Mannitol's role and metabolism in fungi have been widely debated. It has been proposed to serve as carbohydrate storage, neutralize reactive oxygen species, support germination, and increase resistance to stress conditions such as osmotic stress. But some publications demonstrate that mannitol is less critical for osmotic stress resistance than glycerol. We hypothesized that the predominance of Page 2 of 3 mannitol or glycerol in the osmotic stress answer might depend on the environmental conditions. We decided to couple this investigation with a clarification of mannitol metabolism. Mannitol metabolism was described as a cycle by Hult and Gatenbeck in 1978. Their proposed cycle, in Alternaria alternata, comprises four enzymes: a mannitol-1- phosphate dehydrogenase, a mannitol-1-phosphatase, a mannitol dehydrogenase, and a hexokinase. Since then, these enzymes have been identified in many fungi, opening the possibility to generate deletion strains for the “mannitol cycle” enzymes to challenge this model. Such tests performed in Aspergillus niger, A. alternata, or Botrytis cinerea demonstrated that the mannitol cycle model could not fully explain mannitol metabolism. Still, no new model could be built from these results. The tests performed vary from one strain to another, making comparison impossible. To clarify the debate, we decided to explore mannitol metabolism by performing the same tests on two fungi: Trichoderma reesei and Aureobasidium pullulans. We aim to generate single and double deletion strains for mannitol-1-phosphate dehydrogenase and mannitol dehydrogenase. Once developed, the strains are cultivated with different carbon sources with or without osmotic stress. During cultivation, polyols and carbon source amounts are monitored. Biomass is measured at the end of the experiment. Our observations will then be confronted with existing results in other strains to contribute to a new model for mannitol metabolism. These tests will also characterize the osmotic stress answer through polyol production.