Comparative Functional Genomics of Amino Acid Metabolism of Lactic Acid Bacteria

184 Pages·2009·3.85 MB·English

Comparative Functional Genomics of Amino Acid Metabolism of
Lactic Acid Bacteria

Marieke Pastink
Thesis committee

Thesis supervisors Prof. Dr. W. M. de Vos
Professor of Microbiology
Wageningen University

Prof. Dr. J. Hugenholtz
Professor of Industrial Molecular Microbiology
University of Amsterdam

Other members Prof. Dr. T. Abee, Wageningen University
Prof. Dr. P. Hols, Université Catholique de Louvain
Dr. J. E. T. van Hylckama Vlieg, Danone, Palaiseau, France
Prof. Dr. R. J. Siezen, Radboud University Nijmegen

This research was conducted under the auspices of the graduate school VLAG
Comparative Functional Genomics of Amino Acid Metabolism of
Lactic Acid Bacteria

Marieke Pastink

Thesis
submitted in partial fulfilment of the requirements for the degree of doctor
at Wageningen University
by the authority of the Rector Magnificus,
Prof. dr. M. J. Kropff,
in the presence of
Thesis Committee appointed by the Doctorate Board
to be defended in public
on Friday 16 October 2009
at 4 PM in the Aula.
Marieke Pastink
Comparative Functional Genomics of Amino Acid Metabolism of Lactic Acid Bacteria

PhD thesis Wageningen University, Wageningen, the Netherlands (2009)
With references, with summaries in Dutch and English

ISBN 978-90-8585-461-6
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Table of contents

Abstract 9

Chapter 1 Introduction and outline of this thesis 11

Chapter 2 Genomics and high-throughput screening approaches for 35
optimal flavor production in dairy fermentation.

Chapter 3 Genome-scale model of Streptococcus thermophilus LMG18311 55
for metabolic comparison of lactic acid bacteria.

Chapter 4 Effect of amino acid quantity on the metabolism of 81
three lactic acid bacteria: a comparative study

Chapter 5 The role of glutamate dehydrogenase in 115
Streptococcus thermophilus

Chapter 6 General discussion and future perspectives 143

Nederlandse samenvatting 163
List of publications 169
Training and Supervision Plan (VLAG) 171
About the author 173
Acknowledgements 175

Abstract

Abstract
The amino acid metabolism of lactic acid bacteria used as starters in industrial
fermentations has profound effects on the quality of the fermented foods. The work described
in this PhD thesis was initiated to use genomics technologies and a comparative approach to
link the gene content of some well-known lactic acid bacteria to flavor formation and to
increase our general knowledge in the area of amino acid metabolism. The three well-known
lactic acid bacteria that were used in these studies were Streptococcus thermophilus,
Lactococcus lactis and Lactobacillus plantarum. The complete genomes of all these model
bacteria have been sequenced and annotated in detail.
Comparative experimental and in silico studies of Streptococcus thermophilus with the
other two lactic acid bacteria, revealed the low degree of amino acid auxotrophies of this
species; it only needs two amino acids for (minimal) growth and this strain is able to produce
a varied amount of flavors. Lactococcus lactis and Lactobacillus plantarum require more
amino acids and produce fewer flavors than S. thermophilus. Furthermore, S. thermophilus
has a simple primary metabolism; homolactic growth is the only possible route under
anaerobic conditions and, remarkably, it does not have a complete pentose phosphate pathway
in contrast to the other two studied bacteria. This latter property has important consequences
for the redox metabolism of S. thermophilus and particularly its ability to produce NADPH.
A genome-scale metabolic model was developed and predicted that amino acid metabolism,
and especially glutamate degradation, and citrate metabolism are the most obvious
alternatives for NADPH generation. Several of these predictions were confirmed by
constructing a glutamate dehydrogenase mutant of S. thermophilus. This mutant revealed the
importance of the citrate pathway (and other amino acid degradation pathways) in NADPH
generation.
A comparative and functional genomics study of the three lactic acid bacteria showed
that amino acid depletion not only affects amino acid metabolism, but also flavor formation
and overall growth. The comparative genomics approach presented in this thesis can be used
to understand the amino acid metabolism of different lactic acid bacteria and their potential to
produce flavors under different conditions. Finally, it can be applied for optimization of
industrial fermentations.
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