Tuesday, April 30, 2013
Exhibit Hall
The non-conventional yeast Yarrowia lipolytica has been widely used and metabolically engineered for biomanufacuring citric acid, carotenoid and eicosopentaenoic acid (EPA)-rich oil. Y. lipolytica has also attracted much attention as it is able to accumulate significant levels of lipid as an alternative feedstock for producing renewable hydrocarbon. To facilitate the development of synthetic biology of Y. lipolytica for biofuel production, we developed and characterized the molecular tools. The vectors for intra- and extracellular protein expression were constructed. In the recombinants, the expression level of the target genes could be fine-tuned to an extensive range in a constitutive or inducible manner. Thus, our developed expression platform will not only provide the multiple options for the recombinant proteins expression and secretion, but also effective tools for the precise control over the expression level and timing of the genes for pathway engineering. Usually, multiple-gene knockouts or integration are required for metabolic engineering of microorganisms for biofuel production, and the procedure for generation of these mutants is labor-intensive and time-consuming. To overcome this obstacle, we created a new gene disruption cassette for repeated use in Y. lipolytica by incorporating counter-selectable marker, ura3 and an inducible cre/loxp-mediated marker removal procedure. The approach allows us to readily introduce a wide variety of genetic modifications for synthetic biology prospect. Overall, these combined efforts eventually result in the development of a set of toolbox for genetic manipulation of this important strain Y. lipolytica, and pave the way for synthetic biology applications.