Hog1 MAP Kinase Controls Early Riboflavin Overproduction Under Combined Acidic pH and Salinity in the Yeast <em>Debaryomyces hansenii</em>

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 (previously as syn. Candida famata) is a halotolerant flavinogenic yeast previously exploited for riboflavin biosynthesis; however, its biotechnological application has been limited by genetic instability and poor understanding of its regulatory networks. Here, we uncover a novel role for the High Osmolarity Glycerol (HOG) pathway in riboflavin metabolism of D. hansenii. Using the first stable knockout mutant (Dhhog1Δ), we demonstrate that loss of DhHog1 triggers early, premature, and enhanced secretion of riboflavin under acidic and saline conditions, visible as a yellow fluorescent pigment in the culture medium. Accelerated riboflavin accumulation in the mutant 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 consistent up-regulation of RIB1, RIB4, and RIB6 genes and derepression of the iron regulator SEF1 in Dhhog1Δ, supporting a model where DhHog1 negatively controls riboflavin biosynthesis through stress-responsive transcription factors and pseudo iron-starvation signaling. Our findings broaden the functional scope of the HOG pathway by linking osmotic stress adaptation with secondary metabolism and establish DhHog1 as a key negative regulator of early riboflavin overproduction and secretion. This work provides new insights into yeast stress-metabolism crosstalk and highlights D. hansenii as a promising platform for metabolic engineering of industrial riboflavin production.