Riboflavin (vitamin B2), essential in tiny amounts as a precursor for oxidoreductase coenzymes, is a yellow pigment. Although it causes cytotoxicity via photoinduced damage of macromolecules, several microorganisms are striking overproducers. A question, unanswered for decades, is whether riboflavin overproducers can benefit from this property. Here, we report an ultraviolet (UV) protective effect of riboflavin. The spores of Ashbya gossypii, a riboflavin-overproducing fungus, are more sensitive to UV than those of Aspergillus nidulans. The addition of riboflavin to suspensions improves the UV resistance of both spore types. Interestingly, we show that regulation of sporulation and riboflavin overproduction in A. gossypii are linked. In batch culture, both were elevated when growth ceased. At constant growth rates, obtained in a chemostat culture, neither was elevated. Supplementation of cultures by cAMP, a known stress signal, negatively affected sporulation as well as riboflavin overproduction, establishing a second, independent argument for the linkage.

The filamentous fungus Ashbya gossypii is used for riboflavin biosynthesis on an industrial scale, but even the wild type displays overproduction. Because riboflavin overproduction was known to start at the transition between growth and stationary phase, it was suspected that overproduction was induced at low growth rates. However, chemostatic cultivations performed at different growth rates did not result in any detectable riboflavin formation. In this study, we report that it was not the final growth rate that triggered riboflavin overproduction but a decline in growth rate. Therefore, continuous fermenter cultivations with dilution rate shifts were performed. Peaks of riboflavin overproduction were observed in the wild type and in a RIB3placZ reporter strain after downshifts in dilution rate. Accumulation of riboflavin correlated with an increased expression of lacZ reporter activity. The step size of the downshifts corresponded to the peak size of riboflavin formation and reporter activity. Expression of further RIB genes encoding riboflavin biosynthetic enzymes was analyzed by RT-PCR. RIB mRNA levels of the ribulose-5-phosphate branch of the divided riboflavin biosynthesis pathway (RIB3, RIB4, and RIB5) were found to increase in the riboflavin production phase, whereas the RIB2 and RIB7 mRNA levels belonging to the GTP branch remained constant. We propose that a decline in growth rate triggers the increased expression of RIB3, RIB4, and RIB5 resulting in riboflavin overproduction. Because although a reduction in oxygen supply, temperature increase or decrease, or salt stress did affect growth, but neither did lead to riboflavin overproduction nor did induce RIB3 reporter expression, we conclude that declining nutrition must be the stress stimulus. Because about half of the cells in the hyphae of Ashbya gossypii did not accumulate riboflavin, the regulatory response on the cellular level can be estimated to be at least twice as great in comparison to what we detected as overall signals.