Metabolic engineering of exopolysaccharide production in Lactococcus lactis

Exopolysaccharides (EPS) produced by lactic acid bacteria are important structural components in fermented foods. In addition, they may confer health benefits to the consumer, as mouse model studies have indicated that EPS may have immunostimulatory, anti-tumoral, or cholesterol-lowering activity. Lactococcus lactis strain NIZO B40 produces a phosphorylated EPS with a branched repeating unit containing glucose, galactose and rhamnose. The biosynthesis of this polymer depends on both the specific eps genes organized in a plasmid-located gene cluster and on several household genes that are involved in biosynthesis of the primary EPS precursors, the nucleotide-sugars. This work focused on the household genes to induce overproduction and/or structural modification of EPS. Therefore, we cloned, characterized, and controlled expression of the genes that encode enzymes involved (i) in primary sugar metabolism ( glk , pfk , fbp , pgm ), (ii) the biosynthetic pathway from glucose-1P to the EPS precursors UDP-glucose ( galU ), UDP-galactose ( galU and galE ) and dTDP-rhamnose ( rfbACBD) , and (iii) in the specific pathway ( epsA-K ) for the assembly of the repeating unit, export and polymerization of the NIZO B40 EPS. We provide evidence for metabolic control of the gal and rfb genes in EPS precursor and EPS production. Overexpression of the galU , pgm or the rfb genes resulted in a significant increase of EPS-precursors. Moreover, overexpression of the eps genes led to four-fold increased NIZO B40 EPS production. In addition, reduction of the UDP-galactose level by galE disruption abolished EPS production while a rfb conditional knock out yielded an EPS with altered sugar composition and different physical characteristics.The research described in this thesis contributes to the understanding of exopolysaccharide biosynthesis in lactic acid bacteria and provides a starting point for applications in the dairy industry, especially with respect to the texture and health benefits of fermented products