Production of highly polymerized bacterial levan in two eukaryotic hosts of biotechnological interest

Bacterial levan [β(2,6)–linked polyfructan] has potential applications in the food, bio-energetic, medical, pharmaceutical, and other industries. The lack of technically and economically feasible large-scale production systems limits the commercial exploitation of levan. Gluconacetobacter diazotrophicus secretes a levansucrase (LsdA, EC 2.4.1.10) that synthesizes high levels of levan and fructooligosaccharides from sucrose. This bacterium is not attractive for the cost-effective production of LsdA. In this research, we used Pichia pastoris and Nicotiana tabacum as hosts for LsdA production and direct levan synthesis, respectively. The recombinant yeast constitutively expressing the lsdA gene acquired saccharolytic capacity and secreted LsdA to a yield 9-fold higher than the value reported for the natural host. The occurrence of N-glycosylation in the yeast-produced LsdA did not affect the catalytic efficiency, substrate specificity, or product profile compared to the native non-glycosylated enzyme. This finding prompted us to express the lsdA gene in vacuoles, the most physiologically appropriate compartment to direct levan formation within the plant cell. Constitutive expression of LsdA fused to the vacuolar targeting signal of an onion fructosyltransferase allowed the accumulation of highly polymerized levan (above 104 fructose residues) in mature tobacco leaves where the polymer represented between 10 and 70% (w/w) of total dry weight. The latter value is the highest reported in the literature for a levan-producing transgenic plant grown in soil. No drastic physiological changes were observed in tobacco plants with levan yields up to 30% (w/w) in leaves. The polymer production remained stable in the plant progenies pointing for potential application in biotechnology.