Repurposing extracellular phosphatases to enhance thiamine biosynthesis and refine a genetic tool in Schizosaccharomyces pombe.

Thiamine (Vitamin B1) and its active form, thiamine pyrophosphate (TPP), are essential cofactors in all living organisms. However, industrial thiamine production in prokaryotes has been hindered by tight feedback regulation, such as that imposed by TPP riboswitches. This study explores the fission yeast Schizosaccharomyces pombe-which naturally lacks TPP riboswitches-as an alternative production host. We identified the extracellular phosphatases Pho1 and Pho4 as key redundant enzymes that hydrolyze thiamine monophosphate to support the growth of thiamine auxotrophic strains. Intracellular retention of Pho1 and Pho4, engineered by signal peptide deletion, enhanced intracellular thiamine synthesis, as evidenced by strengthened TPP-mediated repression of nmt1+ gene. This modification effectively reduced the leaky basal expression of heterologous genes driven by the thiamine-repressible nmt1 promoter without compromising its strong inducibility, resulting in an improved gene expression system for S. pombe. Our work provides novel insights into thiamine metabolism and an effective engineering strategy for optimizing eukaryotic expression systems.