Biology subjects

Tryfona, T.

Publications and source records attributed to Tryfona, T..

4 recordsLinked to original sources

bioRxiv2024

Hydrothermal pretreatment renders peat susceptible to enzymatic saccharification

Sphagnum peat bogs store a large fraction of biologically-bound carbon, due to a steady accumulation of plant material over millennia. The resistance of Sphagnum biomass to decay is poorly understood but of high importance for preservation efforts and climate models. It is shown that peat cellulose and other glucose-rich polysaccharides are readily degradable by a commercial enzyme cocktail designed for the industrial saccharification of lignocellulose of vascular plants. However, prior hydrothermal pretreatment of peat was required for the enzymes to gain access to the polysaccharides. The pretreatment itself released monosaccharides and glucose-containing soluble oligosaccharides. The monosaccharide profile released from hydrothermal pretreatment was consistent with the expected hemicellulose content of Sphagnum and was clearly different from that seen for pretreated tissue from vascular plants, such as pretreated wheat straw. Cellulose retained in the insoluble part of peat was cleaved at a similar or higher rate compared to cellulose from vascular plant tissues. Confocal laser scanning microscopy showed that the hydrothermal pretreatment disrupted the cells and relocated lignin-like compounds. Peat contains a high concentration of iron, which likely explains the pronounced acidification observed for the pretreated peat slurry at ambient conditions during saccharification assays. The acidification is due to abiotic oxidative reactions that also inactivate the enzymes. Adding catalase to the reactions alleviated enzyme inactivation and essentially stopped acidification during saccharification. This study confirms the importance of considering those abiotic oxidative reactions that take place in drained peat material.

plant biology↗

bioRxiv2023

Altering the substitution and crosslinking of glucuronoarabinoxylans affects cell wall porosity and assembly in Brachypodium distachyon.

O_LIThe Poaceae family of plants provides cereal crops that are critical for human and animal nutrition and also they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion, thus understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. C_LIO_LIWe generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan biosynthesis. Using carbohydrate gel electrophoresis we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. C_LIO_LIBdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are more porous, suggesting the xylan substitutions directed by both BdXAX1A andGUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. C_LIO_LISince xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials. C_LI

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↗

bioRxiv2022

Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood

The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for {beta}-glucuronidase activity. We identified SN243, a genuine {beta}-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase (GH) 3 family member. This GH family had no recorded evidence of {beta}-glucuronidases at the outset of this study, showing that a functional metagenomic approach can shed light on assignments that are currently unpredictable by bioinformatics. Kinetic analyses of SN243 characterised it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous GH3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.

biochemistry↗