Method for producing astaxanthin by using transgenic microalgae

A transgenic and astaxanthin technology, applied in the direction of unicellular algae, introduction of foreign genetic material using a vector, recombinant DNA technology, etc., can solve the problem of not being able to produce astaxanthin, and achieve a short growth cycle, high cell density, and culture technology. mature effect

Inactive Publication Date: 2016-02-10
EAST CHINA UNIV OF SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the GM microbes can't produce the esterified form of astaxanthin
[0010] However, there is no literature report on the production of astaxanthin by expressing β-carotene ketolase (HpBKT) and β-carotene hydroxylase (HpCHYB) in microalgae.

Method used

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  • Method for producing astaxanthin by using transgenic microalgae
  • Method for producing astaxanthin by using transgenic microalgae
  • Method for producing astaxanthin by using transgenic microalgae

Examples

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Embodiment 1

[0093] Example 1: Construction of a microalgae expression vector comprising an endogenous promoter from Chlorella pyrenoidosa

[0094] In this example, a microalgal expression vector containing an endogenous promoter of Chlorella pyrenoidosa was constructed. In this example, the endogenous promoter of Chlorella pyrenoidosa is the endogenous HSP70A promoter of Chlorella pyrenoidosa, and the microalgae expression vector pGreen0029 series of expression vectors.

[0095] 1.1 pEASY-Blunt-Hsp70A plasmid construction

[0096] (1) Take 50 mL of Chlorella pyrenoidosa liquid, refrigerate and centrifuge to remove the supernatant, grind with liquid nitrogen to extract its RNA and store it in a -80°C refrigerator to freeze the algae mud into a block, and transfer it to a research laboratory that has been poured in liquid nitrogen and pre-cooled. Grinding quickly in a bowl, adding an appropriate amount of liquid nitrogen continuously throughout the process to prevent RNA degradation. Ad...

Embodiment 2

[0110] Embodiment 2: Construction of the microalgal expression vector comprising BKT gene

[0111] In this example, a microalgae expression vector containing the BKT gene was constructed. In this embodiment, the BKT gene is the HpBKT gene from Haematococcus pluvialis, and the expression vector is pGreenII0029- containing the endogenous HSP70 promoter of Chlorella pyrenoidosa obtained in Example 1. Hsp-NosT vector.

[0112] Haematococcus pluvialis was provided by Jiaxing Zeyuan Biological Products Co., Ltd. Haematococcus pluvialis is cultured with pluvialis broth / medium. Extract approximately 10 8 For the total RNA of Haematococcus pluvialis cells, refer to the steps in the manual for the method. cDNA was synthesized in 15 minutes using TransScriptOne-StepgDNARemoval and cDNASynthesisSuperMix (purchased from Transgen) kits.

[0113] The registration number of the HpBKT gene sequence in the NCBI database is D45881.1, and the registration number of the HpBKT amino acid seq...

Embodiment 3

[0116] Embodiment 3: Construction of the microalgal expression vector comprising CHYB gene

[0117] In this example, a microalgae expression vector containing the CHYB gene was constructed. In this embodiment, the CHYB gene is the HpCHYB gene from Haematococcus pluvialis, and the expression vector is the pGreenII0029-containing endogenous HSP70 promoter of Chlorella pyrenoidosa obtained in Example 1. Hsp-NosT vector.

[0118] The registration number of the HpCHYB gene sequence in the NCBI database is AY187011.1, and the registration number of the HpCHYB amino acid sequence in the NCBI database is AAO53295.1. Such as image 3 As shown, the CHYB gene fragment was cloned from the cDNA of Haematococcus pluvialis obtained in Example 2 with the upstream primer CHYBF containing the SpeI site and the downstream primer CHYBR containing the NotI site. The resulting HpCHYB gene fragment and the pGreen0029-HSP vector obtained in Example 1 were digested with SpeI and NotI. The digest...

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Abstract

The invention relates to microalgae expression vectors and a method for producing astaxanthin by using beta-carotene ketolase (BKT) and beta-carotene hydroxylase (CHYB) in transgenic microalgae. Particularly, the invention relates to the construction of the microalgae expression vectors, the microalgae expression vectors containing the BKT and CHYB, microalgae containing the microalgae expression vectors, the method for producing the astaxanthin by using the BKT and CHYB in the microalgae, a method for preparing the transgenic microalgae for producing the astaxanthin by using the microalgae expression vectors, application of the microalgae expression vectors to the preparation of the transgenic microalgae, and application of the microalgae expression vectors to the production of the astaxanthin.

Description

technical field [0001] The present application relates to microalgae expression vectors and methods for producing astaxanthin in transgenic microalgae using β-carotene ketolase (BKT) and β-carotene hydroxylase (CHYB). Specifically, the application relates to constructing a microalgae expression vector, comprising a microalgae expression vector of a β-carotene ketolase gene (BKT) and a β-carotene hydroxylase gene (CHYB), comprising the microalgae expression vector The microalgae, the method for producing astaxanthin in microalgae using BKT and CHYB, the method for preparing transgenic microalgae producing astaxanthin using the microalgae expression vector, the microalgae expression vector in the preparation of transgenic microalgae and the application of the microalgae expression vector in the production of astaxanthin. Background technique [0002] The chemical name of astaxanthin is 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene [1] , molecular weight formula C 40 h 52 o 4 ....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N1/13C12P23/00
Inventor 李元广范建华房磊李淑兰王军
Owner EAST CHINA UNIV OF SCI & TECH
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