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A kind of recombinant Escherichia coli producing β-carotene and its construction method and application

A technology for recombining Escherichia coli and carotene, applied in the biological field, can solve problems such as inconvenience of rational design and transformation, and achieve the effect of fewer operating gene sites and simple technology

Active Publication Date: 2021-12-24
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] All above-mentioned engineered Escherichia coli producing β-carotene were obtained after introducing the β-carotene synthesis gene cluster through multi-step combination regulation, involving multiple combinations of multiple gene loci, because the key steps will vary with the genetic background of the strain Changes due to changes, which will bring inconvenience to the subsequent further rational design transformation

Method used

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  • A kind of recombinant Escherichia coli producing β-carotene and its construction method and application
  • A kind of recombinant Escherichia coli producing β-carotene and its construction method and application
  • A kind of recombinant Escherichia coli producing β-carotene and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The specific operation of knocking out the 6-phosphate glucose dehydrogenase coding gene zwf in the pentose phosphate pathway of the β-carotene-producing recombinant Escherichia coli ZF43ΔgdhA strain is as follows:

[0043] Utilize PCR amplification primer pair zwf1 / zwf2 (SEQ ID No.5 and SEQ ID No.6) to carry out PCR amplification with plasmid pTKS / CS as template, obtain the zwf gene knockout DNA fragment (SEQ ID No. 7).

[0044] The zwf gene knockout DNA fragment (5'-end to 3'-end) amplified above consists of: 40bp upstream homology arm sequence (30bp at the 3'-end as a repeat sequence), 18bp homing endonuclease recognition Site sequence, 1277bp tetracycline gene expression cassette sequence, 18bp homing endonuclease recognition site sequence, 30bp repeat sequence and 39bp downstream homology arm sequence.

[0045] The plasmid pTKRED was transferred into the E. coli ZF43ΔgdhA strain and made electrotransfer competent; the zwf gene knockout DNA fragment was transferred...

Embodiment 2

[0049] The specific operation of knocking out the ptsHIcrr gene cluster encoding the phosphate transporter Hpr, the phosphate transporter EI and the glucose-specific transporter EIIA of the ECW1 strain is as follows:

[0050] Utilize PCR amplification primer pair PTS1 / PTS2 (SEQ ID No.8 and SEQ ID No.9) to carry out PCR amplification with plasmid pTKS / CS as template, obtain the ptsHIcrr gene cluster knockout DNA fragment (SEQ ID No. .10).

[0051] The ptsHIcrr gene cluster knockout DNA fragment (5'-end to 3'-end) amplified above consists of: 44bp upstream homology arm sequence (3'-end 30bp as repeat sequence), 18bp homing endonuclease Recognition site sequence, 1277bp tetracycline gene expression cassette sequence, 18bp homing endonuclease recognition site sequence, 30bp repeat sequence and 40bp downstream homology arm sequence.

[0052] The plasmid pTKRED was transferred into E. coli strain ECW1 and made electrotransfer competent; the ptsHIcrr gene cluster knockout DNA fragme...

Embodiment 3

[0056] The specific operation of knocking out the strongly NADPH-dependent alcohol dehydrogenase coding gene yjgB of the ECW2 strain is as follows:

[0057] Utilize PCR amplification primer pair yjgB1 / yjgB2 (SEQ ID No.11 and SEQ ID No.12) to carry out PCR amplification with plasmid pTKS / CS as template, obtain the yjgB gene knockout DNA fragment (SEQ ID No. 13).

[0058] The above amplified yjgB gene knockout DNA fragment (5'-end to 3'-end) consists of: 83bp upstream homology arm sequence (3'-end 30bp as repeat sequence), 18bp homing endonuclease recognition Site sequence, 1277bp tetracycline gene expression box sequence, 18bp homing endonuclease recognition site sequence, 30bp repeat sequence and 59bp downstream homology arm sequence.

[0059] The plasmid pTKRED was transferred into Escherichia coli strain ECW2 and made electrotransfer competent; the yjgB knockout DNA fragment was transferred into electrotransfer competent cells obtained in this step, positive clones were scr...

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Abstract

The invention discloses a recombinant Escherichia coli producing β-carotene and its construction method and application. The construction method is: 1) knocking out the 6-phosphate glucose dehydrogenase encoding of the pentose phosphate pathway in the β-carotene-producing Escherichia coli Gene zwf; 2) Knock out the gene cluster ptsHIcrr encoding phosphate transporter Hpr, phosphate transporter EI and glucose-specific transporter EIIA in the strain obtained in step 1); 3) Knock out the strong NADPH-dependent encoding of the strain obtained in step 2) Alcohol dehydrogenase gene yjgB; 4) import the recombinant plasmid containing gene nadK into the bacterial strain obtained in step 3); the yield of recombinant bacterium β-carotene of the present invention is significantly improved, and the highest yield exceeds 2.5g / L, which has potential industrial application value ; The construction method increases the supply of the cofactor NADPH after knocking out the genes zwf and ptsHIcrr, has fewer gene sites to operate, and is simple in technology.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Escherichia coli producing β-carotene, its construction method and application. Background technique [0002] β-carotene is an orange-yellow fat-soluble compound belonging to carotenoids and a pigment commonly found in higher plants. It is mainly used as food, beverages, and candies together with other natural carotenoids. and pharmaceutical coloring agents. β-carotene has good singlet oxygen scavenging performance, so it has anti-oxidation, detoxification, prevention of cancer, cardiovascular disease and cataract, as well as diseases caused by cell aging and decline. In addition, β-carotene has 11 conjugated double bonds with two retinyl groups, is a source of vitamin A, is an important active substance for human physiological functions, and can treat cytoplasmic keratinization caused by vitamin A deficiency , dry eye disease, night blindness, etc. [0003] There ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P23/00C12R1/19
CPCC12N9/1205C12Y207/01023C12P23/00
Inventor 陈涛吴元庆王智文赵学明
Owner TIANJIN UNIV