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Preparation method of allose 6-phosphate, acetyl phosphate and acetyl coenzyme A

A technology of allose phosphate and allulose phosphate, which is applied in the fields of biotechnology and medicine, can solve the problems of low reaction rate, low efficiency, and difficulty in accumulating high concentrations of glycolaldehyde, and achieves high catalytic activity and reaction efficiency. High and affinity effect

Pending Publication Date: 2022-04-19
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0007] The reaction rate of the preparation method of above-mentioned acetyl phosphate is lower, and the conversion number K of phosphoketolase is for fructose cat Only 0.1 / s, the efficiency is very low, and the long reaction time (18h) will test the stability of the enzyme and product
In addition, the third step, the cracking of D-erythrose into glycolaldehyde is a reversible process, and its reaction rate is affected by the concentration of glycolaldehyde after cracking, making it difficult to accumulate high concentrations of glycolaldehyde in the system, which will lead to the formation of acetyl phosphate The speed is difficult to guarantee
The acetyl phosphate generated by the second enzymatic reaction is likely to affect the yield of the last enzymatic reaction due to product inhibition. If the second enzymatic reaction generates acetyl phosphate and removes it immediately, it will complicate the entire preparation process.

Method used

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  • Preparation method of allose 6-phosphate, acetyl phosphate and acetyl coenzyme A
  • Preparation method of allose 6-phosphate, acetyl phosphate and acetyl coenzyme A
  • Preparation method of allose 6-phosphate, acetyl phosphate and acetyl coenzyme A

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Experimental program
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Effect test

Embodiment 1、6

[0039] Preparation of embodiment 1, allose 6-phosphate

[0040] At an initial concentration of 10 mM of glycolaldehyde GALD, under the action of deoxyribose aldolase (EC 4.1.2.4, NP_418798.1) derived from Escherichia coli, 2.5 mM erythrose 4-phosphate was added to carry out 6-phosphate aldolase For the preparation reaction of Lulose, react at 37°C for 0.5h.

[0041] Detection of Au6P: take 50 μL of the reaction solution, lyophilize the reaction solution, and derivatize with 30 μL of methoxyamine hydrochloride and 90 μL of trimethylsilyltrifluoroacetamide for 1 h respectively, the derivatization temperature is 37 ° C, and the GC-MS Detection of allose 6-phosphate content. The content of allose 6-phosphate quantitatively detected by GC-MS was 1.5 mM, and the average synthesis rate of allose 6-phosphate was 50 μmol (allose 6-phosphate) / min / mg (enzyme protein).

Embodiment 2

[0042] Embodiment 2, the preparation of acetyl phosphoric acid

[0043] Glycoaldehyde GALD at an initial concentration of 10 mM, thiamine pyrophosphate ThDP at a concentration of 1 mM, PO 4 3+ Add fructose-6-phosphate phosphoketolase Fpk (EC 4.1.2.22) from Bifidobacterium adolescentis, deoxyribose aldolase DeoC (EC 4.1.2.4) from Escherichia coli, and allose-6 at a concentration of 2 mM. -Phosphate isomerase RpiB (EC 5.3.1.6), psicose-6-phosphate 3-epimerase AlsE (EC 5.1.3.-) and 2.5mM erythrose 4-phosphate for acetyl phosphate Reactions were prepared to detect the production of acetyl phosphate and the consumption of glycolaldehyde.

[0044] Detection of acetyl phosphate: mix 40μL reaction sample with hydroxylamine solution (2M, pH 6.5), react for 10min, then add trichloroacetic acid solution (15g / 100mL), hydrochloric acid solution 1 (4M) and ferric chloride solution (5g / 100mL of hydrochloric acid solution 2, wherein the concentration of hydrochloric acid solution 2 is 0.1...

Embodiment 3

[0046] Embodiment 3, the construction of the recombinant bacteria of synthesizing poly-3-hydroxybutyrate

[0047] The gene for the synthesis of phosphoketolase was integrated into the genome of Escherichia coli K-12MG1655, and then the genes (PhaA, PhaB and PhaC) for the synthesis of poly-3-hydroxybutyrate (PHB) and the aldolase The plasmid of the gene of DeoC was transferred into the above-mentioned strains. IPTG-inducible promoters were selected for the above plasmids (the rest of the required enzymes already exist in Escherichia coli). The above strains were cultured in LB medium. After 2.5 hours of culture, the bacterial cell OD 600 When the value reaches 0.8-1.0, add IPTG to a final concentration of 0.5mM to induce (16°C, 12h) expression of the target enzyme protein on the above plasmid. After collecting the bacteria by centrifugation (4°C, 8000r / min, 30min), suspend the bacteria with M9 medium without adding carbon source and nitrogen source, and re-centrifuge to colle...

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Abstract

The invention discloses a method for further synthesizing acetyl coenzyme A by reacting glycolaldehyde with 4-erythrose phosphate under the catalysis of aldolase to generate 6-allose phosphate. The method disclosed by the invention is high in catalytic rate, the theoretical carbon yield of a reaction route is 100%, carbon loss is avoided, the 4-erythritol phosphate, the enzyme and the coenzyme can be recycled, and the reaction efficiency is relatively high. The method disclosed by the invention can play more obvious advantages in the production processes of in-vitro continuous multi-enzyme catalysis, fed-batch fermentation, continuous fermentation and the like capable of controlling the substrate level.

Description

technical field [0001] The invention relates to the fields of biotechnology and medical technology, in particular to a method for synthesizing allose-6-phosphate, acetyl phosphate, acetyl-CoA and derivatives thereof by using glycolaldehyde. Background technique [0002] Acetyl coenzyme A (AcCoA) is a key intermediate in the synthesis of essential biological compounds, including polyketides, fatty acids, isoprenoids, alkaloids, vitamins, and amino acids, among others. Metabolites derived from acetyl-CoA are primary and secondary metabolites, which include compounds of industrial utility. Acetyl-CoA can be generated from acetyl phosphate (AcP) (for example, it can be catalyzed by phosphoacetyltransferase Pta (EC 2.3.1.8)), and then generate a series of biological products based on acetyl-CoA, which are widely used in biocatalysis, etc. field. The synthesis method of acetyl phosphate, for example: patent application WO2015 / 144447A1 discloses a method using phosphoketolase (EC...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12P19/24C12P9/00C12P17/18C12N9/88C12N1/21C12N15/60C12N15/53C12N15/54C12R1/19
CPCC12P19/02C12P19/24C12P9/00C12P17/182C12N9/88C12N9/1029C12N9/0006C12Y203/01009C12Y101/01036C12Y203/01
Inventor 马红武袁倩倩毛雨丰杨雪成颖罗家豪
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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