A Microfluidic Multiplex Sorter for Strain Development

Abstract Selecting strains with superior traits from strain improvement strategies is challenging, as it involves navigating the fitness landscape by applying selective pressures that drive variants from peaks of improvement to valleys over time. In recent years, the screening and selection is conducted via droplet microfluidic methods due to its high throughput capabilities. However, the oft‐used binary strategy, targeting only the high levels of improved traits, may not reflect the overall enhancement. A multiplexed sorting method capable of applying an additional threshold to sort traits by phenotypic strength is reported. The novel approach uses a droplet‐digital microfluidic sorter to screen different volumes of droplets using the same device design and sorting parameters. This method is used to sort glucoamylase enzyme mutants with two levels of activity (medium and high) from libraries of diastatic yeast that have been mutated with non‐genetically modified techniques. Using the multiplex system, medium‐performing strains with enhanced (up to 60%) fermentation kinetics in synthetic beverage media, which would have been missed with a binary screening approach, are identified. The multiplex sorting strategy efficiently finds strains with superior fermentation traits in the fitness landscape without requiring extensive screening rounds and mutations.