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Recombinant lactococcus lactis used for generating lycopene, and application of recombinant lactococcus lactis

A technology of Lactococcus lactis and lycopene, applied in the field of recombinant Lactococcus lactis and its construction, can solve the problems such as the inability of Lactococcus lactis to synthesize lycopene, and achieve the effect of broad application prospects

Active Publication Date: 2021-04-13
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the deficiency that the existing Lactococcus lactis cannot synthesize lycopene, the present invention provides a recombinant Lactococcus lactis producing lycopene, and also provides a construction method and application of the recombinant strain

Method used

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  • Recombinant lactococcus lactis used for generating lycopene, and application of recombinant lactococcus lactis
  • Recombinant lactococcus lactis used for generating lycopene, and application of recombinant lactococcus lactis

Examples

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Effect test

Embodiment 1

[0019] Example 1: Elimination of competing branches of lycopene synthesis precursors in Lactococcus lactis

[0020] Using CRISPR / Cas9 combined with single-strand DNA recombination engineering, the lactate dehydrogenase pathway and α-acetolactate synthase pathway, which are competing branches of lycopene synthesis precursors in Lactococcus lactis NZ9000, were eliminated. The method for eliminating the above competition branch is to knock out the partial nucleotide sequence of the ldh gene and the partial nucleotide sequence of the als gene on the chromosome of Lactococcus lactis NZ9000.

[0021] The related method of using CRISPR / Cas9 combined with single-stranded DNA recombination engineering to knock out the chromosome segment of Lactococcus lactis has been disclosed in the paper Microb Cell Fact (2019) 18:22.

[0022] (1) Partial nucleotide sequence of knockout ldh gene

[0023] The specific method of knocking out the partial nucleotide sequence of ldh gene:

[0024] ① Con...

Embodiment 2

[0062] Example 2: Synthesis of Lycopene Gene Expression Recombinant Plasmid

[0063]The recombinant Lactococcus lactis producing lycopene of the present invention eliminates two competing pathways of its lycopene synthesis precursors in Lactococcus lactis: lactate dehydrogenase pathway and α-acetolactate synthase pathway ; then construct and obtain after introducing exogenous expression elements into the strain; wherein the elimination of the lactate dehydrogenase pathway and the α-acetolactate synthase pathway is by knocking out part of the core of the ldh and als genes in the Lactococcus lactis NZ9000 genome The nucleotide sequence is realized; the exogenous expression elements are geranylgeranyl pyrophosphate synthase gene crtE, phytoene synthase gene crtB and phytoene dehydrogenase gene derived from Pantoea pineapple crtI, the nucleotide sequence of crtE, crtB and crtI is represented by crtEBI, with a total of 3372 bases, and its nucleotide sequence is shown in SEQ ID No:3...

Embodiment 3

[0064] Embodiment 3: Construction of Lactococcus lactis producing lycopene according to the present invention

[0065] The recombinant plasmid pSEC:crtEBI was electrotransformed into Lactococcus lactis NZ9000ΔldhΔals to obtain the recombinant lycopene producing strain NZ9000ΔldhΔals / pSEC:crtEBI.

[0066] The above-mentioned electrotransformation method is:

[0067] ① Activate Lactococcus lactis NZ9000ΔldhΔals overnight in GM17 medium, transfer to SolI at a ratio of 2%, and culture statically at 30°C until OD600=0.4. Centrifuge at 6000rpm at 4°C for 10 minutes to collect the bacterial cells, wash the bacterial cells twice with SolII, resuspend the bacterial cells in SolII of 1 / 50 of the original culture volume, and obtain competent cells;

[0068] ② Add 50ng of pSEC:crtEBI to the competent cells, blow and aspirate to mix well, and then transfer them to a pre-cooled electroporation cup.

[0069] ③Set the electrotransformation parameters of the electrotransfer instrument as fol...

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Abstract

The invention discloses a recombinant lactococcus lactis bacterial strain used for generating lycopene, and a construction method and application of the recombinant lactococcus lacti. The recombinant lactococcus lactis bacterial strain is constructed in a way that parts of nucleotide sequences of two competition approaches, i.e., a lactic dehydrogenase gene and an [alpha]-acetolactic acid synthetase gene, of a lycopene synthesis precursor are eliminated, and then, plasmids are used to introduce a geranyl pyrophosphate synthetase gene (crtE), a phytoene synthetase gene (crtB) and a phytoene dehydrogenase gene (crtI) of a Pantoea ananatis source. Experiments prove that after the recombinant lactococcus lactis disclosed by the invention is subjected to shaking culture, the lycopene can be generated, and the experiments also predict that the recombinant lactococcus lactis has wide application potential in the development field of health care products and biological medicines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant Lactococcus lactis producing lycopene and its construction method and application. Background technique [0002] For centuries, Lactococcus lactis has been used in the fermentation of cheese, yogurt, sauerkraut and other foods, and is a safe microorganism recognized by the US Food and Drug Administration. Lactococcus lactis also has some probiotic properties, such as immunomodulatory properties, survivability through the gastrointestinal tract (GIT), but no intestinal colonization, etc., which make Lactococcus lactis a preferred delivery vehicle for biotherapeutic drugs. In 2000, Science magazine reported the use of recombinant Lactococcus lactis to secrete interleukin 10 to treat colitis in mice. In the following 20 years, Lactococcus lactis was used as a carrier for the delivery of nearly 60 therapeutic drugs, targeting diseases including enteritis, Flu, ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P5/00C12R1/01
CPCC12N15/746C12P5/007C12N9/0006C12N9/1022C12Y202/01006C12Y205/01029C12N9/1085C12N9/001C12Y103/99031
Inventor 郭婷婷孔健吴佳鹏辛永平顾心怡
Owner SHANDONG UNIV
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