Enhancing the erythritol production of Yarrowia lipolytica by high-throughput screening based on highly sensitive artificial sensor and anchor protein cwp2

Yarrowia lipolytica is widely used for the industrial production of the natural sweetener erythritol. Despite improvements in fermentation process control and metabolic pathway regulation, bottlenecks still exist in terms of yield and screening technology. Therefore, we constructed an artificial sensor system for effective erythritol detection, established a single-cell droplet-based high-throughput screening system based on fluorescence-activated cell sorting, and obtained Y. lipolytica with improved erythritol production through mutagenesis and high-throughput screening. We used a droplet generator to co-cultivate Y. lipolytica 5-14 with Escherichia coli and used the E. coli fluorescent signal to detect the concentration of erythritol synthesized by Y. lipolytica 5-14 for high-throughput screening. Strains were subjected to UV mutagenesis for 120 s. Under optimized fermentation conditions using Y. lipolytica mutants in 96-well plates, the screening efficiency reached 16.7%. Yarrowia lipolytica 5-14-E6 showed a 21% increase in erythritol to 109.84 g/L. After fermentation at 30 degrees C in a 100-m3 fermenter for 75 hr, the mutant Y. lipolytica 5-14-E6 erythritol yield reached 178 g/L.One-Sentence Summary In this study, we constructed an artificial sensor system for effective erythritol detection, established a single-cell droplet-based high-throughput screening system based on fluorescence-activated cell sorting, and induced and screened mutant strains of Yarrowia lipolytica with improved erythritol production through mutagenesis and high-throughput screening.