Insert front cover on this page. Lactobacillus Genomics and Metabolic Engineering Edited by Sandra M. Ruzal Caister Academic Press Chapter 3 from: Lactobacillus Genomics and Metabolic Engineering Edited by Sandra M. Ruzal ISBN: 978-1-910190-89-0 (paperback) ISBN: 978-1-910190-90-6 (ebook) Caister Academic Press www.caister.com Complex Oligosaccharide Utilization Pathways in Lactobacillus 3 Manuel Zúñiga*, María Jesús Yebra and Vicente Monedero Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Valencia, Spain. *Correspondence: [email protected] https://doi.org/10.21775/9781910190890.03 Abstract the microbes find in the different compartments of Lactobacillus is the bacterial genus that contains the gastrointestinal tract (Gu et al., 2013; Kim and the highest number of characterized probiotics. Isaacson, 2015; Stearns et al., 2011; Tropini et al., Lactobacilli in general can utilize a great variety 2017). For example, Lactobacillaceae is predomi- of carbohydrates. This characteristic is an essential nantly found in the stomach and small intestine of trait for their survival in highly competitive environ- mice whereas anaerobes such as Bacteroidaceae, ments such as the gastrointestinal tract of animals. In Prevotellaceae, Rikenellaceae, Lachnospiraceae particular, the ability of some strains to utilize com- and Ruminococcaceae are mainly found in the plex carbohydrates such as milk oligosaccharides large intestine and faeces (Gu et al., 2013). In the as well as their precursor monosaccharides, confer oral cavity of humans, a neutral pH and aerobic upon lactobacilli a competitive advantage. For this conditions prevail although anaerobic niches are reason, many of these carbohydrates are considered also present. The environment in the stomach is as prebiotics. Genome sequencing of many lacto- acidic and microaerophilic whereas pH increases bacilli strains has revealed a great variety of genes and oxygen availability decreases along the small involved in the metabolism of carbohydrates and intestine and colon. Nutrient availability and some of them have already been characterized. In immune effectors also vary along the gastrointesti- this chapter, the current knowledge at the biochem- nal tract. Furthermore, microbes are not uniformly ical and genetic levels of the catabolic pathways of distributed along the transverse axis of the gut complex carbohydrates utilized by lactobacilli will (Tropini et al., 2017). Species such as Akkermansia be summarized. muciniphila and some Bacteroides spp. are thought to be predominantly associated with the mucus layer whereas closer to the mucosa, aerotolerant Introduction taxa such as Proteobacteria and Actinobacteria are All animals establish symbiotic associations with more abundant due to the radial oxygen gradient microbes. Paramount among these is the estab- established across the intestinal wall (Albenberg et lishment of complex microbial communities in al., 2014). the gastrointestinal tract. These communities may Numerous studies have demonstrated that gut comprise thousands of species and are particular microbiota has a great influence on manifold host to each individual (Eckburg et al., 2005; Ley et al., physiology aspects, from metabolism (Li et al., 2006; Turnbaugh et al., 2009). The composition 2008) to behaviour (Ezenwa et al., 2012). Perturba- and distribution of microbiota changes along the tion of the gut microbiota may markedly affect the gastrointestinal tract reflecting the differing phys- host health (Claesson et al., 2012; Cho and Blaser, icochemical conditions and epithelial surfaces that 2012) and has been related to a number of diseases 32 | Zúñiga et al. such as metabolic disorders (Sonnenburg and and Roberfroid, 1995; Goh and Klaenhammer, Bäckhed, 2016), inflammatory diseases (Blander et 2015). Although not limited by the definition, all al., 2017), diabetes (Membrez et al., 2008; Vaarala prebiotics are glycans. The definition of prebiotic et al., 2008), coeliac disease (Collado et al., 2007), has been revised and the specific stimulatory effect etc. Although the microbiota associated with an of prebiotics on lactobacilli and bifidobacteria has individual may display some resilience to perturba- been challenged (Hutkins et al., 2016). Notwith- tion (Lozupone et al., 2012; Sommer et al., 2017), standing, the original idea gave a strong boost to external environmental factors such as food intake the study of the glycan catabolic pathways of these (Kohl et al., 2014) and composition (David et al., organisms. 2014) can alter it substantially. This aspect is piv- Lactobacillus is a large genus currently compris- otal in the relationship between microbiota and ing over 200 species that have been isolated from host nutrition and health as the gastrointestinal a wide variety of habitats. They are Gram-positive, microbiota provide important metabolic capa- microaerophilic or anaerobic obligate fermentative bilities, including the ability to obtain energy from organisms that produce lactic acid as the major indigestible dietary polysaccharides (Goh and end product of sugar fermentation. Together with Klaenhammer, 2015). The human genome encodes genera Paralactobacillus, Pediococcus and Sharpea only 17 glycosidases, enabling the utilization of a they constitute the family Lactobacillaceae within very limited set of polysaccharides (Cantarel et al., the order Lactobacillales. Lactobacillus strains play 2012; Goh and Klaenhammer, 2015). Therefore, a major role in the production of a wide variety of most complex carbohydrates are available to gut fermented products. Others are naturally associ- microbes able to utilize these compounds. ated with mucosal surfaces of humans and animals Carbohydrates, as oligo- and polysaccharides or and have been considered as probiotics (Tannock, as glycoconjugates constitute an amazingly diverse 2004). Either as foodstuff fermenters or as probiot- group of molecules due to the structural diversity ics, their capacity to utilize glycans is an important and numerous bonding sites of their constituent trait for their performance. This chapter focuses on monosaccharides that allow their assembly among our current knowledge on the pathways of complex themselves or to almost any other organic molecule glycan dissimilation identified in species ofLacto - in a wide array of architectures. Because of this bacillus. structural versatility, carbohydrates fulfil a wide variety of functions in organisms as structural polymers, energy reserve, signalling, etc. Fur- Fructan and thermore, as a major component of human diet, fructooligosaccharide catabolic carbohydrates also have a determining influence pathways on the interactions between host and associated microbiota (Hooper et al., 2002). In addition to Structural characteristics of fructans dietary carbohydrates, host-derived glycans con- Fructans are linear or branched fructose poly- stitute a secondary source of carbohydrates for gut mers and can be broadly divided into inulins microbiota (Hooper et al., 2002). (β-2,1-linked) and levans (β-2,6-linked) (Fuchs, Although the gut microbiota taken as a whole 1991) (Fig. 3.1). Fructans are usually synthesized can utilize a wide variety of glycans, cells belonging from sucrose by repeated fructosyl transfer so that to individual taxa can usually metabolize a limited they have a terminal glucose unit. Levans are pro- set of them. From this fact stems the concept of duced by many microorganisms, including some prebiotic, defined as a non-digestible food ingredi- lactobacilli (Bello et al., 2001; Tieking et al., 2003; ent that beneficially affects the host by selectively Van Geel-Schutten et al., 1999), and a few plant stimulating the growth and/or activity of one or a species (Öner et al., 2016). In contrast, inulins are limited number of bacteria in the colon, and thus relatively common in plants, especially Asteraceae, improves host health (Gibson and Roberfroid, but only a few bacterial species produce them, 1995). Prebiotics were first thought of as a means among them some Lactobacillus and Leuconostoc to selectively enrich probiotic gut microbes, spe- species (Anwar et al., 2008; Olivares-Illana et al., cifically Lactobacillus and Bifidobacterium (Gibson 2003; van Hijum et al., 2002). While bacterial Complex Oligosaccharide Utilization Pathways in Lactobacillus | 33 OH HO OH O O OH HO OH O O OH n HO O HO OH OH O HO O HO Inulin OH HO O OH OH OH HO OH OH OH HO OH O O O O O O OH OH OH n OH Levan Figure 3.1 Chemical structures of inulin and levan. fructans have a very high degree of polymerization -β-d-fructofuranosyl-(2→1)-α-d-glucopyranoside). (DP) up to 105 fructose units, the DP of plant- 1-FFT catalyses the transfer of fructosyl units from derived fructans does generally not exceed DP 100. 1-kestose and any other fructan molecule onto High DP of bacterial fructans is possibly related to 1-kestose and higher DP fructan molecules. 1-FEH, their function as structural components of biofilms, catalyses the degradation of inulin by hydrolysing but they also constitute an extracellular nutrient terminal fructosyl units, which results in the forma- reservoir (Öner et al., 2016). In plants, fructans tion of fructose and lower DP inulin (van Arkel et serve essentially as reserve carbohydrates. al., 2013). Synthesis of fructans is catalysed in bacteria by fructosyltransferases, named inulosucrases (EC Metabolic pathways for fructans 2.4.1.9) when they synthesize inulin, and levan- utilization sucrases (EC 2.4.1.10) when they produce levan. The interest