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Method for generating target product from glycollic acid under action of enzyme

A technology of glycolic acid and acetate kinase, which is applied in biochemical equipment and methods, botanical equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., to achieve the effect of high light efficiency mechanism, improved utilization efficiency, and less accumulation of intermediate by-products

Pending Publication Date: 2022-06-03
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of these pathways avoids the loss of fixed organic carbon

Method used

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  • Method for generating target product from glycollic acid under action of enzyme
  • Method for generating target product from glycollic acid under action of enzyme
  • Method for generating target product from glycollic acid under action of enzyme

Examples

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Effect test

Embodiment 1

[0062] Example 1: Preparation of glycolaldehyde with glycolic acid as raw material

[0063] Sample 1: Add 20 mM glycolic acid, 1 mM NAD(P)H, 1 mM ATP, 1 mM CoA, ACS, GCR at 2 mg / mL each to a 200 μL system. Sample 2: Add 20 mM glycolic acid, 1 mM NAD(P)H, 1 mM ATP, 1 mM CoA, AckA, PTA, GCR at 2 mg / mL each to a 200 μL system. Control: 20 mM glycolic acid, 1 mM NAD(P)H, 1 mM ATP, 1 mM CoA, no enzyme was added to a 200 [mu]L system. After reacting at 30°C for 1 h, after the reaction was completed, the reaction system was lyophilized. 60 μL of pentafluorobenzene hydroxylamine hydrochloride (PFBOA, 200 mM) was then added, vortexed, and incubated for 1 hour at room temperature. 300 μL of hexane was added and allowed to stand at room temperature for 5 min. A 100 μL sample of the organic layer was taken, and 30 μL of trimethylsilyl trifluoroacetamide containing 1% trimethylchlorosilane and 20 μL of pyridine were added to silylate the PFBOA derivative. The derivatized glycolaldehyde...

Embodiment 2

[0066] Example 2: Preparation of Acetyl-CoA with Glycolic Acid as Raw Material

[0067]Sample 1: 20 mM glycolic acid, 1 mM NAD(P)H, 1 mM ATP, 1 mM CoA, 2 mg / mL each of ACS, PTA, GCR, ACPS were added to a 200 μL system. Sample 2: Add 20 mM glycolic acid, 1 mM NAD(P)H, 1 mM ATP, 1 mM CoA, AckA, PTA, GCR, ACPS at 2 mg / mL each to a 200 μL system. Control: 20 mM glycolic acid, 1 mM NAD(P)H, 1 mM ATP, 1 mM CoA, no enzyme was added to a 200 [mu]L system. After 1 h of reaction at 30 °C, acetyl-CoA was detected by liquid-phase mass spectrometry, and the results were as follows image 3 shown. Depend on image 3 know, use figure 1 Middle cofactors and enzymes can realize the synthesis of acetyl-CoA from glycolic acid, and build a complete glycolic acid utilization pathway.

[0068] Liquid mass spectrometry detection conditions: LC conditions: 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, B is ...

Embodiment 3

[0070] Example 3: Application of Glycolic Acid Synthesis Acetyl-CoA Pathway in Plants

[0071] The genes related to the acetyl-CoA pathway of glycolic acid synthesis were transferred into rice by Agrobacterium transfection, so that AckA, PTA, GCR and ACPS or ACS, PTA, GCR and ACPS proteins were localized and expressed in rice chloroplasts. The transgenic plants were passed for another generation, and the seeds were collected and cultured to obtain T1 homozygous plants. 1 g of leaves of wild-type plants (6 plants) and transgenic plants (6 plants) were taken respectively, extracted by Beijing Nuohezhiyuan Technology Co., Ltd. and analyzed for metabolomics in leaves. The result is as Figure 4 shown.

[0072] The results showed that, compared with wild-type plants, transgenic plants contained higher concentrations of amino acids. The results of significant difference analysis showed that the P values ​​were all lower than 0.01, indicating that the difference in amino acid cont...

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Abstract

The invention discloses a method for producing at least one target product from glycollic acid under the action of enzyme. In order to solve the key problem that 25% of fixed organic carbon needs to be lost in the recycling process of natural glycolic acid in C3 plants, a glycolic acid metabolic pathway including acetokinase, phosphoacetyltransferase, glycolic acid coenzyme A reductase and acetyl phosphate synthase is designed; or the pathway comprises a hydroxyacetyl-CoA synthase, a hydroxyacetyl-CoA reductase, an acetyl phosphate synthase, and an acetyl phosphate transferase. The new glycolic acid metabolic pathway greatly reduces the organic carbon loss caused in the glycolic acid reutilization process, so that the by-product glycolic acid generated by photosynthesis is completely converted into acetyl coenzyme A, and a new thought is provided for improving the photosynthesis of plants. The approach also provides a method for preparing glycolaldehyde or acetyl coenzyme A by taking glycollic acid as a raw material.

Description

technical field [0001] The invention relates to a glycolic acid metabolic pathway, in particular to a method for generating a target product from glycolic acid under the action of an enzyme. Background technique [0002] Plant cells can not only perform photosynthesis under light conditions, but also assimilate CO 2 , release O 2 and synthesize organic matter, and at the same time, an absorption of O 2 , release CO 2 metabolic pathway known as photorespiration. In plants such as wheat, rice and soybean, photorespiration reduces photosynthesis efficiency by 20%-50%, mainly due to the immobilization of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) O 2 However, the recycling efficiency of the generated phosphoglycolic acid is low. Phosphoglycolic acid has an inhibitory effect on the key genes of the Calvin cycle - triose phosphate isomerase and sedum heptulose bisphosphatase, which can inhibit the regeneration of ribulose-1,5-bisphosphate and reduce the effici...

Claims

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Application Information

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IPC IPC(8): C12P9/00C12P17/16C12P7/24C12N9/10C12N9/12C12N9/04C12N9/00C12N15/82A01H5/00A01H6/46A01H6/54A01H6/82A01H6/38
CPCC12P9/00C12P17/16C12P7/24C12N9/1217C12N9/1029C12N9/0006C12N9/1288C12N9/93C12N15/8261C12Y207/02001C12Y203/01008C12Y101/01035C12Y207/08007C12Y602/01001A01H5/00A01H6/38A01H6/46A01H6/54A01H6/82C12N9/00C12N9/0004C12N9/10C12N9/12C12N15/82
Inventor 江会锋逯晓云杨巧玉初斋林卢丽娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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