Metabolic Engineering of the Central Carbon Metabolism in Streptococcus thermophilus for Enhanced Exopolysaccharide Production

Sammanfattning: The lactic acid bacterium, Streptococcus thermophilus, is used in the dairy industry in mixed cultures with Lactobacillus delbrueckii subsp. bulgaricus for the production of yoghurt. Many strains of S. thermophilus produce exopolysaccharides (EPSs) which affect the texture and viscosity of fermented food products. However, the production of EPSs by S. thermophilus is low and variable, and better knowledge concerning EPS biosynthesis is therefore required. The overall objectives of this work were to gain a better understanding of the metabolic flux through the central carbon metabolism, which leads to the formation of EPSs and cell wall components, and to metabolically improve the EPS production in S. thermophilus. To make EPS analysis easier, a novel semi-defined medium that supports rapid growth of S. thermophilus and that does not contain interfering polysaccharides has been developed, together with a new EPS quantification method. This medium was used to investigate six S. thermophilus LY03 mutants with altered levels of enzymes in the central metabolism. The Leloir enzymes were found to have a positive effect on EPS biosynthesis. Overexpression of the enzymes phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (GalU) separately did not affect the EPS yield however, overexpression of the two enzymes together resulted in enhanced EPS production. The highest EPS yield was obtained in a mutant with increased activities of PGM, GalU and the Leloir enzymes. An accumulation of intracellular glucose-1-phosphate, found in the other mutants with altered activities of the Leloir enzymes, was not present in this mutant. The constructed S. thermophilus LY03 mutants were cultivated in milk, and it was found that the mutants with an increased metabolic flux towards the GalU reaction in combination with enhanced GalU activity grew more slowly than the parent strain. Rapid growth of these mutants in milk could be restored by the addition of four specific amino acids, i.e. glutamic acid, histidine, methionine and valine. This amino acid requirement was confirmed in a defined medium. Rheological measurements on fermented milk products containing the mutant with the highest EPS production or the parent strain showed that the enhanced EPS production did not affect the viscosity of the product, but lowered the elasticity. Finally, a screening method including transposon mutagenesis and selection of the obtained mutants by staining with ruthenium red was developed. The method was found to be suitable for the identification of cell envelope mutants.