Metabolic Engineering of Yarrowia lipolytica for fuels and chemicals production

The non-conventional yeast Yarrowia lipolytica is an excellent platform for the production of biofuels and biochemical.   However, it is difficult to robustly engineer complex phenotypes in this organism since we are limited in the availability of molecular and synthetic biology tools.  Here, we describe recent advances in both the synthetic control of transcription of this host as well as applications of metabolic engineering.  First, we will discuss our novel approach of hybrid promoter engineering to produce libraries of varied expression for this organism.  We demonstrate that this approach can generate ranges of over 400 fold in mRNA level and led to the creation of the strongest promoters in Y. lipolytica.  Such synthetic parts were critical for gene expression modifications in the context of metabolic engineering.  Second, we demonstrate a large-scale strain engineering effort in this organism that is enabled by the promoter and genetic tools we developed.  Specifically, we investigate the metabolic landscape for lipid production by combining multiple gene deletions and overexpressions simultaneously along with strain directed evolution.  These engineering efforts resulted in saturated cells containing upwards of 90% lipid content and titers higher than 40 g/L lipids, representing a more than 100-fold improvement over parental strain and conditions.  Finally, we discuss recent advances in the metabolic engineering of additional pathways within this organism for the production of non-native metabolites.