Available online at www.sciencedirect.
com Current Opinion in
ScienceDirect
Systems biology of lactic acid bacteria: For food and
thought
Bas Teusink1,2 and Douwe Molenaar1,2
Abstract
Lactic acid bacteria (LAB) ferment plants, fish, meats and milk
and turn them into tasty food products with increased shelf life;
other LAB help digesting food and create a healthy environ-
ment in the intestine. The economic and societal importance of
these relatively simple and small bacteria is immense. In this
review we hope to show that their adaptations to nutrient-rich
environments provides fascinating and often puzzling behav-
iours that give rise to many fundamental evolutionary biological
questions in need of a systems biology approach. We will
provide examples of such questions, compare the (metabolic)
behaviour of LAB to that of other model organisms, and pro-
vide the latest insights, if available.
Addresses
1
Amsterdam Institute for Molecules, Medicines and Systems, Vrije
Universiteit Amsterdam, O|2 Building, Section Systems Bioinformatics,
Location Code 2E51, De Boelelaan 1085, NL-1081HV Amsterdam,
The Netherlands
2
Top Institute Food and Nutrition, 6700 AN Wageningen, The
Netherlands
Corresponding author: Teusink, Bas (b.teusink@vu.nl)
Current Opinion in Systems Biology 2017, 6:7–13
This review comes from a themed issue on Systems biology of model
organisms (2017)
Edited by Jens Nielsen and Kiran Raosaheb Patil
For a complete overview see the Issue and the Editorial
Available online 18 July 2017
http://dx.doi.org/10.1016/j.coisb.2017.07.005
2452-3100/© 2017 The Authors. Published by Elsevier Ltd. This is an
open access ar ticle under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords
Lactic acid bacteria, Lactococcus, Lactobacillus, Food fermentation,
Systems biology, Biotechnology.
Introduction
With over 100 billion Euros annually [1], the economic
value of foods fermented by such small bacteria e LAB
have genome sizes of only 2e3 Mbp-is impressive.
Yogurt, cheese and sauerkraut, but also breads, hams,
and olives, or pickles, soy- and fish sauce require the
metabolic activities of LAB. Many of these foods have a
long history, and the associated industry is rather tradi-
tional and wary of revolutionary changes in production
processes, let alone genetic modifications to improve
traits. Rather, the industry takes advantage of the
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Systems Biology
enormous diversity in species and strains with different
functionalities, such as flavour production profiles or
texturing properties. Furthermore, one may change pH
and temperature a bit, or change some ingredients
perhaps.
It is also not so easy to decide what to change, or how to
change it: foods are chemically complex and undefined,
and often the fermentation is not carried out by a single
strain, but a complex mixture of LABs. Finally, analyses
in sticky, solid and inhomogeneous food matrices can
be tedious, and they often are. Therefore, in contrast
to the industrial biotechnology field that produces
biobased chemicals, largely on the basis of monocultures
in relatively well-defined growth media, research in
LAB has much less adopted engineering and systems
approaches e although metabolic engineering activities
in LAB are ongoing [2e4].
We believe this is a pity, from both sides: Systems
biology has a lot to offer to the LAB field, and the LABs
have a lot to offer to the systems biology field. LABs
provide questions, challenges, and biological examples
of adaptations to environments rich in nutrients and full
of stress [5]. They can offer interesting and relevant
cases to test the generality of findings in other, better
studied, model organisms, Escherichia coli in particular.
Systems biology on the other hand, can provide struc-
ture to, and understanding of, the complex systems
comprising LAB. In this review, we will describe some
features of LAB physiology that we believe are inter-
esting from a systems biology perspective, and we will
describe our current understanding and open questions.
We hope this will attract more systems biologists to
these fascinating microorganisms.
Metabolic adaptation to rich nutrient
environments: auxotrophies and division of
labour
Environments in which LAB thrive are rich in sugars and
protein; fats, vitamins and nucleotides are also often
available. Consequently, most LAB are auxotrophic for a
large number of amino acids and vitamins. The loss of
function in the presence of some specific nutrients may
be caused by genetic drift or provides a selective
advantage. Recent works make strong cases for the
latter. It was shown experimentally in E. coli that
introduction of auxotrophies and subsequent exchange
of amino acids provided the auxotrophic mutant an
increased growth rate over the wildtype [6]. These traits
Current Opinion in Systems Biology 2017, 6:7–13
8 Systems biology of model organisms (2017)
are evolvable in laboratory evolution experiments and
make the mutants depend on cross-feeding [7].
The differences between LAB species and even strains
from the same species, however, is large and although it
was explained in general by niche adaptation for
Lactococcus lactis strains [8], the diversity in amino acid
metabolism and corresponding auxotrophies remains
puzzling as the environments in which LAB are isolated
often appear equally rich in amino acids, peptides and/
or proteins. This matters for LAB applications as their
catabolic products are flavour compounds [9], or bitter
or health-promoting [10] peptides. The physiological
role of such catabolism is not always clear: its wide
spread occurrence may be a consequence of our selec-
tion for flavour production, but it may also contribute to
stress and energy metabolism [11], chemical warfare,
hostemicrobe interactions [12] or possibly geographic
spreading through attracted insects.
In Table 1 the experimentally-determined auxotro-
phies for amino acids are shown for a number of rep-
resentatives of LAB species. However, defining such
auxotrophies is nontrivial by dependencies on the
presence of other nutrients: also in humans the di-
chotomy between essential and non-essential amino
acids was questioned and condition-dependent essen-
tial amino acids introduced [13]. For example, if
glutamine is present, glutamate is not required, but
Table 1
Amino acid requirement for selected LAB and man (in black box). Green indicates that growth is possible (but often at a lower
rate) in the absence of the amino acid, red means no or extremely poor growth is observed. Based on studies of specific strains of
S.t. [18], L. l. [19], L.p. [14], S.p. [16], E.f. [16], H.s. [13] and L.a. [17]. Note that auxotrophies between different strains of the same
species can differ.
Current Opinion in Systems Biology 2017, 6:7–13
almost all LAB require either glutamine or glutamate,
because they cannot synthesize its precursor a-keto-
glutarate de novo, by lack of a complete TCA cycle
[11]. Alternatively, the presence of some amino acids
can provide feedback inhibition on the synthesis of
another amino acid. This happens e.g. for aromatic and
branched chain amino acids [14]. Therefore, auxotro-
phies predicted from genome-scale metabolic models
require careful experimental validation, as recently
done extensively for Enterococcus faecalis [15] and
Streptococcus pyogenes [16].
The similarities between auxotrophies of LAB species
and humans perhaps suggest an underlying cause or
constraint. Methionine or cysteine are always required,
as is histidine (except for Lactobacillus plantarum); Aro-
matic and branched chain amino acids, especially valine,
are often needed for growth, or at least for fast growth
[14]. Why these amino acids? Are these the most
expensive to make, or require complex co-factors or vi-
tamins, and are the genes therefore most easily lost? A
comparative study in lactobacilli showed a great di-
versity for different amino acids in true loss of biosyn-
thetic genes and loss of activity by mutations [17].
However, the growth of all tested lactobacilli could not
be restored without glutamate, even after mutagenesis
and selective plating. The collective loss by LAB of the
ability to synthesize such a key amino acid as glutamate
is mysterious.
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From diversity in auxotrophies to microbial
ecology
Quite a number of lactobacilli that express proteases and
thus can rely on proteolysis, lost the ability to synthesize
or interconvert many more amino acids. As a result,
excess amino acids are excreted that depend on the
protein composition, protease specificity and the amino
acid interconversion metabolism of the protease positive
strain. This in turn may provide specific amino acid
niches for other bacteria. Some LAB-residents of the
human gut even specialized into growth on peptides
derived from gastric proteolysis [20]. And so, perhaps
the specific auxotrophies we observe today are the result
of co-evolution dynamics and testimonies of diversities
arising by chance and necessity [21] in complex eco-
systems. Systems biology may help explain the necessity
[22].
Auxotrophies introduce diversity and dependencies that
result in ecological interactions such as cross feeding,
which was readily observed in spatiotemporal models of
bacterial communities that mimic the gut [23]. The gut
may be too complex and experimentally inaccessible,
however, for deeper quantitative systems approaches.
Many consortia of LAB (sometimes with yeasts) that
turn milk into yogurt or cheese, or wheat into sour-
bread, are of a more manageable complexity, between
two species for yogurt and in the order of a hundred for
cheese and kefir (Kiran Patil, personal communication)
cultures. Moreover, many of these consortia are stable as
they are formed by long histories of back-swapping, and
their fitness may have been optimized [24,25], making
them interesting model systems for systems-level
studies of ecosystem functioning.
One such study pointed to other causes for community
diversity beyond metabolic interactions. For cheese
starter cultures, the dynamics of strain frequencies
during serial cultivation in milk was followed [26], and
genetic heterogeneity in the culture was stabilized by
frequency-dependent phage predation, coined a “kill-
the-winner” mechanism e possibly a specific case of
frequency-dependent predation known in ecology. This
mechanism prevents one lineage to take over the pop-
ulation. Resistance to phages -or bacteriocins, naturally
produced antibiotics-introduce another layer of in-
teractions that is extremely relevant for food production
and evoke fundamental scientific questions, from mo-
lecular mechanistic [27] to evolutionary [28]. The
function of Crispr as a phage immune response was
discovered in a LAB: Streptococcus thermophilus [29].
Metabolic adaptation to rich nutrient
environments: cheating and overflow
Proteases appear useful in protein-rich environments:
indeed, providing a L. lactis plant isolate with casein
(milk protein) protease increased fitness substantially.
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Systems biology of lactic acid bacteria Teusink and Molenaar 9
Yet, subsequent laboratory evolution experiments in
milk showed a loss of the protease and a decrease in
fitness [30]. The trait is evolutionary unstable because
of cheaters that profit from the peptides released by the
efforts of others. The loss of protease activity can be
prevented by cultivation at low densities. Extracellular
sucrose hydrolysis by invertase expression in yeast
showed a similar instability [31]. These examples
demonstrate the need to consider the strategies of
competitors, and to seriously consider “the tragedy of
the commons”, i.e. the idea that common goods drive
individuals to behaviours that are good for themselves,
but not for the population as a whole [32].
This brings us to the metabolic activity that gave LAB
their name: the fermentation of sugars to lactic acid. For
many LAB, lactate is the only major catabolic byproduct,
but for many others, alternative fermentation routes
exist. Of these “mixed acid fermentation” whereby a
mixture of acetate, formate and ethanol is produced is
the most prevalent case (Figure 1A). Lactate fermen-
tation extracts only 2 ATP per glucose, whereas mixed
acid fermentation does slightly better with 3 ATP per
glucose. L. lactis shows the high-ATP yield mixed acid
fermentation strategy at low growth rates, and switches
to lactate formation as the growth rate increases [33].
This is reminiscent of the Crabtree effect in yeast [35],
or of overflow metabolism E coli [36]. Thus, production
of lactate, associated with fast and wasteful growth,
seems the result of a tragedy of the commons. Indeed,
when we privatized the common sugar by propagation in
water-in-oil emulsions, we could select L. lactis mutants
that switched to mixed acid fermentation [37]. The
mechanism by which mutants achieved this switch,
contains an interesting general lesson: we found muta-
tions in the glucose transport system that we hypothe-
size create an internal state that mimics a low-glucose
environment. Such internal states can subsequently
drive metabolic regulation, either through allosteric in-
teractions or gene expression (see Refs. [38,39] for
recent examples in E. coli, or [40] in L. lactis).
In E. coli, it was recently shown that differences in
“proteome-efficiency” can explain overflow metabolism
[36]. Indeed, the perspective of (optimal [25,41])
allocation of limited resources has appeared as a
powerful determinant of regulatory strategies [42,43].
The central idea is that protein is costly [44] and ATP-
efficient pathways cost more resources to implement,
which at higher growth rates becomes too large a burden
and drives cells to switch to cheaper and less ATP-
efficient pathways [36,42]. We tested this idea by a
careful characterisation of the metabolic switch in
L. lactis, and found that the proteome hardly changed
with growth rate [33]. Glycolytic enzyme levels
remained largely constant despite a more than threefold
increase in flux. The apparent overcapacity of glycolytic
enzymes at the lower growth rates, also observed for
Current Opinion in Systems Biology 2017, 6:7–13
10 Systems biology of model organisms (2017)

Bacillus subtilis [45] and yeast [46], is difficult to recon-
cile with optimal proteome management. We performed
prolonged chemostat cultivation studies at different
growth rates, and found that expression of membrane-
associated processes was specifically affected, indi-
cating that under these conditions uptake of nutrient,
rather than its further intracellular processing, limits
growth [Claire Price, Filipe Branco dos Santos, unpub-
lished]. Genome-scale metabolism and expression
modeling in E. coli also suggested a distinction between
nutrient (or uptake) limitation at low growth rates, and
proteome limitation at high growth rates [47].
What remains puzzling, however, is that in the absence
of an apparent proteome limitation, L. lactis still switches
to lactic acid formation as growth rate increases [33].
This strongly suggests a dominant metabolic regulation,
but why? Structural kinetic modeling of L. lactis central
metabolism hinted at a negative control of the acetate
branch on the glycolytic flux, through its impact on
NADH levels [48]. How this subsequently affects
growth rate and fitness is unclear, however, but glyco-
lytic flux as biological objective does fit with an old
observation that the glycolytic flux in L. lactis appears
always on, independent from ATP demand [49]. Alter-
natively, we should also keep in mind that resource
allocation and the associated bacterial growth laws are
steady state relationships. At constant conditions,
metabolic regulation is not needed and costly, which
points to a role under dynamic conditions. Recent
studies have shown how important it is to properly
respond to extracellular dynamics [50], and how cellular
heterogeneity in such responses can create sub-
populations of responders and non-responders, in

Figure 1

Fermentation pathways in L. lactis to lactate or mixed acid fermentation. (A) Overview of the biochemical reactions, with external metabolites in white
boxes, lumped metabolite pools in grey boxes, and positive (green) and negative (red) regulation by F16bP. (B) Is the metabolic regulation of acetate
versus lactate observed in the chemostat (lower figure, based on [33]) a series of still pictures from a normally fast transition to glucose depletion (upper
figure, based on [34])? During a brief period of glucose depletion, growth rate decreases rapidly, and a shift of lactate towards acetate production takes
place.

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L. lactis [51], but also E. coli [52] and Saccharomyces
cerevisiae [53]. If LAB have adapted to conditions of feast
and famine, with relatively fast and frequent transitions
between them, metabolic regulation to rapidly switch
fluxes may make sense. One idea (Frank Bruggeman,
personal communication), is that the titration of growth
rate through the dilution rate in the chemostat may
provide artificial steady-state still pictures of what
normally is a brief transition for the organism
(Figure 1B).
The molecular mechanism by which metabolic regula-
tion achieves the switch between mixed acid and lactate
fermentation, is also not yet resolved, despite much
research in the (distant) past. Complexity arises from
the involvement of moiety conservations and couplings
between enzyme activities through the cofactors
NADH/NAD and ATP/ADP [54]. But similar perhaps to
what has been suggested for yeast [55] and E. coli [56],
regulation by fructose 1,6-bisphosphate seems impor-
tant for the switch. F16bP activates lactate dehydroge-
nase of L. lactis, E. faecalis and S. pyogenes, but not that of
L. plantarum [57]; suggestively, only the latter organism
does not switch to acetate production at low dilution
rate [58]. This activation by F16bP is different from that
on pyruvate kinase, observed from LAB to man: in
L. lactis the current suggestion from computational
studies is that feedforward activation ensures robustness
to feast-famine dynamics [48], possibly through main-
taining adequate phosphoenol pyruvate levels [50], the
substrate required for sugar uptake and phosphorylation.
Conclusion
We have given a brief overview of a number of what we
suggest are adaptive behaviours of lactic acid bacteria to
their rich nutrient environments: a fascinating mix of
chemical warfare, cheating, robustness and rat races. We
focused on current open scientific questions and our lat-
ests insights, and on the commonalities and differences
with other model organisms that are more often studied in
the systems biology field. The models and advanced mo-
lecular and functional genomics tools are available [59].
Acknowledgements
We thank prof J Ellers for useful comments. LAB-related work in our group
is supported by STW grant 13858, TI Food and Nutrition project 16MF01,
EraSysApp project SysMilk: NWO 832.14.001 and the ITN MicroWine:
Horizon2020 grant 643031.
References
Papers of particular interest, published within the period of review,
have been highlighted as:
* of special interest
* * of outstanding interest
1. de Vos WM: Systems solutions by lactic acid bacteria: from
paradigms to practice. Microb Cell Factories 2011, 10:S2.
2. Sauer M, Russmayer H, Grabherr R, Peterbauer CK, Marx H: The
efficient clade: lactic acid bacteria for industrial chemical
www.sciencedirect.com
Systems biology of lactic acid bacteria Teusink and Molenaar 11
production. Trends Biotechnol 2017, http://dx.doi.org/10.1016/
j.tibtech.2017.05.002.
3. Petersen KV, Liu J, Chen J, Martinussen J, Jensen PR, Solem C:
Metabolic characterization and transformation of the non-
dairy Lactococcus lactis strain KF147, for production of
ethanol from xylose. Biotechnol J 2017, http://dx.doi.org/
10.1002/biot.201700171.
4. Song AA-L, In LLA, Lim SHE, Rahim RA: A review on Lacto-
coccus lactis: from food to factory. Microb Cell Factories 2017,
16:55.
5. Papadimitriou K, Alegría Á, Bron PA, de Angelis M, Gobbetti M,
Kleerebezem M, Lemos JA, Linares DM, Ross P, Stanton C,
et al.: Stress physiology of lactic acid bacteria. Microbiol Mol
Biol Rev 2016, 80:837–890.
6. Pande S, Merker H, Bohl K, Reichelt M, Schuster S, de
Figueiredo LF, Kaleta C, Kost C: Fitness and stability of obli-
gate cross-feeding interactions that emerge upon gene loss
in bacteria. ISME J 2014, 8:953–962.
7. D’Souza G, Kost C: Experimental evolution of metabolic de-
* pendency in bacteria. PLoS Genet 2016, 12:e1006364.
Simple but effective set of laboratory evolution experiments in the
presence and absence of exogenous amino acids show that auxotro-
phies and cross feeding are selected for.
8. Kelleher P, Bottacini F, Mahony J, Kilcawley KN, van Sinderen D:
Comparative and functional genomics of the Lactococcus
lactis taxon; insights into evolution and niche adaptation.
BMC Genom 2017, 18:267.
9. Liu M, Nauta A, Francke C, Siezen RJ: Comparative genomics
of enzymes in flavor-forming pathways from amino acids in
lactic acid bacteria. Appl Environ Microbiol 2008, 74:
4590–4600.
10. Rai AK, Sanjukta S, Jeyaram K: Production of angiotensin I
converting enzyme inhibitory (ACE-I) peptides during milk
fermentation and their role in reducing hypertension. Crit Rev
Food Sci Nutr 2017, 57:2789–2800.
11. Fernández M, Zúñiga M: Amino acid catabolic pathways of
lactic acid bacteria. Crit Rev Microbiol 2006, 32:155–183.
12. Goffin P, van de Bunt B, Giovane M, Leveau JHJ, Höppener-
Ogawa S, Teusink B, Hugenholtz J: Understanding the physi-
ology of Lactobacillus plantarum at zero growth. Mol Syst Biol
2010, 6:413.
13. Reeds PJ: Dispensable and indispensable amino acids for
humans. J Nutr 2000, 130:1835S–1840S.
14. Teusink B, van Enckevort FHJ, Francke C, Wiersma A,
Wegkamp A, Smid EJ, Siezen RJ: In silico reconstruction of
the metabolic pathways of Lactobacillus plantarum:
comparing predictions of nutrient requirements with those
from growth experiments. Appl Environ Microbiol 2005, 71:
7253–7262.
15. Veith N, Solheim M, van Grinsven KWA, Olivier BG, Levering J,
Grosseholz R, Hugenholtz J, Holo H, Nes I, Teusink B, et al.:
Using a genome-scale metabolic model of Enterococcus
faecalis V583 to assess amino acid uptake and its impact on
central metabolism. Appl Environ Microbiol 2015, 81:
1622–1633.
16. Levering J, Fiedler T, Sieg A, van Grinsven KWA, Hering S,
Veith N, Olivier BG, Klett L, Hugenholtz J, Teusink B, et al.:
Genome-scale reconstruction of the Streptococcus
pyogenes M49 metabolic network reveals growth re-
quirements and indicates potential drug targets. J Biotechnol
2016, 232:25–37.
17. Morishita T, Deguchi Y, Yajima M, Sakurai T, Yura T: Multiple
nutritional requirements of lactobacilli: genetic lesions affecting
amino acid biosynthetic pathways. J Bacteriol 1981, 148:64–71.
18. Pastink MI, Teusink B, Hols P, Visser S, de Vos WM,
Hugenholtz J: Genome-scale model of Streptococcus ther-
mophilus LMG18311 for metabolic comparison of lactic acid
bacteria. Appl Environ Microbiol 2009, 75:3627–3633.
19. Flahaut NAL, Wiersma A, van de Bunt B, Martens DE, Schaap PJ,
Sijtsma L, Dos Santos VAM, de Vos WM: Genome-scale
Current Opinion in Systems Biology 2017, 6:7–13
12 Systems biology of model organisms (2017)
metabolic model for Lactococcus lactis MG1363 and its
application to the analysis of flavor formation. Appl Microbiol
Biotechnol 2013, 97:8729–8739.
20. Arakawa K, Matsunaga K, Takihiro S, Moritoki A, Ryuto S,
Kawai Y, Masuda T, Miyamoto T: Lactobacillus gasseri re-
quires peptides, not proteins or free amino acids, for growth
in milk. J Dairy Sci 2015, 98:1593–1603.
21. Pál C, Papp B, Lercher MJ, Csermely P, Oliver SG, Hurst LD:
Chance and necessity in the evolution of minimal metabolic
networks. Nature 2006, 440:667–670.
22. Zelezniak A, Andrejev S, Ponomarova O, Mende DR, Bork P,
Patil KR: Metabolic dependencies drive species co-
occurrence in diverse microbial communities. Proc Natl Acad
Sci U S A 2015, 112:6449–6454.
23. MJA van Hoek, Merks RMH: Emergence of microbial diversity
due to cross-feeding interactions in a spatial model of gut
microbial metabolism. BMC Syst Biol 2017, 11:56.
24. Gottstein W, Olivier BG, Bruggeman FJ, Teusink B: Constraint-
based stoichiometric modelling from single organisms to
microbial communities. J R Soc Interface 2016:13.
25. Towbin BD, Korem Y, Bren A, Doron S, Sorek R, Alon U: Opti-
mality and sub-optimality in a bacterial growth law. Nat
Commun 2017, 8:14123.
26. Erkus O, de Jager VCL, Spus M, van Alen-Boerrigter IJ, van
* * Rijswijck IMH, Hazelwood L, Janssen PWM, van Hijum SAFT,
Kleerebezem M, Smid EJ: Multifactorial diversity sustains mi-
crobial community stability. ISME J 2013, 7:2126–2136.
Population dynamics study of a cheese starter culture (a mixture of LAB
strains added to milk to start the cheese making process) that identifies
phage sensitivity as a major driver in maintaining genetic diversity.
27. Mahony J, McDonnell B, Casey E, van Sinderen D: Phage-host
interactions of cheese-making lactic acid bacteria. Annu Rev
Food Sci Technol 2016, 7:267–285.
28. Ferenci T: Trade-off mechanisms shaping the diversity of
bacteria. Trends Microbiol 2016, 24:209–223.
29. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P,
Moineau S, Romero DA, Horvath P: CRISPR provides acquired
resistance against viruses in prokaryotes. Science 2007, 315:
1709–1712.
30. Bachmann H, Molenaar D, Kleerebezem M, van Hylckama
Vlieg JET: High local substrate availability stabilizes a coop-
erative trait. ISME J 2011, 5:929–932.
31. Gore J, Youk H, van Oudenaarden A: Snowdrift game dynamics
and facultative cheating in yeast. Nature 2009, 459:253–256.
32. Hardin G: The tragedy of the commons. The population
problem has no technical solution; it requires a fundamental
extension in morality. Science 1968, 162:1243–1248.
33. Goel A, Eckhardt TH, Puri P, de Jong A, Branco Dos Santos F,
* Giera M, Fusetti F, de Vos WM, Kok J, Poolman B, et al.: Protein
costs do not explain evolution of metabolic strategies and
regulation of ribosomal content: does protein investment
explain an anaerobic bacterial Crabtree effect? Mol Microbiol
2015, 97:77–92.
Careful quantitative study of the shift from mixed acid to lactate
fermentation in the chemostat, showing at the level of mRNA, protein
and enzyme activity that flux increases over threefold without appre-
ciable changes in corresponding protein level.
34. Neves AR, Ventura R, Mansour N, Shearman C, Gasson MJ,
Maycock C, Ramos A, Santos H: Is the glycolytic flux in
Lactococcus lactis primarily controlled by the redox charge?
Kinetics of NAD(+) and NADH pools determined in vivo by
13C NMR. J Biol Chem 2002, 277:28088–28098.
35. van Dijken JP, Weusthuis RA, Pronk JT: Kinetics of growth and
sugar consumption in yeasts. Ant Van Leeuwenhoek 1993, 63:
343–352.
36. Basan M, Hui S, Okano H, Zhang Z, Shen Y, Williamson JR,
* * Hwa T: Overflow metabolism in Escherichia coli results from
efficient proteome allocation. Nature 2015, 528:99–104.
A set of elegant quantitative perturbation studies are interpreted with
phenomenological bacterial growth laws to show that respiration costs
Current Opinion in Systems Biology 2017, 6:7–13
more protein per ATP than overflow metabolism, explaining the latter
phenomenon as a resource allocation strategy.
37. Bachmann H, Fischlechner M, Rabbers I, Barfa N, Branco Dos
Santos F, Molenaar D, Teusink B: Availability of public goods
shapes the evolution of competing metabolic strategies. Proc
Natl Acad Sci U S A 2013, http://dx.doi.org/10.1073/
pnas.1308523110.
38. You C, Okano H, Hui S, Zhang Z, Kim M, Gunderson CW,
Wang Y-P, Lenz P, Yan D, Hwa T: Coordination of bacterial
proteome with metabolism by cyclic AMP signalling. Nature
2013, 500:301–306.
39. Kochanowski K, Gerosa L, Brunner SF, Christodoulou D,
* * Nikolaev YV, Sauer U: Few regulatory metabolites coordinate
expression of central metabolic genes in Escherichia coli.
Mol Syst Biol 2017, 13:903.
This paper combines promoter activity and metabolomics to conclude
that the coordination of gene expression seems relievingly simple. For
a theoretical underpinning, see bioRxiv 115428; doi: https://doi.org/10.
1101/115428.
40. Hove-Jensen B, Andersen KR, Kilstrup M, Martinussen J,
Switzer RL, Willemoe€s M: Phosphoribosyl diphosphate
(PRPP): biosynthesis, enzymology, utilization, and metabolic
significance. Microbiol Mol Biol Rev MMBR 2017:81.
41. Bosdriesz E, Molenaar D, Teusink B, Bruggeman FJ: How fast-
growing bacteria robustly tune their ribosome concentration
to approximate growth-rate maximization. FEBS J 2015, http://
dx.doi.org/10.1111/febs.13258.
42. Molenaar D, van Berlo R, de Ridder D, Teusink B: Shifts in
growth strategies reflect tradeoffs in cellular economics. Mol
Syst Biol 2009, 5:323.
43. Scott M, Gunderson CW, Mateescu EM, Zhang Z, Hwa T: Inter-
dependence of cell growth and gene expression: origins and
consequences. Science 2010, 330:1099–1102.
44. Kafri M, Metzl-Raz E, Jona G, Barkai N: The cost of protein
* production. Cell Rep 2016, 14:22–31.
Elegant study in yeast with reporter constructs to address whether
costs of making a protein are in transcription or translation; this, it
turned out, depends on the conditions.
45. Chubukov V, Uhr M, Le Chat L, Kleijn RJ, Jules M, Link H,
Aymerich S, Stelling J, Sauer U: Transcriptional regulation is
insufficient to explain substrate-induced flux changes in
Bacillus subtilis. Mol Syst Biol 2013, 9:709.
46. Solis-Escalante D, Kuijpers NGA, Barrajon-Simancas N, van den
Broek M, Pronk JT, Daran J-M, Daran-Lapujade P: A minimal set
of glycolytic genes reveals strong redundancies in Saccha-
romyces cerevisiae central metabolism. Eukaryot Cell 2015,
14:804–816.
47. O’Brien EJ, Lerman JA, Chang RL, Hyduke DR, Palsson BØ:
* * Genome-scale models of metabolism and gene expression
extend and refine growth phenotype prediction. Mol Syst Biol
2013, 9:693.
The capabilities of a ME model of E. coli were demonstrated, showing
for the first time how augmenting metabolic networks with protein costs
can enhance their biological interpretation and capabilities, including
overflow metabolism.
48. Murabito E, Verma M, Bekker M, Bellomo D, Westerhoff HV,
Teusink B, Steuer R: Monte-Carlo modeling of the central
carbon metabolism of Lactococcus lactis: insights into
metabolic regulation. PLoS One 2014, 9:e106453.
49. Koebmann BJ, Solem C, Pedersen MB, Nilsson D, Jensen PR:
Expression of genes encoding F(1)-ATPase results in
uncoupling of glycolysis from biomass production in
Lactococcus lactis. Appl Environ Microbiol 2002, 68:
4274–4282.
50. Dolatshahi S, Fonseca LL, Voit EO: New insights into the
complex regulation of the glycolytic pathway in Lactococcus
lactis. II. Inference of the precisely timed control system
regulating glycolysis. Mol Biosyst 2015, 12:37–47.
51. Solopova A, van Gestel J, Weissing FJ, Bachmann H, Teusink B,
Kok J, Kuipers OP: Bet-hedging during bacterial diauxic shift.
Proc Natl Acad Sci U S A 2014, 111:7427–7432.
www.sciencedirect.com

52. Kotte O, Volkmer B, Radzikowski JL, Heinemann M: Phenotypic
bistability in Escherichia coli’s central carbon metabolism.
Mol Syst Biol 2014, 10:736.
53. van Heerden JH, Wortel MT, Bruggeman FJ, Heijnen JJ,
Bollen YJM, Planqué R, Hulshof J, O’Toole TG, Wahl SA,
Teusink B: Lost in transition: start-up of glycolysis yields
subpopulations of nongrowing cells. Science 2014, 343:
1245114.
54. Christensen CD, Hofmeyr J-HS, Rohwer JM: Tracing regulatory
routes in metabolism using generalised supply-demand
analysis. BMC Syst Biol 2015, 9:89.
55. van Heerden JH, Bruggeman FJ, Teusink B: Multi-tasking of
biosynthetic and energetic functions of glycolysis explained
by supply and demand logic. BioEssays News Rev Mol Cell
Dev Biol 2015, 37:34–45.
56. Kochanowski K, Volkmer B, Gerosa L, Haverkorn van
Rijsewijk BR, Schmidt A, Heinemann M: Functioning of a
metabolic flux sensor in Escherichia coli. Proc Natl Acad Sci U
S A 2013, 110:1130–1135.
www.sciencedirect.com
Systems biology of lactic acid bacteria Teusink and Molenaar 13
57. Feldman-Salit A, Hering S, Messiha HL, Veith N, Cojocaru V,
Sieg A, Westerhoff HV, Kreikemeyer B, Wade RC, Fiedler T:
Regulation of the activity of lactate dehydrogenases from
four lactic acid bacteria. J Biol Chem 2013, 288:21295–21306.
58. Teusink B, Wiersma A, Molenaar D, Francke C, de Vos WM,
Siezen RJ, Smid EJ: Analysis of growth of Lactobacillus
plantarum WCFS1 on a complex medium using a genome-
scale metabolic model. J Biol Chem 2006, 281:40041–40048.
59. Grosseholz R, Ching-Chiek K, Veith N, Fiedler T, Strauss M,
* Olivier BG, Collins BC, Schubert OT, Bergman FT,
Kreikemeyer B, et al.: Integrating highly quantitative prote-
omics and genome-scale metabolic modeling to study pH
adaptation in the human pathogen Enterococcus faecalis.
NPJ Syst Biol Appl 2016, http://dx.doi.org/10.1038/
npjsba.2016.17.
Comprehensive time-resolved proteomics data after a pH challenge
that was integrated in a genome-scale metabolic model.
Current Opinion in Systems Biology 2017, 6:7–13