Cross-Feeding among Probiotic Bacterial Strains on Prebiotic Inulin Involves the Extracellular exo-Inulinase of Lactobacillus paracasei Strain W20

Probiotic gut bacteria employ specific metabolic pathways to degrade dietary carbohydrates beyond the capabilities of their human host. Here, we report how individual commercial probiotic strains degrade prebiotic (inulin type) fructans. First, a structural analysis of commercial fructose oligosaccharide-inulin samples was performed. These beta-(2-1)-fructans differ in termination by either glucose (GF) or fructose (FF) residues, with a broad variation in the degrees of polymerization (DPs). The growth of individual probiotic bacteria on short-chain inulin (sc-inulin) (Frutafit CLR), a beta-(2-1)-fructan (DP 2 to DP 40), was studied. Lactobacillus salivarius W57 and other bacteria grew relatively poorly on sc-inulin, with only fractions of DP 3 and DP 5 utilized, reflecting uptake via specific transport systems followed by intracellular metabolism. Lactobacillus paracasei subsp. paracasei W20 completely used all sc-inulin components, employing an extracellular exo-inulinase enzyme (glycoside hydrolase family GH32 [LpGH32], also found in other strains of this species); the purified enzyme converted high-DP compounds into fructose, sucrose, 1-kestose, and F2 (inulobiose). The cocultivation of L. salivarius W57 and L. paracasei W20 on sc-inulin resulted in cross-feeding of the former by the latter, supported by this extracellular exo-inulinase. The extent of cross-feeding depended on the type of fructan, i.e., the GF type (clearly stimulating) versus the FF type (relatively low stimulus), and on fructan chain length, since relatively low-DP beta-(2-1)-fructans contain a relatively high content of GF-type molecules, thus resulting in higher concentrations of GF- type DP 2 to DP 3 degradation products. The results provide an example of how in vivo cross-feeding on prebiotic beta-(2-1)-fructans may occur among probiotic lactobacilli. IMPORTANCE The human gut microbial community is associated strongly with host physiology and human diseases. This observation has prompted research on pre- and probiotics, two concepts enabling specific changes in the composition of the human gut microbiome that result in beneficial effects for the host. Here, we show how fructooligosaccharide-inulin prebiotics are fermented by commercial probiotic bacterial strains involving specific sets of enzymes and transporters. Cross-feeding strains such as Lactobacillus paracasei W20 may thus act as keystone strains in the degradation of prebiotic inulin in the human gut, and this strain-exo-inulinase combination may be used in commercial Lactobacillus-inulin synbiotics

Structural and functional characterization of a family GH53 β-1,4-galactanase from Bacteroides thetaiotaomicron that facilitates degradation of prebiotic galactooligosaccharides

Galactooligosaccharides (GOS) are prebiotic compounds synthesized from lactose using bacterial enzymes and are known to stimulate growth of beneficial bifidobacteria in the human colon. Bacteroides thetaiotaomicron is a prominent human colon commensal bacterial species that hydrolyzes GOS using an extracellular Glycosyl Hydrolase (GH) family GH53 endo-galactanase enzyme (BTGH53), releasing galactose-based products for growth. Here we dissect the molecular basis for GOS activity of this B. thetaiotaomicron GH53 endo-galactanase. Elucidation of its X-ray crystal structure revealed that BTGH53 has a relatively open active site cleft which was not observed with the bacterial enzyme from Bacillus licheniformis (BLGAL). BTGH53 acted on GOS with degree of polymerization ≤3 and therefore more closely resembles activity of fungal GH53 enzymes (e.g. Aspergillus aculeatus AAGAL and Meripileus giganteus MGGAL). Probiotic lactobacilli that lack galactan utilization systems constitute a group of bacteria with relevance for a healthy (infant) gut. The strains tested were unable to use GOS ≥ DP3. However, they completely consumed GOS in the presence of BTGH53, resulting in clear stimulation of their extent of growth. The extracellular BTGH53 enzyme thus may play an important role in carbohydrate metabolism in complex microbial environments such as the human colon. It also may find application for the development of synergistic synbiotics

Using structure to inform carbohydrate binding module function

Generally, non-catalytic carbohydrate binding module (CBM) specificity has been shown to parallel the catalytic activity of the carbohydrate active enzyme (CAZyme) module it is appended to. With the rapid expansion in metagenomic sequence space for the potential discovery of new CBMs in addition to the recent emergence of several new CBM families that display diverse binding profiles and novel functions, elucidating the function of these protein modules has become a much more challenging task. This review summarizes several approaches that have been reported for using primary structure to inform CBM specificity and streamlining their biophysical characterization. In addition we discuss general trends in binding site architecture and several newly identified functions for CBMs. Streams of investigation that will facilitate the development and refinement of sequence-based prediction tools are suggested

Prebiotic galactooligosaccharides activate mucin and pectic galactan utilization pathways in the human gut symbiont Bacteroides thetaiotaomicron

Galactooligosaccharides (GOS) are prebiotic carbohydrates that impart changes in the gut bacterial composition of formula-fed infants to more closely resemble that of breast-fed infants. Consuming human milk oligosaccharides (HMOs) provides specific bacterial strains with an advantage for colonizing the infant intestine. These same effects are seen in infants after GOS consumption, however GOS are very complex mixtures and the underlying molecular mechanisms of how GOS mimic HMOs are relatively unknown. Here we studied the effects of GOS utilization on a prominent gut symbiont, Bacteroides thetaiotaomicron, which has been previously shown to consume HMOs via mucin O-glycan degradation pathways. We show that several pathways for targeting O-mucin glycans are activated in B. thetaiotaomicron by GOS, as well as the galactan utilization sytem. Characterization of the endo-galactanase from this system identified activity on various longer GOS substrates while a subset of GOS compounds were identified as potential activators of mucin glycan metabolism in B. thetaiotaomicron. Our results show that GOS functions as an inducer of mucin-glycan pathways while providing a nutrient source in the form of β-(1 → 4)-galactan. These metabolic features of GOS mixtures may serve to explain the beneficial effects that are seen for GOS supplemented infant formula

Carbohydrate Binding Module 74 is a novel starch binding domain associated with large and multi-domain α-amylase enzymes

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C-terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore we propose that this novel starch binding domain is a new carbohydrate binding module (CBM), the first representative of family CBM74, that assists MaAmyA in efficient pore formation in starch granules. Protein-sequence based BLAST searches revealed that CBM74 occurs widespread, but in Bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches. Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut associated Bifidobacteria where they may assist in raw starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of resistant starch digestion in the mammalian gastrointestinal tract. This article is protected by copyright. All rights reserved

Structural Identity of Galactooligosaccharide Molecules Selectively Utilized by Single Cultures of Probiotic Bacterial Strains

Various beta-galactosidase enzymes catalyze the trans-glycosylation reaction with lactose. The resulting galactooligosaccharide (GOS) mixtures are widely used in infant nutrition to stimulate growth of beneficial gut bacteria. GOS consists mainly of compounds with a degree of polymerization (DP) varying from 2-8 and with diverse glycosidic linkages. In recent years, we have elucidated in detail the composition of several commercial GOS mixtures in terms of DP and the structural identity of the individual compounds. In this work, 13 (single) probiotic strains of gut bacteria, belonging to 11 different species, were grown to stationary phase with a Vivinal GOS-derived sample purified to remove lactose and monosaccharides (pGOS). Growth among the probiotic strains varied strongly between 30 and 100% of OD600nm relative to positive controls with glucose. By identifying the components of the pGOS mixture that remain after growth, we showed that strains varied in their consumption of specific GOS compounds. All strains commonly used most of the GOS DP2 pool. Lactobacillus salivarius W57 also utilized the DP3 branched compound beta-D-Galp-(1 -> 4)-[beta-D-Galp-(1 -> 2)]-D-G1c. Bifidobacterial strains tended to use GOS with higher DP and branching than lactobacilli; Bifidobacterium breve DSM 20091, Lactobacillus acidophilus W37, and Bifidobacterium infantis DSM 20088 were exceptional in using 38, 36, and 35 compounds, respectively, out of the 40 different structures identified in pGOS. We correlated these bacterial GOS consumption profiles with their genomic information and were able to relate metabolic activity with the presence of genome-encoded transporters and carbohydrate-active enzymes. These detailed insights may support the design of synbiotic combinations pairing probiotic bacterial strains with GOS compounds that specifically stimulate their growth. Such synbiotic combinations may be of interest in food/feed and/or pharmacy/medicine applications

Lactobacillus reuteri strains convert starch and maltodextrins into homo-exopolysaccharides using an extracellular and cell-associated 4,6-α-glucanotransferase

Exopolysaccharides (EPS) of lactic acid bacteria (LAB) are of interest for food applications. LAB are well-known to produce α-glucan from sucrose by extracellular glucansucrases. Various <i>Lactobacillus reuteri</i> strains also possess 4,6-α-glucanotransferase (4,6-α-GTase) enzymes. Purified 4,6-α-GTases (e.g., GtfB) were shown to act on starches (hydrolysates), cleaving α1→4 linkages and synthesizing α1→6 linkages, yielding isomalto-/maltopolysaccharides (IMMP). Here we report that also <i>L. reuteri</i> cells with these extracellular, cell-associated 4,6-α-GTases synthesize EPS (α-glucan) from starches (hydrolysates). NMR, SEC, and enzymatic hydrolysis of EPS synthesized by <i>L. reuteri</i> 121 cells showed that these have similar linkage specificities but generally are much bigger in size than IMMP produced by the GtfB enzyme. Various IMMP-like EPS are efficiently used as growth substrates by probiotic <i>Bifidobacterium</i> strains that possess amylopullulanase activity. IMMP-like EPS thus have potential prebiotic activity and may contribute to the application of probiotic <i>L. reuteri</i> strains grown on maltodextrins or starches as synbiotics