Biology subjects

Hollfelder, F.

Publications and source records attributed to Hollfelder, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Deep generative embeddings of gene expression and splicing reposition the interpretation of single-cell transcriptomic signatures

Single-cell transcriptomic analysis predominantly derives cell identity from gene expression analysis, while alternative splicing is processed separately despite its fundamental role for cell homeostasis. To overcome the limits of separate investigations, we developed a probabilistic deep learning framework, Crecerelle, enabling resolution of the contributions of gene expression and alternative splicing in each cell. Crecerelle learns cell embeddings from gene expressions and alternative splicing isoforms, to decipher their mutually dependent impact on the functional characterisation of cells in a data-driven manner, exemplified for the Tabula Muris dataset. This is enabled through a zero-and-N-inflated Dirichlet-Multinomial for a variational autoencoder that learns cell embeddings solely from splicing profiles, as well as a bi-modal variational autoencoder with a relevance-weighted mixture-of-experts variational posterior to consolidate the modality-specific contribution at single-cell level. Crecerelle reveals cell-type-specific isoform markers as well as subpopulations with unique isoforms and uncovers regulatory and disease-associated pathways not detected by gene expression analyses alone. This scalable and interpretable framework thus allows a more holistic study of transcriptomic regulation and will open a route to modality-relevance-weighted investigations across single-cell multiomics datasets and their influence on cellular homeostasis, tissue development and disease phenotypes.

cell biology↗

bioRxiv2026

Latent generative search unlocks de novo design of untapped biomolecular interactions at scale

De novo protein design has advanced rapidly, yet designing binders to polar, solvent-exposed epitopes and small, flexible ligands remains challenging. Such hydrated surfaces and flexible molecules, including carbohydrates, provide few of the hydrophobic contacts favoured by current methods and have largely resisted de novo binders. To address this challenge, here we introduce latent generative search for binder design, a novel framework that uses reward-guided search at inference time to steer the Proteina-Complexa generative model. The model codesigns sequence and structure - generating them together in a continuous latent space - and thereby removes the inverse-folding step on which current methods rely. In a screen of more than one million designs by multiplexed phage display, latent generative search produced more validated binders than every other method tested, its codesigned sequences surpassing post hoc redesign. It delivered high-affinity binders across therapeutic receptors, a viral attachment protein and intracellular signalling targets. Our approach also accessed previously untapped biology, generating the first de novo proteins that bind a free carbohydrate, including one that discriminates between blood-group antigens - a polar, flexible target class beyond the reach of current design methods.

bioengineering↗

bioRxiv2026

Unsupervised protein language models learn patterns of enzyme function

While enormous amounts of sequence information have become available, assignment of sequence to a particular enzymatic function has remained elusive. Here we describe a framework that drives a general protein language model to find a target reaction without specific training, using an initial bridgehead protein. At the heart of this framework is PLM-clust, an algorithm that employs k-means on top of protein language model embeddings to convert sequence space into functional reservoirs of latent space, and samples from these clusters based on accelerated zero-shot scoring. We demonstrate PLM-clust in a recursive discovery process (with enzyme hit rates quickly rising to >90%), segmenting isofunctional reservoirs and exploring them in greater detail. This approach - exemplified for glycosyl hydrolases (a xylanase, >100-fold activity increase) and for imine reductases (IREDs, >100-fold increase in catalytic promiscuity profiles) - reliably brings about novel enzymes that are proficient at the catalytic task at hand, reaching deeply into sequence space with a majority of residues exchanged.

synthetic biology↗

bioRxiv2026

A metagenomic thermostable monomeric meganuclease with novel specificity and unique palindromic 3-prime overhangs

As an alternative to historical enzyme isolation, metagenomic databases (e.g. MGnify) provide information on vast unculturable microbial diversity, especially from extreme environments, and constitute an enormous source of functional proteins. Conservative mining of these data by close sequence homology alone tends to identify merely different versions of known enzymes. Here we present a discovery strategy of meganucleases based on wider capture of less homologous enzymes with new function in metagenomic databases, incorporating metadata with homology, relying on cell-free expression to bypass host incompatibility and the need for purification, along with using deep sequencing for experimental assessment of substrate specificity and cleavage pattern, circumventing classical gel-based profiling. Specifically, we discovered the temperature-stable (>55{degrees}C), intron-encoded LAGLIDADG meganuclease I-MG11 that recognizes a 17 base pair sequence to generate unique 4 base pair palindromic 3'-overhangs -- the first monomeric meganuclease to produce such overhangs. Co-folding models of I-MG11 bound to DNA provide a structural context for enzyme-DNA interactions, highlighting differences from other monomeric LAGLIDADG meganucleases (e.g. I-SceI) shaped by InDels (insertion-deletions) in the DNA binding region that may cause specificity changes. Our strategy streamlines bona fide identification and annotation of meganucleases, while the unique properties of I-MG11 expand the molecular biology toolbox. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=31 SRC="FIGDIR/small/712669v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@199394aorg.highwire.dtl.DTLVardef@806490org.highwire.dtl.DTLVardef@14a533aorg.highwire.dtl.DTLVardef@9e13db_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

A dynamic loop module enables phosphotriesterase function in cysteine-dependent hydrolases

A large disparity has emerged between the availability of massive metagenomic DNA sequence data and the actual functional annotation of enzyme catalysts, their mechanistic classification and insight into catalytic stragies. Exploration by sequence homology is intrinsically conservative, but ultrahigh-throughput functional metagenomics offers the chance to jump into unknown sequence space to provide novel catalysts without precedent. Having identified a novel metal-free phosphotriesterase with an active site cysteine-containing triad, here, we launch an exploration of sequence-structure-function relationships of a range of related proteins from the dienelactone hydrolase (DLH) family, revealing 10 new phosphotriesterases. Four crystal structures provide clues to mechanism, suggesting - based on phylogenetic and structural analysis - that phosphotriesterase activity is mediated by loops surrounding the active site with activity changes over 4 orders of magnitude correlated to loop flexibility. These insights allow protein engineering by loop grafting across family members, resulting in 8-fold increased phosphotriesterase activity in a human enzyme. We thus demonstrate that identification of a single pioneer enzyme can provide starting points for activity engineering strategy yielding new reagents for bioremediation or treatment of organophosphate poisoning.

biochemistry↗

bioRxiv2025

Oogenesis and germinal bed morphology of the brown anole (A. sagrei)

BackgroundThe brown anole is a model species of the genus Anolis, a squamate (encompassing lizards and snakes) group widely studied in evolutionary, behavioral, and developmental biology. Full genome annotation, the establishment of gene editing techniques, and comprehensive description of reproductive tract morphology and embryogenesis in this species, has laid the foundation for functional studies. However, analysis of brown anole oogenesis is still required and vital to optimize genome modification, mutant line establishment, and analyses of the evolution of reproductive developmental mechanisms. ResultsHere, we characterize ovary morphology and gametogenesis in the female brown anole, A. sagrei using brightfield imaging, microCT, histology staining, electron microscopy, and confocal imaging. We define 10 stages of oocyte maturation which commences inside the oogonial nest within the germinal bed and concludes with the mature follicle ready to ovulate based on follicle size, yolk-acquisition, and follicular, cellular, and basement membrane architecture. ConclusionsWe describe the complete oogenesis of the brown anole in 10 stages and report that oogenesis is highly conserved within iguanids, a suborder of lizards. With our staging framework, we lay the foundation for functional studies of oogenesis and optimized gene-editing.

developmental biology↗

bioRxiv2025

Epiblast Lumenogenesis is not a mammalian-specific trait

Epiblast lumenogenesis, which leads to amniotic cavity formation, is a hallmark of mammalian embryogenesis, and required for crucial developmental processes such as anterior-posterior patterning and gastrulation. Based on avian model-organisms, the epiblast in reptiles is thought to form a monolayered flat disc that undergoes anterior-posterior patterning and gastrulation. Here, we report that the squamate, veiled chameleon (Chameleo calyptratus), exhibits epiblast lumenogenesis and amniogenesis prior to anterior-posterior patterning. Using SEM, immunofluorescence, and histology techniques, we demonstrate that chameleon epiblast lumenogenesis occurs via a purse-string-like mechanism involving the formation of supracellular actin cables in concentric rings around the epiblast followed by constriction that closes the epiblast lumen. Through expression analyses of Nodal1, Nodal2, Cerberus, Lefty, Brachyury, Wnt3A, and Bmp2, and immunostaining for Brachyury, we uncovered a Wnt3a- and Brachyury-positive ring at the edge of the epiblast concomitant with lumenogenesis, and preceding anterior-posterior patterning and gastrulation. Furthermore, we report anterior-posterior patterning in veiled chameleons occurs independently of Cerberus and Lefty. These processes that mediate epiblast lumenogenesis in chameleons, result in a morphology remarkably similar to human embryos, despite 300 million years of evolutionary separation. Taken together, we show that pre-gastrulation epiblast lumenogenesis is not mammalian-specific, but also occurs in some non-avian reptiles.

developmental biology↗

bioRxiv2024

Pre-oviposition development of the brown anole (Anolis sagrei)

The brown anole, Anolis sagrei, has emerged as a representative squamate species during the past several decades. Novel functional tools have been established to manipulate embryogenesis through genome editing or the introduction of small molecule inhibitors, and their effective use requires a thorough understanding of early anole embryogenesis. To enable precise and reproducible staging of anole embryos, we need knowledge of the progression of anole embryogenesis and morphogenesis. While post-oviposition development has been described, the pre-oviposition period remains to be explored. Here, we provide the first staging series of pre-oviposition development for the brown anole. Analysing the follicles and embryos through brightfield imaging, SEM, STEM, histology, and DAPI staining, we define 26 distinct developmental stages. Our dataset reveals that peri-gastrulation morphogenesis up to the initiation of neurulation diverges significantly from chicken, the common representative model of reptile embryogenesis. Furthermore, we followed heart development, neural crest cell migration and central nervous system development through immunofluorescence analyses and provide new comparative insights into the morphogenesis of each of these organ systems over time. Our work establishes the brown anole as a squamate model organism for cross clade evolutionary studies of early embryogenesis.

developmental biology↗

bioRxiv2024

Ancestral aryl sulfatases in the alkaline phosphatase family allow for greater increases in catalytic efficiency than extant variants

Characterizing the dynamics and functional shifts during protein evolution is essential, both for understanding protein evolution and for rationalizing efficient strategies for e.g. enzymes with desired and effective functions. Most proteins organize in families, sets of divergent sequences which share a common ancestor and have a similar structural fold. We study aryl sulfatases (ASs), a subfamily of the large and evolutionary old alkaline phosphatase superfamily. In this manuscript we present how ultrahigh-throughput droplet micro-fluidics can be used for studying aryl sulfatases and their computationally reconstructed putative common ancestors. We compare the evolvability and robustness of three ancestors and four extant ASs which all exhibit catalytic promiscuity towards a range of substrate classes. Fourteen libraries with varying mutation rates were expressed in single-cell microdroplets. In general, higher mutation rates resulted in wider distribution of active variants but fewer improved variants overall. However, the impact of mutation rate differed between enzymes, with some benefiting from higher and others from lower mutation rate, underscoring the need to test diverse mutagenesis regimes.

evolutionary biology↗

bioRxiv2024

Microdroplet screening rapidly profiles a biocatalyst to enable its AI-assisted engineering

Engineering enzymes for increased efficiency is key to enabling sustainable, green biocatalytic production processes in the chemical and pharmaceutical industries. This challenge can be tackled from two angles: by directed evolution, based on labour-intensive experimental testing of enzyme variant libraries, or by computational methods, where data-dependent algorithms relating sequence and function are used to predict biocatalyst improvements. Here, we combine both approaches into a two-week, low-cost workflow, in which ultra-high throughput screening of a library of imine reductases (IREDs) in microfluidic devices provides not only selected hits, but also long-read sequence data linked to fitness scores of >17 thousand enzyme variants. We demonstrate the engineering of an IRED for chiral amine synthesis by mapping its local fitness landscape in one go, ready to be used for interpretation and extrapolation by protein engineers with the help of machine learning (ML). We calculate position-dependent mutability and combinability scores of mutations and comprehensively illuminate a complex interplay of mutations driven by synergistic, often positively epistatic effects. When interpreted by easy-to-use regression and tree-based ML algorithms designed for random whole-gene mutagenesis data, 3-fold improved hits initially obtained from experimental screening are extrapolated further to give another order of magnitude improvement (23-fold in kcat) after testing only a handful of designed mutants. Predictions succeed in >80% of cases. The catalytic features discovered in one IRED are shown to be portable and confer activity on IREDs with [~]50% homology. Our campaigns yield biocatalytically efficient IREDs and are paradigmatic for future enzyme engineering efforts that rely on large sequence-function maps, profiling how a biocatalyst responds to mutation. In the age of predictive biology, these maps will chart the way to improved function by exploiting the synergy of rapid experimental screening combined with ML evaluation and extrapolation.

synthetic biology↗

bioRxiv2024

Thermostable in vitro transcription-translation for enzyme screening in microdroplets

BackgroundIn vitro expression involves the utilization of the transcription and translation machinery derived from the cell to produce one or more proteins of interest and has found widespread application in the optimization of gene circuits or metabolic pathways in synthetic biology but also in pharmaceutical manufacturing. Most in vitro expression systems available are active at moderate temperatures but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Moreover, given the fact that the main barrier toward the general use of in vitro expression is its high price compared with host-based recombinant expression, there is a need to develop alternative in vitro expression systems operating at high temperatures and compatible with technologies that enable ultrahigh-throughput screening in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. ResultsTo this end, we produced high-expression cell-free extracts from Thermus thermophilus for in vitro translation and supplemented them with thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable to other thermostable in vitro expression systems, while the preparation procedure is simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Although the composition of these extracts showed a high background in carboxyl esterase assays, {beta}-glucosidase and cellobiose hydrolase activities could be measured with minimal background. ConclusionsCell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

microbiology↗

bioRxiv2023

Sub-single-turnover quantification of enzyme catalysis at ultrahigh throughput via a versatile NAD(P)H coupled assay in microdroplets

Enzyme engineering and discovery are crucial for a future sustainable bioeconomy. Harvesting new biocatalysts from large libraries through directed evolution or functional metagenomics requires accessible, rapid assays. Ultra-high throughput screening formats often require optical readouts, leading to the use of model substrates that may misreport target activity and necessitate bespoke synthesis. This is a particular challenge when screening glycosyl hydrolases, which leverage molecular recognition beyond the target glycosidic bond, so that complex chemical synthesis would have to be deployed to build a fluoro- or chromogenic substrate. In contrast, coupled assays represent a modular plug-and-play system: any enzyme- substrate pairing can be investigated, provided the reaction can produce a common intermediate which links the catalytic reaction to a detection cascade readout. Here, we establish a detection cascade producing a fluorescent readout in response to NAD(P)H via glutathione reductase and a subsequent thiol-mediated uncaging reaction, with a low nanomolar detection limit in plates. Further scaling down to microfluidic droplet screening is possible: the fluorophore is leakage- free and we report a three orders of magnitude improved sensitivity compared to absorbance- based systems, so that less than one turnover per enzyme molecule expressed from a single cell is detectable. Our approach enables the use of non-fluorogenic substrates in droplet-based enrichments, with applicability in screening for glycosyl hydrolases and imine reductases (IREDs). To demonstrate the assays readiness for combinatorial experiments, one round of directed evolution was performed to select a glycosidase processing a natural substrate, beechwood xylan, with improved kinetic parameters from a pool of >106 mutagenized sequences.

biochemistry↗

bioRxiv2023

YeastIT: Reducing mutational bias for in vivo directed evolution using a novel yeast mutator strain based on dual adenine-/cytosine-targeting and error-prone DNA repair

Engineering proteins with new functions and properties often requires navigating large sequence spaces through rounds of iterative improvement. However, a disparity exists between the gradual pace of natural long-term evolution and a typical laboratory evolution workflow that relies on enriching functional variants from highly diverse in vitro generated libraries through very few screening rounds. Laboratory experiments often eschew presumed natural strategies such as neutral/non-adaptive and multi-phase evolution trajectories, and therefore mutagenesis technologies suitable for long nature-like timescales are needed. Here, we introduce YeastIT, a novel in vivo mutagenesis tool for protein engineering that leverages an S. cerevisiae strain engineered to exhibit mutagenic activity directed to the gene of interest, allowing its continuous diversification. Mutagenesis is achieved by generating DNA damage through nucleoside deamination, followed by introduction of mutations by harnessing the process of error-prone DNA translesion synthesis. By eliminating the transformation step, YeastIT allows multiple rounds of screening or selection without interruptions for library diversification, thereby enabling long-term and continuous evolution campaigns. Our characterization of the mutational spectrum and frequency of the YeastIT-generated libraries, and its comparison to other methods (error-prone PCR, PACE, MutaT7, eMutaT7, OrthoRep, TRIDENT, EvolVR) demonstrates comparable mutation rates combined with a significant reduction in mutagenic bias relative to most of the alternatives. To validate YeastIT, we carried out directed evolution of a DARPin binding protein to achieve a 15-fold improved affinity. YeastIT thus provides a tool for exploring different evolutionary trajectories which overcomes previous limitations of variant availability (due to bias and low mutation rates) and emulates the way proteins emerge in Nature.

synthetic biology↗

bioRxiv2023

Ultrahigh throughput evolution of tryptophan synthase in droplets via an aptamer-biosensor

Tryptophan synthase catalyzes the synthesis of a wide array of non-canonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (>107 experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic read-outs which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a biosensor to quantitatively report on tryptophan production in droplets. The utility of the biosensor was validated by identifying 5-fold improved tryptophan synthases from [~]100,000 protein variants. More generally this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.

biochemistry↗

bioRxiv2023

Expansion of novel biosynthetic gene clusters from diverse environments using SanntiS

Natural products biosynthesised by microbes are an important component of the pharmacopeia with a vast array of biomedical and industrial applications, in addition to their key role in mediating many ecological interactions. One approach for the discovery of these metabolites is the identification of biosynthetic gene clusters (BGCs), genomic units which encode the molecular machinery required for producing the natural product. Genome mining has revolutionised the discovery of BGCs, yet metagenomic assemblies represent a largely untapped source of natural products. The imbalanced distribution of BGC classes in existing databases restricts the generalisation of detection patterns and limits the ability of mining methods to recognise a broader spectrum of BGCs. This problem is further intensified in metagenomic datasets, where BGC genes may be incomplete. This work presents SanntiS, a new machine learning-based tool for identifying BGCs. SanntiS achieved high precision and recall in both genomic and metagenomic datasets, effectively capturing a broad range of BGCs. Application of SanntiS to MGnify metagenomic assemblies led to a resource containing 1.9 million BGC predictions with associated contextual data from diverse biomes and demonstrates a significant fraction of novelty compared to equivalent isolate genomes datasets. Subsequent experimental validation of a novel antimicrobial peptide detected solely by SanntiS, further demonstrates the potential of this approach for uncovering novel bioactive compounds.

bioinformatics↗

bioRxiv2023

Rapid quantitative assessment of small molecule leakage from microdroplets by flow cytometry and improvement of fluorophore retention in biochemical assays

Microdroplets are compartments made in the laboratory that allow the miniaturisation of chemical and biological experiments to the femto- to picolitre scale, replacing the classical test tube with a droplet. Ideally containment of the contents of individual droplets would be perfect, but in reality this situation rarely occurs. Instead the leaking of molecules even from intact droplets presents a challenge to the success of miniaturisation and must be assessed on a case-by-case basis. We now present a new method for quantitative determination of leakage: a sheath fluid-free flow cytometer (Guava EasyCyte) is used to directly determine the fluorescence of water-in-oil droplets as a function of time. We validate this method by demonstrating that this assessment of leakage provides a framework for experimental improvements that reduce the leakage of two widely used fluorophores. A 40-fold better retention compared to current protocols is achieved for resorufin with an optimized mix (oil: FC-70, surfactant: 0.1% w/w AZ900C, additive: 1% BSA) to maintain useful retention for up to 130 hours. Likewise leakage of the fluorophore methylumbelliferone is reduced by 75-fold. The availability of a method to quantitate leakage quickly for a variety of experimental conditions will facilitate future applications of droplet-based experiments (e.g. in directed evolution or diagnostics), aid miniaturisation of lab-scale assays into this format, and improve the degrees of freedom in setting up such ultrahigh-throughput experiments.

bioengineering↗

bioRxiv2023

Versatile Product Detection via Coupled Assays for Ultra-high-throughput Screening of Carbohydrate-Active-Enzymes in Microfluidic Droplets

Enzyme discovery and directed evolution are the two major contemporary approaches for the improvement of industrial processes by biocatalysis in various fields. Customization of catalysts for improvement of single enzyme reactions or de novo reaction development is often complex and tedious. The success of screening campaigns relies on the fraction of sequence space that can be sampled, whether for evolving a particular enzyme or screening metagenomes. Ultrahigh-throughput screening (uHTS) based on in-vitro compartmentalization in water-in-oil emulsion of picolitre droplets generated in microfluidic systems allows screening rates >1 kHz (or >107 per day). Screening for Carbohydrate Active Enzymes (CAZymes) catalysing biotechnologically valuable reactions in this format presents an additional challenge, because the released carbohydrates are difficult to monitor in high throughput. Activated substrates with large optically active hydrophobic leaving groups provide a generic optical readout, but the molecular recognition properties of sugars will be altered by incorporation of such fluoro- or chromophores and their typically higher reactivity, as leaving groups with lowered pKa values compared to native substrates make observation of promiscuous reactions more likely. To overcome these issues, we designed microdroplet assays in which optically inactive carbohydrate products are made visible by specific cascades: the primary reaction of an unlabelled substrate leads to an optical signal downstream. Successfully implementing such assays at the picoliter droplet scale allowed us to detect glucose, xylose, glucuronic acid and arabinose as final products of complex oligosaccharide degradation by glycoside hydrolases by absorbance measurements. Enabling the use of uHTS for screening CAZyme reactions that have been thus far elusive will chart a route towards faster and easier development of specific and efficient biocatalysts for biovalorisation, directing enzyme discovery towards catalysts for their natural rather than model substrates. Graphical abstract / TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/534725v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@13063eborg.highwire.dtl.DTLVardef@1efdcfcorg.highwire.dtl.DTLVardef@9562eeorg.highwire.dtl.DTLVardef@1923207_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

The biosynthesis, degradation, and function of cell wall β-xylosylated xyloglucan mirrors that of arabinoxyloglucan

O_LIXyloglucan is an abundant polysaccharide in many primary cell walls and in the human diet. Decoration of its -xylosyl side chains with further sugars is critical for plant growth, even though the sugars themselves vary considerably between species. Plants in the Ericales order--prevalent in human diets--exhibit {beta}1,2-linked xylosyl decorations. The biosynthetic enzymes responsible for adding these xylosyl decorations, as well as the hydrolases that remove them in the human gut, are unidentified. C_LIO_LIGT47 xyloglucan glycosyltransferase candidates were expressed in Arabidopsis and endo-xyloglucanase products from transgenic wall material were analysed by electrophoresis, mass spectrometry, and NMR. The activities of gut bacterial hydrolases BoGH43A and BoGH43B on synthetic glycosides and xyloglucan oligosaccharides were measured by colorimetry and electrophoresis. C_LIO_LICcXBT1 is a xyloglucan {beta}-xylosyltransferase from coffee that can modify Arabidopsis xyloglucan and restore the growth of galactosyltransferase mutants. Related VmXST1 is a weakly active xyloglucan -arabinofuranosyltransferase from cranberry. BoGH43A hydrolyses both -arabinofuranosylated and {beta}-xylosylated oligosaccharides. C_LIO_LICcXBT1s presence in coffee and BoGH43As promiscuity suggest that {beta}-xylosylated xyloglucan is not only more widespread than thought, but might also nourish beneficial gut bacteria. The evolutionary instability of transferase specificity and lack of hydrolase specificity hint that, to enzymes, xylosides and arabinofuranosides are closely resemblant. C_LI

biochemistry↗