Recent advances in droplet microfluidics for enzyme and cell factory engineering.

Enzymes and cell factories play essential roles in industrial biotechnology for the production of chemicals and fuels. The properties of natural enzymes and cells often cannot meet the requirements of different industrial processes in terms of cost-effectiveness and high durability. To rapidly improve their properties and performances, laboratory evolution equipped with high-throughput screening methods and facilities is commonly used to tailor the desired properties of enzymes and cell factories, addressing the challenges of achieving high titer and the yield of the target products at high/low temperatures or extreme pH, in unnatural environments or in the presence of unconventional media. Droplet microfluidic screening (DMFS) systems have demonstrated great potential for exploring vast genetic diversity in a high-throughput manner (>10/h) for laboratory evolution and have been increasingly used in recent years, contributing to the identification of extraordinary mutants. This review highlights the recent advances in concepts and methods of DMFS for library screening, including the key factors in droplet generation and manipulation, signal sources for sensitive detection and sorting, and a comprehensive summary of success stories of DMFS implementation for engineering enzymes and cell factories during the past decade.