Structural and Biochemical Characterization of a Nonbinding SusD-Like Protein Involved in Xylooligosaccharide Utilization by an Uncultured Human Gut Bacteroides Strain.

In the human gut microbiota, Bacteroidetes break down dietary and endogenous glycosides through highly specific polysaccharide utilization loci (PULs). PULs encode a variety of sensor regulators, binding proteins, transporters, and carbohydrate-active enzymes (CAZymes). Surface glycan-binding proteins (SGBPs) are essential for the efficient capture of the glycosides present on the cell surface, providing Bacteroidetes with a competitive advantage in colonizing their habitats. Here, we present the functional and structural characterization of a SusD-like protein encoded by a xylooligosaccharide (XOS) PUL from an uncultured human gut Bacteroides strain. This locus is also conserved in Bacteroides vulgatus, thereby providing new mechanistic insights into the role of SGBPs in the metabolism of dietary fiber of importance for gut health. Various in vitro analyses, including saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, revealed that the SusD-like protein cannot bind to the cognate substrate of the XOS PUL, although its presence is essential for the PUL to function. Analysis of the crystal structure of the SusD-like protein reveals an unfolded binding surface and the absence or inappropriate orientation of several key residues compared with other known SusD-like structures. These results highlight the critical role of the SusD-like protein in the transport of oligosaccharides and provide fundamental knowledge about the structure-function of SusC/D-like transporters, revealing that the binding specificity of SusD-like SGBPs does not necessarily reflect the uptake specificity of the transporter. IMPORTANCE The metabolization of dietary fiber is a crucial function for many gut bacteria, especially Bacteroidetes, which are particularly well adapted for recognizing, binding, transporting, and degrading glycosides. In this study, we report the functional and structural characterization of a SusD-like protein involved in xylooligosaccharide utilization by an uncultured gut Bacteroides strain. We demonstrate that while this protein is structurally similar to many canonical Bacteroidetes surface glycan-binding proteins, it cannot bind the substrate taken up by the cognate SusC-like transporter. This lack of binding might be explained by the absence of several key residues known to be involved in oligosaccharide binding and/or the possible necessity of the SusC-like protein to be present to create a cooperative binding site. The term "surface glycan-binding proteins" generally used for SusD-like proteins is thus not generic. Overall, this study allowed us to revisit the concept of glycoside utilization by Bacteroidetes, in particular those strains that feed on the short fibers naturally present in some dietary compounds or on the leftovers of other microbes.

Identification of Glycoside Transporters From the Human Gut Microbiome.

Transport is a crucial step in the metabolism of glycosides by bacteria, which is itself key for microbiota function and equilibrium. However, most transport proteins are function-unknown or only predicted, limiting our understanding of how bacteria utilize glycosides. Here, we present an activity-based screening method to identify functional glycoside transporters from microbiomes. The method is based on the co-expression in Escherichia coli of genes encoding transporters and carbohydrate-active enzymes (CAZymes) from metagenomic polysaccharide utilization loci (PULs) cloned in fosmids. To establish the proof of concept of the methodology, we used two different metagenomic libraries derived from human gut microbiota to select 18 E. coli clones whose metagenomic sequence contained at least one putative glycoside transporter and one functional CAZyme, identified by screening for various glycoside-hydrolase activities. Growth tests were performed on plant-derived glycosides, which are the target substrates of the CAZymes identified in each PUL. This led to the identification of 10 clones that are able to utilize oligosaccharides as sole carbon sources, thanks to the production of transporters from the PTS, ABC, MFS, and SusCD families. Six of the 10 hit clones contain only one transporter, providing direct experimental evidence that these transporters are functional. In the six cases where two transporters are present in the sequence of a clone, the transporters' function can be predicted from the flanking CAZymes or from similarity with transporters characterized previously, which facilitates further functional characterization. The results expand the understanding of how glycosides are selectively metabolized by bacteria and offers a new approach to screening for glycoside-transporter specificity toward oligosaccharides with defined structures.

α-Galactosidase and Sucrose-Kinase Relationships in a Bi-functional AgaSK Enzyme Produced by the Human Gut Symbiont Ruminococcus gnavus E1.

Plant α-galactosides belonging to the raffinose family oligosaccharides (RFOs) and considered as prebiotics, are commonly degraded by α-galactosidases produced by the human gut microbiome. In this environment, the Ruminococcus gnavus E1 symbiont-well-known for various benefit-is able to produce an original RgAgaSK bifunctional enzyme. This enzyme contains an hydrolytic α-galactosidase domain linked to an ATP dependent extra-domain, specifically involved in the α-galactoside hydrolysis and the phosphorylation of the glucose, respectively. However, the multi-modular relationships between both catalytic domains remained hitherto unexplored and has been, consequently, herein investigated. Biochemical characterization of heterologously expressed enzymes either in full-form or in separated domains revealed similar kinetic parameters. These results were supported by molecular modeling studies performed on the whole enzyme in complex with different RFOs. Further enzymatic analysis associated with kinetic degradation of various substrates followed by high pressure anionic exchange chromatography revealed that catalytic efficiency decreased as the number of D-galactosyl moieties branched onto the oligosaccharide increased, suggesting a preference of RgAgaSK for RFO's short chains. A wide prevalence and abundance study on a human metagenomic library showed a high prevalence of the RgAgaSK encoding gene whatever the health status of the individuals. Finally, phylogeny and synteny studies suggested a limited spread by horizontal transfer of the clusters' containing RgAgaSK to only few species of Firmicutes, highlighting the importance of these undispersed tandem activities in the human gut microbiome.

Investigating host-microbiome interactions by droplet based microfluidics.

BACKGROUND: Despite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota. RESULTS: Here, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries. Thousands of bacterial genomes can be covered in 1 h of work, with less than ten micrograms of substrate. Applied to the screening of the mucosal microbiota for ß-N-acetylgalactosaminidase activity, this approach allowed the identification of pathways involved in the degradation of human gangliosides and milk oligosaccharides, the structural homologs of intestinal mucin glycans. These pathways, whose prevalence is associated with inflammatory bowel diseases, could be the result of horizontal gene transfers with Bacteroides species. Such pathways represent novel targets to study the microbiota-host interactions in the context of inflammatory bowel diseases, in which the integrity of the mucosal barrier is impaired. CONCLUSION: By compartmentalizing experiments inside microfluidic droplets, this method speeds up and miniaturizes by several orders of magnitude the screening process compared to conventional approaches, to capture entire metabolic pathways from metagenomic libraries. The method is compatible with all types of (meta)genomic libraries, and employs a commercially available flow cytometer instead of a custom-made sorting system to detect intracellular or extracellular enzyme activities. This versatile and generic workflow will accelerate experimental exploration campaigns in functional metagenomics and holobiomics studies, to further decipher host-microbiota relationships. Video Abstract.

Harvesting of Prebiotic Fructooligosaccharides by Nonbeneficial Human Gut Bacteria.

Prebiotic oligosaccharides, such as fructooligosaccharides, are increasingly being used to modulate the composition and activity of the gut microbiota. However, carbohydrate utilization analyses and metagenomic studies recently revealed the ability of deleterious and uncultured human gut bacterial species to metabolize these functional foods. Moreover, because of the difficulties of functionally profiling transmembrane proteins, only a few prebiotic transporters have been biochemically characterized to date, while carbohydrate binding and transport are the first and thus crucial steps in their metabolization. Here, we describe the molecular mechanism of a phosphotransferase system, highlighted as a dietary and pathology biomarker in the human gut microbiome. This transporter is encoded by a metagenomic locus that is highly conserved in several human gut Firmicutes, including Dorea species. We developed a generic strategy to deeply analyze, in vitro and in cellulo, the specificity and functionality of recombinant transporters in Escherichia coli, combining carbohydrate utilization locus and host genome engineering and quantification of the binding, transport, and growth rates with analysis of phosphorylated carbohydrates by mass spectrometry. We demonstrated that the Dorea fructooligosaccharide transporter is specific for kestose, whether for binding, transport, or phosphorylation. This constitutes the biochemical proof of effective phosphorylation of glycosides with a degree of polymerization of more than 2, extending the known functional diversity of phosphotransferase systems. Based on these new findings, we revisited the classification of these carbohydrate transporters.IMPORTANCE Prebiotics are increasingly used as food supplements, especially in infant formulas, to modify the functioning and composition of the microbiota. However, little is currently known about the mechanisms of prebiotic recognition and transport by gut bacteria, while these steps are crucial in their metabolism. In this study, we established a new strategy to profile the specificity of oligosaccharide transporters, combining microbiomics, genetic locus and strain engineering, and state-of-the art metabolomics. We revisited the transporter classification database and proposed a new way to classify these membrane proteins based on their structural and mechanistic similarities. Based on these developments, we identified and characterized, at the molecular level, a fructooligosaccharide transporting phosphotransferase system, which constitutes a biomarker of diet and gut pathology. The deciphering of this prebiotic metabolization mechanism by a nonbeneficial bacterium highlights the controversial use of prebiotics, especially in the context of chronic gut diseases.

Adaptation of Syntenic Xyloglucan Utilization Loci of Human Gut Bacteroidetes to Polysaccharide Side Chain Diversity.

Genome sequencing has revealed substantial variation in the predicted abilities of individual species within animal gut microbiota to metabolize the complex carbohydrates comprising dietary fiber. At the same time, a currently limited body of functional studies precludes a richer understanding of how dietary glycan structures affect the gut microbiota composition and community dynamics. Here, using biochemical and biophysical techniques, we identified and characterized differences among recombinant proteins from syntenic xyloglucan utilization loci (XyGUL) of three Bacteroides and one Dysgonomonas species from the human gut, which drive substrate specificity and access to distinct polysaccharide side chains. Enzymology of four syntenic glycoside hydrolase family 5 subfamily 4 (GH5_4) endo-xyloglucanases revealed surprising differences in xyloglucan (XyG) backbone cleavage specificity, including the ability of some homologs to hydrolyze congested branched positions. Further, differences in the complement of GH43 alpha-l-arabinofuranosidases and GH95 alpha-l-fucosidases among syntenic XyGUL confer distinct abilities to fully saccharify plant species-specific arabinogalactoxyloglucan and/or fucogalactoxyloglucan. Finally, characterization of highly sequence-divergent cell surface glycan-binding proteins (SGBPs) across syntenic XyGUL revealed a novel group of XyG oligosaccharide-specific SGBPs encoded within select BacteroidesIMPORTANCE The catabolism of complex carbohydrates that otherwise escape the endogenous digestive enzymes of humans and other animals drives the composition and function of the gut microbiota. Thus, detailed molecular characterization of dietary glycan utilization systems is essential both to understand the ecology of these complex communities and to manipulate their compositions, e.g., to benefit human health. Our research reveals new insight into how ubiquitous members of the human gut microbiota have evolved a set of microheterogeneous gene clusters to efficiently respond to the structural variations of plant xyloglucans. The data here will enable refined functional prediction of xyloglucan utilization among diverse environmental taxa in animal guts and beyond.

Investigating Host Microbiota Relationships Through Functional Metagenomics.

The human Intestinal mucus is formed by glycoproteins, the O- and N-linked glycans which constitute a crucial source of carbon for commensal gut bacteria, especially when deprived of dietary glycans of plant origin. In recent years, a dozen carbohydrate-active enzymes from cultivated mucin degraders have been characterized. But yet, considering the fact that uncultured species predominate in the human gut microbiota, these biochemical data are far from exhaustive. In this study, we used functional metagenomics to identify new metabolic pathways in uncultured bacteria involved in harvesting mucin glycans. First, we performed a high-throughput screening of a fosmid metagenomic library constructed from the ileum mucosa microbiota using chromogenic substrates. The screening resulted in the isolation of 124 clones producing activities crucial in the degradation of human O- and N-glycans, namely sialidases, ß-D-N-acetyl-glucosaminidase, ß-D-N-acetyl-galactosaminidase, and/or ß-D-mannosidase. Thirteen of these clones were selected based on their diversified functional profiles and were further analyzed on a secondary screening. This step consisted of lectin binding assays to demonstrate the ability of the clones to degrade human intestinal mucus. In total, the structural modification of several mucin motifs, sialylated mucin ones in particular, was evidenced for nine clones. Sequencing their metagenomic loci highlighted complex catabolic pathways involving the complementary functions of glycan sensing, transport, hydrolysis, deacetylation, and deamination, which were sometimes associated with amino acid metabolism machinery. These loci are assigned to several Bacteroides and Feacalibacterium species highly prevalent and abundant in the gut microbiome and explain the metabolic flexibility of gut bacteria feeding both on dietary and human glycans.

Sucrose 6F-phosphate phosphorylase: a novel insight in the human gut microbiome.

The human gut microbiome plays an essential role in maintaining human health including in degradation of dietary fibres and carbohydrates further used as nutrients by both the host and the gut bacteria. Previously, we identified a polysaccharide utilization loci (PUL) involved in sucrose and raffinose family oligosaccharide (RFO) metabolism from one of the most common Firmicutes present in individuals, Ruminococcus gnavus E1. One of the enzymes encoded by this PUL was annotated as a putative sucrose phosphate phosphorylase (RgSPP). In the present study, we have in-depth characterized the heterologously expressed RgSPP as sucrose 6F-phosphate phosphorylase (SPP), expanding our knowledge of the glycoside hydrolase GH13_18 subfamily. Specifically, the enzymatic characterization showed a selective activity on sucrose 6F-phosphate (S6FP) acting both in phosphorolysis releasing alpha-d-glucose-1-phosphate (G1P) and alpha-d-fructose-6-phosphate (F6P), and in reverse phosphorolysis from G1P and F6P to S6FP. Interestingly, such a SPP activity had never been observed in gut bacteria before. In addition, a phylogenetic and synteny analysis showed a clustering and a strictly conserved PUL organization specific to gut bacteria. However, a wide prevalence and abundance study with a human metagenomic library showed a correlation between SPP activity and the geographical origin of the individuals and, thus, most likely linked to diet. Rgspp gene overexpression has been observed in mice fed with a high-fat diet suggesting, as observed for humans, that intestine lipid and carbohydrate microbial metabolisms are intertwined. Finally, based on the genomic environment analysis, in vitro and in vivo studies, results provide new insights into the gut microbiota catabolism of sucrose, RFOs and S6FP.

Highly Promiscuous Oxidases Discovered in the Bovine Rumen Microbiome.

The bovine rumen hosts a diverse microbiota, which is highly specialized in the degradation of lignocellulose. Ruminal bacteria, in particular, are well equipped to deconstruct plant cell wall polysaccharides. Nevertheless, their potential role in the breakdown of the lignin network has never been investigated. In this study, we used functional metagenomics to identify bacterial redox enzymes acting on polyaromatic compounds. A new methodology was developed to explore the potential of uncultured microbes to degrade lignin derivatives, namely kraft lignin and lignosulfonate. From a fosmid library covering 0.7 Gb of metagenomic DNA, three hit clones were identified, producing enzymes able to oxidize a wide variety of polyaromatic compounds without the need for the addition of copper, manganese, or mediators. These promiscuous redox enzymes could thus be of potential interest both in plant biomass refining and dye remediation. The enzymes were derived from uncultured Clostridia, and belong to complex gene clusters involving proteins of different functional types, including hemicellulases, which likely work in synergy to produce substrate degradation.

Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening.

Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

Functional characterization of a gene locus from an uncultured gut Bacteroides conferring xylo-oligosaccharides utilization to Escherichia coli.

In prominent gut Bacteroides strains, sophisticated strategies have been evolved to achieve the complete degradation of dietary polysaccharides such as xylan, which is one of the major components of the plant cell wall. Polysaccharide Utilization Loci (PULs) consist of gene clusters encoding different proteins with a vast arsenal of functions, including carbohydrate binding, transport and hydrolysis. Transport is often attributed to TonB-dependent transporters, although major facilitator superfamily (MFS) transporters have also been identified in some PULs. However, until now, few of these transporters have been biochemically characterized. Here, we targeted a PUL-like system from an uncultivated Bacteroides species that is highly prevalent in the human gut metagenome. It encodes three glycoside-hydrolases specific for xylo-oligosaccharides, a SusC/SusD tandem homolog and a MFS transporter. We combined PUL rational engineering, metabolic and transcriptional analysis in Escherichia coli to functionally characterize this genomic locus. We demonstrated that the SusC and the MFS transporters are specific for internalization of linear xylo-oligosaccharides of polymerization degree up to 3 and 4 respectively. These results were strengthened by the study of growth dynamics and transcriptional analyses in response to XOS induction of the PUL in the native strain, Bacteroides vulgatus.

Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont.

UNLABELLED: Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as "dietary fiber." However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the ß-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes IMPORTANCE: The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute "dietary fiber." Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria.

The devil lies in the details: how variations in polysaccharide fine-structure impact the physiology and evolution of gut microbes.

The critical importance of gastrointestinal microbes to digestion of dietary fiber in humans and other mammals has been appreciated for decades. Symbiotic microorganisms expand mammalian digestive physiology by providing an armament of diverse polysaccharide-degrading enzymes, which are largely absent in mammalian genomes. By out-sourcing this aspect of digestive physiology to our gut microbes, we maximize our ability to adapt to different carbohydrate nutrients on timescales as short as several hours due to the ability of the gut microbial community to rapidly alter its physiology from meal to meal. Because of their ability to pick up new traits by lateral gene transfer, our gut microbes also enable adaption over time periods as long as centuries and millennia by adjusting their gene content to reflect cultural dietary trends. Despite a vast amount of sequence-based insight into the metabolic potential of gut microbes, the specific mechanisms by which symbiotic gut microorganisms recognize and attack complex carbohydrates remain largely undefined. Here, we review the recent literature on this topic and posit that numerous, subtle variations in polysaccharides diversify the spectrum of available nutrient niches, each of which may be best filled by a subset of microorganisms that possess the corresponding proteins to recognize and degrade different carbohydrates. Understanding these relationships at precise mechanistic levels will be essential to obtain a complete understanding of the forces shaping gut microbial ecology and genomic evolution, as well as devising strategies to intentionally manipulate the composition and physiology of the gut microbial community to improve health.

Sucrose and invertases, a part of the plant defense response to the biotic stresses.

Sucrose is the main form of assimilated carbon which is produced during photosynthesis and then transported from source to sink tissues via the phloem. This disaccharide is known to have important roles as signaling molecule and it is involved in many metabolic processes in plants. Essential for plant growth and development, sucrose is engaged in plant defense by activating plant immune responses against pathogens. During infection, pathogens reallocate the plant sugars for their own needs forcing the plants to modify their sugar content and triggering their defense responses. Among enzymes that hydrolyze sucrose and alter carbohydrate partitioning, invertases have been reported to be affected during plant-pathogen interactions. Recent highlights on the role of invertases in the establishment of plant defense responses suggest a more complex regulation of sugar signaling in plant-pathogen interaction.

A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes.

A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed 'dietary fibre', from the cell walls of diverse fruits and vegetables. Owing to the paucity of alimentary enzymes encoded by the human genome, our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut. The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear. Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.