obtained.

(84) Detection method: 75 μL of the above reaction liquid is taken to react with 75 μL of 2M pH7.5 hydroxylamine hydrochloride solution for 10 min at 30° C. After that, 50 μL of each of developer 1, developer 2, and developer 3 is added. The reaction liquid after development is centrifuged at 12000 rpm for 2 min, and 130 μL of the supernatant is taken to measure its (acetyl phosphoric acid) absorbance at 505 nm.

(85) The result of BFD mutant W86R-N87T-L109G-L110E-H281V-Q282F-A460M is shown in FIG. 5. As can be seen, the above BFD mutant protein along with F/XPK protein catalyzes formaldehyde to obtain acetyl phosphoric acid.

Example 8. Three-Enzyme Catalyzing of Formaldehyde with a BFD Mutant W86R-N87T-L109G-L110E-H281V-Q282F-A460M, F/XPK and PTA to Generate Acetyl Coenzyme A

(86) 0.2 mg/ml BFD mutant W86R-N87T-L109G-L110E-H281V-Q282F-A460M, 0.2 mg/ml F/XPK, 0.2 mg/ml PTA, 0.1 g/L formaldehyde, 10 mM potassium phosphate, 2 mM CoA, 50 mM potassium phosphate buffer, 5 mM MgCl.sub.2 and 0.5 mM TPP, at pH7.5, are subjected to the shaking reaction for 2 h at 37° C. to obtain a reaction liquid.

(87) Detection method I: 1 mL of the reaction liquid is taken to measure its absorbance at 233 nm. The results are seen in Table 3.

Table 3 Shows Detection Data for the Synthesis of Acetyl Coenzyme A by Formaldehyde

(88) TABLE-US-00003 Reaction time OD.sub.233  0 min 0.033 120 min 0.147

(89) Calculation Method:

(90) OD.sub.233 for the reaction for 0 min is taken as Ea, and OD.sub.233 for the reaction for 120 min is taken as Eb. The difference of the molar extinction coefficients between acetyl coenzyme A and CoA is Δε. C.sub.acetyl coenzyme A is the concentration of acetyl coenzyme A.
C.sub.acetyl coenzyme A=10*(Eb−Ea)/Δε

(91) As can be seen, the concentration of acetyl coenzyme A obtained by using formaldehyde as a substrate is 0.257 mM.

(92) The catalyzing of 0.1 g/L formaldehyde by a wild-type BFD obtains no hydroxyl acetaldehyde and 1,3-dihydroxyacetone, thus, no acetyl coenzyme A is detected by a reaction of a wild-type BFD with the other two enzymes.

(93) Detection method II: 100 μL of the reaction liquid is taken to be added with 300 μL of acetonitrile for LC-MS detection.

(94) LC Conditions:

(95) Instrument: Shimadzu LC-30A; chromatographic column: Merck zic-HILIC (100 mm×2.1 mm, 3.5 μm); mobile phase A is 10 mM ammonium acetate, and mobile phase B is 100% acetonitrile. Condition for a gradient liquid chromatography is: 0-3 min, 90% B; 3-25 min, 90%-60% B; 25-30 min, 60% B; 30-38 min, 90% B; flow rate: 0.3 mL/min.

(96) MS Conditions:

(97) Instrument: ABSciex TripleTOF5600; ESI source; positive ion detection mode; voltage 5500V; ion source temperature 600° C.; GS1 gas pressure: 55 psi; GS2 gas pressure: 55 psi; curtain gas pressure: 35 psi; IDA collection mode, primary scanning range 50-1200 Da, secondary scanning range 30-1200 Da.

(98) The results are seen in FIG. 6. As can be seen, formaldehyde can be catalyzed to acetyl coenzyme A with three enzymes: a BFD mutant, F/XPK and PTA.

Example 9. To Obtain Biosynthesis Pathways of Acetyl Coenzyme A with Formaldehyde

(99) As can be seen from above, biosynthesis pathways of acetyl coenzyme A with formaldehyde can be any one of the following pathways:

(100) ##STR00001##

(101) The experiments of the present invention demonstrate that there does not exist an enzyme in the nature for catalyzing formaldehyde to be condensed to hydroxyl acetaldehyde or 1,3-dihydroxyacetone, nor one for converting 1,3-dihydroxyacetone to acetyl phosphoric acid. In the present invention, by means of site-directed mutation of BFD, a mutant of this enzyme is found. The mutant can catalyze formaldehyde to be condensed to hydroxyl acetaldehyde, which is first found and achieves the highly effective polymerization of the formaldehyde; meanwhile, by means of F/XPK, generation of acetyl phosphoric acid from the hydroxyl acetaldehyde or 1,3-dihydroxyacetone (DHA) is achieved; in combination with phosphotransacetylase (Pta), a route from the formaldehyde to acetyl coenzyme A is achieved in three steps, thereby creating a new pathway for assimilation of formaldehyde-synthesis of acetyl coenzyme A from formaldehyde in three steps. This pathway has a short route without a carbon loss and an input for ATP.