Heterologous production of structurally distinct levans in an optimized Paenibacillus polymyxa chassis

Levan is a bacterial β-fructan exopolysaccharide that is highly valued for its applications as a prebiotic, natural sweetener, and adhesive. Levan is synthesized by levansucrases (EC 2.4.1.10), which utilize the energy from sucrose hydrolysis to polymerize fructose. To establish an efficient microbial platform for levan production, we employed the non-pathogenic soil bacterium, Paenibacillus polymyxa, a natural producer of the exopolysaccharides paenan and levan. By sequential deletion of genes involved in paenan biosynthesis, extracellular proteolysis, levan degradation, and foam formation, we created a production chassis with enhanced levan production. In batch fermentation, the optimized strain produced 30.2 g⋅L⁻¹ of ultrapure levan (yield: 0.30 g⋅g⁻¹), which was further increased by 82 % using a fed-batch strategy. To expand the structural diversity of levans, levansucrases from various bacterial strains were integrated into the genome of P. polymyxa. Seven of the eight variants successfully produced high-purity levan at titers from 1.8 to 26.3 g⋅L⁻¹. The obtained levans were structurally diverse, with molecular weights from 5.5 to 30.9 MDa and branching degrees of 9 to 16 %. This engineered P. polymyxa chassis enables the production of structurally tailored levans under standardized conditions and provides a versatile platform for recombinant exopolysaccharide biosynthesis.