Enhanced fructooligosaccharides synthesis by engineered Trichoderma atroviride β-fructofuranosidase

Here we report the first β-fructofuranosidase from the Trichoderma genus producing fructooligosaccharides (FOS). The novel enzyme from Trichoderma atroviride (TaINV) here characterized was heterologously expressed, purified, and biochemically analyzed. TaINV exhibited hydrolytic activity mainly toward sucrose and other substrates containing β-(2 → 1) linkages, with minor activity toward β-(2 → 6) bonds. In addition to hydrolysis, it catalyzed the synthesis of FOS of all three structural series (1F-FOS, 6F-FOS, and 6G-FOS). At the maximal production point, TaINV synthesized 252 g/L of total FOS, representing 50.3% (w/w) of the total sugars in the reaction mixture, with 1-kestose as the major product, representing ~ 85% of the total products synthesized. Structural analysis based on AlphaFold-predicted TaINV model and comparative superimposition with GH32-substrate complexes revealed conserved catalytic motifs and residues located in positions associated with substrate binding and specificity in characterized GH32 enzymes. Site-directed mutagenesis confirmed the essential role of the catalytic triad (Asp63, Asp201, Glu277) and identified additional residues shaping transfructosylation specificity. Variants including substitutions W60Y and N62S increased total FOS production, reaching 62.7% and 57.4% (w/w) of total sugars, respectively, which are comparable to yields obtained with commercial enzymes. Overall, TaINV represents a distinct intracellular fungal β-fructofuranosidase with strong transfructosylation capacity and preference for short-chain FOS. These findings expand the current knowledge of GH32 enzyme diversity and highlight TaINV as a promising biocatalyst for the efficient production of low-degree polymerization FOS with potential prebiotic applications. KEY POINTS: • Novel Trichoderma β-fructofuranosidase with high transfructosylation activity. • Catalytic residues defining FOS synthesis identified by structure-function analysis. • Engineered variant boosting FOS yield to 62% of total sugars.