OsTH1 is a key player in thiamin biosynthesis in rice.

Thiamin is a vital nutrient that acts as a cofactor for several enzymes primarily localized in the mitochondria. These thiamin-dependent enzymes are involved in energy metabolism, nucleic acid biosynthesis, and antioxidant machinery. The enzyme HMP-P kinase/thiamin monophosphate synthase (TH1) holds a key position in thiamin biosynthesis, being responsible for the phosphorylation of HMP-P into HMP-PP and for the condensation of HMP-PP and HET-P to form TMP. Through mathematical kinetic model, we have identified TH1 as a critical player for thiamin biofortification in rice. We further focused on the functional characterization of OsTH1. Sequence and gene expression analysis, along with phylogenetic studies, provided insights into OsTH1 bifunctional features and evolution. The indispensable role of OsTH1 in thiamin biosynthesis was validated by heterologous expression of OsTH1 and successful complementation of yeast knock-out mutants impaired in thiamin production. We also proved that the sole OsTH1 overexpression in rice callus significantly improves B1 concentration, resulting in 50% increase in thiamin accumulation. Our study underscores the critical role of OsTH1 in thiamin biosynthesis, shedding light on its bifunctional nature and evolutionary significance. The significant enhancement of thiamin accumulation in rice callus upon OsTH1 overexpression constitutes evidence of its potential application in biofortification strategies.