Transformed yarrowia lipolytica, and building method and application thereof
A technology of Yarrowia lipolytica and its construction method, which is applied in the field of transformation of Yarrowia lipolytica and its construction, can solve the problems of limited improvement range and low yield, and achieve reduced production procedures, low production cost, and mild conditions Effect
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Embodiment 1
[0034] (1) With the lipase lip (having the nucleotide sequence shown in SEQ ID NO: 1) derived from the lipase family of Yarrowia lipolytica as the target lipase gene, using pINA1317 as the carrier, by enzymatic digestion Construct the recombinant plasmid pINA1317-lip;
[0035] (2) Linearize the obtained recombinant plasmid, an expression cassette (hp4d-XPR2pre-lip-XPR2t) consisting of hp4d promoter, XPR2pre signal peptide, lip gene and XPR2t terminator; transform the recombinant plasmid into ura-deficient Y.lipolytica Competent cells;
[0036] (3) Using Ura3d1 as a screening marker, the recombinant engineering of Yarrowia lipolytica in Example 1 was obtained by screening with YNBD selective medium (6.7g / L yeast nitrogen base without amino acids, 10g / L glucose, 20g / L agar). strain.
Embodiment 2
[0038] The recombinant engineering strain of Yarrowia lipolytica obtained in Example 1 was fermented in a medium with 5% molasses as a carbon source, the liquid volume in the shaker bottle was 10%, the shaking table speed was 250 rpm, the culture temperature was 28°C, and the culture time was 84h. The output of lipase reaches 3200U / ml, the dry weight of the bacteria reaches 33g / L, and the protein content of the bacteria reaches 61%.
Embodiment 3
[0040] (1) With the lipase lip (having the nucleotide sequence shown in SEQ ID NO: 1) derived from the lipase family of Yarrowia lipolytica as the target lipase gene, using pINA1297 as the carrier, by enzymatic digestion Construct the recombinant plasmid pINA1297-lip;
[0041] (2) Linearize the recombinant plasmid to obtain an expression cassette (hp4d-XPR2pre-lip-XPR2t) consisting of hp4d promoter, XPR2pre signal peptide, lip gene and XPR2t terminator; transform the recombinant plasmid into ura-deficient Y.lipolytica State cells;
[0042](3) Using Ura3d4 as a screening marker, the recombinant engineering of Yarrowia lipolytica in Example 3 was obtained by screening with YNBD selective medium (6.7g / L yeast nitrogen base without amino acids, 10g / L glucose, 20g / L agar). strain.
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