Engineered Yeast Strains for Lipid Production

Knockout mutants were constructed by homologous recombination (transformation with linearized knockout cassette) and marker rescue (Cre-recombinase based uracil marker deletion) as previously described (Fickers et al., 2003 (link); Wang et al., 2016 (link)). We first constructed the strain YL-1 (PO1f-△PEX10) through the deletion of PEX10 in the Y. lipolytica PO1f. The strain YL-1was used for episomal expression of pJN44-DGA1, pJN44- DGA1-12D, pJN44-DGA1-SCD, pJN44-DGA1-15D, pJN44-DGA1-12D-SCD, pJN44-DGA1-15D-SCD, and pJN44-DGA1-12D-15D, utilizing a selective marker leucine. Then, the integrative cassette plasmid ura-△MFE1:DGA1-12D-15D was linearized (12D-15D-DGA1-MFE1-up-loxp-down), inserted into the YL-1 strain, and the transformants were screened using uracil dropout plate to create the strain YL-9. Then the plasmid pJN44-ACC1 was expressed in YL-9, resulting in YL-10. The transformation of Y. lipolytica was performed using Zymogen Frozen EZ yeast transformation kit II (Zymo Research Corporation; United States) according to the manufacturer’s instruction.

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Yan F.X., Dong G.R., Qiang S., Niu Y.J., Hu C.Y, & Meng Y.H. (2020). Overexpression of △12, △15-Desaturases for Enhanced Lipids Synthesis in Yarrowia lipolytica. Frontiers in Microbiology, 11, 289.

Publication 2020

Zymogen Frozen EZ yeast transformation kit II

Acc1 Cre recombinase Deletion Episomal Frozen Homologous recombination Leucine Plasmid Strain Uracil Y lipolytica Yeast Zymogen

Corresponding Organization : Shaanxi Normal University

Other organizations : Northwest A&F University, Hunan New Wellful (China), University of Hawaiʻi at Mānoa

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Variable analysis

independent variables

Deletion of PEX10 in Y. lipolytica PO1f to create strain YL-1
Episomal expression of pJN44-DGA1, pJN44-DGA1-12D, pJN44-DGA1-SCD, pJN44-DGA1-15D, pJN44-DGA1-12D-SCD, pJN44-DGA1-15D-SCD, and pJN44-DGA1-12D-15D in YL-1 strain
Insertion of the linearized integrative cassette plasmid ura-ΔMFE1:DGA1-12D-15D into YL-1 strain to create YL-9
Expression of pJN44-ACC1 in YL-9 strain to create YL-10

dependent variables

Not explicitly mentioned

control variables

Selective marker leucine used for episomal expression
Uracil dropout plate used to screen for transformants containing the integrative cassette plasmid

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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