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Pseudomonas rhodesiae engineering bacteria and application thereof in preparation of 2, 5-furandicarboxylic acid

A technology of furandicarboxylic acid and Pseudomonas, applied in application, genetic engineering, bacteria and other directions

Active Publication Date: 2021-09-14
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no single enzyme or single wild-type strain that can simultaneously oxidize HMF to FDCA through two pathways
There is no report on the one-pot synthesis of FDCA using mixed bacteria and multicellular as a biocatalyst

Method used

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  • Pseudomonas rhodesiae engineering bacteria and application thereof in preparation of 2, 5-furandicarboxylic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Screening and preservation of Pseudomonas halleri.

[0033] The soil at 5 different locations was selected on the back hill of Nanjing Forestry University, and HMF was irrigated irregularly for a long time. The concentration of HMF used was low at the beginning, and gradually increased until the concentration of HMF reached 5g / L. Afterwards, soil samples were collected at the above-mentioned 5 locations, and each 1 g of the mixed soil sample was mixed with 9 mL of sterile normal saline evenly, and 500 microliters were inoculated into 50 mL of LB medium containing 15 mM HMF. Incubate for 14 hours. Inoculate 500 microliters of the culture solution into 50 mL of LB medium containing 30 mM DFF, and incubate at 30°C and 200 r / min for 14 hours. Dilute the culture medium with 10 -4 、10 -5 、10 -6 Three dilutions were spread on solid LB medium containing 30mM HMF, and cultured at 30°C until microbial colonies appeared. Pick a single colony and inoculate it into 5...

Embodiment 2

[0035] Embodiment 2: Construction of Pseudomonas halleri engineering bacteria A.

[0036] (1) Genomic DNA of Pseudomonas hallii CCTCC NO:M 2021356 was prepared by conventional methods. For this process, please refer to the method for small-scale preparation of bacterial genomes in the "Guidelines for Molecular Biology" published by Science Press. Use synthetic primers xdhC1-f and xdhC1-r to PCR amplify from the above genomic DNA to obtain the putative upstream homology arm of the XdhC family protein coding gene 1; use synthetic primers xdhC2-f and xdhC2-r to obtain The downstream homology arm of putative XdhC family protein-coding gene 1 was amplified by PCR. Recombination PCR was performed using the obtained upper and lower homology arms as templates, and then the recombination fragment was amplified by PCR using primers xdhC1-f and xdhC2-r.

[0037] The recombinant fragment and the suicide plasmid pK18mobSacB were respectively digested with EcoR I and Hind III, and the dige...

Embodiment 3

[0058] Embodiment 3: Construction of Pseudomonas halleri engineering bacteria B.

[0059] Chemically synthesized 5-hydroxymethylfurfural / furfural oxidoreductase gene sequence, the nucleotide sequence of which is shown in SEQ ID NO.3. And according to conventional molecular biology methods, using -Uni Seamless Cloning and Assembly Kit (purchased from Quanshijin) was cloned into pBBR1MCS-2 plasmid (purchased from Addgene). The specific operation is as follows:

[0060] (1) Design two pairs of primers, which are used to amplify the target gene and plasmid respectively.

[0061] (2) The primers for amplifying the target gene are:

[0062] Upstream primer: 5'-TAACAATTTCACACAGGAAACAGCTATGCTGAACAGGCAGGAGAC-3'

[0063] Downstream primers:

[0064] 5'-GAATTTTAACAAAATATTAACGCTCATTGCAAGGGGATGGCCG-3'

[0065] (3) The primers for amplifying the plasmid are:

[0066] Upstream primer: 5'-GCGTTAATATTTTGTTAAAATTC-3'

[0067] Downstream primer: 5'-AGCTGTTTCCTGTGTGAAATTGTTA-3'

[0068]...

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Abstract

The invention discloses two pseudomonas rhodesiae engineering bacteria and application thereof in preparation of 2, 5-furandicarboxylic acid. The 2, 5-furandicarboxylic acid is prepared by constructing a mixed bacteria multi-cell catalytic system by constructing genetic engineering bacteria of pseudomonas rhodesiae and oxidizing 5-hydroxymethylfurfural as a biocatalyst. The pseudomonas rhodesiae engineering bacteria and the application thereof in preparation of the 2, 5-furandicarboxylic acid have the advantages that (1) the mixed bacteria multi-cell catalytic system is adopted for the first time, and FDCA is synthesized by a one-pot method; and (2) the FDCA synthesis process is simple and controllable, three-step continuous oxidation reaction is carried out in the same system, the reaction system is simple, the reaction conditions are mild, expensive cofactors are not needed, intermediate oxidation products are not accumulated, the yield and the productivity of the FDCA are high, and the industrial application potential is achieved.

Description

technical field [0001] The invention relates to the field of biocatalytic conversion, in particular to engineering bacteria of Pseudomonas halleri and its application in the preparation of 2,5-furandicarboxylic acid. Background technique [0002] Biomass is considered to be the most promising raw material to replace petrochemical resources in the production of high-value chemicals due to its abundance, renewability, and environmental friendliness. 5-Hydroxymethylfurfural (HMF) is an important platform compound that can be prepared from biomass. HMF can be oxidized to a variety of furan carboxylic acids, among which 2,5-furandicarboxylic acid (FDCA) has attracted special attention from researchers because of its potential to replace terephthalic acid, a petroleum-based bulk chemical. [0003] FDCA is a bio-based aromatic monomer that can be used to synthesize high-performance polyesters, polyamides, and epoxy resins. It has been identified as one of the 12 most potential bio...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/53C12P17/04C12R1/38
CPCC12N9/0006C07K14/21C12P17/04
Inventor 郑兆娟刘青谭黄虹欧阳嘉王静
Owner NANJING FORESTRY UNIV
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