Design and Development of Molecular Tools for Synthetic Biology of Yarrowia lipolytica Towards Production of Lipid-based Industrial Oils

The yeast Yarrowia lipolytica with long history for industrial application has attracted growing attention due to its capability of accumulating lipid as an alternative feedstock for producing renewable hydrocarbon. To facilitate synthetic biology of Y. lipolytica, we developed and characterized the molecular tools. A “Biobrick” library consisting of more than 35 natural and engineered promoters was constructed, and the expression level of the target genes could be fine-tuned to an extensive range in a constitutive or copper ion-inducible manner in the recombinants. This expression platform developed can provide effective tools for the precise control over the expression level and timing of the genes for pathway engineering. Usually, multiple-gene knockout or integration is required for metabolic engineering of microorganisms for biofuel production, and procedure for generation of these mutants is labor-intensive and time-consuming. To overcome this obstacle, we increased the frequency of homologous recombination in the strains by blocking non-homologous end joining pathway, allowing us to introduce a wide variety of genetic modifications for synthetic biology operations. Altogether, these combined efforts eventually result in the development of a set of tools for genetic manipulation of this important strain Y. lipolytica. After establishment of the genetic manipulation platform, biosynthesis pathway of ricinoleic acid was engineered in this strain, and the fatty acid with same structure of the major component present in castor oil was produced by the recombinants. This demonstrated the feasibility of design and production of tailored lipid-based oils and products through interception and modification of Yarrowia’s lipid metabolism.