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A gene encoding lycopene dehydrogenase

A lycopene and dehydrogenase technology, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., can solve problems such as low yield and poor genetic stability of strains

Active Publication Date: 2021-05-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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 fermentative production of β-carotene, but the strains constructed by this method have poor genetic stability Defects

Method used

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  • A gene encoding lycopene dehydrogenase
  • A gene encoding lycopene dehydrogenase
  • A gene encoding lycopene dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Construction of uracil auxotrophic Candida tropicalis strain (ZZ)

[0052] Specific steps are as follows:

[0053] (1) Design primers URA3-F1: 5'-TACTCTAACGACGGGTACAAC-3' (SEQ ID NO.2) and URA3-R1: 5'-ACATCGGTACCAGTCAGAATTGTGCTAACCATTGCGAAT according to the URA3 gene sequence (SEQ ID NO.1) of Candida tropicalis in NCBI -3' (SEQ ID NO.3), URA3-F2: 5'-ATTCGCAATGGTTAGCACAATTCTGACTGGTACCGATGT-3' (SEQ ID NO.4) and URA3-R2: 5'-ACCCGATTTCAAAAGTGCAGA-3' (SEQ ID NO.5); then Using Candida tropicalis ATCC 20336 chromosomal DNA as a template, the 5' end fragment (1URA3, 436bp in length) and the 3' end fragment (2URA3, 488bp in length) of the URA3 gene were amplified by PCR;

[0054] (2) Using URA3-F1 and URA3-R2 as primers and 1URA3 and 2URA3 as templates, amplify the URA3 gene deletion cassette 1URA3-2URA3 by fusion PCR;

[0055] (3) The above-mentioned URA3 gene deletion cassette was passed through the lithium chloride conversion method (the lithium chloride conve...

Embodiment 2

[0056] Embodiment 2: the construction of gene expression cassette

[0057] Specific steps are as follows:

[0058] 1. Construction of carRP gene integration expression cassette:

[0059] (1) Design primers PGAP-F: 5'-GCTCTAGAAACGTGGTATGGTTGTAAGAAAC-3' (SEQ ID NO.7) and TGAP-R: 5'-GCTCTAGATCTGGTTTAGAAGTAGGGACTGTATG according to the GAPDH gene sequence (SEQ ID NO.6) of Candida tropicalis in NCBI -3' (SEQ ID NO.8); Using Candida tropicalis ATCC 20336 chromosomal DNA as a template, amplify the GAPDH gene by PCR, and then insert it into the pMD19-T Simple commercial vector to obtain the recombinant plasmid Ts-GAPDH; then Using PGAP-R: 5'-CCCAAGCTTTGTTTAAATTCTTTAATTG-3' (SEQ ID NO.9) and TGAP-F: 5'-GCGTCGACGCTATCCAACAAACTCTAG-3' (SEQ ID NO.10) as primers, using the plasmid Ts-GAPDH as a template, by A linearized DNA fragment carrying the GAPDH promoter and GAPDH terminator was obtained by PCR (GAPDH promoter-pMD19-TSimple vector-GAPDH terminator, abbreviated as P GAPDH -Ts-T GAP...

Embodiment 3

[0070] Embodiment 3: Construction of recombinant bacterial strain

[0071] Specific steps are as follows:

[0072] (1) The integrated expression cassette of the carRP gene obtained in Example 2 was transformed by the lithium chloride conversion method (the lithium chloride conversion method is described in the document "Lihua Zhang, et al. 2016, Development of an efficient genetic manipulation strategy for sequential gene disruption and expression Ofdifferent heterologous GFP genes in Candida tropicalis. Appl Microbiol Biot, 100 (22): 9567-9580 "in) transformed the uracil auxotrophic Candida tropicalis strain ZZ obtained in Example 1, and then screened transformants by minimal medium plates , the positive transformants were named ZZ-RP; the ZZ-RP strain was inoculated in liquid SM medium at 30°C, 200r min -1 In culture, direct repeats in chromosomes (i.e., carRP expression cassette POX4-gda324-URA3-P GAPDH -carRP -T GAPDH - The gda324 fragment in POX4 and the 3' end sequenc...

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Abstract

The invention discloses a gene that can be used for encoding lycopene dehydrogenase, and belongs to the technical field of genetic engineering. The nucleotide sequence of the gene of the present invention is shown in SEQ ID NO.24; this gene is integrated into the co-expressed gene encoding lycopene cyclase and lycopene synthase (nucleotide sequence is shown in SEQ ID NO.12 Shown) in Candida tropicalis, can make Candida tropicalis can highly express β-carotene; Ferment this Candida tropicalis for 96h, can make the output of β-carotene up to 4.24g L ‑1 .

Description

technical field [0001] The invention relates to a gene for encoding lycopene dehydrogenase, which belongs to the technical field of genetic 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 by chemical synthesis is very low,...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P23/00C12R1/74
CPCC12N9/001C12P23/00
Inventor 刘宇飞陈献忠谭云鹰王江月汪闫申欣艳张利华沈微
Owner JIANGNAN UNIV
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