Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus

Abstract

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal ؉ ) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal ؉ strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal ؉ strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal ؉ strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of many fermented milk products. There is great diversity in the composition and structure of these EPSs, which results in different properties (for a review, see reference 12). A variety of EPSs with different repeating units and chain lengths have been found in Streptococcus thermophilus, and diversity is also found in the increasing number of eps operons that are being sequenced (1, 7, 32). The operons consist of a more conserved region with genes that are thought to be involved in regulation, chain length determination, and export, which is followed by a variable region with genes coding for glycosyltransferases and enzymes involved in polymerization and export of the EPSs. The information being accumulated could form the basis for the construction of new tailor-made polysaccharides by genetic engineering (35). However, before the variety of EPSs from lactic acid bacteria can be fully exploited, ways of enhancing the low levels of production must be found. For example, the amount of EPS produced by S. thermophilus normally is less than 1% of the amount of the carbohydrate source (12). EPSs are synthesized from activated nucleotide sugars, and a potential bottleneck in EPS production could be the low levels of these precursors. In S. thermophilus, which possesses the Leloir pathway, UDP glucose (UDPglc) and UDP galactose (UDPgal) can be formed from either the glucose or the galactose moiety ...

Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp. longum CRC 002

“…EPS yields have been enhanced in S. thermophilus by overexpressing genes coding for the Leloir enzymes (Levander et al, 2002;Svensson et al, 2005). EPS biosynthesis pathways and regulation mechanisms are generally complex, and enhancement of EPS production when metabolizing lactose may not be explained only by sugar nucleotide biosynthesis.…”

“…To increase EPS production, metabolic engineering has been attempted to overcome regulation in the basal metabolic reactions involved in sugar nucleotide biosynthesis in Lactococcus lactis and Streptococcus thermophilus (Boels et al, 2001a(Boels et al, , 2003aLevander & Rådström, 2001;Levander et al, 2002;Looijesteijn et al, 1999). EPS yields have been enhanced in S. thermophilus by overexpressing genes coding for the Leloir enzymes (Levander et al, 2002;Svensson et al, 2005).…”

Optimization of Exopolysaccharide Overproduction byLactobacillus confususin Solid State Fermentation under High Salinity Stress

“…Due to competition, a reduction in growth was observed with increasing EPS production. 28,29) There is a report the induction of EPSs under salt stress conditions, the desert soil alga Microcoleus vaginatus Gom. It was found that the presence of NaCl in the culture led to an accumulation of low molecular weight carbohydrates.…”

Genetically Engineered Lactobacilli for Technological and Functional Food Applications

“…Genetic engineering strategies could be applied to one or more of those stages involved in the EPS biosynthesis in order to increase the EPS production or to modify its composition, however, until now only strains of L. lactis and Streptococcus thermophilus have been genetically modified to enhance EPS biosynthesis. In S. thermophilus the modification of the levels of the GalU, PgmA and the Leloir route enzymes resulted in increased levels of EPS (Levander et al, 2002). Homologous overexpression of a complete eps operon in L. lactis resulted in about fourfold increase in EPS production (Boels et al, 2003).…”