Fluorescence-activated droplet sorting for enhanced pyruvic acid accumulation by Candida glabrata

One of the goals of metabolic engineering is to engineer strains that can optimally produce target metabolites. However, the current workflow for rational engineering of the metabolic pathway is sometimes time-consuming and labor-intensive. Here, we have established a cost-effective approach for screening for variants secreting metabolites. Different surface display systems were adopted and verified, which anchored pHluorin to the Candida glabrata cell surface to associate pyruvic acid detection with the read out of this reporter. A generalizable simulation approach based on computational fluid dynamics and regularity of generated droplet dimension was presented, which was found to be an efficient design tool to explore microfluidic characteristics or optimization. Finally, a microfluidic platform based on simulation coupled with surface display system was constructed. A mutant exhibiting a 73.6% increase in pyruvic acid production was identified. This ultrahigh-throughput screening pattern offers a practical guide for identifying microbial strains with many traits of interest.