Advanced

Metabolic Engineering of Disaccharide Catabolism for Polysaccharide Formation in Lactococcus lactis and Streptococcus thermophilus

Levander, Fredrik LU (2001)
Abstract
Exopolysaccharides (EPSs) produced by the lactic acid bacteria (LAB) <i>Lactococcus lactis</i> and <i>Streptococcus thermophilus</i> are important for the rheological behaviour and texture of a variety of fermented food products. Furthermore, EPSs from food-grade LAB have potential as food additives and functional food ingredients with both health and economic benefits. However, the production levels of EPSs from LAB are low, and this must be overcome before EPSs can be fully exploited. The objective of the work presented in this thesis was to improve the production of EPSs from disaccharides by <i>L. lactis</i> and <i>S. thermophilus</i>. The disaccharide metabolism and EPS biosynthesis of... (More)
Exopolysaccharides (EPSs) produced by the lactic acid bacteria (LAB) <i>Lactococcus lactis</i> and <i>Streptococcus thermophilus</i> are important for the rheological behaviour and texture of a variety of fermented food products. Furthermore, EPSs from food-grade LAB have potential as food additives and functional food ingredients with both health and economic benefits. However, the production levels of EPSs from LAB are low, and this must be overcome before EPSs can be fully exploited. The objective of the work presented in this thesis was to improve the production of EPSs from disaccharides by <i>L. lactis</i> and <i>S. thermophilus</i>. The disaccharide metabolism and EPS biosynthesis of these organisms have been investigated by the construction of strains with different levels of central enzymes and the study of those strains under controlled growth conditions.



The role of the enzyme beta-phosphoglucomutase (beta-PGM) was assessed in <i>L. lactis</i> by comparing a mutant lacking the enzyme with the parent strain. It was found that the enzyme was important for growth on maltose and essential for growth on trehalose. Furthermore, maltose-grown cells of the beta-PGM mutant accumulated polysaccharides. The predominant role of beta-PGM in trehalose metabolism was found to depend on another enzyme, trehalose 6-phosphate phosphorylase (TrePP). This novel enzyme, which phosphorylates trehalose 6-phosphate into beta-glucose 1-phosphate and glucose 6-phosphate, was characterised on both biochemical and genetical levels.



Different EPS-producing strains of <i>S. thermophilus</i> were genetically typed, and the strains were found to group according to their EPS structure. The role of enzymes in the carbohydrate metabolism for EPS production in <i>S. thermophilus</i> was further investigated. First the pgmA gene, encoding phosphoglucomutase (PGM), was identified and cloned. Strains lacking PGM activity and those strains with elevated levels of PGM activity were found to produce similar levels of EPS. However, pgmA was essential for growth on glucose. The results implied that the Leloir pathway and the enzyme UDP-glucose pyrophosphorylase (GalU) were important for EPS production. A new semi-defined growth medium was developed and used for studies of strains with different levels of these enzymes. The Leloir pathway could be overexpressed in spontaneous mutants, while the gene encoding GalU had to be identified and cloned. The results indicate that simultaneous overexpression of PGM and GalU has a positive effect on EPS production. The highest EPS production could be achieved by inactivating pgmA and overexpressing the Leloir pathway. The results suggest ways of enhancing the EPS production with genetic engineering or through the natural selection of mutants. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Dr Renault, Pierre, Institut National de la Recherche Agronomique, Jouy en Josas, France
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Kemiteknik och kemisk teknologi, Chemical technology and engineering, metabolic engineering, exopolysaccharide, polysaccharide, disaccharide, Streptococcus thermophilus, lactic acid bacteria, Lactococcus lactis, Biochemical technology, Biokemisk teknik, Microbiology, bacteriology, virology, mycology, Mikrobiologi, bakteriologi, virologi, mykologi
pages
104 pages
publisher
Applied Microbiology (LTH)
defense location
Lecture Hall C at the Center for Chemistry and Chemical Engineering
defense date
2001-12-07 10:15
external identifiers
  • Other:ISRN: LUTKDH/TMB-00/1036-SE
language
English
LU publication?
yes
id
a1b3111a-8b05-4afb-b3f5-39ff197c15e9 (old id 42200)
date added to LUP
2007-08-01 13:05:05
date last changed
2016-09-19 08:45:15
@phdthesis{a1b3111a-8b05-4afb-b3f5-39ff197c15e9,
  abstract     = {Exopolysaccharides (EPSs) produced by the lactic acid bacteria (LAB) &lt;i&gt;Lactococcus lactis&lt;/i&gt; and &lt;i&gt;Streptococcus thermophilus&lt;/i&gt; are important for the rheological behaviour and texture of a variety of fermented food products. Furthermore, EPSs from food-grade LAB have potential as food additives and functional food ingredients with both health and economic benefits. However, the production levels of EPSs from LAB are low, and this must be overcome before EPSs can be fully exploited. The objective of the work presented in this thesis was to improve the production of EPSs from disaccharides by &lt;i&gt;L. lactis&lt;/i&gt; and &lt;i&gt;S. thermophilus&lt;/i&gt;. The disaccharide metabolism and EPS biosynthesis of these organisms have been investigated by the construction of strains with different levels of central enzymes and the study of those strains under controlled growth conditions.<br/><br>
<br/><br>
The role of the enzyme beta-phosphoglucomutase (beta-PGM) was assessed in &lt;i&gt;L. lactis&lt;/i&gt; by comparing a mutant lacking the enzyme with the parent strain. It was found that the enzyme was important for growth on maltose and essential for growth on trehalose. Furthermore, maltose-grown cells of the beta-PGM mutant accumulated polysaccharides. The predominant role of beta-PGM in trehalose metabolism was found to depend on another enzyme, trehalose 6-phosphate phosphorylase (TrePP). This novel enzyme, which phosphorylates trehalose 6-phosphate into beta-glucose 1-phosphate and glucose 6-phosphate, was characterised on both biochemical and genetical levels.<br/><br>
<br/><br>
Different EPS-producing strains of &lt;i&gt;S. thermophilus&lt;/i&gt; were genetically typed, and the strains were found to group according to their EPS structure. The role of enzymes in the carbohydrate metabolism for EPS production in &lt;i&gt;S. thermophilus&lt;/i&gt; was further investigated. First the pgmA gene, encoding phosphoglucomutase (PGM), was identified and cloned. Strains lacking PGM activity and those strains with elevated levels of PGM activity were found to produce similar levels of EPS. However, pgmA was essential for growth on glucose. The results implied that the Leloir pathway and the enzyme UDP-glucose pyrophosphorylase (GalU) were important for EPS production. A new semi-defined growth medium was developed and used for studies of strains with different levels of these enzymes. The Leloir pathway could be overexpressed in spontaneous mutants, while the gene encoding GalU had to be identified and cloned. The results indicate that simultaneous overexpression of PGM and GalU has a positive effect on EPS production. The highest EPS production could be achieved by inactivating pgmA and overexpressing the Leloir pathway. The results suggest ways of enhancing the EPS production with genetic engineering or through the natural selection of mutants.},
  author       = {Levander, Fredrik},
  keyword      = {Kemiteknik och kemisk teknologi,Chemical technology and engineering,metabolic engineering,exopolysaccharide,polysaccharide,disaccharide,Streptococcus thermophilus,lactic acid bacteria,Lactococcus lactis,Biochemical technology,Biokemisk teknik,Microbiology,bacteriology,virology,mycology,Mikrobiologi,bakteriologi,virologi,mykologi},
  language     = {eng},
  pages        = {104},
  publisher    = {Applied Microbiology (LTH)},
  school       = {Lund University},
  title        = {Metabolic Engineering of Disaccharide Catabolism for Polysaccharide Formation in <i>Lactococcus lactis</i> and <i>Streptococcus thermophilus</i>},
  year         = {2001},
}