Analysis of Growth of Lactobacillus plantarum WCFS1 on a Complex Medium Using a Genome-scale Metabolic Model

Douwe Molenaar,Bas Teusink,Eddy J. Smid,Willem M. de Vos,Roland J. Siezen,Anne Wiersma,Christof Francke

doi:10.1074/jbc.m606263200

Abstract

A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.

Highlights

Metabolic engineering [2, 3], and from the bioinformatics field [4, 5]
Constraint-based modeling techniques have mainly been applied to microorganisms that grow on a minimal salt medium containing a single carbon source [1]
We used constraint-based modeling methods to explore the metabolism of Lactobacillus plantarum, a lactic acid bacterium (LAB)2 used in a variety of industrial food fermentations, and marketed as a probiotic [6]

Summary

EXPERIMENTAL PROCEDURES

Fermentation and Growth Conditions—L. plantarum WCFS1 was grown at 37 °C in chemically defined medium [7] in a 10-ml tube, and used as inoculum of 500 ml of pH-controlled (pH 5.5) cultures in a 1.0-liter fermentor (Applikon Biotechnology BV, the Netherlands). The amount of ATP needed to make biomass components (defined as x), is explicitly accounted for by the model reactions and will be calculated below. We first seek to find y, which can be done with a genome-scale model by setting y ϭ 0 in the biomass equation and instead introduce an ATP dissipation reaction, vATP (ATP ϩ H2O 3 ADP ϩ Pi ϩ H). The problem is defined as: maximize vATP subject to, ΆS 1⁄7 v ϭ 0 v៮ i Ϫ SDi Ͻ vi Ͻ v៮ i ϩ SDi ᭙ measured fluxes vi vj,min Ͻ vj Ͻ vj,max ᭙ fluxes vjj i ␮ϭD (Eq 1). The result is the maximal rate of ATP production that the system can attain at the measured fluxes and at the growth rate. The amount of ATP needed for maintenance and assembly is left open, i.e. vATP was allowed to vary between 0 and max(vATP)

RESULTS

Linear dependencies of metabolitesa

Citric acid

Absolute reduced cost

DISCUSSION