Hog1 MAP kinase modulates early riboflavin accumulation under low-pH and saline conditions in Debaryomyces hansenii

Riboflavin (vitamin B2) is an essential precursor of flavin cofactors involved in redox metabolism, and its industrial production increasingly relies on microbial fermentation. Debaryomyces hansenii is a halotolerant flavinogenic yeast previously exploited for riboflavin biosynthesis; however, its biotechnological application has been limited by genetic instability and incomplete understanding of its regulatory networks. Here, we reveal a novel connection between the High Osmolarity Glycerol (HOG) pathway and riboflavin metabolism in D. hansenii . Using a stable Dhhog1 Δ mutant, we demonstrate that loss of Dh Hog1 leads to earlier secretion of riboflavin under acidic and saline conditions, visible as a yellow fluorescent pigment in the culture medium. This early riboflavin accumulation was accompanied by altered assimilation of phosphorus, sulfur, and magnesium but not iron, suggesting that regulation extends beyond classical iron limitation. Gene expression analyses showed up-regulation of RIB1 , RIB4 , and RIB6 , together with derepression of SEF1 , indicating that Dh Hog1 modulates the timing of riboflavin production. These findings uncover a previously unrecognized role of the HOG pathway in coordinating stress responses with secondary metabolism and highlight D. hansenii as a promising platform for metabolic engineering of riboflavin production.