(317b) A Comparative Analysis of Single Cell and Droplet-Based FACS for Improving Production Phenotypes: Riboflavin Overproduction in Yarrowia Lipolytica

Evolutionary approaches to strain engineering inherently require the identification of suitable selection techniques for the product and phenotype of interest. Here, we undertake a comparative analysis of two related but functionally distinct methods of high-throughput screening: traditional single cell fluorescence activated cell sorting (single cell FACS) and microdroplet-enabled FACS (droplet FACS) using water/oil/water (w/o/w) emulsions. To do so, we first engineer and evolve the non-conventional yeast Yarrowia lipolyticafor high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. Following mutagenesis and adaptive evolution, a direct parity-matched comparison of these two selection strategies was conducted. Both strategies led to significant increases in total riboflavin titer (32 and 54 fold relative to the parental PO1f strain, respectively). However, single cell FACS favored intracellular riboflavin accumulation compared with droplet FACS that favored extracellular product accumulation. We also find that for the test case of riboflavin, the extent of secretion and total production were highly correlated. The resulting differences in production modes and levels clearly demonstrate the significant impact that selection approaches can exert on final evolutionary outcomes in strain engineering and provide a cautionary tale when intracellular read-outs of product concentration (including signals from biosensors) are used as surrogates for total production of potentially secreted products.