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A recombinant bacterium that utilizes xylose to produce glycolic acid and its construction method and application

A technology of recombinant bacteria and glycolic acid, applied in the field of genetic engineering

Active Publication Date: 2020-02-21
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But still do not take xylose as substrate in the prior art, utilize the report of this pathway to successfully synthesize glycolic acid

Method used

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  • A recombinant bacterium that utilizes xylose to produce glycolic acid and its construction method and application
  • A recombinant bacterium that utilizes xylose to produce glycolic acid and its construction method and application
  • A recombinant bacterium that utilizes xylose to produce glycolic acid and its construction method and application

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Experimental program
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Embodiment 1

[0054] The cloning of embodiment 1 exogenous gene

[0055] Xylose dehydrogenase gene: (xdh) (Gene ID: 7329904) was cloned using C. crescentus as a template and obtained by PCR amplification. The primer sequence was: xdh-F 5'-GGGAATTCCATATGTCCTCAGCCATCTATCCC-3', xdh-R5 '-CGGGGTACCTCAACGCCAGCCGGCGTCGAT-3'; the clone of xylonolactonase gene (xylC) (Gene ID: 7329903) is obtained by PCR amplification using C. crescentus as a template, and the primer sequence is:

[0056] xylC-F 5'-CCGGAATTCTAATACGACTCACTATAGGGGAATTG-3', xylC-R 5'-AAGGAAAAAAGCGGCCGCTTAAACCAGACGAACTTCGTGCTG-3'; xylonate dehydratase gene (yjhG) (Gene ID: 946829) was cloned using E.coli as a template and amplified by PCR Obtained, the primer sequence is: yjhG-F 5'-GGAATTCCATATGTCTGTTCGCAATATTTTGC-3', yjhG-R5'-CCGCTCGAGTCAGTTTTTTATTCATAAAATCGCG-3'; ) clone is obtained by PCR amplification using E.coli as a template. The primer sequence is: yjhH-F5'-CCGCCATGGCATGAAAAAATTCAGCGGCAT-3', yjhH-R 5'-CCGGAATTCTCAGACTGGTAAAATGC...

Embodiment 2

[0057] The construction of embodiment 2 recombinant plasmids

[0058] 1. Construction process of recombinant plasmid pETDuet-1-yjhH-xdh-xylC

[0059] 1) The xylose dehydrogenase gene xdh cloned in Example 1 and the carrier pETDuet-1 were double-digested with NdeI and KpnI, and the target fragment xdh and the carrier pETDuet-1 after digestion were recovered with a recovery kit, and then ligated. The ligation product was transformed into E.coli DH5α, and positive clones were screened to obtain the recombinant plasmid pETDuet-1-xdh;

[0060] 2) After the xylC gene cloned in Example 1 and the recombinant plasmid pETDuet-1-xdh were digested with EcoRI and NotI, the target fragment xylC and the vector pETDuet-1-xdh after digestion were recovered using a recovery kit, and then ligated. The product was transformed into E.coli DH5α, and positive clones were screened to obtain the recombinant plasmid pETDuet-1-xdh-xylC;

[0061] 3) After the cloned yjhH gene obtained in Example 1 and ...

Embodiment 3

[0065] Example 3 Recombinant strain construction

[0066] Prepare the wild-type control strain E.coliBL21 (DE3) competent according to the operation steps of the TAKARA Competent Preparation Kit, and transform the recombinant plasmids pETDuet-1-yjhH-xdh-xylC and pACYCDuet-1-aldA-yjhG into Competent cells of the host strain E.coliBL21 (DE3) were used to obtain a recombinant strain, numbered ZG-2562.

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Abstract

The invention discloses recombinant bacteria using xylose to produce glycollic acid and a building method and application of the recombinant bacteria and belongs to the technical field of genetic engineering. The xylose dehydrogenase gene, xylonic acid lactonase gene, xylonic acid dehydratase gene, 3-deoxygenation-D-glycerin ketopentose acid aldolase gene and glycolic aldehyde dehydrogenase gene in the recombinant bacteria using the xylose to produce the glycollic acid are overexpressed. Meanwhile, the invention further provides a preparation method of the recombinant bacteria and a method using the recombinant bacteria to produce the glycollic acid. By the recombinant bacteria, the biological synthesizing path using D-xylose as the carbon source to form the glycollic acid through glycolic aldehyde conversion is achieved for the first time.

Description

technical field [0001] The invention relates to a recombinant bacterium for producing glycolic acid by using xylose, its construction method and application, and belongs to the technical field of genetic engineering. Background technique [0002] Organic acids are a class of compounds that are currently in great demand in the market. The annual production of organic acids is about millions of tons, and they are used in many fields such as food, medicine, cosmetics, and chemical industry. At the same time, organic acids are also an important class of bio-based products synthesized by metabolically modified microorganisms. Glycolic acid is the simplest α-hydroxy acid, which has important use value in many fields, such as in the cosmetic industry: Glycolic acid can effectively protect the flexibility of the skin and improve the skin's disease resistance; Glycolic acid compound metal Ions can be used as a decontamination cleaning agent; glycolic acid monomers or polymers with o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/53C12N15/55C12N15/60C12P7/42C12R1/19
CPCC12N9/0006C12N9/0008C12N9/18C12N9/88C12P7/42C12Y101/01C12Y102/01021C12Y301/01C12Y401/02C12Y402/01082
Inventor 赵广咸漠刘敏
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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