Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus.

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Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus. / Levander, Fredrik; Svensson, Malin; Rådström, Peter.

In: Applied and Environmental Microbiology, Vol. 68, No. 2, 2002, p. 784-790.

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TY - JOUR

T1 - Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus.

AU - Levander, Fredrik

AU - Svensson, Malin

AU - Rådström, Peter

PY - 2002

Y1 - 2002

N2 - 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.

AB - 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.

KW - Genetic Engineering/methods

KW - Molecular Sequence Data

KW - Phosphoglucomutase/genetics/metabolism

KW - UTP-Glucose-1-Phosphate Uridylyltransferase/genetics/metabolism

KW - Non-U.S. Gov't

KW - Support

KW - Streptococcus/enzymology/genetics/growth & development

KW - Polysaccharides

KW - Bacterial/biosynthesis

KW - Galactose/metabolism

KW - Molecular

KW - Culture Media

KW - Cloning

KW - Base Sequence

U2 - 10.1128/AEM.68.2.784-790.2002

DO - 10.1128/AEM.68.2.784-790.2002

M3 - Article

VL - 68

SP - 784

EP - 790

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 2

ER -