Method for producing acetyl phosphoric acid
By converting D-xilose into ethanol aldehyde and dihydroxyacetone, and using aldolase and phosphotransferase to generate acetylphosphate, the complex problems of carbon loss and pathways in the preparation of acetyl-CoA in the prior art are solved. Achieve efficient and low-cost preparation of acetylphosphate.
Patent Information
- Application Number
- CN202310735236.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-20
- Publication Date
- 2024-12-20
AI Technical Summary
The process of converting D-xylose to acetyl-CoA in the prior art has problems with carbon loss and complex synthesis pathways, and requires additional ATP energy input.
By converting D-xilose to alcohol aldehyde and/or dihydroxyacetone and converting it into acetylphosphate using aldolase and phosphotransferase, then acetyl-CoA is generated without additional energy input throughout the process And no carbon loss.
An efficient, simple and low-cost acetylphosphate preparation method is realized. The carbon yield of the reaction route is 100%, and the enzymes can be recycled, which reduces production costs and increases waste utilization.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of enzyme preparation, in particular to a method for producing acetyl phosphate. Background Art
[0002] D-xylose is the second most abundant monosaccharide in lignocellulose hydrolysate after glucose, accounting for about 35%. Lignocellulose comes from wood waste from forestry and agriculture, as well as industrial waste with wood products as raw materials. The annual output of lignocellulose reaches more than 100 billion tons, so the utilization of cellulose biomass can greatly reduce the competition with food crops. In addition, compared with sugar crops, cellulose can grow more efficiently and produce more stably. Lignocellulose cannot be directly used by microorganisms and cannot be used as a natural substrate for microorganisms; and lignocellulose is more difficult to use than corn starch, etc. Most microorganisms cannot fully absorb xylose in cellulose hydrolysate due to the inhibitory effect of carbon decomposition metabolites, so the application of lignocellulose is greatly limited. The key to fully utilizing lignocellulose is to be able to utilize xylose, so the effective utilization of xylose has always been a hot topic in industrial biotechnology research.
[0003] The catabolism of D-xylose mainly includes the pentose phosphate pathway (PPP) and the phosphoketolase pathway (phosphoketolase pathway) (Int J Mol Sci. 2016; 17(3): 207). D-xylose is first converted into D-xylulose through the xylose isomerase pathway or the redox pathway. The xylose isomerase pathway refers to the direct conversion of D-xylose into D-xylulose under the catalysis of xylose isomerase. The redox pathway is also called the xylose reductase-xylitol dehydrogenase pathway. The first step is that the xylose reductase dependent on the cofactor NADPH reduces D-xylose to xylitol, and xylitol is converted into D-xylulose by the xylitol dehydrogenase dependent on NAD+. D-xylulose is converted into xylulose-5-phosphate under the action of xylulokinase (XKS), and then xylulose-5-phosphate enters the pentose phosphate pathway or the phosphoketolase pathway. The two pathways of pentose phosphate and phosphoketolase are not only complex, but also involve carbon loss when converting D-xylose into acetyl-CoA derivatives.
[0004] CN107429269A utilizes ketohexokinase C (KhkC), rhamnokinase (rhaB) and fucokinase (fucK) to phosphorylate (D)-xylulose at position 1 to obtain pentose-1-phosphate, and then utilizes aldolase B (Aldo-B) or fructose-1,6-bisphosphate aldolase (FbaB) to cleave pentose-1-phosphate to obtain glycolaldehyde and dihydroxyacetone phosphate (DHAP), and then reduces to obtain ethylene glycol or oxidizes to obtain glycolic acid. This method is a natural way to utilize pentose phosphate, and there are the following three problems: 1) ATP is consumed; 2) carbon loss will occur; 3) DHAP needs to go through the sugar degradation pathway to reach acetyl-CoA, and the synthesis pathway is complicated.
[0005] Therefore, it is necessary to seek a more efficient, simpler, lower-cost method for preparing acetyl phosphate without carbon loss. Summary of the invention
[0006] In order to improve the above technical problems, the present invention provides a method for producing acetyl phosphate, which solves the problems in the prior art of carbon loss and complex synthesis pathway in converting D-xylose into acetyl-CoA derivatives.
[0007] In one aspect, the present invention provides a method for producing acetyl phosphate, comprising the following steps: (1) preparing ethanolaldehyde and / or dihydroxyacetone using D-xylulose as a substrate; and (2) preparing acetyl phosphate using ethanolaldehyde and / or dihydroxyacetone as a substrate.
[0008] Further, the following steps are included:
[0009] S1: converting D-xylulose into glycolaldehyde and / or dihydroxyacetone using aldolase;
[0010] S2: Conversion of glycolaldehyde and / or dihydroxyacetone into acetyl phosphate using phosphoketolase.
[0011] Further, in step S1, the acetalase comprises D-fructose-6-phosphate acetalase and / or its mutant, preferably, the mutant is FSA-A129S; and / or
[0012] In step S2, the conversion product of dihydroxyacetone also includes formaldehyde, and the formaldehyde is further converted into dihydroxyacetone by ethanolaldehyde synthase and / or formaldehyde condensing enzyme.
[0013] In a specific embodiment of the present invention, formaldehyde is converted into dihydroxyacetone under the action of formaldehyde condensing enzyme, wherein the formaldehyde concentration is 5-20mM, the formaldehyde condensing enzyme (FLS) concentration is 1-3mg / ml, the thiamine pyrophosphate (TPP) concentration is 0.1-1mM, 1mM MgSO4, 50mM K3PO4, pH 8.0, and the reaction time is 1-3 hours.
[0014] Furthermore, the D-xylulose is prepared from D-xylose.
[0015] In a specific embodiment of the present invention, D-xylulose is prepared from D-xylose under the catalysis of xylose isomerase (XI).
[0016] Furthermore, the method comprises step S3: preparing acetyl-CoA using acetyl phosphate as a substrate,
[0017] Preferably, acetyl phosphate and coenzyme A are reacted to generate acetyl-CoA using phosphate acetyltransferase.
[0018] Furthermore, the method comprises step S4: preparing polyhydroxybutyrate using acetyl-CoA as a substrate.
[0019] Further, acetyl-CoA is converted into succinyl-CoA using acetyl-CoA acetyltransferase; succinyl-CoA is converted into hydroxybutyryl-CoA using acetoacetyl-CoA reductase; and hydroxybutyryl-CoA is converted into polyhydroxybutyrate using polyhydroxybutyrate synthetase.
[0020] Further, it is prepared by a one-pot reaction,
[0021] Preferably, the concentration of D-xylulose is 20-25 mM, the concentration of aldolase is 2-4 mg / ml; the concentration of phosphoketolase is 1-3 mg / ml; the concentration of formaldehyde condensing enzyme is 1-3 mg / ml; the concentration of glycolaldehyde synthase is 2-4 mg / ml; the concentration of coenzyme A is 1-3 mg / ml; the concentration of phosphate acetyltransferase is 1-3 mg / ml; the concentration of acetyl-CoA acetyltransferase is 1-3 mg / ml; the concentration of acetyl-CoA reductase is 1-3 mg / ml; the concentration of polyhydroxybutyrate synthase is 1-3 mg / ml;
[0022] Preferably, the buffer used in the reaction comprises a phosphate buffer, wherein the phosphate buffer comprises Mg 2+ ;
[0023] Preferably, the reaction pH is 7.2-7.6;
[0024] Preferably, the reaction temperature is 37°C and the reaction time is 10-14h.
[0025] In another aspect, the present invention provides a biomaterial for preparing acetyl phosphate, wherein the biomaterial expresses aldolase and phosphoketolase, or contains genes of aldolase and phosphoketolase;
[0026] Preferably, the biological material comprises a gene cassette, an expression vector, a microorganism or a plant;
[0027] Preferably, the biomaterial also expresses the phosphoketolase coenzyme.
[0028] In another aspect, the present invention provides the use of a biological material for preparing acetyl phosphate in preparing acetyl phosphate, acetyl coenzyme A and polyhydroxybutyrate.
[0029] The aldolase, phosphoketolase, glycolaldehyde synthase, formaldehyde condensing enzyme, phosphate acetyltransferase, acetyl-CoA acetyltransferase, acetoacetyl-CoA reductase and polyhydroxybutyrate synthase used in the present invention include enzymes from various species and also include various mutants.
[0030] In a specific embodiment of the present invention, fructose-6-phosphate aldolase (FSA) is from Escherichia coli; phosphoketolase (PK) is from Bifidobacterium, Chloroflexi Nitrolancea, AlphaproteobacteriaAgrobacterium, Alphaproteobacteria Sphingomonas, GammaproteobacteriaMarinicella, Fungi; glycolaldehyde synthase (GALS) is from Pseudomonas putida; formaldehyde condensing enzyme (FLS) is from Pseudomonas fluorescens biovar I; phosphate acetyltransferase (PTA) is from Escherichia coli; acyl-CoA acetyltransferase (PhaA) is from R.eutropha; acetoacetyl-CoA reductase (PhaB) is from R.eutroph; polyhydroxybutyrate synthase is from R.eutroph.
[0031] The technical principle of the present invention is as follows: in the prior art, D-xylulose is generally converted into 5-xylulose phosphate under the action of xylulokinase (XKS), and then acetyl phosphate is generated under the catalysis of phosphoketolase. Alternatively, D-xylulose is phosphorylated to generate D-xylulose-1-phosphate, and then ethanolaldehyde and dihydroxyacetone phosphate are generated under the action of aldolase. This process not only requires ATP energy input, but also has carbon loss. After repeated experiments and screening, the inventors found that D-xylulose can be efficiently converted into ethanolaldehyde and dihydroxyacetone under the action of aldolase (D-fructose-6-phosphate acetalase or its mutant), not only without consuming additional energy, but also without carbon loss.
[0032] Furthermore, when the inventors screened aldolases, they found that fructose-6-phosphate lyase could effectively convert D-xylose ketone into ethanolaldehyde and dihydroxyacetone, compared to aldolase B (Aldo-B) or fructose-1,6-bisphosphate aldolase (FbaB), which could hardly achieve the conversion.
[0033] Furthermore, ethanolaldehyde and dihydroxyacetone can be converted into acetyl-CoA in just two steps under the action of phosphoketolase, and the synthetic pathway is simple. In the reaction process, the byproduct formaldehyde can generate dihydroxyacetone under the action of ethanolaldehyde synthase (GALS) or formaldehyde condensing enzyme (FLS), which can be reused to produce acetyl phosphate, which not only reduces the emission of formaldehyde harmful gas, is safe and environmentally friendly, but also improves the utilization rate of waste and reduces carbon loss.
[0034] Compared with the prior art, the present invention has the following beneficial effects:
[0035] (1) The present invention constructs a new pathway for synthesizing acetyl phosphate, acetyl coenzyme A and polyhydroxybutyrate using D-xylose or D-xylulose, which does not require the addition of expensive ATP energy supplement. The theoretical carbon yield of the reaction route is 100%, there is no carbon loss, the enzymes can be recycled, and the cost is reduced.
[0036] (2) In the method of the present invention, D-xylose can be converted into acetyl-CoA by only five steps of enzyme catalysis, with fewer reaction steps, mild conditions, simple method and high reaction yield of 42%.
[0037] (3) The aldolase in the present invention does not rely on coenzymes and does not require consumption of additional energy.
[0038] (4) The catalytic substrates of the phosphoketolase in the present invention are ethanolaldehyde and dihydroxyacetone. The catalytic process is irreversible, so that the system is not limited by the reaction equilibrium, which can not only increase the rate of aldolase, but also maximize the yield of acetyl phosphate.
[0039] (5) The present invention utilizes formaldehyde, a byproduct of dihydroxyacetone, for recycling, which not only saves costs but also improves yield and reduces carbon loss.
[0040] Therefore, this patent develops a simple and efficient artificial D-xylose utilization pathway to overcome the defects of the natural D-xylose pathway, such as complex pathway, carbon loss, and the need for additional ATP energy supplementation. DETAILED DESCRIPTION
[0041] 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.
[0042] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
[0043] Example 1 Construction of expression vector
[0044] 1. Construction of a vector expressing phosphoketolase (PK)
[0045] The phosphoketolase gene species is derived from Bifidobacterium, and the gene is inserted into the NdeI and XhoI restriction sites of the pET-28a vector to obtain a vector named pET-28a-PK. The nucleotide sequence of the phosphoketolase gene is shown in SEQ ID NO.1.
[0046] 2. Construction of vectors expressing phosphate acetyltransferase (PTA) and D-fructose-6-phosphate aldolase (FSA)
[0047] Phosphate acetyltransferase (PTA) and D-fructose-6-phosphate aldolase (FSA) genes are both from Escherichia coli. The two genes are inserted between the NdeI and XhoI restriction sites of the pET-28a vector to obtain vectors, which are named pET-28a-FSA and pET-28a-PTA. The nucleotide sequence of the phosphate acetyltransferase gene is shown in SEQ ID NO.2, and the nucleotide sequence of the D-fructose-6-phosphate aldolase gene is shown in SEQ ID NO.3.
[0048] 3. Construction of vectors expressing acetyl-CoA acetyltransferase (PhaA), acetoacetyl-CoA reductase (PhaB), and polyhydroxybutyrate synthase (PhaC)
[0049] The genes of acetyl-CoA acetyltransferase (PhaA), acetoacetyl-CoA reductase (PhaB), and polyhydroxybutyrate synthase (PhaC) are all from R.eutropha. The three genes are inserted between the NdeI and XhoI restriction sites of the pET-28a vector to obtain vectors named pET-28a-PhaA, pET-28a-PhaB, and pET-28a-PhaC. The nucleotide sequence of the acetyl-CoA acetyltransferase gene is shown in SEQ ID NO.4, the nucleotide sequence of the acetoacetyl-CoA reductase gene is shown in SEQ ID NO.5, and the nucleotide sequence of the polyhydroxybutyrate synthase gene is shown in SEQ ID NO.6.
[0050] Example 2 Protein expression and purification
[0051] For in vitro reactions, all enzymes in Example 1 were exogenously expressed and purified in E. coli.
[0052] 1. Protein expression
[0053] (1) The E. coli expression recombinant plasmids pET-28a-FSA, pET-28a-PK, pET-28a-FLS, pET-28a-PhaA, pET-28a-PhaB, pET-28a-PhaC, and pET-28a-PTA prepared in Example 1 were respectively transferred into E. coli BL21 (DE3) to obtain recombinant bacteria expressing the protein in Example 1. Positive clones were screened using kanamycin resistance plates (Kan+, 100 mg / mL) and cultured overnight at 37°C;
[0054] (2) Pick single clones into 5 mL 2YT liquid medium (16 mg / mL tryptone, 10 mg / mL yeast extract, 5 mg / mL sodium chloride, 100 mg / mL Kan+), and culture at 37°C and 220 rpm / min until OD600 is 0.6-0.8. Transfer the bacterial solution in 5 mL 2YT medium to 800 mL 2YT medium (Kan+, 100 mg / mL), and culture at 37°C and 220 rpm until OD600 is 0.6-0.8, then cool to 16°C, add IPTG to a final concentration of 0.5 mM, and induce expression for 16 h;
[0055] (3) Collect the above culture solutions into collection bottles and centrifuge at 5000 rpm for 15 min;
[0056] (4) Discard the supernatant and wash with 35 mL of protein buffer (50 mM phosphate buffer containing 5 mM Mg 2+ , pH 7.4) and the obtained bacterial precipitate was suspended and placed on ice for crushing and purification.
[0057] 2. Protein purification
[0058] (1) Bacterial disruption: The bacteria in step 1 were disrupted twice using a high-pressure low-temperature disruptor at a pressure of 1200 bar and 4°C. The cells were centrifuged at 4°C and 10,000 rpm for 45 min, and the supernatant was collected;
[0059] (2) Purification: The supernatant is purified by nickel affinity chromatography, and the specific steps are as follows:
[0060] a: Column equilibration: Before loading the supernatant, wash the column with ddH2O for 2 column volumes, then use protein buffer (50mM phosphate buffer containing 5mM Mg 2+ , pH 7.4) to equilibrate a Ni affinity chromatography column for 1 column volume;
[0061] b: Sample loading: slowly pass the supernatant through the Ni affinity chromatography column, repeat once;
[0062] c: Elute impurities: Use protein buffer (50 mM phosphate buffer containing 5 mM Mg 2+ , pH 7.4) for one column volume, and then 50 mL of protein buffer containing 50 mM and 100 mM imidazole were used to elute strongly bound impurities;
[0063] d: Elute the target protein: Use 20 mL of protein buffer containing 250 mM imidazole (50 mM phosphate buffer containing 5 mM imidazole). 2+ , pH 7.4) to elute the target protein, take a few drops of flow-through, prepare samples, and detect them by 12% SDS-PAGE.
[0064] (3) Replacement of liquid: The collected target protein was concentrated by centrifugation (4°C, 3400 r / min) using a 50 mL Amicon ultrafiltration tube (10 kDa, Millipore) to 1 mL. 15 mL of protein buffer without imidazole (50 mM phosphate buffer containing 5 mM Mg 2+ , pH 7.4), concentrated to 1 mL, repeated once, and obtained FSA, FLS, PK, PTA, PhaA, PhaB, and PhaC proteins respectively.
[0065] (4) The concentrated protein is subjected to SDS-PAGE detection and the protein is determined according to the size of the target band.
[0066] Example 3 Production of polyhydroxybutyrate (PHB)
[0067]
[0068] Reaction conditions: D-xylulose final concentration is 20mM, D-fructose-6-phosphate acetalase mutant (FSA-A129S) final concentration is 3mg / ml, formaldehyde condensing enzyme (FLS) final concentration is 2mg / ml, phosphoketolase (PK) final concentration is 2mg / ml, thiamine pyrophosphate (TPP, PK coenzyme) final concentration is 1mM, coenzyme A (CoA) final concentration is 1mM, NADP+ final concentration is 1mM, 6-phosphoglucose (G6P) final concentration is 100mM, glucose-6-phosphate dehydrogenase (G6PD) final concentration is 1U / ml, PTA final concentration is 1mg / ml, PhaA final concentration is 2mg / ml, PhaB final concentration is 1mg / ml, PhaC final concentration is 1mg / ml. Buffer: 50mM phosphate buffer containing 5mM Mg 2+ , pH 7.4. Reaction temperature: 37°C, reaction time: 12h.
[0069] Product detection: PHB was detected by liquid phase. After the reaction was terminated, the supernatant was taken by centrifugation, and the remaining precipitate was dried at 37°C to ensure that there was no remaining liquid. Then 1ml of concentrated sulfuric acid was added and placed at 95°C for 30min. During this period, the concentrated sulfuric acid and the precipitate were mixed evenly. After heating, it was diluted 10 times for liquid phase detection. The liquid phase column used was 87H column. The mobile phase was 5mMH2SO4, the flow rate was 0.6ml / min, the column temperature was 40°C, and the detection was performed at 210nm. The peak time was about 28min.
[0070] Test results: With 20 mM D-xylulose as substrate, 10.58 mM PHB was finally generated, and the conversion rate was 42%.
[0071] Formaldehyde generates 1,3-dihydroxyacetone:
[0072] Reaction conditions: formaldehyde concentration 20 mM, formaldehyde polymerase (FLS) concentration 3 mg / ml, thiamine pyrophosphate (TPP) concentration 1 mM, 1 mM MgSO4, 50 mM K3PO4, pH 8.0, reaction time 3 hours.
[0073] 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. A method for producing acetyl phosphate, characterized in that: The following steps are involved: (1) Using D-xylulose as a substrate to prepare glycolaldehyde and / or dihydroxyacetone; (2) Using glycolaldehyde and / or dihydroxyacetone as a substrate to prepare acetyl phosphate.
2. A method for producing acetyl phosphate as claimed in claim 1, characterized in that: The following steps are involved: S1: converting D-xylulose into glycolaldehyde and / or dihydroxyacetone using aldolase; S2: Conversion of glycolaldehyde and / or dihydroxyacetone into acetyl phosphate using phosphoketolase.
3. A method for producing acetyl phosphate as claimed in claim 2, characterized in that: In step S1, the acetalase comprises D-fructose-6-phosphate acetalase and / or a mutant thereof, preferably, the mutant is FSA-A129S; and / or In step S2, the conversion product of dihydroxyacetone also includes formaldehyde, and the formaldehyde is further converted into dihydroxyacetone by ethanolaldehyde synthase and / or formaldehyde condensing enzyme.
4. A method for producing acetyl phosphate as claimed in claim 2, characterized in that: The D-xylulose is prepared from D-xylose.
5. A method for producing acetyl phosphate as claimed in claim 2, characterized in that: The method further comprises step S3: preparing acetyl-CoA using acetyl phosphate as a substrate, Preferably, acetyl phosphate and coenzyme A are reacted to generate acetyl-CoA using phosphate acetyltransferase.
6. A method for producing acetyl phosphate as claimed in claim 5, characterized in that: The method further comprises step S4: preparing polyhydroxybutyrate using acetyl-CoA as a substrate.
7. A method for producing acetyl phosphate as claimed in claim 6, characterized in that: Acetyl-CoA is converted to succinyl-CoA using acetyl-CoA acetyltransferase; succinyl-CoA is converted to hydroxybutyryl-CoA using acetoacetyl-CoA reductase; and hydroxybutyryl-CoA is converted to polyhydroxybutyrate using polyhydroxybutyrate synthetase.
8. A method for producing acetyl phosphate as described in any one of claims 1 to 7, characterized in that: Prepared by one-pot reaction. Preferably, the concentration of D-xylulose is 20-25 mM, the concentration of aldolase is 2-4 mg / ml; the concentration of phosphoketolase is 1-3 mg / ml; the concentration of formaldehyde condensing enzyme is 1-3 mg / ml; the concentration of glycolaldehyde synthase is 2-4 mg / ml; the concentration of coenzyme A is 1-3 mg / ml; the concentration of phosphate acetyltransferase is 1-3 mg / ml; the concentration of acetyl-CoA acetyltransferase is 1-3 mg / ml; the concentration of acetyl-CoA reductase is 1-3 mg / ml; the concentration of polyhydroxybutyrate synthase is 1-3 mg / ml; Preferably, the buffer used in the reaction comprises a phosphate buffer, wherein the phosphate buffer comprises Mg 2+ ; Preferably, the reaction pH is 7.2-7.6; Preferably, the reaction temperature is 37°C and the reaction time is 10-14h.
9. A biomaterial for preparing acetyl phosphate, the biomaterial expressing aldolase and phosphoketolase, or comprising genes of aldolase and phosphoketolase; Preferably, the biological material comprises a gene cassette, an expression vector, a microorganism or a plant; Preferably, the biomaterial also expresses the phosphoketolase coenzyme.
10. Use of the biomaterial for preparing acetyl phosphate according to claim 9 in preparing acetyl phosphate, acetyl coenzyme A and polyhydroxybutyrate.