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Construction method and application of engineering bacteria for producing muconic acid by using lignin derivatives

A construction method and technology of engineering bacteria, applied in the field of bioengineering, to achieve good economic value and industrial application prospects, and to avoid the effect of direct use

Pending Publication Date: 2021-03-02
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biocatalytic production of muconic acid using lignin-derived vanillic acid as a substrate has not been reported yet

Method used

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  • Construction method and application of engineering bacteria for producing muconic acid by using lignin derivatives
  • Construction method and application of engineering bacteria for producing muconic acid by using lignin derivatives
  • Construction method and application of engineering bacteria for producing muconic acid by using lignin derivatives

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 constructs recombinant escherichia coli

[0033] 1. Construction of pACYC-VanAB plasmid

[0034]Using the Pseudomonas putida KT2440 genome as a template, with the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 as primers (Table 1), PCR amplification VanAB gene fragment (VanA: SEQ ID NO: 9, VanB: SEQ ID NO: 10), PCR amplification conditions: 98°C 2min, 98°C 10s, 58°C 30s, 72°C 1min, cycle 30 times; 72°C 2min, Cut the gel to recover the target band; then digest with BsaI at 37°C for 2 hours; purify and recover the digested fragment by PCR; connect the purified fragment to the expression vector pACYCDuet-1 that has been digested with BamHI and XhoI, and ligate at 16°C Transfer to Escherichia coli TOP10 competent cells for 1 hour, and then verify the positive clone colonies, and extract the pACYC-VanAB plasmid.

[0035] 2. Construction of pRSF-GDC-CatA plasmid

[0036] Using the plasmid containing the GDC / CatA gene as a template (f...

Embodiment 2

[0042] Example 2 Whole Cell Biotransformation Recombinant Strain

[0043] The Escherichia coli recombinant strain prepared in Example 1 was inserted into the TB culture medium at a volume ratio of 1:100 and expanded to OD 600 After reaching 0.4-0.6, add 0.5mM IPTG, induce protein expression at low temperature for 24 hours, and obtain bacteria after centrifugation at 7000rpm for 10min. Catalyze according to 1mL reaction system (10g / L bacterial mass, 5-25mM vanillic acid substrate), and finally add phosphate buffer (200mM, pH=8.0) to make up, at 30°C, 250rpm, the whole cell is catalyzed for 1-24h Afterwards, the production of muconic acid was detected by liquid chromatography.

[0044] Quantitative analysis of the product: the conversion solution was detected and analyzed by Shimadzu high performance liquid chromatography, using photodiode array detector (working wavelength 210nm), Shimadzu C18 column (4.6×250mm, 5μm), flow rate 1mL / min, column temperature 40°C, Injection volu...

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Abstract

The invention relates to a construction method and application of engineering bacteria for producing muconic acid by using lignin derivatives. The method comprises the following steps: transforming recombinant plasmids containing vanillic acid demethylase genes VanAB and recombinant plasmids containing decarboxylase genes GDC and catechol dioxygenase genes CatA into escherichia coli. According tothe embodiment of the invention, the engineering bacterium bacillus obtained by the method can be used for producing muconic acid from 50mM lignin-derived vanillic acid at the conversion efficiency ofmore than 87%, and has a wide industrial application prospect.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for constructing engineering bacteria for efficiently producing muconic acid by using lignin derivatives and its application. Background technique [0002] Adipic acid is an important chemical precursor raw material, which can be used in the synthesis of nylon-6,6. Adipic acid currently used in industry is usually derived from petroleum-based raw materials, which faces problems such as non-renewability and generation of three wastes. The microbial production of adipic acid is usually obtained by using muconic acid as an intermediate and hydrogenation reduction, which has the characteristics of regeneration and less environmental pollution. The currently reported microbial synthesis method usually adopts fermentation method to produce muconic acid. For example, Karen M.Draths et al. reported using Escherichia coli to ferment and produce muconic acid, which can be...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/60C12N15/53C12N1/21C12P7/44C12R1/19
CPCC12N15/70C12N15/52C12N9/0073C12N9/88C12N9/0069C12P7/44C12Y401/01059C12Y113/11001
Inventor 袁吉锋
Owner XIAMEN UNIV
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