Supplement bij veertiende jaargang, april 2006 - NVMM

eduction, was applied. The DGGE profiles showed a
relatively high SRB diversity and similar profiles for
lakes with similar salinity and pH. Individual DGGE
bands were sequenced and phylogenetic analysis was
performed. Firstly, the closest relatives were determined
using the BLAST search tool, subsequently the sequences
were imported into the ARB software program, manually
aligned and added into an existing tree. All our sequences
grouped with those from Deltaproteobacteria and in
particular with sequences of Desulfonatronum hydrogenovorans
and Desulfonatronum lacustre, two low salt-tolerant
alkaliphilic sulfate reducers isolated from soda lakes.
These results give for the first time insight in an important
functional group of microorganisms in soda lakes, which
is essential for a better understanding of the sulfur cycle
in these ecosystems.
06.14
Metabolic engineering of folate biosynthesis in Lactobacillus
plantarum
A. Wegkamp1,3 , B. Teusink 1,2,3 , A. Mars1,3 , J. Hugenholtz1,3 ,
W.M. de Vos1 , E.J. Smid1,2,3 1 2 Wageningen Centre for Food Sciences, Wageningen, NIZO
Food Research, Ede, 3Kluyver Centre for Genomics of Industrial
Fermentation, Delft
Many plants and micro-organisms have the ability to
synthesise folate, whereas animals lack this ability. Folate
is an essential cofactor for the synthesis of DNA, RNA
and certain amino acids. Folate-deficiency in humans
is often associated with anaemia, neural tube defects
and cardiovascular diseases. A number of folate related
deficiencies in humans can be overcome by uplifting
the folate consumption levels. One way of doing this is
to produce fermented food products with elevated folate
levels. As an alternative for traditional fortification, this
could be achieved by using folate (over)producing lactic
acid bacteria in the fermentation process.
Here we present a metabolic engineering strategy that
yields a Lactobacillus plantarum strain producing elevated
folate pools. The genes involved in folate biosynthesis in
L. plantarum were identified and cloned on a vector and
subsequently transferred to the host strain L. plantarum
WCFS1. The transformed L. plantarum strains were found
to produce high folate levels (10 mg/l) upon the supplementation
of para-aminobenzoic acid (a building block in
the folate biosynthesis). In the folate overproducing strain
the transcriptome and metabolome were compared to that
of the wild type using DNA-microarrays and deferential
LC-MS, respectively. The transcriptome and metabolome
data will subsequently be projected on metabolic pathway
maps specifically designed for L. plantarum WCFS1. The
transcription profiling coupled with the metabolic map
Ned Tijdschr Med Microbiol 2006; 4:Supplement
S37
will facilitate the formulation of improved metabolic
engineering strategies to further increase folate levels in
the host strain. In addition, this approach will lead to a
better understanding of regulatory aspects of folate biosynthesis
in L. plantarum.
06.15
Measuring yeasts intracellular pH upon sorbic acid stress
in vivo
R. Orij, J. Postmus, S. Brul, G. Smits
Swammerdam Institute for Life Sciences (SILS), Molecular
Biology & Microbial Food Safety, Amsterdam
Sorbic acid, a weak organic, is the most widespread food
preservative used in the industry. Yeast and other fungi
are, to a certain extent, able adapt to this acid and resume
growth in the presence of the highest concentrations
allowed in foods. This can result in product spoilage and
thus create substantial economic losses. Quite a lot is
known with respect to the end point of the yeasts’ response
to sorbic acid stress, i.e. when growth is resumed, from
genome-wide transcript analyses and studies with yeast
knockout mutants. However currently we still do not
know why the cells initially arrest growth upon the weakacid
challenge. Also the molecular physiological events
that occur during the adaptation phase and finally lead
to a resumption of growth are poorly understood. Thus,
to understand the mechanisms of growth limitation and
adaptation we perform time-resolved studies of yeast cells
exposed to sorbic acid in an integrated way. That is, we
perform analyses at the level of gene expression, protein
composition, and cellular metabolism. By calculating
energy generating capacity, we try to map the cost and
benefit of the various aspects of the stress response
towards weak acids. In practice this means we determine
metabolic fluxes, ATP/ADP ratios and ultimately try to
construct a mathematical model of the response to the
stress. Currently we are working on an experiment to
measure intracellular pH upon sorbate stress in vivo. We
use a pH sensitive GFP originally developed in mammals
called a ‘pHluorin’ (Miesenböck et al., 1998). This GFP has
been constructed in such a way that it can monitor the pH
of its environment without disturbing cell function. We
targeted this GFP to different compartments of the cell
using different targeting sequences. The first results show
that the system works in yeast and online pH measurements
without applying a stress are possible.