We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose-and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDPglucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDPgalactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.Lactic acid bacteria are widely used for production of fermented foods, where they are responsible not only for production of lactic acid as a food preservative but also for generation of flavor and texture. Several lactic acid bacteria produce exopolysaccharides (EPS). These EPS contribute to the rheological properties and texture of fermented products and are therefore of interest for food applications as natural biothickeners (46). Moreover, it has been suggested that EPS may confer health benefits to the consumer, and mouse model studies have indicated that EPS may have immunostimulatory (22), antitumoral (23), or cholesterol-lowering activity (34).Microbial polysaccharides can be present as constituents of cell walls, as parts of lipopolysaccharides (LPS) often referred to as O-antigens, or as capsular polysaccharides (CPS) associated with the cell surface, or they can be secreted as EPS in the environment of the cell. Detailed knowledge concerning microbial polysaccharide biosynthesis and the biophysical characteristics of these molecules has accumulated over the years (46). Different classes of EPS can be distinguished on basis of their biosynthesis mechanisms and the precursors required (45). They can be synthesized either extracellularly from exogenous substrates or intracellularly from sugar nucleotide precursors. Many EPS contain repeating units, the biosynthesis of which involves glycosyltransferases that sequentially link sugars from intracellular nucleotide sugars to a lipid carrier. This mechanism resembles the mechanism of production of O-antigens and several types o...

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps ؉ ) and in nonproducer strain MG5267 (Eps ؊ ) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31 P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps ؉ strain than in the Eps ؊ strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoylpentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps ؉ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, ␣-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains.13 C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation.The exopolysaccharides (EPS) include a diverse range of molecules that play vital roles in a variety of biological processes. Insight into how these molecules are synthesized and exported is crucial for exploitation of microorganisms in order to produce EPS of industrial or medical importance (24). Over the last few decades, studies on EPS biosynthesis have focused mainly on gram-negative bacteria, such as Escherichia coli, Xanthomonas campestris, Klebsiella spp., and Pseudomonas spp. (35). However, EPS-producing lactic acid bacteria have received growing attention in recent years because the EPS which they produce are food grade and have applications as food stabilizers, gelling agents, or immunostimulants (4, 7, 11). Biosynthesis of EPS includes assembly of the repeating monosaccharide unit on a lipid carrier by sequential transfer of monosaccharides from sugar nucleotides by glycosyltransferases and subsequent polymerization and export (27,35). Lactococcus lactis subsp. cremoris B40 produces an EPS composed of glucose, galactose, rhamnose, and phosphate at a ratio of 2:2:1:1 (18, 28). The genetic clusters for production of EPS by L. lactis B40 and Streptococcus thermophilus Sfi6 were characterized and found to encode proteins that have signi...

G.J. GROBBEN, J. SIKKEMA, M.R. SMITH A N D J. A.M. DE BONT. 1995. Lactobacillusdelbrueckiissp. bulgaricus NCFB 2772 produced an extracellular polysaccharide when grown in a chemically defined medium with glucose or lactose as the substrate carbohydrate. T h e isolated extracellular polysaccharide had a sugar composition of glucose, galactose and rhamnose in a ratio of 1 : 6.8 : 0.7. T h e production of extracellular polysaccharides increased a t higher temperatures, but the bacterium rapidly lost its polysaccharide producing ability at 47°C.