Metabolic engineering of exopolysaccharide production in Lactococcus lactis

I.C. Boels

Research output: Thesisexternal PhD, WU

Abstract

<strong><FONT FACE="Arial"></strong><p>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. <em>Lactococcus lactis</em> 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 <em>eps</em> 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 ( <em>glk</em> , <em>pfk</em> , <em>fbp</em> , <em>pgm</em> ), (ii) the biosynthetic pathway from glucose-1P to the EPS precursors UDP-glucose ( <em>galU</em> ), UDP-galactose ( <em>galU</em> and <em>galE</em> ) and dTDP-rhamnose ( <em>rfbACBD)</em> , and (iii) in the specific pathway ( <em>epsA-K</em> ) for the assembly of the repeating unit, export and polymerization of the NIZO B40 EPS. We provide evidence for metabolic control of the <em>gal</em> and <em>rfb</em> genes in EPS precursor and EPS production. Overexpression of the <em>galU</em> , <em>pgm</em> or the <em>rfb</em> genes resulted in a significant increase of EPS-precursors. Moreover, overexpression of the <em>eps</em> genes led to four-fold increased NIZO B40 EPS production. In addition, reduction of the UDP-galactose level by <em>galE</em> disruption abolished EPS production while a <em>rfb</em> conditional knock out yielded an EPS with altered sugar composition and different physical characteristics.</p><p>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.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • de Vos, W.M., Promotor
  • Kleerebezem, Michiel, Promotor
Award date11 Oct 2002
Place of PublicationS.l.
Publisher
Print ISBNs9789058086938
Publication statusPublished - 2002

Keywords

  • polysaccharides
  • lactic acid bacteria
  • lactococcus lactis
  • production
  • biosynthesis
  • metabolism

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