Oligosaccharide Metabolism and Lipoteichoic Acid Production in Lactobacillus gasseri and Lactobacillus paragasseri.

Lactobacillus gasseri and Lactobacillus paragasseri are human commensal lactobacilli that are candidates for probiotic application. Knowledge of their oligosaccharide metabolic properties is valuable for synbiotic application. The present study characterized oligosaccharide metabolic systems and their impact on lipoteichoic acid (LTA) production in the two organisms, i.e., L. gasseri JCM 1131T and L. paragasseri JCM 11657. The two strains grew well in medium with glucose but poorly in medium with raffinose, and growth rates in medium with kestose differed between the strains. Oligosaccharide metabolism markedly influenced their LTA production, and apparent molecular size of LTA in electrophoresis recovered from cells cultured with glucose and kestose differed from that from cells cultured with raffinose in the strains. On the other hand, more than 15-fold more LTA was observed in the L. gasseri cells cultured with raffinose when compared with glucose or kestose after incubation for 15 h. Transcriptome analysis identified glycoside hydrolase family 32 enzyme as a potential kestose hydrolysis enzyme in the two strains. Transcriptomic levels of multiple genes in the dlt operon, involved in D-alanine substitution of LTA, were lower in cells cultured with raffinose than in those cultured with kestose or glucose. This suggested that the different sizes of LTA observed among the carbohydrates tested were partly due to different levels of alanylation of LTA. The present study indicates that available oligosaccharide has the impact on the LTA production of the industrially important lactobacilli, which might influence their probiotic properties.

Characterization of fructooligosaccharide metabolism and fructooligosaccharide-degrading enzymes in human commensal butyrate producers.

Butyrate produced by gut microbiota has multiple beneficial effects on host health, and oligosaccharides derived from host diets and glycans originating from host mucus are major sources of its production. A significant reduction of butyrate-producing bacteria has been reported in patients with inflammatory bowel diseases and colorectal cancers. Although gut butyrate levels are important for host health, oligosaccharide metabolic properties in butyrate producers are poorly characterized. We studied the metabolic properties of fructooligosaccharides (FOSs) and other prebiotic oligosaccharides (i.e. raffinose and xylooligosaccharides; XOSs) in gut butyrate producers. 1-Kestose (kestose) and nystose, FOSs with degrees of polymerization of 3 and 4, respectively, were also included. Fourteen species of butyrate producers were divided into four groups based on their oligosaccharide metabolic properties, which are group A (two species) metabolizing all oligosaccharides tested, group F (four species) metabolizing FOSs but not raffinose and XOSs, group XR (four species) metabolizing XOSs and/or raffinose but not FOSs, and group N (four species) metabolizing none of the oligosaccharides tested. Species assigned to groups A and XR are rich glycoside hydrolase (GH) holders, whereas those in groups F and N are the opposite. In total, 17 enzymes assigned to GH32 were observed in nine of the 14 butyrate producers tested, and species that metabolized FOSs had at least one active GH32 enzyme. The GH32 enzymes were divided into four clusters by phylogenetic analysis. Heterologous gene expression analysis revealed that the GH32 enzymes in each cluster had similar FOS degradation properties within clusters, which may be linked to the conservation/substitution of amino acids to bind with substrates in GH32 enzymes. This study provides important knowledge to understand the impact of FOS supplementation on the activation of gut butyrate producers. Abbreviations: SCFA, short chain fatty acid; FOS, fructooligosaccharide; XOS, xylooligosaccharide; CAZy, Carbohydrate Active Enzymes; CBM, carbohydrate-binding module; PUL, polysaccharide utilization locus; S6PH sucrose-6-phosphate hydrolase.

Crystal structure of a glycoside hydrolase family 68 ß-fructosyltransferase from Beijerinckia indica subsp. indica in complex with fructose.

An enzyme belonging to glycoside hydrolase family 68 (GH68) from Beijerinckia indica subsp. indica NBRC 3744 was expressed in Escherichia coli. Biochemical characterization showed that the enzyme was identified to be a ß-fructosyltransferase (BiBftA). Crystallization of a full-length BiBftA was initially attempted, but no crystals were obtained. We constructed a variant in which 5 residues (Pro199-Gly203) and 13 residues (Leu522-Gln534) in potentially flexible regions were deleted, and we successfully crystallized this variant BiBftA. BiBftA is composed of a five-bladed ß-propeller fold as in other GH68 enzymes. The structure of BiBftA in complex with fructose unexpectedly indicated that one ß-fructofuranose (ß-Fruf) molecule and one ß-fructopyranose molecule bind to the catalytic pocket. The orientation of ß-Fruf at subsite -1 is tilted from the orientation observed in most GH68 enzymes, presenting a second structure of a GH68 enzyme in complex with the tilted binding mode of ß-Fruf.

In Vitro Evaluation of Different Prebiotics on the Modulation of Gut Microbiota Composition and Function in Morbid Obese and Normal-Weight Subjects.

The gut microbiota remains relatively stable during adulthood; however, certain intrinsic and environmental factors can lead to microbiota dysbiosis. Its restoration towards a healthy condition using best-suited prebiotics requires previous development of in vitro models for evaluating their functionality. Herein, we carried out fecal cultures with microbiota from healthy normal-weight and morbid obese adults. Cultures were supplemented with different inulin-type fructans (1-kestose, Actilight, P95, Synergy1 and Inulin) and a galactooligosaccharide. Their impact on the gut microbiota was assessed by monitoring gas production and evaluating changes in the microbiota composition (qPCR and 16S rRNA gene profiling) and metabolic activity (gas chromatography). Additionally, the effect on the bifidobacterial species was assessed (ITS-sequencing). Moreover, the functionality of the microbiota before and after prebiotic-modulation was determined in an in vitro model of interaction with an intestinal cell line. In general, 1-kestose was the compound showing the largest effects. The modulation with prebiotics led to significant increases in the Bacteroides group and Faecalibacterium in obese subjects, whereas in normal-weight individuals, substantial rises in Bifidobacterium and Faecalibacterium were appreciated. Notably, the results obtained showed differences in the responses among the tested compounds but also among the studied human populations, indicating the need for developing population-specific products.

Impact of kestose supplementation on the healthy adult microbiota in in vitro fecal batch cultures.

Prebiotics are widely used to shape a balanced microbiota in humans and animals. 1-Kestose (kestose) is one of the major components in commercialized short-chain fructooligosaccharide and is a promising prebiotic for infants. We herein studied the impact of kestose on the healthy adult microbiota in an in vitro fecal batch culture model. Stool samples obtained from seven healthy adults were diluted, inoculated into broth supplemented with or without 0.5% (w/v) kestose (kestose group and control group, respectively), and cultured under anaerobic conditions. Microbiota in the groups and stool samples were analyzed using 16S rRNA gene sequencing. At the phylum level, the kestose group showed increases in Bacteroidetes, whereas the control group showed increases in Proteobacteria. At the species level, Bifidobacterium longum was the only species showing significantly higher levels in the kestose group than in the control group and stool samples. On the other hand, levels of Escherichia coli were significantly higher in the control group than in stool samples, while the levels were not significantly different between the kestose group and stool samples. Quantitative PCR assays also revealed significantly higher levels of B. longum and lower tendency of E. coli in the kestose group than in the control group. These results suggest that supplementation with kestose increased the levels of beneficial microorganism and prevented the growth of risk-associated microorganisms related to disease development. Further interventional studies are needed to understand the health benefits of kestose in adult humans.

In vitro assembly and cellulolytic activity of a ß-glucosidase-integrated cellulosome complex.

The cellulosome is a supramolecular multi-enzyme complex formed by protein interactions between the cohesin modules of scaffoldin proteins and the dockerin module of various polysaccharide-degrading enzymes. In general, the cellulosome exhibits no detectable ß-glucosidase activity to catalyze the conversion of cellobiose to glucose. Because ß-glucosidase prevents product inhibition of cellobiohydrolase by cellobiose, addition of ß-glucosidase to the cellulosome greatly enhances the saccharification of crystalline cellulose and plant biomass. Here, we report the in vitro assembly and cellulolytic activity of a ß-glucosidase-coupled cellulosome complex comprising the three major cellulosomal cellulases and full-length scaffoldin protein of Clostridium (Ruminiclostridium) thermocellum, and Thermoanaerobacter brockii ß-glucosidase fused to the type-I dockerin module of C. thermocellum. We show that the cellulosome complex composed of nearly equal numbers of cellulase and ß-glucosidase molecules exhibits maximum activity toward crystalline cellulose, and saccharification activity decreases as the enzymatic ratio of ß-glucosidase increases. Moreover, ß-glucosidase-coupled and ß-glucosidase-supplemented cellulosome complexes similarly exhibit maximum activity toward crystalline cellulose (i.e. 1.7-fold higher than that of the ß-glucosidase-free cellulosome complex). These results suggest that the enzymatic ratio of cellulase and ß-glucosidase in the assembled complex is crucial for the efficient saccharification of crystalline cellulose by the ß-glucosidase-integrated cellulosome complex.

Characterization of fructooligosaccharide-degrading enzymes in human commensal Bifidobacterium longum and Anaerostipes caccae.

Kestose and nystose are short chain fructooligosaccharides (scFOSs) with degrees of polymerization of 3 and 4, respectively. A previous study revealed that these scFOSs have different growth stimulation properties against two human commensals, i.e. Bifidobacterium longum subsp. longum and butyrogenic Anaerostipes caccae. The present study characterized genes involved in FOS metabolism in these organisms. A. caccae possesses a single gene cluster consisting of four genes, including a gene encoding the putative FOS degradation enzyme sucrose-6-phosphate hydrolase (S6PH). B. longum possesses two gene clusters consisting of three genes each, including genes encoding ß-fructofuranosidase (CscA) and sucrose phosphorylase (ScrP). In A. caccae, the genes were highly transcribed in cells cultured with sucrose or kestose but poorly in cells cultured with glucose or nystose. Heterologously expressed S6PH degraded sucrose and kestose but not nystose. In B. longum, transcription of the genes was high in cells cultured with sucrose or kestose but was poor or not detected in cells cultured with glucose or nystose. Heterologously expressed CscA degraded sucrose, kestose and nystose, but ScrP degraded only sucrose. These data suggested that the different growth stimulation activities of kestose and nystose are due to different substrate specificities of FOS degradation enzymes in the organisms and/or induction activity of the genes in the two scFOSs. This is the first study characterizing the FOS metabolism at the transcriptional level and substrate-specificity of the degradation enzyme in butyrogenic human gut anaerobes.

Comparative Biochemical Analysis of Cellulosomes Isolated from Clostridium clariflavum DSM 19732 and Clostridium thermocellum ATCC 27405 Grown on Plant Biomass.

The cellulosome is a supramolecular multienzyme complex formed via species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Here, we report a comparative analysis of cellulosomes prepared from the thermophilic anaerobic bacteria Clostridium (Ruminiclostridium) clariflavum DSM 19732 and Clostridium (Ruminiclostridium) thermocellum ATCC 27405 grown on delignified rice straw. The results indicate that the isolated C. clariflavum cellulosome exhibits lower activity for insoluble cellulosic substrates and higher activity for hemicellulosic substrates, especially for xylan, compared to the isolated C. thermocellum cellulosome. The C. clariflavum cellulosome was separated into large and small complexes by size exclusion chromatography, and the high xylanase activity of the intact complex is mainly attributed to the small complex. Furthermore, both C. clariflavum and C. thermocellum cellulosomes efficiently converted delignified rice straw into soluble sugars with different compositions, whereas a mixture of these cellulosomes exhibited essentially no synergy for the saccharification of delignified rice straw. This is the first study to report that isolated C. clariflavum cellulosomes exhibit greater xylanase activity than isolated C. thermocellum cellulosomes. We also report the effect of a combination of intact cellulosome complexes isolated from different species on the saccharification of plant biomass.

Rapid evaluation of 1-kestose producing ß-fructofuranosidases from Aspergillus species and enhancement of 1-kestose production using a PgsA surface-display system.

1-Kestose is a key prebiotic fructooligosaccharide (FOS) sugar. Some ß-fructofuranosidases (FFases) have high transfructosylation activity, which is useful for manufacturing FOS. Therefore, obtaining FFases that produce 1-kestose efficiently is important. Here, we established a rapid FFase evaluation method using Escherichia coli that display different FFases fused to a PgsA anchor protein from Bacillus subtilis. E. coli cell suspensions expressing the PgsA-FFase fusion efficiently produce FOS from sucrose. Using this screening technique, we found that the E. coli transformant expressing Aspergillus kawachii FFase (AkFFase) produced a larger amount of 1-kestose than those expressing FFases from A. oryzae and A. terreus. Saturation mutagenesis of AkFFase was performed, and the mutant G85W was obtained. The E. coli transformant expressing AkFFase G85W markedly increased production of 1-kestose. Our results indicate that the surface display technique using PgsA is useful for screening of FFases, and AkFFase G85W is likely to be suitable for 1-kestose production. ABBREVIATIONS: AkFFase: Aspergillus kawachii FFase; AoFFase: Aspergillus oryzae FFase; AtFFase: Aspergillus terreus FFase; FFase: ß-fructofuranosidase; FOS: fructooligosaccharide; fructosylnystose: 1F-ß-fructofuranosylnystose.

The ability of human intestinal anaerobes to metabolize different oligosaccharides: Novel means for microbiota modulation?

Prebiotic oligosaccharides are known to have significant impacts on gut microbiota and are thus widely used to program healthy microbiota composition and activity from infants to the elderly. Bifidobacteria and lactobacilli are among the major target microorganisms of oligosaccharides, but the metabolic properties of oligosaccharides in other predominant gut microbes have not been well characterized. In the present study, we demonstrated the metabolic properties of six oligosaccharides in 31 key gut anaerobes. Bifidobacteria readily metabolized fructooligosaccharide (FOSs) with degree of polymerization (DP) 3, i.e. 1-kestose, but several strains used did not actively metabolize FOSs with DP4 and DP5, i.e. nystose and fructosylnystose. Akkermansia muciniphila, a potential new probiotic against obesity, did not show significant growth with any of the oligosaccharides tested. The butyrate producer Anaerostipes caccae grew well on 1-kestose but poorly on FOS mixtures, whereas it contained 1-kestose at 30%. Bacteroides-Parabacteroides group species were separated into two groups based on oligosaccharide metabolic properties. One group metabolized well most of the oligosaccharides tested, but the others metabolized only 1 or 2 selected oligosaccharides. Oligosaccharide profiles after culturing revealed that Bifidobacterium spp. preferentially metabolized shorter oligosaccharides (DP3) in the mixtures, whereas Bacteroides-Parabacteroides spp. did not show oligosaccharide selectivity for metabolism or rather preferred longer oligosaccharides (>DP4). The fermentation profiles indicated specific links between the microbial end-products and specific gut microbes. Available carbohydrates had a significant impact on the accumulation of amino acid-derived bacterial metabolites (i.e. phenol, p-cresol, indole and skatole) and short chain fatty acids. The results assist in predicting the impact of oligosaccharides in human intervention and gut microbiota modulation.

Crystal structure of a ß-fructofuranosidase with high transfructosylation activity from Aspergillus kawachii.

ß-Fructofuranosidases belonging to glycoside hydrolase family (GH) 32 are enzymes that hydrolyze sucrose. Some GH32 enzymes also catalyze transfructosylation to produce fructooligosaccharides. We found that Aspergillus kawachii IFO 4308 ß-fructofuranosidase (AkFFase) produces fructooligosaccharides, mainly 1-kestose, from sucrose. We determined the crystal structure of AkFFase. AkFFase is composed of an N-terminal small component, a ß-propeller catalytic domain, an α-helical linker, and a C-terminal ß-sandwich, similar to other GH32 enzymes. AkFFase forms a dimer, and the dimerization pattern is different from those of other oligomeric GH32 enzymes. The complex structure of AkFFase with fructose unexpectedly showed that fructose binds both subsites -1 and +1, despite the fact that the catalytic residues were not mutated. Fructose at subsite +1 interacts with Ile146 and Glu296 of AkFFase via direct hydrogen bonds.

Enzymatic diversity of the Clostridium thermocellum cellulosome is crucial for the degradation of crystalline cellulose and plant biomass.

The cellulosome is a supramolecular multienzyme complex comprised of a wide variety of polysaccharide-degrading enzymes and scaffold proteins. The cellulosomal enzymes that bind to the scaffold proteins synergistically degrade crystalline cellulose. Here, we report in vitro reconstitution of the Clostridium thermocellum cellulosome from 40 cellulosomal components and the full-length scaffoldin protein that binds to nine enzyme molecules. These components were each synthesized using a wheat germ cell-free protein synthesis system and purified. Cellulosome complexes were reconstituted from 3, 12, 30, and 40 components based on their contents in the native cellulosome. The activity of the enzyme-saturated complex indicated that greater enzymatic variety generated more synergy for the degradation of crystalline cellulose and delignified rice straw. Surprisingly, a less complete enzyme complex displaying fewer than nine enzyme molecules was more efficient for the degradation of delignified rice straw than the enzyme-saturated complex, despite the fact that the enzyme-saturated complex exhibited maximum synergy for the degradation of crystalline cellulose. These results suggest that greater enzymatic diversity of the cellulosome is crucial for the degradation of crystalline cellulose and plant biomass, and that efficient degradation of different substrates by the cellulosome requires not only a different enzymatic composition, but also different cellulosome structures.

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose, as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome.

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.

Classification of chitosanases by hydrolytic specificity toward N¹,N4-diacetylchitohexaose.

The hydrolytic specificities of chitosanases were determined using N¹,N4-diacetylchitohexaose [(GlcN)2-GlcNAc-(GlcN)2-GlcNAc]. The results for the hydrolytic specificities of chitosanases belonging to subclasses I, II, and III toward chitohexaose and N¹,N4-diacetylchitohexaose agreed with previous results obtained by analysis of the hydrolysis products of partially N-acetylated chitosan. N¹,N4-Diacetylchitohexaose is a useful substrate to determine the hydrolytic specificity of chitosanase. On the other hand, chitosanases from Amycolatopsis sp. CsO-2 and Pseudomonas sp. A-01 showed broad cleavage specificity. They cleaved both the GlcNAc-GlcN and the GlcN-GlcNAc bonds in addition to the GlcN-GlcN bond in the substrate. Thus, both enzymes were new chitosanases. The chitosanases were divided into four subclasses according to their specificity for hydrolysis of the ß-glycosidic linkages in partially N-acetylated chitosan.

Characterization of a chitosanase from Aspergillus fumigatus ATCC13073.

Chitosanase II was purified from the culture filtrate of Aspergillus fumigatus ATCC13073. The purified enzyme had a molecular mass of 23.5 kDa. The N-terminal amino acid sequence of chitosanase II was identical to those of other Aspergillus chitosanases belonging to glycoside hydrolase family 75. The optimum pH and temperature were pH 6.0 and 40 °C. Chitosanase II hydrolyzed 70% deacetylated chitosan faster than fully deacetylated chitosan. Analysis of the degradation products generated from partially N-acetylated chitosan showed that chitosanase II split GlcN-GlcN and GlcNAc-GlcN bonds but not GlcNAc-GlcNAc or GlcN-GlcNAc, suggesting that it is a subclass I chitosanase. It degraded (GlcN)(6) to produce (GlcN)(3) as main product and small amounts of (GlcN)(2) and (GlcN)(4). Reaction rate analyses of mono-N-acetylated chitohexaose suggested that the (+3) site of chitosanase II recognizes the GlcNAc residue rather than the GlcN residue of its substrate.