Multifunctional enzyme engineering by computational design for lignocellulosic valorization

Biomass- acting enzymes are vital components of biorefinery processes that aim to convert complex, lignocellulosic biomass into fuels, chemicals and materials and therefore, much effort has focused on the improvement of their characteristics (activity, stability, cost of production, etc) as well as on the discovery and development of novel enzymes. Metagenomic approaches revealed that in the Bacteroidetes phylum functionally related genes are often organized in characteristic clusters, known as Polysaccharide Utilization Loci (PUL) reflecting that biomass- acting enzymes act in synergy and that enzyme proximity is important to target complex substrates. In this study we designed a tailored made multifunctional enzyme, combining enzymes isolated from a xylan PUL (1). Computational simulations were performed to define and optimize engineered versions of a multi-domain GH10 endo- xylanase by replacing carbohydrate binding module (CBM) and grafting two new catalytic domains: either a GH43 xylosidase or a CE1 carbohydrate-esterase activities also present in the same PUL. The multifunctional enzymes were then experimentally assessed, demonstrating that chimeric GH10-GH43 had both activities and thus represents a powerful biological tool for hemicellulose deconstruction. Bastien, G., et al. (2013). Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics. Biotechnology for biofuels 6(1): 7

Characterization and Enzyme Engineering of a Hyperthermophilic Laccase toward Improving Its Activity in Ionic Liquid

Ionic liquids (ILs) are organic salts molten at room temperature that can be used for a wide variety of applications. Many ILs, such as 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]), have been shown to remove a significant fraction of the complex biopolymer lignin from biomass during pretreatment. Valorizing lignin via biological pathways (e.g., enzymes) holds promise but is limited by the low biocompatibility of many ILs used for pretreatment. The discovery of thermostable enzymes and the application of enzyme engineering techniques have yielded biocatalysts capable of withstanding high concentrations of ILs. Converting lignin from a waste product to value-added chemicals is vital to the success of future cellulosic biorefineries. To that end, we screened the activity of the lignolytic enzyme laccase from a hyperthermophilic bacterium (Thermus thermophilus) in aqueous [C2C1Im][OAc]. Despite the thermophilicity (Topt \u3e 90°C) of this laccase, significant activity loss (\u3e 50%) was observed in only 2% (w/v) [C2C1Im][OAc]. Kinetics studies show that the IL can bind to the free enzyme and the enzyme-substrate complex. Docking simulations suggest that the cation favors binding to a region close to the active site. We then used a rational design strategy to improve the activity of the laccase in [C2C1Im][OAc]. A total of 8 single amino acid mutations were made; however, there were no significant improvements in the activity of the mutants in [C2C1Im][OAc] compared to the wild type. The results of this study shed light on the complex nature of enzyme-IL interactions and the challenges faced when designing a biological lignin valorization strategy

GH62 arabinofuranosidases: Structure, function and applications

Motivated by industrial demands and ongoing scientific discoveries continuous efforts are made to identify andcreate improved biocatalysts dedicated to plant biomass conversion.α-1,2 and α-1,3 arabinofuranosyl specific α-L-arabinofuranosidases (EC 3.2.1.55) are debranching enzymes catalyzing hydrolytic release of α-L-arabinofur-anosyl residues, which decorate xylan or arabinan backbones in lignocellulosic and pectin constituents of plantcell walls. The CAZy database classifies α-L-arabinofuranosidases in Glycoside Hydrolase (GH) families GH2,GH3, GH43, GH51, GH54 and GH62. Only GH62 contains exclusively α-L-arabinofuranosidases and these are offungal and bacterial origin. Twenty-two GH62 enzymes out of 223 entries in the CAZy database have beencharacterized and very recently new knowledge was acquired with regard to crystal structures, substrate spe-cificities, and phylogenetics, which overall provides novel insights into structure/function relationships of GH62.Overall GH62 α-L-arabinofuranosidases are believed to play important roles in nature by acting in synergy withseveral cell wall degrading enzymes and members of GH62 represent promising candidates for biotechnologicalimprovements of biofuel production and in various biorefinery application

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

Spatial organisation of enzymes in the biosynthetic limonene production pathway in Escherichia coli

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Post-transcriptional association of proteins to study spatial organisation within multi-enzyme complexes

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PACER : a novel 3D plant cell wall model for the analysis of non-catalytic and enzymatic responses

Background Substrate accessibility remains a key limitation to the efficient enzymatic deconstruction of lignocellulosic biomass. Limited substrate accessibility is often addressed by increasing enzyme loading, which increases process and product costs. Alternatively, considerable efforts are underway world-wide to identify amorphogenesis-inducing proteins and protein domains that increase the accessibility of carbohydrate-active enzymes to targeted lignocellulose components. Results We established a three-dimensional assay, PACER (plant cell wall model for the analysis of non-catalytic and enzymatic responses), that enables analysis of enzyme migration through defined lignocellulose composites. A cellulose/azo-xylan composite was made to demonstrate the PACER concept and then used to test the migration and activity of multiple xylanolytic enzymes. In addition to non-catalytic domains of xylanases, the potential of loosenin-like proteins to boost xylanase migration through cellulose/azo-xylan composites was observed. Conclusions The PACER assay is inexpensive and parallelizable, suitable for screening proteins for ability to increase enzyme accessibility to lignocellulose substrates. Using the PACER assay, we visualized the impact of xylan-binding modules and loosenin-like proteins on xylanase mobility and access to targeted substrates. Given the flexibility to use different composite materials, the PACER assay presents a versatile platform to study impacts of lignocellulose components on enzyme access to targeted substrates.Peer reviewe

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