Engineering better biomass-degrading ability into a GH11 xylanase using a directed evolution strategy

Background: Improving the hydrolytic performance of hemicellulases on lignocellulosic biomass is of considerable importance for second-generation biorefining. To address this problem, and also to gain greater understanding of structure-function relationships, especially related to xylanase action on complex biomass, we have implemented a combinatorial strategy to engineer the GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn). Results: Following in vitro enzyme evolution and screening on wheat straw, nine best-performing clones were identified, which display mutations at positions 3, 6, 27 and 111. All of these mutants showed increased hydrolytic activity on wheat straw, and solubilized arabinoxylans that were not modified by the parental enzyme. The most active mutants, S27T and Y111T, increased the solubilization of arabinoxylans from depleted wheat straw 2.3-fold and 2.1-fold, respectively, in comparison to the wild-type enzyme. In addition, five mutants, S27T, Y111H, Y111S, Y111T and S27T-Y111H increased total hemicellulose conversion of intact wheat straw from 16.7%(tot. xyl) (wild-type Tx-Xyn) to 18.6% to 20.4%(tot. xyl). Also, all five mutant enzymes exhibited a better ability to act in synergy with a cellulase cocktail (Accellerase 1500), thus procuring increases in overall wheat straw hydrolysis. Conclusions: Analysis of the results allows us to hypothesize that the increased hydrolytic ability of the mutants is linked to (i) improved ligand binding in a putative secondary binding site, (ii) the diminution of surface hydrophobicity, and/or (iii) the modification of thumb flexibility, induced by mutations at position 111. Nevertheless, the relatively modest improvements that were observed also underline the fact that enzyme engineering alone cannot overcome the limits imposed by the complex organization of the plant cell wall and the lignin barrier

Permutations whose reverse shares the same recording tableau in the RSK correspondence

The RSK correspondence is a bijection between permutations and pairs of standard Young tableaux with identical shape, where the tableaux are commonly denoted $P$ (insertion) and $Q$ (recording). It has been an open problem to demonstrate $$|\{w \in \mathfrak{S}_n | \, Q(w) = Q(w^r)\}| = \begin{cases} \displaystyle 2^{\frac{n-1}{2}}{n-1 \choose \frac{n-1}{2}} & n \text{ odd} \newline \displaystyle 0 & n \text{ even} \end{cases},$$ where $w^r$ is the reverse permutation of $w$. First we show that for each $w$ where $Q(w) = Q(w^r)$ the recording tableau $Q(w)$ has a symmetric hook shape and satisfies a certain simple property. From these two results, we succeed in proving the desired identity.Comment: 14 pages, 4 figure

The GH51 α-l-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans.

Background: Conceptually, multi functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi functional enzymes are quite rare and are generally multi domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α l arabinofuranosidase and β d xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality. Results: THSAbf is a GH51 α l arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4–7). THSAbf preferentially releases para nitrophenyl from the l arabino furanoside ( k cat / K M = 1050 s − 1 mM − 1 ) and to some extent from d galactofuranoside and d xyloside. THSAbf is active on 4 O methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg − 1 , respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg − 1 ). Further investigation revealed that like the Alicyclo - bacillus sp. A4 α l arabinofuranosidase, THSAbf also displays endo xylanase activity, cleaving β 1,4 bonds in heteroxy lans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α l arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi functionality

Combined approaches provide an anatomical and transcriptomic fingerprint of maize cell wall digestibility

Understanding cell wall biosynthesis and degradation in grasses has become a major aim in plant biology. Although independent previous reports have focused on specific features that dictate cell wall digestibility, cytological, biochemical, and gene regulation parameters have never been integrated within the same study. Herein, we applied a combination of state-of-the-art technologies and different scales of observation on two maize lines that are characterized by highly contrasted forage digestibility. Comparative image analysis of internode sections allow to get an anatomical fingerprint associated with high digestibility: a thin peripheral rind of lignified parenchyma, small numerous vascular bundles, and low proportion of PeriVascular Sclerenchyma (PVS). This cell type patterning led to enhanced digestibility when internode sections were treated with Celluclast, a commercially cell wall degrading enzyme. At a lower scale of observation, Laser Capture Microdissection (LCM) followed by thioacidolysis of PVS revealed a higher proportion of Syringyl (S) unit lignins in the low digestible line while the high digestible line was p-Hydroxyphenyl (H)-rich. Moreover, cytological observation of internodes of the two lines point out that this difference in composition is associated with a delayed lignification of PVS. At the same time, comparative transcriptomics on internodes indicated differential expression of several genes encoding enzymes along the phenylpropanoid pathway and known cell wall-associated Transcription Factors (TFs). Together, these results give an integrative view of different factors which could aim in designing a maize silage ideotype and provide a novel set of potential regulatory genes controlling lignification in maize

The development of (biorefineries) biomanufacturing and prospects for the future

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Biotech potential set to soar with the help of digital technology

International audienceBiotech potential set to soar with the help of digital technologyMicrobes and fungi have long been nature's helpers in producing fine food, drinks and medicine, but new digital technologies could unlock far greater potential for the European biotech sector.</div

Biotechnology and biomanufacturing: core elements of Europe’s industrial future?

International audienceThe recent communication by the European Commission entitled “Building the future with nature: boosting Biotechnology and Biomanufacturing in the EU” is highly welcome [1]. However, while this communication has been much awaited by the European biotechnology sector, global competitors have not remained idle. Currently, the global market is dominated by the USA, with Europe lagging somewhat behind.In my presentation, using an incomplete set of indicators, I will review the current status of biotechnology and biomanufacturing, focusing on opportunities and threats, notably throwing some light on the European situation.In the second part of my presentation, I will focus on IBISBA, a distributed European infrastructure for biotechnology and biomanufacturing. I will provide a brief overview of this strategic instrument, describing its current status and activities.1.Biotechnology and biomanufacturing: core elements of Europe’s industrial future? COM (2024) 137 final. Brussels 20/03/202

IBISBA: A strategic R&amp;D infrastructure to support EU green growth

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Biomass-derived pentoses Foreword

Forewordabsen