Chemical Constituents and Molecular Mechanism of the Yellow Phenotype of Yellow Mushroom (Floccularia luteovirens)

(1) Background: Yellow mushroom (Floccularia luteovirens) is a natural resource that is highly nutritional, has a high economic value, and is found in Northwest China. Despite its value, the chemical and molecular mechanisms of yellow phenotype formation are still unclear. (2) Methods: This study uses the combined analysis of transcriptome and metabolome to explain the molecular mechanism of the formation of yellow mushroom. Subcellular localization and transgene overexpression techniques were used to verify the function of the candidate gene. (3) Results: 112 compounds had a higher expression in yellow mushroom; riboflavin was the ninth most-expressed compound. HPLC showed that a key target peak at 23.128 min under visible light at 444 nm was Vb2. All proteins exhibited the closest relationship with Agaricus bisporus var. bisporus H97. One riboflavin transporter, CL911.Contig3_All (FlMCH5), was highly expressed in yellow mushrooms with a different value (log(2) fold change) of −12.98, whereas it was not detected in white mushrooms. FlMCH5 was homologous to the riboflavin transporter MCH5 or MFS transporter in other strains, and the FlMCH5-GFP fusion protein was mainly located in the cell membrane. Overexpression of FlMCH5 in tobacco increased the content of riboflavin in three transgenic plants to 26 μg/g, 26.52 μg/g, and 36.94 μg/g, respectively. (4) Conclusions: In this study, it is clear that riboflavin is the main coloring compound of yellow mushrooms, and FlMCH5 is the key transport regulatory gene that produces the yellow phenotype.