Cloning of Yarrowia lipolytica extracellular lipase 2 gene in Saccharomyces cerevisiae

Introduction and Objectives: Lipases have attracted a high domain of biocatalytic processes in many industries such as biodiesel production, food industry and synthetic pharmaceuticals. Microbial production of lipases, specially yeast lipases plays an important role in industry due to the capability of large-scale production with lower costs of production. Yarrowia lipolytica yeast is one of the most important species that produces several intracellular, cell-bound, and extracellular lipases. Among several lipases of this yeast, Y. lipolytica extracellular lipase (YlLIP2) has significant importance since it has high biocatalytic activity. Recently, systems and synthetic biology tools have taken for developing microorganisms such as Escherichia coli and Saccharomyces cerevisiae. In this study, we developed a shuttle vector containing Y. lipolytica extracellular lipase gene for cloning in S. cerevisiae as a cell factory, by synthetic biology method. Materials and Methods: The LIP2 lipase genes were isolated from Yarrowia lipolytica strain DSM3286 by PCR method and inserted in Saccharomyces cerevisiae CEN. PK113-7D strain. The purified LIP2 gene and USER cloning vectors, PCFB3035 were prepared by with FastDigest AsiSI. Digested lipase genes and recombinants vectors were ligated. Qualitative analyses of lipases expression were tested on MSM with 10 mL/L tributyrin plats by the halo assay. After incubation at 29 °C for 72 h, the clear halos are observed where extracellular tributyrin was degraded. Results: After incubation, the halos around of the engineered strain colonies had been extended, and it was about 17 mm diameter. However, no halo was observed for wild strain which due to the absence of any extracellular lipase activity. Conclusion: The recombinant strain efficiently degrades hydrophobic substrates such as fatty acids, fats and oils by these lipases.

Keywords:

Saccharomyces cerevisiae , Lipase , Yarrowia lipolytica , Synthetic biology.