Physiological responses to folate overproduction in Lactobacillus plantarum WCFS1

RESEARCH Open Access
Physiological responses to folate overproduction
in Lactobacillus plantarum WCFS1

Arno Wegkamp1,2, Astrid E Mars1,6, Magda Faijes1,5, Douwe Molenaar1,2, Ric CH de Vos3, Sebastian MJ Klaus4,9,
Andrew D Hanson4, Willem M de Vos1,7, Eddy J Smid1,8*
Abstract
Background: Using a functional genomics approach we addressed the impact of folate overproduction on
metabolite formation and gene expression in Lactobacillus plantarum WCFS1. We focused specifically on the
mechanism that reduces growth rates in folate-overproducing cells.
Results: Metabolite formation and gene expression were determined in a folate-overproducing- and wild-type
strain. Differential metabolomics analysis of intracellular metabolite pools indicated that the pool sizes of 18
metabolites differed significantly between these strains. The gene expression profile was determined for both
strains in pH-regulated chemostat culture and batch culture. Apart from the expected overexpression of the 6
genes of the folate gene cluster, no other genes were found to be differentially expressed both in continuous and
batch cultures. The discrepancy between the low transcriptome and metabolome response and the 25% growth
rate reduction of the folate overproducing strain was further investigated. Folate production per se could be ruled
out as a contributing factor, since in the absence of folate production the growth rate of the overproducer was
also reduced by 25%. The higher metabolic costs for DNA and RNA biosynthesis in the folate overproducing strain
were also ruled out. However, it was demonstrated that folate-specific mRNAs and proteins constitute 8% and 4%
of the total mRNA and protein pool, respectively.
Conclusion: Folate overproduction leads to very little change in metabolite levels or overall transcript profile, while
at the same time the growth rate is reduced drastically. This shows that Lactobacillus plantarum WCFS1 is unable
to respond to this growth rate reduction, most likely because the growth-related transcripts and proteins are
diluted by the enormous amount of gratuitous folate-related transcripts and proteins.
Background
Microorganisms are often used as cell factories to produce
a wide range of metabolites and proteins. Metabolic
engineering is a suitable method to increase the
production levels of these desired compounds. Feasibility
studies with lactic acid bacteria have been performed
in which strains were constructed with increased
production of metabolites such as D-alanine, sorbitol,
riboflavin, and folate [1-4]. In Lactococcus lactis, overproduction
of alanine dehydrogenase in a lactate dehydrogenase
(LDH) deficient strain resulted rerouting the
glycolytic flux towards alanine [3]. In another case, overexpression
of the complete riboflavin gene cluster in L.
lactis resulted in a high riboflavin producing L. lactis
strain [2]. A third example is the combined overexpression
of the folate gene cluster and the p-aminobenzoate
(pABA) gene cluster in L. lactis which resulted in a high
folate producing strain [1]. The latter strain was able to
produce 100-fold more folate (total folate levels) when
compared to control strains. Folate biosynthesis proceeds
via the conversion of GTP in seven consecutive
steps towards the biologically active cofactor tetrahydrofolate
(THF). The biosynthesis of THF includes two
condensation reactions. The first is the condensation
of pABA with 2-amino-4-hydroxy-6-hydroxymethyl-
7,8-dihydropteridine to produce dihydropteroate. Subsequently,
glutamate is attached to dihydropteroate to
form dihydrofolate [5]. Without pABA, no THF can be
produced and THF is needed as the donor and acceptor
of one-carbon groups (i.e methyl, formyl, methenyl and
* Correspondence: eddy.smid@wur.nl
1TI Food & Nutrition, Wageningen, Nieuwe Kanaal 9A, 6709 PA, Wageningen,
The Netherlands
Full list of author information is available at the end of the article
Wegkamp et al. Microbial Cell Factories 2010, 9:100
http://www.microbialcellfactories.com/content/9/1/100
© 2010 Wegkamp et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.