Method for preparing 10-hydroxyl-2-decenoic acid with decylic acid as raw material and by utilizing escherichia coli engineering bacteria

A technology of Escherichia coli and engineering bacteria, which is applied in the field of preparing 10-hydroxy-2-decenoic acid, can solve the problems of related technologies that have not been reported, and achieve the effect of improving conversion rate and efficient assembly

Active Publication Date: 2019-06-18
QILU UNIV OF TECH
View PDF2 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report on the production of 10-hydroxyl-2-decenoic acid by fermentation of cheap raw materials such as capric acid

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for preparing 10-hydroxyl-2-decenoic acid with decylic acid as raw material and by utilizing escherichia coli engineering bacteria
  • Method for preparing 10-hydroxyl-2-decenoic acid with decylic acid as raw material and by utilizing escherichia coli engineering bacteria
  • Method for preparing 10-hydroxyl-2-decenoic acid with decylic acid as raw material and by utilizing escherichia coli engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: PCR amplification of Escherichia coli acyl-CoA thioesterase gene ydiI, MCAD, FadK, P450 fusion enzyme.

[0073] According to Escherichia coli ester acyl-CoA thioesterase gene P450 fusion enzyme, FadK, MCAD, ydiI design PCR amplification primer, the nucleotide sequence of upstream primer is shown in SEQ ID NO.1,3,5,7 respectively, downstream The nucleotide sequences of the primers are shown in SEQID NO.2, 4, 6, and 8;

[0074] Wherein, the nucleotide sequences of the Escherichia coli acyl-CoA thioesterase gene P450 fusion enzyme, FadK, MCAD, and ydiI are shown in SEQ ID NO.9, 10, 11, and 12 respectively, and the expressed small molecule thioesterase The amino acid sequence is shown in SEQ ID NO.13, 14, 15 and 16.

[0075] The construction contains recombinant plasmid pBbB5K-P450 fusion enzyme, comprising the following steps:

[0076] The codon-optimized fusion enzyme gene of alkane hydroxylase CYP153A of Marinobacter aquaeolei and P450 NADH reductase of Baci...

Embodiment 2

[0101] Embodiment 2: Construction of recombinant plasmid pBbB5K-ydiI, pBbB5K-MCAD, pBbB5K-FadK, pBbB5K-P450 fusion enzyme

[0102] The PCR product recovered in Example 1 and the double enzyme digestion reaction of the pBbB5K-GFP plasmid vector, the reaction system is as follows:

[0103]

[0104] Reaction conditions: react at 37°C for 1.5h.

[0105] 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.

[0106] Ligate the digested PCR product with the plasmid vector that has also been digested, and the ligation reaction system is as follows:

[0107]

[0108] Mix the above ligation reaction system thoroughly and centrifuge for a few seconds, collect the drop on the tube wall to the bottom of the tube, and ligate at 22°C for 10 minutes to obtain recombinant plasmids pBbB5K-ydiI, pBbB5K-MCAD, pBbB5K-FadK, pBbB5K-P450 fusion enzyme.

Embodiment 3

[0109] Embodiment 3: Transformation of recombinant plasmid pBbB5K-ydiI, recombinant plasmid pBbB5K-MCAD, recombinant plasmid pBbB5K-FadK, recombinant plasmid pBbB5K-P450 fusion enzyme:

[0110] (1) Preparation of Competent Cells

[0111] 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;

[0112] 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;

[0113] ③ Place the bacterial solution on the ice-water mixture for 10 minutes, and pre-cool the 50ml centrifuge tube at the same time;

[0114] ④ Transfer the bacterial solution to a centrifuge tube, centrifuge at 3700 rpm for 10 minutes at 4°C to collect the bacterial cells;

[0115] ⑤Add 10mL of pre-cooled 0.1M CaCl2 solution to each centrifuge tube, resuspend the bacteria, then add 30mL of pre-cooled 0.1M Ca...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a method for preparing 10-hydroxyl-2-decenoic acid by utilizing escherichia coli engineering bacteria. The method comprises the following steps that (1) a recombinant plasmidpBbB5K-P450 fusion enzyme, recombinant plasmids pBbB5K-FadK, recombinant plasmids pBbB5K-MCAD and recombinant plasmids pBbB5K-YdiI are constructed; (2) pBbB5K-ydiI-MCAD-FadK-P450 fusion enzyme recombinant plasmids are constructed; (3) the fusion enzyme recombinant plasmids are converted into escherichia coli, the escherichia coli are screened and subjected to induction culture, and thus inductioncells are prepared; and (4) the induction cells are cultured by a transformation culture medium, thus resting cells are prepared, then decylic acid is added into the culture medium, culturing is conducted, and thus the 10-hydroxyl-2-decenoic acid is prepared. The 10-hydroxyl-2-decenoic acid is produced in a fermented mode by utilizing the decylic acid as a raw material, and the process of producing the 10-hydroxyl-2-decenoic acid with the high added-value through the low-value decylic acid is realized.

Description

technical field [0001] The invention relates to a method for preparing 10-hydroxy-2-decenoic acid by using 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, the molecular formula is C 10 h 18 o 3 . 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 methods of 10-HDA...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N15/70C12R1/19
Inventor 苏静王瑞明王芬孙淑慧汪俊卿杨晓慧
Owner QILU UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap