Combining adaptive laboratory evolution and metabolic engineering for efficient erythritol production from crude glycerol by Yarrowia lipolytica.

Erythritol is a natural zero-calorie sweetener with potential for sustainable healthy diets. While yeasts can convert biodiesel-derived glycerol into erythritol, the low production and by-product accumulation limit its industrial commercialization. Here, we isolated different morphologically Yarrowia lipolytica mutants with higher erythritol production through adaptive laboratory evolution under hyperosmotic stress, in which mutant Z exhibited superior growth performance and membrane-related genetic variants compared to the parent strain. We then systematically engineered strain Z to optimize the flux towards erythritol by improving glycerol utilization, reducing the synthesis of competing sugar alcohols, complementing auxotrophic markers, and boosting precursor supply. After fermentation condition optimization and two-stage fed-batch fermentation in a 5-L bioreactor, the final engineered strain Z12 produced 250.76g/L erythritol from pure glycerol and 232g/L erythritol from crude glycerol, which is the highest reported titers for both feedstocks. This study demonstrates the effective integration of adaptive evolution with metabolic remodeling for efficient erythritol biosynthesis.