Genetically engineered bacterium with high yield of trans-aconitic acid as well as construction method and application of genetically engineered bacterium

A technology of trans-aconitic acid and genetic engineering strains, applied in the field of genetic engineering, can solve the problems of no large-scale production, many by-products, complex processes, etc., and achieve strong operability, strong application value, and easy screening Effect

Active Publication Date: 2020-12-01
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, trans-aconitic acid is mainly produced by chemical synthesis, the process is complicated, there are many by-products, the cost is high, and no large-scale production has been formed. The effective supply of trans-aconitic acid will promote the downstream application and product development. factor

Method used

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  • Genetically engineered bacterium with high yield of trans-aconitic acid as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium with high yield of trans-aconitic acid as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium with high yield of trans-aconitic acid as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1. Construction of a genetically engineered strain with high yield of trans-aconitic acid

[0035] 1. Construction of targeting elements for knocking out CICC_3028g

[0036] according to figure 1 The targeting element strategy diagram of CICC_3028g, and the construction method of the recombinant Aspergillus terreus described in the present invention are described.

[0037] Amplification of the upstream and downstream homology arms of the cytochrome P450 monooxygenase gene: U-3028g-F (5'-CGACGGCCAGTGAATTCGAGctggtgggatgctgggtttagtct-3') and U-3028g-R (hph2) (5'-ctttacgcttgcgatcccgaagtcttcggaagacggcca-3') As a primer pair, the genome of Aspergillus terreus CICC40205 was used as a template for PCR amplification, and the upstream homology arm fragment of the CICC_3028g gene was obtained after the product was purified and recovered; D-3028g-R (5'-agatgtgcggcagatcagatccatcg-3') was used as a primer pair, and the genome of Aspergillus terreus CICC40205 was used as a t...

Embodiment 2

[0049] Example 2. Fermentation of recombinant Aspergillus terreus to produce trans-aconitic acid

[0050] 1. Shake flask screening of recombinant Aspergillus terreus strains producing trans-aconitic acid

[0051] The stably passaged recombinant Aspergillus terreus strains obtained in Example 2 were respectively inoculated into Aspergillus terreus sporulation medium, and cultured at 32° C. for 6 days to obtain mature spores. Then respectively inoculate the spores cultivated to maturity into the organic acid fermentation medium IPM (55ml IPM medium is housed in a 500ml Erlenmeyer flask), make two parallel bottles for each strain, and ferment for 72 hours in a shaker at 37°C with a rotation speed of 220rpm . The hyphae in the fermentation broth were removed by filtration, and the fermentation supernatant was properly diluted for HPLC analysis.

[0052] 2. Analysis of the content and purity of trans-aconitic acid in the fermentation broth

[0053] The fermentation supernatants ...

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Abstract

The invention discloses a genetically engineered bacterium with high yield of trans-aconitic acid as well as a construction method and application of the genetically engineered bacterium, and belongsto the field of genetic engineering. Chemical synthesis of trans-aconitic acid has the defects of more byproducts and complex process. Aiming to overcome the production defects of the trans-aconitic acid, the invention constructs the genetically engineered bacterium for producing the trans-aconitic acid. The method comprises the following steps: subjecting Cytochrome P450 monooxygenase gene CICC_3028g in At-delta cadA aspergillus terreus to gene mutation to obtain recombinant aspergillus terreus. The content of the trans-aconitic acid produced by fermentation of the recombinant aspergillus terreus is remarkably increased compared with that of trans-aconitic acid produced by the At-delta cadA aspergillus terreus.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetically engineered bacterium with high yield of trans-aconitic acid, its construction method and application. Background technique [0002] Aconitic acid (propylene-1,2,3-tricarboxylic acid, 1,2,3-Propenetricarboxylic acid) is named after it is extracted from aconitum, and is an unsaturated tricarboxylic acid. Because it contains unsaturated double bonds and abundant hydroxyl groups, it can be used as a monomer compound for the preparation of polymer materials, and can also be used as a precursor for the synthesis of other compounds, such as trimethyltrans-aconitic acid, etc. Aconitic acid has two configurations, cis and trans. Among them, cis-aconitic acid (CAS: 585-84-2) is an intermediate in the isomerization of citric acid catalyzed by aconitase in the tricarboxylic acid cycle to generate isocitrate. product. Aconitic acid can be degraded by cis-aconitic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/53C12N15/10C12P7/48C12R1/66
CPCC12N9/0071C12Y114/14001C12N15/102C12P7/48Y02A50/30
Inventor 吕雪峰耿策黄雪年王敏靳志刚曹海峰郭强李继彬
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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