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A kind of recombinant escherichia coli producing glycolic acid and its construction method and application

A technology for recombining Escherichia coli and Escherichia coli, applied in microorganism-based methods, other methods of inserting foreign genetic materials, applications, etc., to achieve the effects of enhancing synthesis, enhancing expression, and reducing the loss of carbon and sulfur

Active Publication Date: 2022-06-14
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]The main technical limitation of biological synthesis of glycolic acid is still the low theoretical conversion rate of the pathway adopted, so a new route with high conversion rate needs to be developed urgently

Method used

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  • A kind of recombinant escherichia coli producing glycolic acid and its construction method and application
  • A kind of recombinant escherichia coli producing glycolic acid and its construction method and application
  • A kind of recombinant escherichia coli producing glycolic acid and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0219] The construction of embodiment 1 recombinant escherichia coli engineering strain GA00-GA07

[0220] 1. Construction of a recombinant plasmid (pYB1s-ghrA) expressing glyoxylate reductase derived from large intestine.

[0221] (1) Extraction of Escherichia coli genomic DNA and PCR amplification of ghrA gene.

[0222] Genomic DNA of Escherichia coli BW25113 was extracted using a bacterial genome extraction kit (Tiangen Biochemical Technology Co., Ltd., product catalog DP302). Using the total genomic DNA extracted from Escherichia coli as a template, using ghrA-F and ghrA-R as primers (Table 1), high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog AP221) was used for PCR amplification. The PCR product was obtained, and the PCR product was subjected to agarose gel electrophoresis, and the target fragment was recovered to obtain the gene fragment ghrA-NX comprising the gene ghrA (nucleotide sequence shown in SEQ ID No.2...

Embodiment 2

[0259] Example 2 Production of Glycolic Acid Using Recombinant Escherichia coli Strains GA00, GA06, and GA07 Using Glucose as a Raw Material

[0260] 1. The formula of the culture medium

[0261] (11) The composition of LB medium is as follows:

[0262] Yeast powder: 5g / L

[0263] Peptone: 10g / L

[0264] NaCl: 10g / L

[0265] (12) The formula of self-induction medium ZYM is as follows:

[0266] 100mL A+2mL B+2mL C+200μL D+100μL E (the following are mass percentage concentrations);

[0267] A.ZY: 1% tryptone, 0.5% yeast powder;

[0268] B.50×M: 1.25M Na 2 HPO 4 , 1.25M KH 2 PO 4 , 2.5M NH 4 Cl and 0.25M Na 2 SO 4 ;

[0269] C.50×5052: 25% glycerol, 2.5% glucose, 10% L-arabinose;

[0270] D.500×MgSO 4 : 1M MgSO 4

[0271] E.1000×trace elements: 50mM FeCl 3 , 20mM CaCl 2 , 10mM MnCl 2 , 10mM ZnSO 4 , CoCl 2 、NiCl 2 、Na 2 Mo 4 、Na 2 SeO 3 and H 3 BO 3 2mM each.

[0272] (13) The composition of the conversion solution is as follows:

[0273] Glucose 10...

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Abstract

The invention discloses a glycolic acid-producing recombinant Escherichia coli and its construction method and application. The present invention firstly discloses recombinant Escherichia coli, compared with recipient Escherichia coli, the expression amount and / or content and / or activity of the protein A gene, and / or the expression amount and / or content and / or activity of the protein B gene are reduced / or activity; said protein A is at least one of the following: malate thiokinase; malyl-CoA lyase; glyoxylate reductase; isocitrate lyase; isocitrate dehydrogenase kinase / phosphatase; The protein B is at least one of the following: NAD-dependent-malic enzyme; NADP-dependent-malic enzyme; malate synthase A; glyoxylate pathway transcriptional repressor; glycolate oxidase. Further provided are methods of making glycolic acid. In the present invention, the recombinant Escherichia coli constructed by using glucose as a raw material makes the glucose have a higher conversion rate and greatly improves the yield of glycolic acid.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a glycolic acid-producing recombinant Escherichia coli and its construction method and application. Background technique [0002] Glycolic acid (English name Glycolic acid), also known as glycolic acid, is the simplest α-hydroxy acid, which is widely used in industrial processing, cosmetic industry and polymer (PGA, PLGA) synthesis. The price of high-purity glycolic acid is 30,000-40,000 yuan / ton. [0003] At present, the synthesis of glycolic acid mainly includes chemical synthesis and biosynthesis. Chemical synthesis mainly includes chloroacetic acid method, formaldehyde carbonylation method, cyanidation method, transesterification method, etc., but chemical reactions have disadvantages such as harsh reaction conditions, difficult recycling of catalysts, and environmental pollution. Biosynthesis can be subdivided into bioenzyme synthesis and total biosynthesis. The enzymes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N15/60C12N15/53C12N15/54C12N15/52C12N15/31C12P7/42C12R1/19
CPCC12N15/52C12N9/88C12N9/0006C12N9/12C12N9/1025C12N9/93C07K14/245C12N15/70C12N15/902C12P7/42C12Y101/01026C12Y207/11005C12Y401/03001C12Y203/03009C12Y602/01009C12Y401/03C12Y101/0104C12Y101/01038C12Y101/03015
Inventor 刘伟丰杨梦杰朴晓宇陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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