Abstract
Levan-type fructooligosaccharides (LFOSs) possess valuable bioactivities but are produced in low yields by Gram-negative bacterial levansucrases. Here, we rationally engineered the levanbiose-binding site of Erwinia tasmaniensis levansucrase (EtLsc) to enhance LFOS synthesis. Eleven residues surrounding the levanbiose-binding site were substituted to disrupt levan chain binding or to modulate loop flexibility. The results indicate that D82A, R377A, and G379P do not synthesize levan polymers. In addition, R377A and G379P largely produced LFOS, converting 35% and 24% of sucrose into LFOS. Affinity PAGE confirmed the loss of the levan-binding ability of the R377A and G379P variants. MD simulations revealed that G379P induces a structural rearrangement of loop II, altering the orientation of the key residue R377. Prebiotic activity analysis demonstrated that LFOS synthesized by the R377A variant stimulated the growth of probiotic bacteria, including Limosilactobacillus fermentum, and Lacticaseibacillus casei, more effectively than inulin. Together, these results identify R377 and G379 as critical determinants of levan elongation and demonstrate a promising strategy for enhancing LFOS production with Gram-negative levansucrases.