Adaptive Laboratory Evolution and Scale-Up of <i>Rhodosporidium toruloides</i> for Commercial Lipid Production

There is increasing demand for microbial lipids as a sustainable alternative to mitigate the detrimental environmental and societal impacts associated with the production of palm oil, including tropical deforestation, significant contributions to global warming, and extensive land and water use. Microbial lipid production has the potential to address these concerns and meet the growing demand for palm oil through strain engineering and innovative fermentation and downstream process development. However, the current cost to produce microbial oils is not competitive with traditional sources of palm oil, and improvements in fermentation titer, rate, and yield are needed. We report here the application of Adaptive Laboratory Evolution to improve lipid production by the oleaginous yeast Rhodosporidium toruloides, multi-omic characterization of the genome-wide metabolic rewiring resulting from its evolutionary adaptation, and scale-up of a high-cell density fed-batch fermentation process to 50 m 3 commercial scale. The methods, strains, and process improvements described here represent significant steps toward commercialization of a fermentation-based palm oil substitute.