Yeast for producing artemisia apiacea diene and construction method of yeast

A technology of artemisinene and construction method, applied in the field of yeast for producing artemisinene and construction, can solve the problems of unfriendly environment of artemisia annua, unstable hereditary traits of artemisia annua, and no industrial extraction value, etc. The effect of broad market prospects

Active Publication Date: 2015-02-04
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Except for parts of Hainan, Fujian, Chongqing, and Guangxi in my country, the content of artemisinin in Artemisia annua in most parts of the world is very low (less than 1%), which does not have industrial extraction value, and the genetic traits of Artemisia annua are unstable. The production is greatly affected by the climate, which leads to very unstable production of artemisinin; the separation and extraction process of plant raw materials uses a large amount of industrial acid and alkali, and produces seriously excessive waste water
At the same time, residues that cannot be effectively reused can easily cause eutrophication of the water body, and cutting down large quantities of Artemisia annua is an environmentally unfriendly behavior; although the method of plant cell culture has solved the problem of large-scale felling of Artemisia annua, it is difficult for plant cells to grow. The cycle is long, so the efficiency of obtaining artemisinin is not high; the chemical synthesis method not only has complicated synthesis steps, but also has many by-products, and is expensive

Method used

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  • Yeast for producing artemisia apiacea diene and construction method of yeast
  • Yeast for producing artemisia apiacea diene and construction method of yeast
  • Yeast for producing artemisia apiacea diene and construction method of yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Acquisition of genes related to the synthesis pathway of artemisinin in yeast

[0040] A. Acquisition of ERG20 gene (yeast FPP and GPP synthase gene)

[0041] Primers were designed according to the yeast ERG20 gene sequence, SEQ ID NO: 1E20-F: 5'-CGGGATCCATGGCTTCAGAAAAAGAAATTA-3' and SEQ ID NO: 2E20-R: 5'-CGGAATTCCTATTTGCTTCTCTTGTAAAC-3', based on the genome of W303a strain (purchased from ATCC Company) As a template, use pfu enzyme to perform PCR (95°C, 3min; 95°C, 30s, 50°C, 35s, 72°C, 75s, 32cycles; 72°C, 5min; 4°C, +∞) amplification to obtain a 1059bp fragment. Shown in SEQ ID NO:3, it was cloned into the pMD18-T vector and sequenced to confirm that no mutation occurred. The amino acid sequence expressed by the fragment shown in SEQ ID NO:3 uses SEQ ID NO:4.

[0042] B. Acquisition of tHMGR gene (yeast truncated HMG-CoA reductase gene)

[0043]Primers were designed according to the yeast HMG-CoA reductase gene sequence, SEQ ID NO: 5tHM-F: 5'-CGGAATTCAT...

Embodiment 2

[0047] Embodiment 2: the construction of carrier

[0048] A. Construction of vector SyBE_001317 (ERG20 gene expression vector)

[0049] The yeast promoter TDH3p, ERG20 gene, and yeast terminator CYC1t were sequentially ligated into the integrated vector pRS403 by restriction endonuclease ligation to obtain the vector SyBE_001317 (see figure 1 );

[0050] B. Construction of vector SyBE_001318 (expression vector of truncated HMG-CoA synthase gene)

[0051] The yeast promoter TDH3p, tHMGR gene, and yeast terminator CYC1t were sequentially ligated into the integrated vector pRS304 by restriction endonuclease ligation to obtain the vector SyBE_001318 (see figure 2 );

[0052] C. Construction of vector SyBE_001338 (a fusion protein expression vector containing ERG20 gene and optimized artemisinin synthase gene ADS):

[0053] The yeast promoter PGK1p, ERG20 gene, fusion protein linking peptide (GGGS, whose nucleotide sequence is shown in SEQ ID NO: 11), artemisinin synthase gene...

Embodiment 3

[0056] Example 3: Acquisition of yeast producing artemisinin

[0057] Yeast transformation of vectors was performed using the lithium acetate method.

[0058] The vectors SyBE_001317 and SyBE_001318 were linearized in advance, integrated into the corresponding sites of the genome of Saccharomyces cerevisiae strain W303a, and then SyBE_001338 was directly transformed. The transformed yeast was screened with SD-drop solid medium (deaminated yeast nitrogen source, 6.7g / L; glucose, 20g / L; Dropout mix, 0.2%; agar powder, 2%), and the obtained transformants were transferred to SD -drop liquid culture medium for 36 hours, extract the yeast plasmid or genome as a template, and perform PCR verification to exclude interference from false positives. Confirm the correct positive strain, plate streak or glycerol bacteria preservation.

[0059] Using the MEV pathway existing in yeast itself, the key genes involved in the pathway, HMG-CoA reductase gene (HMGR) and FPP synthase gene (ERG20)...

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Abstract

The invention discloses yeast for producing artemisia apiacea diene and a construction method of the yeast. The construction method of the yeast for producing artemisia apiacea diene comprises the following steps of: constructing a carrier SyBE_001317; constructing a carrier SyBE_001318; constructing a carrier SyBE_001338; and introducing the carriers SyBE_001317, SyBE_001318 and SyBE_001338 into brewer yeast W303a or W303 alpha to obtain the yeast for producing artemisia apiacea diene. A fusion protein functional module of the artemisia apiacea diene is constructed by modifying the endogenous mevalonate approach of the brewer yeast by using a synthesis biotechnology, so that the artificial synthesis of the artemisia apiacea diene is successfully realized; and the yeast is green, clean, convenient and rapid in production processes and has great advantages and wide market prospects. The yeast for producing artemisia apiacea diene, disclosed by the invention, provides a feasible method for heterologous production of the artemisia apiacea diene.

Description

technical field [0001] The invention relates to a yeast for producing artemisinin and a construction method. Background technique [0002] Artemisinin is an effective antimalarial drug. The traditional method of obtaining it includes extracting the plant Artemisia annua. Although Artemisia annua is widely distributed all over the world, the content of artemisinin in it varies greatly depending on the place of origin. Except for parts of Hainan, Fujian, Chongqing, and Guangxi in my country, the content of artemisinin in Artemisia annua in most parts of the world is very low (less than 1%), which does not have industrial extraction value, and the genetic traits of Artemisia annua are unstable. The production is greatly affected by the climate, resulting in very unstable production of artemisinin; the separation and extraction process of plant raw materials uses a large amount of industrial acid and alkali, and produces seriously excessive waste water. At the same time, residue...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/81C12N1/19C12R1/865
Inventor 元英进贾云婧丁明珠
Owner TIANJIN UNIV
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