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Catechol 1,2-dioxygenase gene and application thereof in synthesis of 4-position substituted cis,cis-muconic acid

A catechol and dioxygenase technology, which is applied in the fields of biotechnology and genetic engineering to achieve the effects of high yield, low pollution and low cost

Active Publication Date: 2020-06-09
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the production of 4-ethylmuconic acid and 4-propylmuconic acid catalyzed by Arthrobacter CatA

Method used

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  • Catechol 1,2-dioxygenase gene and application thereof in synthesis of 4-position substituted cis,cis-muconic acid
  • Catechol 1,2-dioxygenase gene and application thereof in synthesis of 4-position substituted cis,cis-muconic acid
  • Catechol 1,2-dioxygenase gene and application thereof in synthesis of 4-position substituted cis,cis-muconic acid

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Experimental program
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Effect test

Embodiment 1

[0048] The cloning of CatA gene and the construction of recombinant vector in embodiment 1 joint bacillus

[0049] The gene encoding CatA comes from Arthrobacter sp.CGMCC No.3584, which has been disclosed in Chinese invention patent application 201010191515.6. Using the above-mentioned Arthrobacter genome as a template, the CatA gene was amplified by PCR.

[0050] The primer sequences are as follows:

[0051] Upstream primers:

[0052] 5'-TGCCGCGCGGCAGC CATATG ACTGAGACCCAAGTGGATATC-3' (contains Nde I restriction site, see the horizontal line)

[0053] Downstream primers:

[0054] 5'-TTGTCGACGGAGCTC GAATTC CTACTTGGTCTCGCCTTCCTT-3' (contains EcoR I restriction site, see the horizontal line)

[0055] The PCR reaction system and reaction conditions are shown in Table 1.

[0056] Table 1 PCR reaction system and reaction conditions of CatA gene

[0057]

[0058]

[0059] The vector pET28a was linearly digested, and the restriction system and reaction conditions are s...

Embodiment 2

[0065] Embodiment 2 Construction of recombinant Escherichia coli BL21 (DE3) (pET28a-ArcatA)

[0066] The constructed recombinant plasmid pET28a-ArcatA was transformed into E.coli BL21(DE3) (purchased from Sangon Bioengineering (Shanghai) Co., Ltd. ), the specific operation is as follows: take 9 μL of the recombinant plasmid (obtained in Example 1), add it to 100 μL of E.coli BL21 (DE3) competent cell liquid, place it on ice for 30 minutes, heat shock at 42°C for 90 seconds, take it out and put it on ice immediately On 2min. Add 900 μL of LB liquid medium, at 37°C, 200 rpm, cultivate for 40 minutes, take 200 μL of culture solution and spread it on LB solid medium containing kanamycin, and the single colony obtained after culturing at 37°C for 12 hours is the recombinant strain E .coli BL21(DE3)(pET28a-ArcatA). Pick a single colony, culture overnight in LB liquid medium, extract the transformant plasmid with a plasmid DNA mini-extraction kit (purchased from AxyPrep Company, C...

Embodiment 3

[0067] The expression condition optimization of embodiment 3 recombinant CatA protein

[0068] 1. The effect of the concentration of inducer IPTG on the expression of recombinant CatA protein

[0069] The recombinant bacterium E.coli BL21 (DE3) (pET28a-ArcatA) constructed in Example 2 was inoculated into the LB liquid medium containing 50mg / L kanamycin, 37°C, 200rpm was cultivated overnight, and then 1% (v The inoculum of / v) was inoculated into a 500mL Erlenmeyer flask containing 200mL LB medium, and the OD 600nm When it reaches 0.6-0.8, no IPTG induction is used as a blank control, and IPTG with a final concentration of 0.05-1.0mM is added respectively (IPTG concentration gradients with final concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0mM are selected) to induce , 20°C, 150rpm induction for 6h. After induction, the bacteria sludge was collected by centrifugation at 8000 rpm for 10 min at 4°C and stored at -20°C for later use.

[0070] Wash the collected sludge twic...

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Abstract

The invention belongs to the field of biotechnology and gene engineering, and particularly discloses a catechol 1,2-dioxygenase gene and an application thereof in synthesis of 4-position substituted cis,cis-muconic acid. The nucleotide sequence of a catechol 1,2-dioxygenase coding gene is shown in SEQ ID NO.1. The expression product catechol 1,2-dioxygenase of the gene can efficiently catalyze 4-position substituted catechol to generate the corresponding 4-position substituted cis,cis-muconic acid. The invention also provides a method for performing whole-cell catalysis to prepare the 4-position substituted cis,cis-mucoconic acid by using a batch feeding strategy; and the method has the advantages of simple process, mild reaction conditions, environmental friendliness, accords with the concept of green economic development, and provides a simple and convenient way for safe and mass production.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and relates to a catechol 1,2-dioxygenase coding gene in the synthesis of 4-substituted cis, cis-muconic acid (4-ethyl muconic acid and 4- Propyl muconic acid) specifically relates to a recombinant bacterium expressing catechol 1,2-dioxygenase and a method for catalyzing the synthesis of 4-position substituted cis, cis-muconic acid by whole cells. Background technique [0002] Muconic acid, also known as adienedioic acid, can be used as the basic raw material of resin, medicine, food and pesticide, and is considered as a valuable intermediate, which can be used to produce such as adipic acid, terephthalic acid Such bulk chemicals. These two products can be used to make nylon-66, industrial plastics, polyester synthetic fibers, etc. Due to the regularity of the main chain of the polymer, most of the nylon presents a white translucent appearance, and the poor transparency limi...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/70C12N1/21C12N9/02C12P7/44C12R1/19
CPCC12N9/0069C12N15/70C12P7/44C12Y113/11001
Inventor 应汉杰宋佳睿牛欢青冷静胡瑞佳朱晨杰陈勇柳东
Owner NANJING UNIV OF TECH
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