Method for preparing 10-hydroxy-2-caproleic acid through escherichia coli engineering bacteria resting cells

A technology of Escherichia coli and resting cells, applied in the field of biological fermentation, can solve problems such as related technologies that have not been reported, achieve the effect of improving conversion rate and realizing industrial production

Active Publication Date: 2019-03-01
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no report on the related technology of producing 10-hydroxy-2-decenoic acid by biological fermentation method

Method used

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  • Method for preparing 10-hydroxy-2-caproleic acid through escherichia coli engineering bacteria resting cells
  • Method for preparing 10-hydroxy-2-caproleic acid through escherichia coli engineering bacteria resting cells
  • Method for preparing 10-hydroxy-2-caproleic acid through escherichia coli engineering bacteria resting cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: PCR amplification of Escherichia coli acyl-CoA thioesterase gene ydiI

[0051] According to Escherichia coli acyl-CoA thioesterase gene ydiI design PCR amplification primers, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2;

[0052] Wherein, the nucleotide sequence of Escherichia coli acyl-CoA thioesterase gene ydiI is shown in SEQ ID NO.3, and the amino acid sequence of the expressed small molecule thioesterase is shown in SEQ ID NO.4.

[0053] Using the above primers for PCR amplification, the reaction system is as follows:

[0054]

[0055] The PCR reaction conditions are as follows:

[0056]

[0057] PCR product recovery:

[0058] After PCR amplification, the length of the fragment was analyzed by 1% agarose gel electrophoresis, and the results were as follows: figure 2 As shown, the target band was excised according to the size of the fragment, a...

Embodiment 2

[0059] Embodiment 2: Construction of recombinant plasmid pET-28a-ydiI

[0060] The double enzyme digestion reaction of the PCR product recovered in Example 1 and the pET-28a plasmid vector containing the sumo tag, the reaction system is as follows:

[0061]

[0062] Reaction conditions: react at 37°C for 1.5h.

[0063] PCR products and plasmid vectors were digested and purified by 1% agarose gel electrophoresis, and the target fragments were recovered using a DNA gel recovery kit.

[0064] Ligate the digested PCR product with the plasmid vector that has also been digested, and the ligation reaction system is as follows:

[0065]

[0066] The above ligation reaction system was thoroughly mixed and then centrifuged for a few seconds, and the droplet on the tube wall was collected at the bottom of the tube, and ligated overnight at 16°C to obtain the recombinant plasmid pET-28a-ydiI.

Embodiment 3

[0067] Embodiment 3: Transformation of recombinant plasmid pET-28a-ydiI:

[0068] (1) Preparation of Competent Cells

[0069] ①Pick a single colony of Escherichia coli BL21 (DE3) (or pick a preserved strain) and inoculate it into 10ml of liquid LB medium, culture overnight at 37°C and 210rpm;

[0070] ② Inoculate 5ml of the bacterial solution into 500ml of LB medium, and cultivate at 37°C and 210rpm until the OD600 of the bacterial solution is about 0.375;

[0071] ③ Place the bacterial solution on the ice-water mixture for 10 minutes, and pre-cool the 50ml centrifuge tube at the same time;

[0072] ④ Transfer the bacterial solution to a centrifuge tube, centrifuge at 3700 rpm for 10 minutes at 4°C to collect the bacterial cells;

[0073] ⑤Add 10mL pre-cooled 0.1M CaCl to each centrifuge tube 2 solution, resuspend the bacteria, and then add 30mL pre-cooled 0.1MCaCl 2 solution, mix it upside down, and let it stand on ice for 20 minutes;

[0074] ⑥Collect the cells by centr...

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Abstract

The invention relates to a method for preparing 10-hydroxy-2-caproleic acid through escherichia coli engineering bacteria resting cells. The method includes the following steps that (1) escherichia coli engineering bacteria containing recombinant plasmids pET-28a-ydiI is constructed; (2) the escherichia coli engineering bacteria containing the recombinant plasmids pET-28a-ydiI is taken to prepareinduced cells; and (3) the induced cells are cultured in a transformed culture medium to obtain the resting cells, and 10-hydroxy capric acid is then added into the culture medium to prepare the 10-hydroxy-2-caproleic acid. According to the method for preparing the 10-hydroxy-2-caproleic acid through the escherichia coli engineering bacteria resting cells, positive recombinant escherichia coli, containing thioesterase, modified through genetic engineering is disclosed for the first time, after special induction treatment, the 10-hydroxy-2-caproleic acid can be produced by fermentation throughthe resting cells, and a large quantity of biosynthesis of the 10-hydroxy-2-caproleic acid is realized.

Description

technical field [0001] The invention relates to a method for preparing 10-hydroxy-2-decenoic acid by using resting cells of Escherichia coli engineering bacteria, and belongs to the technical field of biological fermentation. Background technique [0002] 10-hydroxy-2-decenoic acid (10-hydroxy-2-decenoic acid, 10-HDA) is a monounsaturated fatty acid containing a hydroxyl group, and its molecular formula is C10H18O3. So far, it has only been found in royal jelly and propolis in nature, so it is also called royal jelly acid. Studies have shown that 10-HDA has many important physiological functions such as antibacterial, immune regulation, anti-oxidation, anti-tumor, and lowering blood sugar. It has extremely high medical and health care values, and has a wide application prospect. The structure of the compound is as follows: [0003] In view of the extensive and important application value of 10-HDA, the research on finding efficient, convenient and low-cost production metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N1/21C12R1/19
CPCC12N9/16C12P7/42C12Y301/02
Inventor 王瑞明孙淑慧苏静汪俊卿杨晓慧
Owner QILU UNIV OF TECH
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