Construction and fermentation optimization method of amino-bis-demethoxycurcumin high-yield strain

A kind of technology of demethoxycurcumin and aminobis, applied in the field of metabolic engineering

Active Publication Date: 2021-03-12
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This compound has not been reported before, so it cannot be obtained using natural plant extraction methods

Method used

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  • Construction and fermentation optimization method of amino-bis-demethoxycurcumin high-yield strain
  • Construction and fermentation optimization method of amino-bis-demethoxycurcumin high-yield strain
  • Construction and fermentation optimization method of amino-bis-demethoxycurcumin high-yield strain

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Experimental program
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Embodiment 1

[0036] Embodiment 1, construction and module division of aminobisdemethoxycurcumin synthetic pathway

[0037] In the biosynthetic pathway of curcumin, dimeric ketone synthase DCS and curcumin synthase CURS can use two molecules of coumaryl-CoA and one molecule of malonyl-CoA as substrates to synthesize a molecule of bis-nor Oxycurcuminoids. The present invention intends to use p-aminocinnamic acid as a feeding substrate in order to obtain aminobisdemethoxy curcumin. Therefore, the present invention plans the whole biosynthetic pathway into three modules ( figure 1 ). Module I contains the 4-coumaroyl-CoA ligase gene 4cl from Arabidopsis, which encodes a 4CL protein that converts the substrate p-aminocinnamic acid to aminocinnamoyl-CoA. Module Ⅱ contains dimeric ketone synthase gene dcs and curcumin synthase gene curs3 from turmeric, which is the core module of curcumin synthesis. Module III contains the acetyl-CoA carboxylase gene (including two genes, accBC and dtsR1) f...

Embodiment 2

[0040] Embodiment 2, the construction of modular engineering bacteria

[0041] The above five genes were codon-optimized in Escherichia coli and fully synthesized. Construction of module I: 4cl was ligated with pACYCDuet vector digested with NdeI and XhoI to construct plasmid 4CL-PACYC. Construction of module II: connect dcs and curs3 to pRSFDuet vector digested with BamHI, HindIII, NdeI and XhoI to obtain plasmid DCS-CURS3-pRSF. Construction of module III: Refer to the construction of module II to construct the plasmid AccBC-DtsR1-pCDF. For the schematic diagram of each plasmid, see image 3 . The three recombinant plasmids were co-electrotransformed into MG1655(DE3) competent cells, and screened on LB solid plates containing 50 μg / mL spectinomycin, 50 μg / mL kanamycin and 25 μg / mL chloramphenicol. The grown single clones were verified by PCR to ensure the correctness of the positive clones ( Figure 4 ). The recombinant strain obtained from the final screening was nam...

Embodiment 3

[0044] Embodiment 3, fermentation produces the method for amino bis-demethoxy curcumin

[0045] The recombinant strain HXJE109 obtained in Example 2 was inoculated in LB liquid medium, and after culturing overnight at 37°C, it was inoculated in 100 mL of fresh LB liquid medium at an inoculation ratio of 1:100. Cultivate to OD at 37°C and 220rpm 600 When ≈0.6, add 0.5mM IPTG and 0.5mM p-aminocinnamic acid at a final concentration, turn to 25°C, 220rpm and continue culturing for 48h. Take 500 μL of fermentation broth, add 500 μL of methanol, mix well, and then ultrasonically disrupt the bacteria. Centrifuge at 12000rpm for 5min, and take the supernatant for HPLC detection. Use 4.6μm×250mm Agilent TC C18 reverse-phase column, use 0.1% formic acid water as phase A, pure methanol as phase B, flow rate 1mL / min, carry out elution according to the following conditions: 0min, 10% B; 9min, 100% B; 15 min, 100% B. The detection wavelength is 450nm. The final calculation shows that...

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Abstract

The invention relates to construction and fermentation optimization methods of an amino-bis-demethoxycurcumin high-yield strain. The invention discloses an escherichia coli engineering strain for synthesizing aminodidemethoxycurcumin by taking p-aminocinnamic acid as a substrate, and belongs to the field of synthetic biology or metabolic engineering. A synthetic route composed of a 4-coumaroyl coenzyme A ligase gene 4cl, a dimer ketone synthase gene dcs, a curcumin synthase gene curs3 and acetyl coenzyme A carboxylase genes accBC and dtsR1 is transferred into Escherichia coli MG1655 (DE3), andthe synthetic route is subjected to modular division and optimization to obtain an engineering bacterium with optimal yield. Through optimization of fermentation conditions, the yield of amino didemethoxycurcumin reaches 33mg / L after the strain is fermented for 48h by taking p-aminocinnamic acid as a substrate. The invention lays a foundation for industrial production and application of the high-activity amino didemethoxycurcumin.

Description

technical field [0001] The invention belongs to the field of metabolic engineering, and relates to the construction of a high-yield aminobisdemethoxy curcumin strain and its fermentation optimization method; in particular, it relates to a high-yield aminobisdemethoxy curcumin synthesized by using p-aminocinnamic acid as a substrate Strain construction method and optimization of fermentation conditions. Background technique [0002] Curcumin compounds are the main active ingredients with pharmacological effects in the traditional Chinese medicine turmeric. According to their different structures, they are mainly divided into three types: curcumin (curcumin, C 21 h 20 o 6 ), demethoxycurcumin (demethoxycurcumin, C 20 h 18 o 5 ) and bisdemethoxycurcumin (bisdemethoxycurcumin, C 19 h 16 o 4 ). Curcumin compounds have good anti-inflammatory, anti-tumor, anti-oxidation and anti-cardiovascular activities. On the other hand, curcumin is also widely used as food additive in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/66C12N15/52C12N15/54C12P13/00C12R1/19
CPCC12N9/93C12N9/1029C12N15/70C12N15/66C12P13/001C12Y602/01012C12Y604/01002C12N2800/22
Inventor 康前进胡晓婧欧一新白林泉
Owner SHANGHAI JIAO TONG UNIV
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