Dynamic metabolic modeling uncovers systems-level strategies to simultaneously maximize levan yield and substrate...

Biopolymers such as levan have attracted growing interest in recent years due to their environmental sustainability and biocompatibility. Nevertheless, industrial-scale production remains constrained by the inherently low yields of bacterial synthesis under mass fermentation conditions. Bacillus subtilis LY7.16 was recently identified as a high-performing levan producer, achieving up to 101.9 grams of levan with a substrate use efficiency of 40.7% (w/w). Its scalability is strongly influenced by environmental parameters, particularly the initial sucrose concentration, which regulates both the equilibrium of exopolysaccharide biosynthesis and intracellular metabolic activity. To overcome these limitations, a dynamic metabolic modeling framework (ly716-Bs-dMM) was developed to simulate the complex effects of sucrose concentration on substrate conversion to levan. This integrated approach, combining dynamic and constraint-based modeling, incorporates both environmental variables and cellular metabolism to identify strategies for yield enhancement. Model simulations showed strong agreement with experimental data, demonstrating that elevated sucrose concentrations promote levan production while driving a metabolic shift from biomass synthesis toward levansucrase expression, mediated by sacB upregulation. Furthermore, the simulations demonstrate transition in extracellular levansucrase activity-from hydrolysis dominance under low sucrose conditions to transfructosylation dominance under high sucrose abundance. Collectively, ly716-Bs-dMM underscores a systems metabolic engineering strategy that integrates microbial physiology with operating conditions to simultaneously maximize levan yield and substrate use efficiency. Scenario-based analysis of a predictive hypothesis further suggests that integrating amino acid supplementation with fed-batch operation, alongside energy pathway engineering, can overcome the current tradeoff between levansucrase activity and cell biomass formation, thereby advancing levan production potential.