A kind of recombinant yeast strain and its construction method and application
A construction method and yeast technology, applied in the field of genetic engineering, can solve problems such as gaps, and achieve the effect of improving synthetic yield
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Embodiment 1
[0099] Example 1: Construction of gene knockout strains
[0100] Using Saccharomyces cerevisiae CEN.PK2-1C as the starting strain, a four-gene knockout strain CEN.PK2-1C△gal1,△gal7,△gal10::DR,△ypl062w::kanMX was constructed. The specific process is as follows:
[0101]First construct △gal1, △gal7, △gal10::DR-Kl URA3-DR knockout cassettes, that is, knockout cassette fragment 1, and use plasmid pWJ1042 as a template to design upstream and downstream primers for PCR amplification with the same 40 bp upstream and downstream of the gene. The knockout box fragment of the source arm and the DR-K1 URA3-DR nutritional label was integrated into the yeast genome by using the homologous recombination mechanism of yeast itself through yeast transformation with lithium acetate method. After transformation, SD-URA solid plate (synthetic Yeast nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L lacking uracil, 2% agar powder) were screened, and the obtained transformants ...
Embodiment 2
[0104] Embodiment 2: Construction of gene knockout strain
[0105] Using Saccharomyces cerevisiae CEN.PK2-1C as the starting strain, a double gene knockout strain CEN.PK2-1C△gal80::DR,△ypl062w::kanMX was constructed. The specific process is as follows:
[0106] First construct the △gal80::DR-Kl URA3-DR knockout box, that is, the knockout box fragment 2, and use the plasmid pWJ1042 as a template to design the upper and lower primers for PCR amplification with the upper and lower 40bp homology arms of the gene and DR-Kl The fragment of the knockout cassette of the URA3-DR nutritional label is integrated into the yeast genome by using the homologous recombination mechanism of the yeast itself through yeast transformation with lithium acetate method. After transformation, SD-URA solid plate (synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, the mixed amino acid powder 2g / L of single-lactility uracil, 2% agar powder) carry out screening, the transformant obtained carries...
Embodiment 3
[0108] Embodiment 3: Construction of gene fragment 1
[0109] Amplify the CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa crtB, PGK1 terminator and splice them sequentially by OE-PCR to obtain a fragment T containing HindIII and XhoI restriction sites at both ends CYC1 -crtI-P GAL10 -P GAL1 -crtB-T PGK1 ;
[0110] At the same time, the homologous 631bp sequence upstream of the yeast trp1 site and the 733bp homologous sequence downstream of the yeast trp1 site were amplified and spliced sequentially by OE-PCR to obtain SacI and ApaI restriction sites at both ends, and in yeast trp1 The fragment containing the HindIII and XhoI restriction sites between the upstream and downstream homologous sequences of the site is then connected into the vector pRS405 through the SacI and ApaI restriction sites (see SEQ ID NO: 8 for the complete gene sequence, and see Figure 8 ), to obtain the TRP1 integration plasmid pRS405-TRP, the above obtained fragment T CYC1 -crtI-P G...
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