Expanding the Substrate Range and Product Portfolio of Y. Lipolytica through Metabolic Pathway Engineering and Synthetic Biology Tool Development

Yarrowia lipolytica is an industrially attractive, non-conventional yeast due to advantageous traits such as its ability to bypass tight pathway regulation, grow on hydrocarbons and other diverse carbon sources, grow at lower temperatures, and tolerate natural inhibitors like salts.  Several efforts of metabolic engineering have demonstrated the flexibility and potential of this platform.  As an example, our lab has demonstrated the lipogenesis potential of this yeast by engineering a strain capable of accumulating greater than 90% dry cell weight lipid with titers exceeding 40 g/L.  This high lipogenic potential, and more broadly, the high flux through CoA precursor molecules and key TCA cycle intermediates, makes this organism an attractive host for the production of a wide breadth of chemicals.  However, several tools and pathways require improvements to further engineering capacity in this host.  To further harness the full metabolic potential of Y. lipolytica, we have established a suite of synthetic biology tools to facilitate metabolic engineering of this host.  We demonstrate here some recent successes in this effort to create synthetic promoters and terminators capable of tuning gene expression.  Initial efforts from our lab have focused on a hybrid promoter approach capable of varying gene expression by tuning the number of repeated upstream activating sequences.  Additionally, a collection of synthetic terminators expands this toolset to create tunable expression at levels that were previously unachievable in this yeast.  More recent efforts from our lab, including creating synthetic transcription control elements, will be described.  Finally, we demonstrate the utility of these parts and approaches through several applications.  First, we developed a workflow to enhance utilization of alternative carbon substrates in Y. lipolytica.  We demonstrate this through a case study of producing lipids (suitable for biodiesel) from sorghum syrup.  Second, we demonstrate a diversified product portfolio demonstrating that high levels of CoA precursor molecules can be converted and rewired into valuable commodity and specialty chemicals including modified fatty acids, organic acids, and other industrially attractive small molecules at high titers (i.e. g/L levels).  To this end, our expanded synthetic biology toolkit enables both an enhanced substrate range and expanded product portfolio for this industrially attractive host organism.