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Construction method and application of strain for efficiently synthesizing beta-carotene

A carotene, purpose technology, applied in the field of metabolic engineering, can solve the problems of poor genetic stability of strains, low yield, improved synthesis ability and the like

Active Publication Date: 2022-04-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there have been studies to improve the production of β-carotene by modifying the genes in E.coli by CRISPR-Cas9 gene editing technology, but this method has the defect of low production (currently the highest reported in the literature is 3.2g L -1 ); There are also studies by trying to obtain three genes of CrtE, CrtYB and CrtI from Phaffia rhodozyma, and then transfer to Saccharomyces cerevisiae cells to construct recombinant bacteria for fermentation and production of β-carotene, but the strains constructed by this method have poor genetic stability Defects
In addition, the current use of metabolic engineering to construct terpene-synthesizing yeast engineering strains is still limited to localizing all metabolic pathways to the cytoplasm or to specific subcellular organelles, and the synthetic ability of the constructed engineering strains is limited.

Method used

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  • Construction method and application of strain for efficiently synthesizing beta-carotene
  • Construction method and application of strain for efficiently synthesizing beta-carotene
  • Construction method and application of strain for efficiently synthesizing beta-carotene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1: Construction and application of expression cassettes expressing genes in the cytoplasm

[0099] Specific steps are as follows:

[0100] 1. Construction of integrated expression cassettes for simultaneous expression of carB, carRP, ERG20 and BTS1:

[0101] (1) Using the recombinant plasmid Ts-PGAPDH-carRP-TGAPDH (the construction method is described in the Chinese invention patent text with the publication number CN109652388B) as a template, the primers CarRP-F and CarRP-R are amplified by PCR to obtain the carRP gene (nuclear The nucleotide sequence is shown in SEQ ID NO.2), and then the carRP gene was inserted into the pMD19-T Simple commercial vector to obtain the recombinant plasmid Ts-carRP;

[0102] (2) Using Candida tropicalis ATCC 20336 chromosomal DNA as a template, P GAP1 -F and P GAP1 -R is a primer, amplifies the GAPDH gene promoter by PCR (the P GAP1 The genebank accession number of the promoter is: HQ171163.1), and then inserted into the XbaI...

Embodiment 2

[0144] Example 2: Construction and application of expression cassettes expressing genes in mitochondria

[0145] The nucleotide sequence of the mitochondrial localization signal peptide coding sequence MLS involved in the examples is shown in Table 2.

[0146] 1. Construction of an integrated expression cassette for simultaneous expression of carB, carRP, ERG20 and BTS1 in mitochondria

[0147] (1) Add the mitochondrial localization signal peptide coding sequence MLS to the 5' end of the carB and carRP genes in the recombinant plasmid PBRP01 by using the one-step cloning kit C113 (Novazyme) to construct the recombinant plasmid PBRP02;

[0148] (2) Add the mitochondrial localization signal peptide coding sequence MLS to the 5' end of the ERG20 and BTS1 genes in the recombinant plasmid PBE01 by using the one-step cloning kit C113 to construct the recombinant plasmid PBE02;

[0149] (3) Recover ERG20 and BTS1 gene expression cassette P with NotI digestion plasmid pBE02 GAP1 -ML...

Embodiment 3

[0169] Example 3: Construction and application of expression cassettes expressing genes in peroxisomes

[0170] The nucleotide sequence of the peroxisome localization signal peptide coding sequence ePTS involved in the examples is shown in SEQ ID NO.1.

[0171] 1. Construction of an integrated expression cassette for the simultaneous expression of carB, carRP, ERG20 and BTS1 in peroxisomes

[0172] (1) Add the peroxisome localization signal peptide coding sequence ePTS to the 3' end of the carB and carRP genes in the recombinant plasmid PBRP01 by using the one-step cloning kit C113 to construct the recombinant plasmid PBRP03;

[0173] (2) Add the peroxisome localization signal peptide coding sequence ePTS to the 3' end of the ERG20 and BTS1 genes in the recombinant plasmid PBE01 by using the one-step cloning kit C113 to construct the recombinant plasmid PBE03;

[0174] (3) Recover ERG20 and BTS1 gene expression cassette P with NotI digestion plasmid pBE03 GAP1-ERG20-ePTS-T7s...

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Abstract

The invention discloses a construction method and application of a strain for efficiently synthesizing beta-carotene, and belongs to the field of metabolic engineering. According to the construction method, related genes synthesized from acetyl coenzyme A to mevalonic acid in a beta-carotene synthesis route are expressed in yeast cytoplasm, and related genes synthesized from IPP and DMAPP to beta-carotene are overexpressed in yeast peroxisome. Related genes synthesized from mevalonic acid to IPP and DMAPP are over-expressed in cytoplasm or peroxisome. The yield of beta-carotene in the engineering strain constructed by the method is obviously improved. After the genetic engineering strain constructed by the invention is fermented for 9 days, the dry weight of thalli in fermentation liquor can reach 213g / L, and the yield of beta-carotene can reach 6.5 g.L <-1 >. The engineering strain constructed by the invention can efficiently synthesize beta-carotene, and has a good application prospect.

Description

technical field [0001] The invention relates to a construction method and application of a strain for efficiently synthesizing β-carotene, belonging to the field of metabolic engineering. Background technique [0002] β-carotene is the precursor of vitamin A synthesized by the human body. It is also a type A nutritional pigment unanimously recognized by the Food and Agriculture Organization of the United Nations and the World Health Organization. It has strong anti-oxidation, anti-aging and anti-tumor functions. Therefore, it is widely used Used in the preparation of food, medicine, cosmetics and health care products. [0003] At present, β-carotene is mainly produced by chemical synthesis, but the β-carotene produced by chemical synthesis is not easy to be completely absorbed by the human body, and will cause certain toxic and side effects to the human body. Long-term use will cause irreversible lesions to the human body, and, The biological activity of β-carotene obtained...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N1/19C12P23/00C12R1/74
CPCY02A50/30
Inventor 张利华陈献忠夏媛媛沈微杨海泉
Owner JIANGNAN UNIV
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