Unique synergistic and compensatory interaction between β-1,3-glucosyltransferase isoenzyme in Ganoderma lucidum...

Two β-1,3-glucosyltransferase isoenzymes, designated GL20535 and GL24465, are present in Ganoderma lucidum polysaccharide (GLPS) biosynthesis. Previous studies have demonstrated that the expression level of gl20535 significantly influences the yield of GLPS, and silencing gl20535 results in upregulation of gl24465. However, the specific function of GL24465 in polysaccharide biosynthesis and its interaction mechanism with GL20535 remain poorly understood. In this study, the roles and interrelationship between GL24465 and GL20535 in GLPS biosynthesis were systematically investigated through individual and combinatorial gene silencing and overexpression approaches. Results indicated that individual modulation of gl24465 exerted minimal effects on polysaccharide production. In contrast, co-overexpression of both genes markedly enhanced the accumulation of intracellular polysaccharides (IPS) and extracellular polysaccharides (EPS), with increases of 9.92% and 89.15%. Conversely, co-silencing led to significant reductions in cell biomass, cell wall thickness, and EPS and IPS contents, by 13.81%, 50.25%, 20.52%, and 35.02%, respectively. Co-regulation also affected GLPS monosaccharide composition and glycosidic linkage ratios by altering the transcription of key enzymes in sugar donor synthesis, including phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGP), as well as glucosyltransferases Transcriptional analysis revealed that changes in gl24465 expression did not significantly affect the transcription of gl20535. Integrating these findings with existing evidence indicating that GL20535 functions as a major enzyme in polysaccharide production, while GL24465 provides compensatory support for maintaining both yield and structural integrity, this study offers novel insights into the functional cooperation of glycosyltransferase isoenzymes in edible and medicinal fungi，with potential implications in biomanufacturing optimized polysaccharides.