Metabolic Engineering of Disaccharide Catabolism for Polysaccharide Formation in Lactococcus lactis and Streptococcus thermophilus

Abstract: Exopolysaccharides (EPSs) produced by the lactic acid bacteria (LAB) Lactococcus lactis and Streptococcus thermophilus are important for the rheological behaviour and texture of a variety of fermented food products. Furthermore, EPSs from food-grade LAB have potential as food additives and functional food ingredients with both health and economic benefits. However, the production levels of EPSs from LAB are low, and this must be overcome before EPSs can be fully exploited. The objective of the work presented in this thesis was to improve the production of EPSs from disaccharides by L. lactis and S. thermophilus. The disaccharide metabolism and EPS biosynthesis of these organisms have been investigated by the construction of strains with different levels of central enzymes and the study of those strains under controlled growth conditions. The role of the enzyme beta-phosphoglucomutase (beta-PGM) was assessed in L. lactis by comparing a mutant lacking the enzyme with the parent strain. It was found that the enzyme was important for growth on maltose and essential for growth on trehalose. Furthermore, maltose-grown cells of the beta-PGM mutant accumulated polysaccharides. The predominant role of beta-PGM in trehalose metabolism was found to depend on another enzyme, trehalose 6-phosphate phosphorylase (TrePP). This novel enzyme, which phosphorylates trehalose 6-phosphate into beta-glucose 1-phosphate and glucose 6-phosphate, was characterised on both biochemical and genetical levels. Different EPS-producing strains of S. thermophilus were genetically typed, and the strains were found to group according to their EPS structure. The role of enzymes in the carbohydrate metabolism for EPS production in S. thermophilus was further investigated. First the pgmA gene, encoding phosphoglucomutase (PGM), was identified and cloned. Strains lacking PGM activity and those strains with elevated levels of PGM activity were found to produce similar levels of EPS. However, pgmA was essential for growth on glucose. The results implied that the Leloir pathway and the enzyme UDP-glucose pyrophosphorylase (GalU) were important for EPS production. A new semi-defined growth medium was developed and used for studies of strains with different levels of these enzymes. The Leloir pathway could be overexpressed in spontaneous mutants, while the gene encoding GalU had to be identified and cloned. The results indicate that simultaneous overexpression of PGM and GalU has a positive effect on EPS production. The highest EPS production could be achieved by inactivating pgmA and overexpressing the Leloir pathway. The results suggest ways of enhancing the EPS production with genetic engineering or through the natural selection of mutants.