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
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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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