Phosphate transketolase mutant and application thereof

Abstract

The invention provides a phosphoketolase mutant and application thereof. Comprising the following steps: (a) compared with an amino acid sequence as shown in SEQ ID NO.1, the phosphoketolase mutant contains at least one of the following sites: H142, Q321 and E520, and an amino acid sequence which is subjected to amino acid mutation and has a phosphoketolase activity function; or (b) an amino acid sequence which is obtained by substitution and / or deletion and / or addition of one or more conserved amino acid residues of the phosphoketolase mutant and the amino acid sequence shown in (a) and has the same function. The invention provides a new way for converting C1-6 carbon sources into acetyl phosphate or acetyl coenzyme A without carbon loss. The catalytic activity of the phosphorylated transketolase mutant is higher than that of wild type phosphorylated transketolase.

Description

Technical Field

[0001] The invention relates to the technical field of biological enzymes, in particular to a phosphoketolase mutant and an application thereof. Background Art

[0002] In industrial biotechnology, feedstock costs account for approximately 50% of total production costs. Achieving the highest carbon yield is the goal of metabolic engineering. However, carbon loss is inevitable in obtaining acetyl-CoA derivatives such as fatty acids, alcohols, alkanes, isoprenoids, and polyketides through sugar degradation pathways. Thiamine pyrophosphate (ThDP)-dependent phosphoketolase (PK) can bypass the pyruvate decarboxylation process and directly convert fructose-6-phosphate (F6P) or xylulose-5-phosphate (Xu5P) into erythrose-4-phosphate (E4P) or glyceraldehyde-3-phosphate (G3P) and acetyl phosphate (AcP), the precursor of acetyl-CoA. Phosphoketolase pathway engineering has been successfully used to improve the yield of acetyl-CoA-derived products in different microorganisms, including Corynebacterium glutamicum, Saccharomyces cerevisiae, Aspergillus niger, Escherichia coli, and methanotrophic bacteria.

[0003] However, the metabolic flux of acetyl-CoA is still limited by the conversion of erythrose 4-phosphate or glyceraldehyde 3-phosphate to acetyl-CoA. The nonoxidative glycolysis pathway (NOG) solves the problem of carbon loss from erythrose 4-phosphate or glyceraldehyde 3-phosphate to acetyl-CoA by combining phosphoketolase with the pentose phosphate pathway (PPP), and fructose 6-phosphate (F6P) or xylulose 5-phosphate can stoichiometrically synthesize acetyl-CoA. Although great progress has been made, the nonoxidative glycolysis pathway still has inherent defects, such as complex metabolic pathways and kinetic traps caused by metabolic flux imbalance.

[0004] Moreover, for one-carbon carbon sources, the main metabolic pathways include the ribulose monophosphate cycle, the xylulose monophosphate cycle, the Calvin cycle, etc., and using these pathways to convert one-carbon carbon sources (carbon dioxide, formic acid, methanol, methane, etc.) into derivatives of acetyl-CoA will release carbon dioxide, resulting in carbon loss. For three-carbon carbon sources (mainly glycerol) and six-carbon carbon sources (mainly glucose), the main metabolic pathway is the sugar degradation pathway, and converting the carbon source into derivatives of acetyl-CoA will release carbon dioxide, resulting in carbon loss. For five-carbon carbon sources (D-xylose), the main metabolic pathways include the pentose phosphate pathway (PPP) and the phosphoketolase pathway. These natural pathways metabolize D-xylose to produce derivatives of acetyl-CoA, which will also release carbon dioxide, also resulting in carbon loss. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a phosphoketolase mutant and its application, which solves the problem of carbon loss in the conversion of various carbon sources (C1-C6) to acetyl-CoA in the prior art.

[0006] In a first aspect of the present invention, a phosphoketolase mutant is provided, comprising: (a) the phosphoketolase mutant contains at least one of the following sites compared with the amino acid sequence shown in SEQ ID NO.1: H142, Q321, E520, an amino acid sequence that has undergone amino acid mutation and has phosphoketolase activity; or

[0007] (b) The phosphoketolase mutant has the same function as the amino acid sequence shown in (a) through substitution and / or deletion and / or addition of one or more conservative amino acid residues.

[0008] Furthermore, the phosphoketolase mutant comprises at least one site substitution selected from the group consisting of: H142N, Q321A, and E520I.

[0009] Furthermore, the amino acid sequence of the phosphoketolase mutant is as follows: SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7; or

[0010] The amino acid sequence of the phosphoketolase mutant has at least 95%, preferably 98%, and more preferably 99% sequence identity with SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.7.

[0011] In a second aspect, the present invention provides a DNA molecule encoding the phosphoketolase mutant.

[0012] Furthermore, the nucleotide sequence of the DNA molecule is shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 or

[0013] The nucleotide sequence of the DNA molecule is a sequence having at least 95%, preferably 98%, and more preferably 99% sequence identity with SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8.

[0014] The third aspect of the present invention provides a vector, an expression cassette or a host cell containing the above-mentioned DNA molecule.

[0015] In a fourth aspect, the present invention provides the use of the mutant, DNA molecule, vector, expression cassette or host cell, the phosphoketolase shown in SEQ ID NO.1, the DNA molecule shown in SEQ ID NO.2, the vector, expression cassette or host cell expressing the phosphoketolase shown in SEQ ID NO.1 or containing the DNA molecule shown in SEQ ID NO.2 in catalyzing the production of acetyl phosphate or acetyl-CoA from a carbon source;

[0016] Preferably, the carbon source comprises one or more of formaldehyde, methanol, ethylene glycol, ethanolamine, hydroxyacetaldehyde, glycerol, glyceraldehyde, hydroxyacetone, erythrulose, erythritol, pentose, pentose phosphate, hexose, and hexose phosphate;

[0017] More preferably, the carbon source comprises erythrulose or erythritol.

[0018] In a fifth aspect, the present invention provides a biomaterial for catalyzing the generation of acetyl phosphate from a carbon source, wherein the biomaterial expresses the above-mentioned phosphoketolase mutant, or contains the above-mentioned DNA molecule; the biomaterial is one or more of a vector, an expression cassette or a host cell.

[0019] Preferably, the biomaterial further expresses one or more of glycanase, phosphoketolase, phosphohydrolase, isomerase, and alkaline phosphatase.

[0020] In a sixth aspect, the present invention provides a method for generating acetyl phosphate using a carbon source, wherein acetyl phosphate is generated using a carbon source as a substrate under the action of phosphoketolase or a mutant thereof or a biological material expressing the phosphoketolase or a mutant thereof.

[0021] In a seventh aspect, the present invention provides a method for producing acetyl-CoA using a carbon source, comprising the following steps:

[0022] S1: generating acetyl phosphate using the above method;

[0023] S2: Acetyl phosphate generates acetyl-CoA under the action of phosphate acetyltransferase.

[0024] The term "carbon source" refers to a nutrient rich in carbon that can meet the needs of microbial growth, and its effective component is an organic compound with a single molecular formula and molecular structure that is easily utilized by microorganisms. A one-carbon carbon source refers to a carbon source with one carbon atom in the molecule, including but not limited to methanol and formaldehyde; a two-carbon carbon source refers to a carbon source with two carbon atoms in the molecule, including but not limited to ethylene glycol, ethanolamine, and hydroxyacetaldehyde; a three-carbon carbon source refers to a carbon source with three carbon atoms in the molecule, including but not limited to glycerol, glyceraldehyde, and hydroxyacetone; a four-carbon carbon source refers to a carbon source with four carbon atoms in the molecule, including but not limited to erythrulose and erythritol; a five-carbon carbon source refers to a carbon source with five carbon atoms in the molecule, including but not limited to xylose, xylulose, and xylulose-5-phosphate; a six-carbon carbon source refers to a carbon source with six carbon atoms in the molecule, including but not limited to fructose, fructose-6-phosphate, glucose, and glucose-6-phosphate.

[0025] The technical principle of the present invention is as follows: in the prior art, L-erythrulose and D-erythrulose are generally converted into glycolaldehyde under the action of erythrulose isomerase, and then acetyl phosphate is further synthesized. After repeated screening and experiments, the inventors found that phosphoketolase or its mutants can directly catalyze the conversion of L-erythrulose and D-erythrulose into acetyl phosphate, providing a new pathway for the conversion of erythrulose into acetyl phosphate. Moreover, the inventors further found through experiments that the enzyme activity of these mutants is significantly higher than that of the wild-type phosphoketolase, and the catalytic efficiency of converting various carbon sources into acetyl phosphate is significantly improved.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention first discovered that phosphorylating transketolase and its mutants can realize the conversion of carbon one to carbon six sources to acetyl phosphate or acetyl CoA without carbon loss, with a theoretical carbon yield of 100% and an actual carbon yield of up to 95%, thus constructing and realizing a utilization route from carbon one to carbon six sources.

[0028] (2) The present invention first discovered that phosphoketolase (BbPK) can catalyze L-erythrulose and / or D-erythrulose to produce acetyl phosphate, providing a new pathway for converting erythrose into acetyl phosphate.

[0029] (3) The catalytic activity of the phosphorylating transketolase mutant of the present invention is higher than that of the wild-type phosphorylating transketolase. The catalytic activity of the mutants BbPK-E520I and BbPK-Q321A on the substrate dihydroxyacetone is 2.3 times and 2.8 times higher than that of the wild-type, respectively. The catalytic activity of the mutant BbPK-H142N on the substrate hydroxyacetaldehyde is 4.9 times higher than that of the wild-type, and the catalytic activity on the substrate D-erythrulose is 3.8 times higher than that of the wild-type.

[0030] (4) The method of the present invention is simple and efficient, realizes the efficient synthesis of acetyl phosphate, and is conducive to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 3 is a diagram of various carbon source utilization pathways in Example 3 of the present invention, wherein A is a one-carbon carbon source utilization pathway; B is a two-carbon carbon source utilization pathway; C is a three-carbon carbon source utilization pathway; D is a four-carbon carbon source utilization pathway; E is a five-carbon sugar utilization pathway; F is a six-carbon sugar utilization pathway, and the reaction represented by the dotted line was not tested.

[0032] Figure 2 This is a graph showing the utilization rate test results of one-carbon carbon source and six-carbon carbon source. DETAILED DESCRIPTION

[0033] 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.

[0034] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. EcTIM is glyceraldehyde 3-phosphate isomerase from Escherichia coli; CpHAD is phosphohydrolase from Candida parapsilosis; EcHAD is phosphohydrolase from Escherichia coli; PsLRhI is erythrose isomerase from Pseudomonas stutzeri.

[0035] Example 1 Obtaining a phosphoketolase mutant

[0036] The encoding gene of phosphoketolase BbPK is from Actinobacteria Bifidobacterium (refer to Lu.X et al Nature communication 10,1378(2019)), the amino acid of phosphoketolase is shown in SEQ ID NO.1, and the nucleotide sequence of the phosphoketolase encoding gene is shown in SEQ ID NO.2.

[0037] Single-point mutation or multi-point mutation was performed on phosphoketolase to obtain the following phosphoketolase mutants.

[0038] The phosphoketolase mutant BbPK_H142N is obtained by mutating the histidine (H) at position 142 of the phosphoketolase amino acid sequence (SEQ ID NO.1) to asparagine (N). Its amino acid sequence is shown in SEQ ID NO.3.

[0039] The coding gene of the phosphoketolase mutant BbPK_H142N is a sequence obtained by mutating the CAC at positions 424-426 of the phosphoketolase coding gene nucleotide sequence (SEQ ID NO.2) to AAC, while keeping the other bases unchanged. The nucleotide sequence is shown in SEQ ID NO.4.

[0040] The phosphoketolase mutant BbPK_Q321A is a mutant in which the glutamine (Q) at position 321 of the phosphoketolase amino acid sequence (SEQ ID NO.1) is mutated to alanine (A). Its amino acid sequence is shown in SEQ ID NO.5.

[0041] The coding gene of the phosphoketolase mutant BbPK_Q321A is a sequence obtained by mutating the CAG at positions 961-963 of the phosphoketolase coding gene nucleotide sequence (SEQ ID NO.2) to GCG, while keeping the other bases unchanged. The nucleotide sequence is shown in SEQ ID NO.6.

[0042] The phosphoketolase mutant BbPK_E520I is a mutant in which the glutamic acid (E) at position 520 of the phosphoketolase amino acid sequence (SEQ ID NO.1) is mutated to isoleucine (I). Its amino acid sequence is shown in SEQ ID NO.7.

[0043] The coding gene of the phosphoketolase mutant BbPK_E520I is obtained by mutating the GAA at positions 1558-1560 of the phosphoketolase coding gene nucleotide sequence (SEQ ID NO.2) to ATC, while keeping the other bases unchanged. The nucleotide sequence is shown in SEQ ID NO.8.

[0044] Example 2 Construction, expression and purification of expression vector

[0045] 1. Construction of expression vector

[0046] The coding genes of the phosphoketolase mutants BbPK_H142N, BbPK_Q321A, and BbPK_E520I in Example 1 were respectively inserted between the Nde I and Xho I restriction sites of the pet-28a vector to obtain vectors expressing the phosphoketolase mutants BbPK_H142N, BbPK_Q321A, and BbPK_E520.

[0047] The coding gene of phosphoketolase BbPK in Example 1 (SEQ ID NO. 2) was inserted between the NdeI and XhoI restriction sites of the pet-28a vector to obtain a vector expressing phosphoketolase.

[0048] 2. Expression and purification of phosphoketolase and its mutants

[0049] The vectors expressing the phosphoketolase mutants BbPK_H142N, BbPK_Q321A, and BbPK_E520 prepared in step 1 and the vector expressing phosphoketolase were transformed into Escherichia coli BL21 (DE3), respectively, and resistance screening was performed, and single clones were selected to 2YT medium for induction of expression. The culture solution was centrifuged, and the supernatant was discarded to obtain the bacterial cells expressing BbPK and the bacterial cells expressing each mutant. The phosphoketolase BbPK and the phosphoketolase mutants BbPK_H142N, BbPK_Q321A, and BbPK_E520I were obtained by breaking the bacteria and purifying.

[0050] Example 3 Functional detection of phosphoketolase and its mutant proteins

[0051] Detection method of acetyl phosphate: Since acetyl phosphate is easy to decompose, we convert acetyl phosphate into acetic acid and test the amount of acetic acid. Specifically, add an equal volume of 5% sulfuric acid to the sample to completely decompose acetyl phosphate into acetic acid. Then use high performance liquid chromatography (HPLC) to detect acetic acid. HPLC conditions: column is Aminex HPX-87H (Bio-Rad); detection wavelength is 210nm; mobile phase is 5mM sulfuric acid; flow rate is 0.6mL min-1; sample volume is 20μL; column temperature is 40℃.

[0052] 1. The reaction conditions for phosphoketolase (BbPK) catalyzing C1-C6 carbon sources are as follows:

[0053] One-carbon and three-carbon source reaction conditions: 200 μL reaction system contains 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO4, 2 mg mL -1 Phosphoketolase (BbPK), 2 mg mL -1 Glucanase (FLS), 1 mM thiamine pyrophosphate (ThDP), 20 mM formaldehyde or 10 mM hydroxyacetone. The reaction was carried out at 37°C overnight. Acetic acid was detected by high performance liquid chromatography. The reaction process is as follows Figure 1 A, as shown in 1C.

[0054] Reaction conditions for C2 and C4 carbon sources: 200 μL reaction system contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO4, 2 mg mL -1 Phosphoketolase (BbPK), 1 mM ThDP, 10 mM hydroxyacetaldehyde or 5 mM D-erythrulose. The reaction was carried out at 37°C overnight. Acetic acid was detected by high performance liquid chromatography (HPLC). Figure 1 B, as shown in 1D.

[0055] Five carbon source reaction conditions: 1 mL reaction system contains 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO4, 2 mg mL -1 Phosphoketolase (BbPK), 1 mg mL -1 Phosphohydrolase (CpHAD), 0.1 mg mL -1 Glyceraldehyde 3-phosphate isomerase (EcTIM), 2 mg mL -1glycanase (FLS), 1 mM ThDP and 10 mM xylulose-5-phosphate. The reaction was carried out at 37°C for 10 hours. The reaction was terminated by heating at 95°C for 5 minutes and then cooled to 37°C. 5 U of alkaline phosphatase was added and the reaction was carried out at 37°C for 4 hours and terminated with an equal volume of acetonitrile. Acetic acid was detected by HPLC. The reaction process is shown in 1E.

[0056] Hexacarbon source reaction conditions: 1 mL reaction system contains 50 mM potassium phosphate buffer (pH 7.5), 5 mM MgSO4, 2 mg mL -1 Phosphoketolase (BbPK), 0.5 mg mL -1 Phosphohydrolase (EcHAD), 1 mg mL -1 Erythrose isomerase (PsLRHI), 1mM ThDP, 10mM fructose-6-phosphate. The reaction was carried out at 37°C for 10 hours. The reaction was terminated by heating at 95°C for 5 minutes and then cooling to 37°C. 5U alkaline phosphatase was added and the reaction was carried out at 37°C for 4 hours and terminated with an equal volume of acetonitrile. Acetic acid was detected by HPLC. The reaction process is shown in 1F.

[0057] The utilization rate test results of one carbon source and six carbon sources are as follows: Figure 2 As shown. The carbon source utilization rate is calculated as: (actual product molar concentration / theoretical product molar concentration) × 100%. For example, the theoretical product amount of acetic acid generated by 1 mol / L fructose-6-phosphate catalyzed by BbPK is 3 mol / L. The actual molar concentration of acetic acid product detected by high performance liquid chromatography is 2.7 mol / L. After calculation, 2.7 mol / L divided by 3 mol / L is 0.9, and then multiplied by 100%, the obtained 90% is the carbon yield. Figure 2 It can be seen that the carbon yields of C1 to C6 are 83%, 95%, 88%, 93%, 90% and 90% respectively.

[0058] Compared with the natural pathway of carbon source utilization. In the natural pathway of carbon source utilization, one molecule of glucose is converted into two molecules of acetyl-CoA through sugar degradation, with the loss of two molecules of carbon dioxide, and the theoretical carbon yield is only 67%. One molecule of xylose is converted into two molecules of acetyl-CoA through the phosphoketolase pathway, with the loss of one molecule of carbon dioxide, and the theoretical carbon yield is 80%. One molecule of glycerol is converted into one molecule of acetyl-CoA through sugar degradation, with the loss of one molecule of carbon dioxide, and the theoretical carbon yield is 67%. Three molecules of formaldehyde are converted into one molecule of acetyl-CoA through the ribulose monophosphate cycle or the xylulose monophosphate cycle, with the loss of one molecule of carbon dioxide, and the theoretical carbon yield is 67%. The artificial phosphoketolase pathway constructed by the present invention can efficiently utilize C1 to C6 carbon sources, with a theoretical carbon yield of 100%, and an actual carbon yield of 83% to 95%, far exceeding the theoretical carbon yield of the natural pathway.

[0059] 2. Phosphoketolase (BbPK) and its mutants BbPK-E520I, BbPK-Q321A, PK-H142N activity assay conditions for dihydroxyacetone (DHA), L-erythrulose (L-EUS), D-erythrulose (D-EUS), and hydroxyacetaldehyde (GALD) (other ketose substrates are natural substrates or have been reported): Phosphoketolase (BbPK and mutants) 1 mg mL -1 ; Reaction volume 200 μL; The reaction system contains 10 mM dihydroxyacetone (or L-erythrulose, D-erythrulose, hydroxyacetaldehyde), 1 mM ThDP coenzyme, 5 mM MgSO4, 50 mM phosphate buffer (pH 7.4), react at 37°C for half an hour, add 200 μL acetonitrile to terminate the reaction, and detect the product. The test results are shown in Table 1.

[0060] Table 1 Catalytic results of enzymes and mutants on different substrates

[0061]

[0062]

[0063] As shown in Table 1, the catalytic activity of mutants BbPK-E520I and BbPK-Q321A on substrate dihydroxyacetone (DHA) was 2.3 and 2.8 times higher than that of wild-type BbPK, respectively. The catalytic activity of mutant PK-H142N on substrate hydroxyacetaldehyde (GALD) was 4.9 times higher than that of wild-type BbPK, and the catalytic activity on substrate D-erythrulose (D-EUS) was 3.8 times higher than that of wild-type BbPK.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Phosphoketolase mutants, Features: Including (a) the phosphoketolase mutant contains at least one of the following sites compared with the amino acid sequence shown in SEQ ID NO.1: H142, Q321, E520, an amino acid sequence with phosphoketolase activity after amino acid mutation; or (b) The phosphoketolase mutant has the same function as the amino acid sequence shown in (a) through substitution and / or deletion and / or addition of one or more conservative amino acid residues.

2. The phosphoketolase mutant according to claim 1, Features: The phosphoketolase mutant comprises at least one site substitution selected from the group consisting of: H142N, Q321A, and E520I.

3. The phosphoketolase mutant according to claim 2, Features: The amino acid sequence of the phosphoketolase mutant is as follows: SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7; or The amino acid sequence of the phosphoketolase mutant has at least 95%, preferably 98%, and more preferably 99% sequence identity with SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.

7.

4. A DNA molecule, Features: The DNA molecule encodes the phosphoketolase mutant according to any one of claims 1 to 3.

5. The DNA molecule according to claim 4, Features: The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.4, SEQ ID NO.6, or SEQ ID NO.8; or The nucleotide sequence of the DNA molecule is a sequence having at least 95%, preferably 98%, and more preferably 99% sequence identity with SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.

8.

6. A vector, expression cassette or host cell containing the DNA molecule according to claim 4 or 5.

7. Use of the mutant according to any one of claims 1 to 3, the DNA molecule according to claim 4 or 5, the vector, expression cassette or host cell according to claim 6, the phosphoketolase shown in SEQ ID NO.1, the DNA molecule shown in SEQ ID NO.2, the vector, expression cassette or host cell expressing the phosphoketolase shown in SEQ ID NO.1 or containing the DNA molecule shown in SEQ ID NO.2 in catalyzing the production of acetyl phosphate or acetyl-CoA from a carbon source; Preferably, the carbon source comprises one or more of formaldehyde, methanol, ethylene glycol, ethanolamine, hydroxyacetaldehyde, glycerol, glyceraldehyde, hydroxyacetone, erythrulose, erythritol, pentose, pentose phosphate, hexose, and hexose phosphate; More preferably, the carbon source comprises erythrulose or erythritol.

8. A biomaterial for catalyzing the generation of acetyl phosphate from a carbon source, wherein the biomaterial expresses the phosphoketolase mutant according to any one of claims 1 to 3, or contains the DNA molecule according to claim 4 or 5; the biomaterial is one or more of a vector, an expression cassette or a host cell; Preferably, the biomaterial further expresses one or more of glycanase, phosphoketolase, phosphohydrolase, isomerase, and alkaline phosphatase.

9. A method for generating acetyl phosphate using a carbon source, Features: With a carbon source as a substrate, acetyl phosphate is generated under the action of phosphoketolase or its mutant or a biological material expressing the phosphoketolase or its mutant.

10. A method for producing acetyl-CoA using a carbon source, Features: The following steps are involved: S1: generating acetyl phosphate using the method of claim 9; S2: Acetyl phosphate generates acetyl-CoA under the action of phosphate acetyltransferase.

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