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Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof

A technology of chalcone synthase and flavonoids, applied in botany equipment and methods, applications, genetic engineering, etc.

Inactive Publication Date: 2011-01-19
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the recombination of the key enzyme genes of the flavonoid synthesis pathway completely derived from plants into lactic acid bacteria

Method used

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  • Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof
  • Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof
  • Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1 The acquisition of Lactococcus lactis subsp. cremoris pMG26e-4GS

[0054] For the whole process of obtaining Lactococcus lactis subsp. cremoris pMG26e-4GS, see figure 1 .

[0055] (1) Pretreatment of young soybean leaves and extraction of total RNA

[0056] Soybean (Glycine max L.Merr.) cultivar is Northeast Spring Soybean Series (Beifeng 12) (preserved in our laboratory or purchased from the market), and cultivated under light until 10 to 15 young leaves grow, then transfer to the ultraviolet lamp intermittently. In the irradiated culture room, cultivate for one week, take young leaves as materials, and extract total RNA from leaves by Trizol method (Trizol reagent is purchased from Invitrogen); UV irradiation conditions: wavelength, 254nm; power: 20W; distance about 1.5m;

[0057] (2) RNA reverse transcription into a cDNA library: using the total RNA obtained in step (1) as a template, the first-strand cDNA was synthesized under the action of M-MLV revers...

Embodiment 2

[0075] Example 2 Production of Liquiritigen by Fermentation of Lactococcus lactis subsp. cremoris MG1363-4GS

[0076] (1) The recombinant bacteria MG1363-4GS whose preservation number is CGMCC No.4086 is grown overnight, and the fresh bacterial solution (growth to the logarithmic growth phase, OD 600 =2.0) Inoculate in 50ml MRS liquid medium (containing 5 μg / ml erythromycin) according to the inoculation amount of 1wt%, add the phenylalanine and tyrosine that final concentration is 500mg / L simultaneously, grow 48h at 37 ℃;

[0077] (2) Centrifuge the culture solution of step (1) at 6000rmp for 10min, take the supernatant, then extract it with an equal volume of ethyl acetate, take the organic phase, dry it with a rotary evaporator, and dissolve the residue in 2.0ml anhydrous Methanol, promptly obtains flavonoid crude product solution;

[0078] (3) adopt HPLC to carry out component analysis to the flavonoid crude product that step (2) obtains, carry out HPLC chromatogram under ...

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Abstract

The invention discloses engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof, belonging to the field of lactobacillus reformation of genetic engineering. The engineering lactobacilli is produced by the steps of: synthesizing flavonoid completely from Chinese locally planted soybeans (Glycine max(L.)Merr.) into related enzyme genes PAL, 4CL, CHS and CHI which are connected into a polycistron structure PLA-4CL-CHS-CHI; then constructing the polycistron structure onto a reformed lactobacillus expression vector pMG26e to form a recombinant vector pMG26e-4GS; and then electrically shocking and converting the pMG26e-4GS into lactobacilli to obtain the engineering lactobacilli for producing plant flavonoid to synthesize related enzymes. The preservation number of the recombinant lactobacilli is CGMCC (China General Microbiological Culture Collection Center) No.4086, and the engineering lactobacilli has more abundant physiological functions compared with original lactobacilli and can be applied to the field of food. Through fermentation, the flavonoid output of the obtained recombinant strain is in the level of mg / l, which is higher than the fermentation levels of like recombinant escherichia coli and recombinant yeast.

Description

field of invention [0001] The invention relates to engineering lactic acid bacteria producing enzymes related to plant flavonoid synthesis and its construction and application. The obtained functional engineering lactic acid bacteria strain can be used to produce flavonoid substances, and the strain has huge potential market, economic benefit and social benefit in the food industry. Background technique [0002] Flavonoids (Flavonoids) are a group of polyphenols widely present in plants, which belong to secondary metabolites and are natural pigments present in leaves, flowers, and fruits of plants. They are called flavonoids because they are mostly yellow. . Flavonoids have broad application prospects in medicine, food and other fields due to their important physiological functions, especially scavenging free radicals and anti-cancer and anti-cancer effects. However, the content of flavonoids in plants is very low, the extraction cost is high, and the yield is low; flavono...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/52C12N15/61C12N15/63C12P17/06C12R1/46
Inventor 陈尚武左芳雷卢晓明赵建云郑超郝彦玲李平兰马会勤
Owner CHINA AGRI UNIV
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