Community Research and Development Information Service - CORDIS

Galactose-removal from fermented (dairy) products

L. lactis with high Leloir activity
A strain with significant increase in the galactose consumption rate was delivered.
L. lactis strains with high Leloir activity (overexpression of galP or galPMKT) did not show improved galactose utilisation, but instead poor growth on galactose was observed. Using in vivo NMR, accumulation of the Leloir pathway intermediates galactose-1-phosphate and glucose-1-phosphate was observed, suggesting a bottleneck at the level of ƒÑ-PGM. It was expected that this obstruction could be overcome by overexpressing a gene encoding ƒÑ-PGM, which was unknown in L. lactis (new deliverable). Overexpression of S. thermophilus pgmA together with galPMKT resulted in reduced amounts of the phosphorylated metabolites and increased galactose consumption rates, showing that £\-PGM was indeed a major bottleneck in galactose metabolism. A manuscript describing our results is under preparation (see Articles). This work is the result of collaboration with partner 2 (RUG, Oscar Kuipers).

Identification and characterization of L. lactis £\-PGM.
A unique bacterial phosphoglucomutase was identified and fully characterized.
Lactococcus lactis contains Ą-phosphoglucomutase (Ą-PGM) activity, which catalyzes the specific conversion of Ą glucose 1P into glucose 6P, reversibly. However, overexpression of femD, the only homologue in the sequenced genome of this organism with significant identity to Ą-D-phosphohexomutases, did not result in the expected enhanced Ą-PGM activity. The Ą-PGM activity was purified and its N-terminal determined. A gene coding for the isolated protein was identified by homology search. Cloning and overexpression of that gene under the nisin promoter in pNZ8048 led to a 30-fold increase of Ą-PGM activity. Biochemical characterization of the enzyme was performed and the effect of its overproduction on glucose and galactose metabolism was investigated. A manuscript describing our results is under preparation (see Articles). This work is the result of collaboration with partner 2 (RUG, Oscar Kuipers).

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Contact

Jeroen HUGENHOLTZ, (Principal Scientist Food Fermentations)
Tel.: +31-31-8659540
Fax: +31-31-8650400
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