Adaptive Laboratory Evolution of Ashbya gossypii in Sugarcane Molasses: Biomass-Driven Riboflavin Overproduction

The utilization of sugarcane molasses as a low-cost carbon source for riboflavin production is hindered by the reactive oxygen species (ROS) stress induced by its complex components, which suppresses microbial metabolism. To address this, we employed adaptive laboratory evolution (ALE) under progressively increasing stress to develop a sugarcane molasses-tolerant and high-yielding Ashbya gossypii. The adapted strain achieved a riboflavin titer of 298.39 ± 2.01 mg/L, representing a 99.4% increase over the parental strain (149.66 ± 4.97 mg/L), accompanied by a 96% increase in biomass (dry cell weight). Notably, the specific riboflavin production per unit biomass showed no significant difference between the two strains, indicating that the improved total yield was primarily driven by enhanced biomass accumulation. Transcriptomic analysis revealed the molecular basis for this enhanced biomass accumulation—the elevated expression of antioxidant enzymes (SOD1, PRDX5) mitigated ROS levels to support cellular growth, while the coordinated upregulation of the pentose phosphate pathway (E2.2.1.1) and purine metabolism genes (PPAT, ADE5, PFAS, ADSL) enhanced the supply of biosynthetic precursors, ribulose-5-phosphate (Ru5P) and GTP, for nucleotide biosynthesis and cell proliferation. These metabolic adjustments collectively enabled the adapted strain to achieve robust growth under sugarcane molasses stress, thereby driving the overall increase in riboflavin production. This study elucidates the molecular mechanism underlying ALE-improved riboflavin production and provides a promising strategy for its industrial fermentation using sugarcane molasses.