**Optimisation of Cell Bioenergetics in Food-Associated Microorganisms**

Diego Mora and Stefania Arioli

*University of Milan, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Milano, Italy* 

## **1. Introduction**

218 Bioenergetics

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> Microorganisms display a considerable versatility, with mechanisms that govern cell bioenergetics and a large number of redox active molecules being used as electron donors or acceptors. We will not review the basis of microbial bioenergetics here, but instead focus attention on the metabolic systems that microorganisms have evolved to optimise the efficiency of cell catabolism and cell energy homeostasis. The mechanisms that act in the regulation of cell bioenergetics belong to the complexity of biological systems in which large networks of metabolic pathways interact to govern the life and responsiveness of cells towards environmental fluctuations. During growth, all microorganisms determine considerable changes in the environmental concentration of nutrients, organic acids and other molecules generated by cell catabolism. As a consequence, microorganisms are constantly faced with different environmental stimuli and stresses. The natural habitats of some microorganisms may fluctuate erratically, whereas others which are more predictable offer the opportunity to prepare in advance for the next environmental change. In this context, microorganisms may have evolved the bioenergetic machinery to anticipate environmental fluctuations by adapting to their temporal order of appearance. Food matrixes represent an example of 'predictable' fluctuating environments, generated by anthropic activities and able to drive the speciation of several microorganisms. The nutrient's richness, and specifically the abundance of mono- and disaccharides that characterise several food matrixes (such as milk and grape juice), have allowed the speciation of lactic acid bacteria (LAB) and yeasts with a high fermentation capacity. The bakers' yeast *Saccharomyces cerevisiae* degrades sugars to two-carbon components – in particular, ethanol – even in the presence of excess oxygen, thus using a fermentation metabolism instead of the energetically favourable respiration metabolism (2 mol versus about 32 mol of ATP per mol of glucose respectively). *S. cerevisiae* alcoholic fermentation has been exploited for several millennia throughout the world in a variety of food processes of crucial importance for humans, such as the making of beer, wine and bread. Moreover, LAB species have partially lost the genetic information need in order to carry out a respiratory metabolism on behalf of a homofermentative pathway in which lactic acid is the primary product, or a heterofermentative pathway in which lactic acid, CO2, acetic acid and/or ethanol are produced (Kandler, 1983). The seemingly simplistic metabolism of LAB has been exploited throughout history for the preservation of foods and beverages in nearly all societies, and dates back to the origins of agriculture. The domestication of LAB strains passed down through various

Optimisation of Cell Bioenergetics in Food-Associated Microorganisms 221

Both LAB and *S. cerevisiae* have definitely evolved their energetic metabolism to reach maximum fitness in a defined environmental niche characterised by a high carbohydrates concentration. Milk, the proposed evolutionary environmental niche for the LAB *Streptococcus thermophilus*, *Lactobacillus delbrueckii* subsp. *bulgaricus*, and the "domesticated" strains of *Lactococcus lactis* (Bolotin *et al.*, 2004; van de Guchte, 2006; Passerini *et al*., 2010), and the man-made niches, grape juice, has driven the evolution of the domesticated strains of *S. cerevisiae* (Martini, 1993, Fay & Benavides, 2005). These two are environments in which mono- and disaccharides resources are both large and dense. In these environmental contexts, fast sugar consumption, lactic acid or ethanol production, accumulation and tolerance, and the ability to propagate without oxygen are some of the 'winning' traits, and they have apparently evolved and become specialised to perfection in these fermenting microorganisms. In other words, energetic limitation is an important factor for organisms in their natural environment and therefore the ATP-production pathways have been under strong selection pressure during evolution (Pfeiffer *et al.*, 2001). Similarly, we can hypothesise that all mechanisms acting in the regulation and optimisation of the ATP-

The complexity of the understanding of how metabolic fluxes are modulated arises from multiple overlapping regulatory mechanisms and metabolic feedback into regulatory networks (Figure 1). The *in vivo* capacity of an enzyme to govern and modulate a metabolic flux is a function of its abundance and kinetic properties. Both abundance and the kinetic properties of enzymes are governed by three layers of cellular regulatory mechanisms: i) *gene expression*, acting on enzyme abundance, ii) *post-translational modification*, modulating enzyme abundance and kinetic parameters, and iii) *allosteric modulation*, exclusively affecting the kinetic parameters. Moreover, the *in vivo* metabolic flux depends also on the *in vivo reactant concentrations* (Gerosa & Sauer, 2011) which are function of thermodynamics and

This chapter examines the mechanisms regulating the primary metabolism by using as model organisms the dairy species *L. lactis* among prokaryotes, and the bakers' yeast *S. cerevisiae* among eukaryotes. Moreover, some enzymatic activities and metabolic pathways are described and their physiological role is revisited, taking into consideration the optimisation of the cellular bioenergetics as a result of an environment-dependent selection

Despite the wide use of LAB in food production and the role of some species for their health benefits for the human gastro intestinal tract, the regulatory mechanisms that govern the main energetic metabolism of these bacteria have still not been completely disclosed. Most of the studies have been carried out on the 'domesticated' *L. lactis* species, a member of the LAB widely used in the industrial manufacture of milk-fermented products. The most important industrial application of *L. lactis* is based on its energetic metabolism, which leads mainly to the production of high amounts of lactic acid. Anaerobic glycolysis is the principal energy-generating process of *L. lactis*, it is thus considered exclusively as a fermenting microorganism. Nevertheless, in aerobic conditions and in presence of an exogenous source of heme, *L. lactis* may be able to carry out oxidative phosphorylation (Duwat *et al.*, 2001). This cofactor-dependent respiration capacity has also been discovered in other LAB species (Lechardeur *et al.*, 2011). Although named and used for their capacity to produced lactic

production pathways are subjected to the same selection pressures.

reaction kinetics, i.e. parameters that a cell may modulate only indirectly.

**2. The regulation of the energetic metabolism in lactic acid bacteria** 

pressure.

culinary traditions and the continuous passage of food stuffs has resulted in modern-day cultures that are able to carry out these fermentations (Makarova *et al.*, 2006).

Fig. 1. View of the overlapping regulatory mechanisms modulating metabolic fluxes. A) Example of a metabolic network and schematic representation of the three layers of cellular regulatory mechanisms. The metabolic flux and metabolite pools' concentrations are subjected to the three layers of regulation. The regulation mechanisms act as a response to environmental stimuli. B) Different environmental stimuli (blue and green areas) affect the metabolic fluxes thereby determining the accumulation or depletion of intermediate metabolites.

culinary traditions and the continuous passage of food stuffs has resulted in modern-day

Fig. 1. View of the overlapping regulatory mechanisms modulating metabolic fluxes. A) Example of a metabolic network and schematic representation of the three layers of cellular regulatory mechanisms. The metabolic flux and metabolite pools' concentrations are subjected to the three layers of regulation. The regulation mechanisms act as a response to environmental stimuli. B) Different environmental stimuli (blue and green areas) affect the metabolic fluxes thereby determining the accumulation or depletion of intermediate

metabolites.

cultures that are able to carry out these fermentations (Makarova *et al.*, 2006).

Both LAB and *S. cerevisiae* have definitely evolved their energetic metabolism to reach maximum fitness in a defined environmental niche characterised by a high carbohydrates concentration. Milk, the proposed evolutionary environmental niche for the LAB *Streptococcus thermophilus*, *Lactobacillus delbrueckii* subsp. *bulgaricus*, and the "domesticated" strains of *Lactococcus lactis* (Bolotin *et al.*, 2004; van de Guchte, 2006; Passerini *et al*., 2010), and the man-made niches, grape juice, has driven the evolution of the domesticated strains of *S. cerevisiae* (Martini, 1993, Fay & Benavides, 2005). These two are environments in which mono- and disaccharides resources are both large and dense. In these environmental contexts, fast sugar consumption, lactic acid or ethanol production, accumulation and tolerance, and the ability to propagate without oxygen are some of the 'winning' traits, and they have apparently evolved and become specialised to perfection in these fermenting microorganisms. In other words, energetic limitation is an important factor for organisms in their natural environment and therefore the ATP-production pathways have been under strong selection pressure during evolution (Pfeiffer *et al.*, 2001). Similarly, we can hypothesise that all mechanisms acting in the regulation and optimisation of the ATPproduction pathways are subjected to the same selection pressures.

The complexity of the understanding of how metabolic fluxes are modulated arises from multiple overlapping regulatory mechanisms and metabolic feedback into regulatory networks (Figure 1). The *in vivo* capacity of an enzyme to govern and modulate a metabolic flux is a function of its abundance and kinetic properties. Both abundance and the kinetic properties of enzymes are governed by three layers of cellular regulatory mechanisms: i) *gene expression*, acting on enzyme abundance, ii) *post-translational modification*, modulating enzyme abundance and kinetic parameters, and iii) *allosteric modulation*, exclusively affecting the kinetic parameters. Moreover, the *in vivo* metabolic flux depends also on the *in vivo reactant concentrations* (Gerosa & Sauer, 2011) which are function of thermodynamics and reaction kinetics, i.e. parameters that a cell may modulate only indirectly.

This chapter examines the mechanisms regulating the primary metabolism by using as model organisms the dairy species *L. lactis* among prokaryotes, and the bakers' yeast *S. cerevisiae* among eukaryotes. Moreover, some enzymatic activities and metabolic pathways are described and their physiological role is revisited, taking into consideration the optimisation of the cellular bioenergetics as a result of an environment-dependent selection pressure.
