Genomic Identification of the Levansucrase Operon in Novel Bacillus velezensis HL25 in Sucrose Utilizing Pathway and Functional Characterization of Its Levansucrase

Levan and levan-type fructooligosaccharides (L-FOSs) are non-digestible fructans with prebiotic properties that promote gut microbiota growth. This study presents the first genomic analysis of a Bacillus velezensis HL25 strain with high fructan-producing efficiency, revealing genes involved in sucrose utilization and fructan biosynthesis. A putative levansucrase operon was identified in the HL25 genome, consisting of the sacB levansucrase gene classified in GH68 subfamily 1 and the following three GH32 genes: endo-levanase (lev), β-fructofuranosidase (ffase), and sucrose-6-phosphate hydrolase (scrB). Remarkably, sugars involved in levan biosynthesis are proposed to be transported through three distinct systems: a multiple-component ABC sugar transporter, a glucose/H+ symporter, and glucose- and fructose-specific phosphotransferase systems (PTS). Subsequently, recombinant HL25SacB levansucrase exhibited optimal activity at 40 °C and pH 5.0 in 50 mM sodium acetate buffer. The enzyme demonstrates high specificity in converting sucrose into a mixture of short-chain FOSs (DP 2–4) and levan, achieving a 62.5% conversion rate at 30 °C with 200 g/L sucrose over 24 h. These findings demonstrate the potential of this B. velezensis HL25 strain as an efficient whole-cell biocatalyst and highlight the applicability of the recombinant HL25SacB enzyme as a promising tool for sustainable production of FOSs and levan.