Establishment of picodroplet-based co-culture system to improve erythritol production in Yarrowia lipolytica

Fluorescence-activated droplet sorting (FADS) combined with transcription factor (TF)-based biosensors allows for the high-throughput isolation of clones that overproduce extracellular metabolites. Here, we developed a FADS-TF coculture pipeline for improving erythritol production in Yarrowia lipolytica. The pipeline comprises three droplet operation steps: the generation and incubation of droplets that encapsulate yeast mutants with single-cell resolution, the pico-injection of fluorescence-based erythritol-biosensing Escherichia coli, and the sorting of the highest fluorescence droplets with an approximately 1 parts per thousand survival ratio. With the picodroplet-based workflow, we initially carried out a two-stage temperature and pH control strategy on the droplets to separate the erythritol production and detection processes. We then eliminated the background expression of biosensors by utilizing pH-sensitive erythromycin, finally establishing a complete version of the coculture screening system. The optimal strain, Yarrowia lipolytica S4-9, contributed 231.2 g/L erythritol after a 114-h fermentation and was successfully screened from the fourth round of the mutant library. Compared to the parent, the erythritol yield and production rate of Yarrowia lipolytica S4-9 increased by 16.97 % and 26.09 %, respectively, in a 5-L bioreactor. Our results indicated that FADS-TFs have great potential for the high-throughput screening of extracellular products as additional biosensors become available.