Analysis of sugar metabolism in an EPS producing Lactococcus lactis by 31P NMR.

Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31P NMR. Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energization by the addition of glucose. EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former. They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium. They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls. By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars. The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.

Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?

Over the last years, important advances have been made in the study of the production of exopolysaccharides (EPS) by several lactic acid bacteria, including Lactococcus lactis. From different EPS-producing lactococcal strains the specific eps gene clusters have been characterised. They contain eps genes, which are involved in EPS repeating unit synthesis, export, polymerisation, and chain length determination. The function of the glycosyltransferase genes has been established and the availability of these genes opened the way to EPS engineering. In addition to the eps genes, biosynthesis of EPS requires a number of housekeeping genes that are involved in the metabolic pathways leading to the EPS-building blocks, the nucleotide sugars. The identification and characterisation of several of these housekeeping genes (galE, galU, rfbABCD) allows the design of metabolic engineering strategies that should lead to increased EPS production levels by L. lactis. Finally, model development has been initiated in order to predict the physicochemical consequences of the addition of a EPS to a product.