In silico identification of metabolic engineering strategies for improved lipid production in Yarrowia lipolytica by genome-scale metabolic modeling

Yarrowia lipolytica , an oleaginous yeast, is a promising platform strain for production of biofuels and oleochemicals as it can accumulate a high level of lipids in response to nitrogen limitation. Accordingly, many metabolic engineering efforts have been made to develop engineered strains of Y. lipolytica with higher lipid yields. Genome-scale model of metabolism (GEM) is a powerful tool for identifying novel genetic designs for metabolic engineering. Several GEMs for Y. lipolytica have recently been developed; however, not many applications of the GEMs have been reported for actual metabolic engineering of Y. lipolytica . The major obstacle impeding the application of Y. lipolytica GEMs is the lack of proper methods for predicting phenotypes of the cells in the nitrogen-limited condition, or more specifically in the stationary phase of a batch culture. In this study, we showed that environmental version of minimization of metabolic adjustment (eMOMA) can be used for predicting metabolic flux distribution of Y. lipolytica under the nitrogen-limited condition and identifying metabolic engineering strategies to improve lipid production in Y. lipolytica . Several well-characterized overexpression targets, such as diglyceride acyltransferase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase, were successfully rediscovered by our eMOMA-based design method, showing the relevance of prediction results. Interestingly, the eMOMA-based design method also suggested non-intuitive knockout targets, and we experimentally validated the prediction with a mutant lacking YALI0F30745g, one of the predicted targets involved in one-carbon/methionine metabolism. The mutant accumulated 45% more lipids compared to the wild-type. This study demonstrated that eMOMA is a powerful computational method for understanding and engineering the metabolism of Y. lipolytica and potentially other oleaginous microorganisms.