Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

Abstract

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...

Catabolism of mannitol in Lactococcus lactis MG1363 and a mutant defective in lactate dehydrogenase

“…Samples (5 ml) of the LDH d or MG1363 cultures grown in medium containing mannitol or glucose were taken at different growth stages, centrifuged (2000 g, 5 min, 4 mC) and supernatant solutions were stored at k20 mC until analysis by HPLC using a refractive index detector (LKB2142). Glucose, mannitol, acetate, ethanol, formate, lactate, acetoin and 2,3-butanediol were quantified using an HPX-87H anion exchange column (Bio-Rad) at 60 mC, with 5 mM H # SO % as the elution fluid and a flow rate of 0n5 ml min − " (Hugenholtz & Starrenburg, 1992 (Ramos & Santos, 1996 ;Ramos et al, 2001) or by spiking the NMR samples with the suspected pure compounds. The reported values for intracellular phosphorylated compounds are means of two independent growth experiments and the accuracy varied from 10 to 15 %.…”

“…For the quantitative analysis of NMR sample extracts by "$C-NMR, a repetition delay of 60n5 s was used. The $"P-NMR spectra of the ethanol extracts were obtained as described by Ramos et al (2001). For the determination of LDH activity in cell extracts, lactate production was monitored by "H-NMR using a pulse width of 6 µs (90m flip angle) and a recycle delay of 3n1 s. For the quantification of lactate, formate was used as a concentration standard, the recycle delay was increased to 45n7 s and 96 transients were acquired.…”

Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-d-Glucose 1,6-(Bis)phosphate Intermediate

“…The retention times of G1P; G6P; and G1,6bisP are 8, 15, and 21 min, respectively. 2 No distinction between the R-and -anomers of glucose-6-phosphate is offered because the solution epimerization of the G6P C(1)OH is rapid (1). The radioactivity associated with the respective [ 14 C] G1P; [ 14 C]G6P; and [ 14 C] G1,6bisP fractions was used to calculate the mole fraction of each species present in the reaction mixture at termination.…”

“…2 This reaction is mediated by an active site aspartate (Asp8), which forms an acyl phosphate as the covalent enzyme intermediate (12). Kinetic methods were used to demonstrate the intermediacy of G16bisP in G6P formation and to show that the G16bisP intermediate is reoriented in the active site by dissociation into solvent and then binding in the opposite orientation.…”

Genomic Insights into Bifidobacteria

“…Most LAB produce heteropolysaccharides consisting of glucose, galactose, and rhamnose, generally at a 2:1.3:1 ratio (201,243,261,320). While EPS production in various LAB has been studied extensively, there are very few studies on EPS production in bifidobacteria.…”