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Method of modifying phenol substances through CO2 biological conversion method and application of the method

A technology for biotransformation and phenolic substances, applied in the field of synthetic biology, can solve problems such as reduction of carboxyl groups, and achieve the effects of reducing carbon emissions, shortening synthesis steps, and reducing separation burden

Inactive Publication Date: 2017-07-04
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no report of using the same strain of engineering bacteria to add carboxyl to the phenolic ring and reduce the carboxyl to the corresponding alcohol or aldehyde.

Method used

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  • Method of modifying phenol substances through CO2 biological conversion method and application of the method
  • Method of modifying phenol substances through CO2 biological conversion method and application of the method
  • Method of modifying phenol substances through CO2 biological conversion method and application of the method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Carboxylase gene from Bacillus subtilis yclBCD (gene sequence 1), salicylate decarboxylase gene of yeast sdc (gene sequence 2) and the carboxylic acid reductase gene in Nocardia car (Gene sequence 3) clone

[0054] (1) Look up Carboxylate Reductase in GenBank car and phosphopantethein transferase sfp The gene sequence of Car was derived from Nocardia NRRL5646 and sfp from Bacillus subtilis ， The sequence names are: AY495697.1 and WP_015715234.1, respectively. The carboxylic acid reductase and phosphopantetheinyl transferase genes were synthesized by a bio company and named as car– sfp, car – sfp The fragment of gene size is 4343bp (gene sequence is 3).

[0055] (2) Check yclBCD The gene sequence in GenBank is: 2632649, 2632650, 2632651 three-segment gene, which is derived from Bacillus subtilis 168 ( B. subtilis subsp. subtilis 168), obtained from the Bacillus subtilis 168 genome by PCR. Cloning of Bacillus subtilis 168 genome by PCR reaction yclBC...

Embodiment 2

[0064] Construction of Escherichia coli BL21(DE3) Engineering Bacteria Recombinantly Expressing Carboxylase and Carboxylate Reductase Genes

[0065] (1) Construction of expression vector

[0066] The cloned carboxylase gene yclBCD fragment and sdc Fragment, with restriction endonucleases Kpn I and Nde I respectively for the carboxylase gene yclBCD fragment and sdc The fragment was subjected to double enzyme digestion, and the 100 μL enzyme digestion system was as follows:

[0067] PCR carboxylase gene product 40 μL, 10×H buffer 10 μL, 10×BSA 10 μL, Kpn I 15 μL, Nde I 15 μL, ddH 2 O 10 μL. After digesting at 37°C for 4 hours, it was recovered by agarose gel electrophoresis.

[0068] The expression vector pETDuet-1 was double digested with restriction endonucleases Kpn I and Nde I, and the 100 μL digestion system was as follows: expression vector pETDuet-1 40 μL, 10×H buffer 10 μL, 10×BSA 10 μL, Kpn I 15 μL, Nde I 15 μL, ddH 2 O 10 μL. After digesting at 37°C for 4 ho...

Embodiment 3

[0077] CO 2 Preparation of p-Hydroxybenzyl Alcohol by Carboxylation and Reduction of Phenol by Biotransformation Method

[0078] (1) Take the recombinant pETDuet obtained in Example 2 of the overnight culture – yclBCD - car –sfp Inoculate genetically engineered bacteria into 50ml LB medium, at 37°C, 200r / min -1 When the absorbance OD600 of the bacterial solution reaches 0.6-0.8, add isopropylthiogalactoside (IPTG) to make the final concentration 0.1mM, at 37°C, 200r / min -1 Under culture for 4-4.5h. Then add 15mmol / L of phenol and 100mmol / L of sodium bicarbonate. After culturing for 12 hours, centrifuge and filter to obtain the fermentation broth.

[0079] The reaction formula is as follows:

[0080]

[0081] (2) HPLC analysis of fermentation products

[0082] Agilent liquid chromatography was used for detection, and the detection conditions were: Kromstar C 18 (250 mm×4. 6mm, 5 μm) chromatographic column, mobile phase methanol (A)-0.1% acetic acid aqueous solution ...

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Abstract

The invention discloses a method of modifying phenol substances through a CO2 biological conversion method. The method comprises the steps of: by means of gene-engineering bacteria, combining CO2 onto an aromatic ring of a phenol substance to obtain hydroxybenzoic acid and a derivative; and reducing the carboxyl group, which is generated after carboxylation of the phenol substance, thereby generating corresponding aldehydes or alcohols. The method not only completely reduces carbon emission, but also technically avoid introduction of solid or liquid impurities due to addition of other carbon sources, so that the method reduces synthesis steps, reduces work load of separation and is more economical.

Description

technical field [0001] The present invention is in the field of synthetic biology and involves the use of CO 2 A method for modifying phenolic substances by biotransformation. By constructing the decarboxylase gene and carboxyl reductase gene of hydroxybenzoic acid and its derivatives in the same strain of bacteria, the obtained genetically engineered bacteria containing two functional enzymes can be converted into CO by one-step fermentation. 2 Add to the aromatic ring of phenolic substances to obtain the corresponding carboxylated products, and further reduce the carboxylated products to obtain the corresponding aldehydes or alcohols. Background technique [0002] Various phenols and their derivatives are widely used in the fields of flavor, food, medicine, and chemical industry. Many of the flavors are low-molecular-weight phenolic substances, such as vanillin and ethyl vanillin. Due to safety reasons, such substances obtained by biotransformation are more acceptable to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12P7/24C12N1/21C12N15/70C12R1/19
CPCC12N9/0008C12N9/1288C12N9/88C12N15/70C12P7/22C12P7/24C12Y102/99C12Y207/08007C12Y401/01061C12Y401/01091
Inventor 戴玉杰师坤澎胡彦营凌君张会图张秀利贾士儒高年发吕和鑫
Owner TIANJIN UNIV OF SCI & TECH
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