High-Throughput Screening and Dual Feeding Fed-Batch Strategy for Enhanced Single-Cell Oil Accumulation in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Herein, we combined metabolic evolution with fluorescence-activated cell sorting (FACS) of cells stained with the lipophilic dye BODIPY for isolation of SCO-overproducing strains of Yarrowia lipolytica. Metabolic evolution was implemented for enrichment of high SCO-accumulating mutant population which were then sorted by fluorescence signals using flow cytometry coupled with FACS. A mutant isolated by this approach exhibited 1.5- and 1.2-fold higher SCO titer and content, respectively, than the wild type under batch culture of sugarcane bagasse hydrolysate complex media. In addition, the mutant had whole-cell fatty acid composition different from that of the wild type with higher oleic and linoleic acids. Dual-stage fed-batch process applied to the mutant yielded high SCO titer of 49.7 g/L from hydrolysates, a fourfold improvement over batch process. This study highlights evolution-based in conjunction with fluorescence-based high-throughput screening as a powerful strategy for attaining high single-cell oil-accumulating phenotype in Y. lipolytica exploited for sustainable biodiesel and oleochemicals synthesis.