Enhanced levansucrase expression in Bacillus subtilis via regulatory element optimization for high-yield lactosucrose...

Lactosucrose is a functional oligosaccharide with prebiotic properties and broad applications in the food and nutraceutical industries. In this study, a systematic strategy was developed to enhance lactosucrose production by optimizing levansucrase expression in Bacillus subtilis. Six levansucrases were screened, and levansucrase from Brenneria goodwinii (BgLS) exhibited the highest solubility and whole-cell catalytic activity. Whole-cell reaction conditions were optimized, identifying pH 6.0 and 50 °C as optimal. BgLS expression was further enhanced through translational regulation by engineering synthetic 5'-untranslated regions (5'-UTRs) and N-terminal coding sequences (NCSs), followed by combinatorial optimization. Subsequent transcriptional optimization using endogenous promoters further increased enzyme expression. The best engineered strain achieved a fermentation activity of 190.7 U/mL and produced lactosucrose with a 46.5% conversion rate from 200 g/L lactose and 300 g/L sucrose. This work demonstrates an efficient platform for high-level lactosucrose biosynthesis and highlights the effectiveness of multi-level gene expression regulation.