Background <p>Konjac glucomannan (KGM) is a high-viscosity, water-soluble dietary fiber that accumulates predominantly in the corms of Amorphophallus konjac (A. konjac). Due to its excellent gel-forming and water-retention properties, KGM is widely used in the food industry. The biosynthesis of KGM involves the coordinated action of cellulose synthase-like (CSL) enzymes and mannan synthesis-related (MSR) proteins. However, the regulatory mechanisms and functional interactions between these components remain poorly understood in A. konjac.</p> Results <p>In this study, we systematically characterized the AkCSLA gene family in A. konjac and identified 11 members. Transcriptomic analysis revealed that AkCSLA2 and AkCSLA3 were highly expressed during corm expansion and maturation, coinciding with active KGM accumulation. Subcellular localization assays in Nicotiana benthamiana (N. benthamiana) demonstrated that both AkCSLA2 and AkCSLA3 proteins localized to the plasma membrane, endoplasmic reticulum, and Golgi apparatus. Protein interaction analyses using yeast two-hybrid and co-immunoprecipitation assays showed that AkMSR1, a homologue of Arabidopsis thaliana MSR1, specifically interacted with AkCSLA3 but not with AkCSLA2. Functional studies in Pichia pastoris (P. pastoris) revealed that co-expression of AkCSLA3 and AkMSR1 significantly increased free mannose and glucose accumulation, with mannose levels elevated more than threefold compared to AkCSLA3 alone. These findings demonstrate that AkMSR1 modulates monosaccharide pools in an AkCSLA3-dependent manner in yeast.</p> Conclusion <p>This study demonstrates the physical interaction between AkCSLA3 and AkMSR1 and reveals that their co-expression in P. pastoris significantly reshapes monosaccharide content. These findings provide novel insights into the molecular regulation of KGM biosynthesis and identify potential targets for the genetic enhancement of KGM content in A. konjac.</p>