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Gene synthesizing method of luteolin-producing yeast strain as well as strain and application

A yeast strain, luteolin technology, applied in the biological field, to achieve low cost, short time, no tailing effect

Pending Publication Date: 2018-07-24
CHUXIONG MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this study is not an example of large-scale and complete production of luteolin, so how to realize the industrial production of luteolin has become a hot spot that people pay more and more attention to.

Method used

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  • Gene synthesizing method of luteolin-producing yeast strain as well as strain and application
  • Gene synthesizing method of luteolin-producing yeast strain as well as strain and application
  • Gene synthesizing method of luteolin-producing yeast strain as well as strain and application

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preparation example Construction

[0030] The gene synthesis method of the luteolin-producing yeast strain of the present invention comprises the steps of DNA extraction from scutellaria breviscapus tissue, gene cloning of Saccharomyces cerevisiae, construction of recombinant vector, gene recombination, integration of soluble fusion protein, and screening steps, and the specific steps are as follows:

[0031] A. DNA extraction from breviscapine tissue: Obtain scutellaria breviscapus genome cDNA according to conventional methods, and clone breviscapenda genome cDNA through primer design to obtain phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase, and p-coumaric acid-coA linkage The genes encoding the enzymes, chalcone synthase, chalcone isomerase and flavonoid synthase II;

[0032] B. Saccharomyces cerevisiae gene cloning: the genes encoding alcohol dehydrogenase, acetaldehyde dehydrogenase, acetyl-CoA synthase and acetyl-CoA carboxylase 1 were cloned from the Saccharomyces cerevisiae genome by conventiona...

Embodiment

[0060] 1 Extraction of DNA from Erigeron breviscapus tissue

[0061] 1.1 Materials

[0062] Table 1 Extraction materials

[0063]

[0064] 1.2 Method

[0065] Take out the centrifuge tubes containing three parallel samples of each tissue of Breviscapus preserved in liquid nitrogen, and take 2g (the following units are the minimum units can be equipped in multiples, the same below) tissue samples from each centrifuge tube to obtain a 6g (the following The unit is the smallest unit and can be equipped in multiples, the same below) for tissue mixed samples for RNA extraction. Quickly weigh 100mg (the following unit is the smallest unit and can be equipped in multiples, the same below), put it into a mortar, add liquid nitrogen and quickly grind it into a powder. Total RNA was extracted using the RNRN12-PLANTeasy Reagent Kit provided by Beijing Aidelai Biotechnology Co., Ltd. according to the procedure. The extraction process is as follows:

[0066] 1.2.1 Steps

[0067]St...

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Abstract

The invention discloses a gene synthesizing method of a luteolin-producing yeast strain as well as the strain and application. The gene synthesizing method is characterized in that genes PAL, C4H, 4CL, CHS, CHI and FSII are obtained by an erigeron breviscapus genome cDNA (complementary deoxyribonucleic acid) via primer cloning; genes ADH2, ALD6, ACS and ACCI are obtained by a saccharomyces cerevisiae genome via cloning; recombinant carriers T1 to T5 are constructed by the genes and the primer-amplified promoter and terminator plasmids by a Golden gate cloning technique; bidirectional gene amplification segments of the recombinant carriers are integrated into the conventional integrating sites of the saccharomyces cerevisiae genome by a conventional method, so as to obtain recombinant strains; genes F3'H and CPRI are integrated, recombined with shuttle plasmids, and integrated into chromosomes of the recombinant bacterial strains by homologous arms, so as to obtain the integrated recombinant bacterial strains; the integrated recombinant bacterial strains are screened by an auxotroph culture medium CM-TrP, so as to obtain the luteolin-producing yeast strain. The gene synthesizing method has the characteristics that the yield rate of luteolin is high, and the green and environment-friendly effects are realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a gene synthesis method of a luteolin-producing yeast strain transformed by gene synthesis, which has a high yield of luteolin, low cost, and an environmentally friendly synthesis method, as well as the strain and its application. Background technique [0002] Saccharomyces cerevisiae is the first eukaryotic model organism whose genome has been sequenced. Saccharomyces cerevisiae is a frequently used platform strain due to its clear genetic background, high homologous recombination efficiency, better expression of P450 enzymes, good biological safety, and genetic stability during fermentation. [0003] Luteolin (Luteolin) is a flavonoid compound produced by plants in response to environmental stress, etc. It is widely distributed in nature, and mainly exists in many natural medicinal materials such as tea, honeysuckle, chrysanthemum, ginkgo, perilla, and scutellaria. Lut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12N1/19C12P17/06C12R1/865
CPCC12N15/66C12N15/81C12P17/06
Inventor 张泽渊王晓明昝雪峰马鹏罗永贤张明李光富谢忠祥张荷兰路则宝李洪文韩朝志张志琴尹书
Owner CHUXIONG MEDICAL COLLEGE
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