Exopolysaccharide production by Lactobacillus reuteri, involving sucrase type of enzymes

Exopolysaccharides (EPSs) find numerous applications in the food as well as in the non-food industries. They can be used as for instance viscosifying, thickening, gelling or water binding agents. Furthermore certain EPSs are known to exert health promoting effects such as cholesterol lowering, immunomodulating, antitumoral and prebiotic activities. Using a new method, a large collection of Lactobacillus strains was screened on the production of EPS. One of the positive strains, strain 121, produced two different soluble homopolysaccharides during growth on sucrose, a fructan and glucan. This strain was identified as Lactobacillus reuteri, a probiotic strain and an excellent colonizer of the gastro-intestinal tract of a broad variety of hosts, including humans. L. reuteri 121 was selected for further research. Structure analysis of the polysaccharides produced by L. reuteri 121 revealed that the fructan was a linear levan with beta(2-6) linked fructosyl units. This was the first example of fructan synthesis by lactobacilli. The glucan possessed a unique highly branched structure with alpha(1-4) and alpha(1-6) linkages with alpha(1-4,6) branching points. Both polymers were synthesized by sucrase type of enzymes (glucosyl- and fructosyltransferases). These enzymes only need sucrose as substrate; the energy released by the cleavage of the glycosidic bond in sucrose is subsequently used for the polysaccharide synthesis reaction. During growth of L. reuteri on sucrose or maltose, the sucrases responsible for the synthesis of the glucan and the levan appeared to be completely bound to the cell wall, whereas during growth on sucrose part of the enzymes was released into the culture medium. The EPS production was not a stable characteristic in continuous cultures. Different spontaneous mutants appeared, such as the EPS negative mutant strain K24, lacking both the glucansucrase (a glucosyltransferase) and the levansucrase (a fructosyltransferase). Mutant 35-5, lacking levansucrase, appeared after a pH shift-down. Using PCR techniques with degenerated primers based on known glucansucrase or fructosyltransferase amino acid sequences, chromosomal fragments containing a glucansucrase (gtfA) or a fructosyltransferase (ftfA) were amplified. Both fragments were sequenced and characterized at the amino acid level and phylogenetic trees of both types of sucrases were constructed. Both the gtfA and the ftfA were cloned separately in Escherichia coli. Cell free extracts of the E. coli strain harboring the ftfA gene produced an inulosucrase, which synthesized inulin and fructose-oligosaccharides from sucrose. The recombinant glucansucrase and the L. reuteri glucansucrase synthesized the same unique glucan. These were the first examples of the isolation, characterization and cloning of Lactobacillus glucansucrase and fructosyltransferase genes