Forståelse og optimalisering av rollen til lytisk polysakkarid monooksygenaser i enzymatisk nedbrytning av lignocellulosisk biomasse

Lignocellulosic biomass holds great potential for production of biofuels and other chemicals traditionally produced from fossil fuels. However, its significant recalcitrance presents a substantial challenge in industrial biorefining, where chemical and/or physical pretreatment methods and enzymatic saccharification are used to convert the polysaccharides within the lignocellulosic structure to fermentable sugars. One way of overcoming this innate recalcitrance is by developing strategies for improved enzymatic conversion, via process optimization or by exploring new enzyme activities. The discovery of lytic polysaccharide monooxygenases (LPMOs) and their role in plant biomass degradation, and, more recently, the ability of these enzymes to catalyze a fast and specific peroxygenase reaction, has altered our understanding of the mechanisms of lignocellulose depolymerization, and revealed new avenues for potential improvement of this process. However, achieving such improvement requires in-depth understanding of how LPMOs function, including their substrate specificities, their catalytic mechanism, and the conditions under which they perform best, both alone and during synergistic action with hydrolytic enzymes. In addition, for process improvement and general understanding of LPMO activity, we must be able to analytically interpret and quantify the complex product mixtures generated by LPMOs. The work presented in this thesis has addressed several of these topics both from a fundamental and an applied perspective. Paper I of this thesis describes the implementation of a recently developed chromatographic platform for high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using dual electrolytic eluent generation in analytical methods for separation and quantification of LPMO-generated carbohydrate products. In this platform, the eluents are based on potassium methanesulfonate and potassium hydroxide. We established methods for simultaneous detection of native, C1-, and C4-oxidized cello-oligosaccharides, as well as separate methods for analysis of gluconic and glucuronic acid. The developed methods showed increased sensitivity and precision in detection and quantification of LPMO products compared to traditional HPAEC-PAD using manually prepared eluents based on sodium hydroxide and sodium acetate. In the study described in Paper II, we evaluated the use of a relatively simple, LPMO-rich fungal enzyme cocktail produced by Thermoascus aurantiacus in saccharification of pretreated lignocellulosic biomass, and showed that this cocktail performs nearly as well in saccharification of lignin-poor sulfite-pulped spruce at 60°C as the commercial cellulase preparation Cellic CTec2 at 50°C. These results underpin the potential of the T. aurantiacus fungus as a producer of enzymes for use in industrial biorefining processes, where maintaining higher temperatures during saccharification can be advantageous. Furthermore, addition of H2O2-producing glucose oxidase to saccharification reactions with Cellic CTec2 showed that in situ H2O2 production can drive LPMO activity in saccharification of lignin-poor substrates. The results obtained in this study were substrate-dependent: when using a lignin-rich substrate, the T. aurantiacus cocktail was less advantageous, and addition of glucose oxidase was detrimental to the saccharification efficiency of Cellic CTec2. These results highlight the importance of adapting process conditions to individual lignocellulosic feedstocks and enzyme preparations. Paper III and Paper IV of this thesis describe the functional characterization of AA9 LPMOs. In Paper III, we demonstrate the C4-oxidizing activity of ScLPMO9A from Schizophyllum commune on a range of hemicellulosic substrates and soluble cellooligomeric substrates, including cellotetraose and cellohexaose (with apparent preferential -3 - +3 binding), and its strong peroxygenase activity when acting on soluble and insoluble amorphous substrates. In contrast to the traditionally-perceived role of LPMOs in depolymerization of crystalline substrates, ScLPMO9A appeared to have little-to-no activity on crystalline cellulose. Although further investigation of these observations is needed, including determination of potential active site residues that may contribute to the observed substrate preferences, these results are intriguing and may aid in providing insight into hitherto unknown biological roles of LPMOs. The research presented in Paper IV uncovered the glucuronoxylanolytic activity of two LPMOs from Neurospora crassa. By quantification of cellulose- and xylan-derived oxidized products, this study demonstrated that preferential cleavage of xylan or cellulose in a mixture of these two polysaccharides can vary substantially between xylan-active LPMOs, suggesting that these LPMOs may have evolved to target different co-polymeric structures within plant biomass. Phylogenetic analysis and structural modeling also enabled the identification of additional putatively xylan-active LPMOs. The results reported in this thesis add to our understanding of LPMO action and how best to leverage this action for current and future academic and industrial applications.Lignocellulose innehar et stort potensial for produksjon av biobrensel og andre kjemikalier som tradisjonelt produseres fra fossile kilder. Men dens komplekse sammensetning gir betydelige utfordringer i industriell bioraffinering, hvor kjemiske og/eller fysiske forbehandlingsmetoder og enzymatisk nedbrytning brukes for å omdanne polysakkaridene i lignocellulosen til fermenterbare sukkere. En måte å løse denne kompleksiteten på er å utvikle strategier for forbedret enzymatisk omdanning, via prosessoptimalisering eller ved å utforske nye enzymaktiviteter. Oppdagelsen av lytisk polysakkarid monooksygenaser (LPMOer) og deres rolle i nedbrytning av plantebiomasse, og, nylig, evnen disse enzymene har til å katalysere en hurtig og spesifikk peroksygenasereaksjon, har endret vår forståelse av mekanismene for nedbrytning av lignocellulose, og åpnet opp for nye mulige forbedringer av denne prosessen. Dog, det å få til slike forbedringer krever mer kunnskap om hvordan LPMOer fungerer, inkludert deres substratspesifisiteter, den katalytiske mekanismen og om hvilke forhold de presterer best under, både alene og i synergi sammen med hydrolytiske enzymer. I tillegg, for å kunne forbedre prosessen og generelt forstå LPMO-aktiviteten, må vi kunne analysere og kvantifisere de komplekse produktblandingene som LPMOer gir. Arbeidet presentert i denne avhandlingen adresserer flere av disse temaene, både fra et fundamentalt og et anvendt perspektiv. Artikkel I beskriver implementeringen av en nylig utviklet kromatografisk plattform for høypresisjons-ionebytterkromatografi med pulserende amperometrisk deteksjon (HPAEC-PAD) ved bruk av dobbel elektrolytisk eluentgenerering i analytiske metoder for separasjon og kvantifisering av karbohydratprodukter produsert av LPMOer. I denne plattformen er eluentene basert på kaliummetansulfonat og kaliumhydroksid. Vi etablerte metoder for samtidig deteksjon av native, C1- og C4-oksiderte cello-oligosakkarider, samt separate metoder for analyse av glukonsyre og glukuronsyre. Metodene som ble utviklet her viste økt sensitivitet og presisjon ved deteksjon og kvantifisering av LPMO-produkter sammenlignet med tradisjonell HPAEC-PAD, hvor eluentene lages manuelt og er basert på natriumhydroksid og natriumacetat. I studien beskrevet i Artikkel II, evaluerte vi bruken av en relativt enkel, LPMO-rik enzymblanding produsert av soppen Thermoascus aurantiacus i sakkarifiseringen av forbehandlet lignocellulose, og viste at denne blandingen presterer nesten like godt i sakkarifisering av ligninfattig sulfitt-prosessert gran ved 60°C som det kommersielle cellulasepreparatet Cellic CTec2 ved 50°C. Disse resultatene understreker potensialet til T. aurantiacus som produsent av enzymer for bruk i industrielle bioraffineringsprosesser hvor det å opprettholde høye temperaturer kan være fordelaktig. Tilsetting av H2O2-produserende glukoseoksidase til sakkarifiseringsreaksjonene med Cellic CTec2 viste i tillegg at in situ H2O2 produksjon kan drive LPMO-aktiviteten ved sakkarifisering av ligninfattige substrater. Resultatene oppnådd i denne studien var substratavhengige: ved bruk av et ligninrikt substrat var enzymblandingen fra T. aurantiacus mindre fordelaktig, og tilsetting av glukoseoksidase var uheldig for sakkarifiseringseffektiviteten til Cellic CTec2. Disse resultatene fremhever viktigheten av å tilpasse prosessbetingelsene til individuelle lignocellulosesubstrater og enzympreparater. Artikkel III og IV beskriver den funksjonelle karakteriseringen av AA9 LPMOer. I Artikkel III demonstrerte vi C4-oksideringsaktiviteten til ScLPMO9A fra Schizophyllum commune på en rekke hemicelluloser og løselige cello-oligomer-substrater, inkludert cellotetraose og celloheksaose (med tilsynelatende -3 – +3 binding), og enzymets sterke peroksygenaseaktivitet på løselige og uløselige amorfe substrater. I motsetning til den tradisjonelt antatte rollen til LPMOer i depolymeriseringen av krystallinske substrater, ser ScLPMO9A ut til å ha liten til ingen aktivitet på krystallisk cellulose. Selv om videre undersøkelser av disse observasjonene er nødvendig, inkludert bestemmelse av mulige aminosyrer i det aktive setet som kan bidra til de observerte substratpreferansene, er disse resultatene interessante og kan gi innsikt i LPMOers hittil ukjente biologiske roller. Forskningen presentert i Artikkel IV avdekket aktivitet på glukuronoxylan for to LPMOer fra Neurospora crassa. Ved kvantifisering av oksiderte produkter fra cellulose og xylan, demonstrerte denne studien at foretrukket kløyving av xylan eller cellulose i en blanding av disse to polysakkaridene kan variere betydelig mellom xylan-aktive LPMOer, noe som antyder at disse LPMOene kan ha utviklet seg til å virke på ulike co-polymeriske strukturer i plantebiomasse. Fylogenetisk analyse og modellering av strukturer gjorde det også mulig å identifisere andre mulige xylan-aktive LPMOer. Resultatene rapportert i denne avhandlingen gir utvidet kunnskap om vår forståelse av LPMO-aktivitet og hvordan best bruke dette i nåværende og fremtidige akademiske og industrielle anvendelser

Microbial strategies for deconstruction of bark components

Bark is the outermost part of a tree, and it is a protective layer against external threats such as microorganisms and environmental stressors. Bark consists of various polymers including lignin, cellulose, hemicellulose, and it also contains a large fraction of compounds known as extractives. The polymers and extractives are assembled into a heterogenous complex matrix, forming a highly recalcitrant material. Despite its protective role, bark is degraded in nature by microorganisms, yet little is known about the specific microorganisms involved and how they affect bark composition.In this thesis, I have investigated different strategies that individual species and microbial communities employ to degrade bark and how enzymes hydrolyze pure polysaccharides and extractive compounds, focusing on spruce bark degradation. I analyzed a microbial community growing on spruce bark over six months and observed significant effects on the extractives, especially resin acids at the start of the cultivation. The community was dominated by bacteria, and guided by metagenomics, a new\ua0Pseudomonas\ua0species was isolated, sequenced, and shown to degrade the major resin acids present in spruce bark. The role of filamentous fungi in the microbial community was unclear, despite their reputation as exceptional lignocellulose degraders. Therefore, I studied fungi from the Basidiomycota and Ascomycota phyla known to employ different lignocellulose degradation strategies. I showed that the Basidiomycetes can degrade/modify resin acids, while the Ascomycetes instead appeared to tolerate resin acids. All fungi investigated were able to degrade the bark polysaccharides, with significant differences in pectin and xylan degradation. To understand xylan degrading mechanisms in more detail, I studied the growth of taxonomically different yeasts and biochemically characterized their xylanases. One of the yeasts, Wickerhamomyces canadensis, grew poorly on xylan but its growth was boosted when co-cultured with another yeast, Blastobotrys mokoenaii. This suggests that W. canadensis is a secondary degrader of xylan. For in-depth studies of extractive-degrading enzymes, I biochemically characterized three tannases from the bacterium Clostridium butyricum and demonstrated their ability to cleave oak bark tannins. My work contributes to our understanding of the microbial degradation of bark and the strategies employed by microbial communities, individual species, and enzymes to degrade bark

Establishing the developmental function of the rhamnogalacturonan II component of pectin

The primary plant cell wall consists of a complex set of polysaccharides including pectin, cellulose and hemicelluloses that are critical for normal plant development. There are three major forms of pectin, rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and homogalacturonan (HGA). Of these, the pectic polysaccharide RG-II, is the least abundant but the most complex. Despite this, RG-II is highly conserved among vascular plants, suggesting animportant function which is dependent upon structure. RG-II consists of four structurally welldefined side chains attached to a backbone of 1,4-linked galacturonic acid (GalA) residues and exists predominately as a dimer in plant cell walls. RG-II function has yet to be identified; however, mutations affecting RG-II structure have severe growth and development defects. 3-deoxy-D-manno-2-octulosonic acid (Kdo) is a rarely found sugar and is a component of the RGII side chain C. Kdo biosynthesis has been well characterised and a number of Kdo synthesis genes identified in Arabidopsis. Traditional gene knockout approaches to study the effect of disrupting Kdo biosynthesis have been limited by the apparent lethality of these mutants. Alternative approaches using partial knockout, inducible gene silencing and chemical approaches have being employed with the primary aim of specifically altering the structure of RG-II to determine the developmental function of RG-II. By combination of a GAL4/VP16 expression system and ALCR/alcA ethanol-switch to achieve temporal and spatial control of transgene expression, it has been possible to generate a genetic tool kit consisting of a series of Arabidopsis lines in which it should be possible to disrupt Kdo biosynthesis in specific tissues at strictly defined developmental stages. As a proof of concept the J0951/iKdsB line, in which expression of an AtKdsB antisense sequence is restricted to the epidermis and root cap in the presence of ethanol, is shown to be almost completely devoid of root hairs when grown under induced conditions. This result is suggestive of a role for RG-II in tip growth processes and is consistent with the phenotypes of null mutants in which a failure in pollen tube elongation results in gametophyte lethality. In silico and in vitro approaches are used to investigate the potential application of an inhibitor of AtKdsB, 2?-deoxy Kdo, as a tool for the disruption of CMP-Kdo synthesis in plants. Using homology modelling the Arabidopsis and E. coli enzymes are shown to have a near identical active site conformation. Using recombinantly expressed AtKdsB in enzyme kinetic and inhibition studies the substrate analogue 2?-deoxy-Kdo was shown to be a potent in vitro inhibitor of AtKdsB with a Ki of 1.26 ± 0.15 ??, consistent with measures of the Kd made by isothermal titration calorimetry (ITC) analysis. The 2?-deoxy-Kdo was subsequently applied in vivo and results in a severe inhibition of cell elongation of Arabidopsis root cells that can be partially rescued by either Kdo or boron. It is likely that 2?-deoxy-Kdo application disrupts CMP-Kdo biosynthesis with consequences for RG-II structure and dimer formation

BIOCHEMICAL FUNCTIONAL CHARACTERIZATION AND MOLECULAR BIOLOGY OF PLANT INHIBITOR PROTEINS ACTING AGAINST GLYCOSIDE HYDROLASE

Plant cell walls are composed mostly of polysaccharides and it consist of three layers (the primary cell wall, the secondary cell wall and the middle lamella) that are made up of different percentage of cellulose, pectins and hemicelluloses. These latter are composed of a linear backbone made up of (1,4)-\u3b2-D-glycans with an equatorial configuration. Based on type of glycans forming the backbone it is possible to distinguish: mannans contain \u3b2-(1,4)-linked mannose; in xyloglucan \u3b2-1,4 glucans can be substituted with a diverse array of glycosyl and nonglycosyl residues and xylans are composed by \u3b2-(1,4)-linked xylose residues. The seeds of many legumes are known to accumulate galactomannan in their endospermic cell walls. In many dicots xyloglucans constitute the major hemicellulose of growing cell walls, comprising ~20% of the dry mass of primary cell walls. Grasses - but not monocots in general - have a reduced xyloglucan content. Cell wall polysaccharide biogenesis includes polymer synthesis, secretion, assembly, and rearrangement during development. All of these modification demands the reversible \u2018loosening\u2019 of the cellulose\u2013 hemicellulose\u2013pectin network. Glycoside hydrolase (GH) enzymes located in the wall or in the plasma membrane play a crucial role in the degradation of different cell wall polysaccharides. On the other hand, pathogenic microorganisms secrete glycoside hydrolase to penetrate plant cell walls. As a response, plants produce glycoside hydrolase inhibitor proteins (GHIPs). Xyloglucan- specific endo-\u3b2-1,4-glucanase inhibitor proteins-like (XEGIPs-like) are typical of dicots, they inhibit the hydrolytic activity of a xyloglucan-specific \u3b2-1,4-endoglucanase isolated from GH12 family. XEGIPs-like have been found widespread in dicots: they were detected in the medium of cultured tomato cells, purified from carrot callus, isolated from the nectar of ornamental tobacco, when overexpressed they were capable of protecting potato from disease caused by endo-\u3b2-1,4- glucanase GH12 from Phytophthora infestans, enhanced in apple in response to infection of Botryosphaeria dothidea, they have distinct roles in defence mechanisms in Humulus lupus. In cereals three types of GHIPs occur in a fairly coordinated fashion throughout grain development and germination: Triticum aestivum L. endoxylanase inhibitors (TAXIs-like), xylanase inhibitor proteins (XIPs-like), thaumatin-like xylanase inhibitors (TLXIs-like). The accumulation of GHIPs during the early stages of germination is consistent with the phenomenon of germination-based resistance and their highest concentrations occur in the aleuronic layer. The apoplastic localization of GHIPs in cereals may be favourable for their action as inhibitors of microbial xylanases GH10 and/or GH11 from Aspergillus niger, Bacillus subtilis and Hypocrea jecorina intruding the host plant. GHIPs homologous are also present in legume (LACGs-like): \u3b3-conglutin is largely expressed and accumulated in Lupinus spp. and Bg7S in Glycine spp. GHIPs have common structural features. In particular, the alignment of the primary structurer showed that the position of the 12 cysteines is fully conserved, so various GHIPs have similar three- dimensional structures. Cys10-Cys11 is located at the C-terminal region of the proteins, the sequence amongst them is called \u201cinhibition loop 1\u201d and a conserved arginine, in XEGIPs-like, or leucine, in TAXI-I, is involved in the bond GH12 or GH11, respectively. In the LAGCs-like it\u2019s also present the loop, but a deletion of about five amino acids involve this region, otherwise highly conserved. The superimposition of TAXI-I 3D structure (PDB accession number: 1T6E) and EDGP 3D structure (PDB accession number: 3VLA) with \u3b3-conglutin 3D structure (PDB accession number: 4PPH) confirm the structural difference of the loop responsible for the interaction with the enzyme as a consequence of the five amino acid long deletion. This may be the cause of an unfavourable local spatial conformation of the protein for the correct interaction with the enzyme. The disulphide bridge Cys9-Cys12 define another region called \u201cinhibition loop 2\u201d where a conserved arginine, in XEGIPs-like, or histidine, in TAXIs-like and LACGs-like, is involved in the bond with a specific GH. The sequence of the IL2 loop of \u3b3-conglutin is more similar to the sequence of the IL2 loop of TAXI-I, rather than to the one of XEGIPs. In particular, a His residue, considered a key amino acid for the inhibitory activity of TAXI-I is also present in \u3b3-conglutin sequence, but not in XEGIPs. \u3b3-conglutin was expressed in Pichia pastoris. Unexpectedly, this recombinant \u3b3-conglutin (r\u3b3c) was able to inhibit a GH11 enzyme, but not GH12. In lupin, \u3b3-conglutin is naturally cleaved in two subunits, whereas in P. pastoris it is not. Most likely, the proteolytic processing influences the structural conformation of \u3b3-conglutin and small local rearrangements may be the cause of the observed activity. Also a set of \u3b3-conglutin mutants was designed upon TAXIs-like and XEGIPs-like sequences and expressed in Pichia pastoris. The mutants were able to modulate the inhibition capacity. The enzymatic assays and the bioinformatics analysis confirmed that the presence of IL1 is not strictly required to manifest inhibition, even if the specifically inserted amino acid stretches enhanced the activity. On the other hand, histidine in IL2 is confirmed to be necessary and sufficient to manifest the inhibitory competence of r\u3b3c. The LACGs-like among GHIPs remains the less characterized. For this reason, we undertook experiments aimed to study the inhibitory specificity of wild type \u3b3-conglutin. Inhibitory capacity was tested against under different condition using selected GH2 and GH5 members. GH11 and GH12 were not tested since previous results evidenced no capacity. In Arabidopsis thaliana, which is a small flowering plant widely used as a model organism in plant biology, we have found five genes coding for GHIPs belonging to each of three aforementioned groups. At1g03220 and At1g03230 are closer related to XEGIPs-like proteins: the cysteines pathway is totally conserved, they show arginine in the inhibition loop 1 and inhibition loop 2, which interact with glycoside hydrolases family 12, and along the sequences there are some potential glycosylation sites. At5g19110 is the unique putative proteins that shows leucine and histidine in the inhibition loop 1 and inhibition loop 2, respectively, the same region used by TAXIs-like to interact with glycoside hydrolases family GH11. At5g19100 and At5g19120 show the characteristic LACGs-like deletion at the IL1 and only in At5g19100 sequence at the IL2 we recover the histidine like both LACGs-like and TAXIs-like. We have studied the expression of these genes to deep into the biology of the plant response to pathogen attack. The results of this set of experiment contributed to unveil the role of each. The seeds were germinated in Petri dishes with MS medium and held in growth chambers with constant temperature of 21\ub0C, under long-day photoperiod, for different times (0-23-48-96-144 hours) and in different conditions simulating biotic stresses. Western-blot with antibody anti-C\u3b3 show the presence of a similar \u3b3-conglutin protein in A. thaliana seeds at 96h of germination in MS medium and then incubate in a chitosan (150 mg/L) solution for 3h. For the first time we show that proteolytic processing of LACGs-like occurs in organism other than legume. In parallel, total RNAs were extracted and RT-qPCR has been set up to quantify the relative expression levels of gene expression. The GHIPs homologues genes after 48, 96 and 144 hours of germination aren\u2019t expressed under basal condition. Seeds germinated for 96 hours and then exposed to incubation with aqueous solution or 150mg/l chitosan solution (mimic a biotic stress) shown an expression of gene, greater if the biotic stress was applied at seeds contaminated. In the seeds germinated in dishes that after 96 hours shown a spontaneous growth of Paenibacillus polymyxa, an endophytic bacterium exploited as biocontrolling agent. In this case the expression of all analysed genes increased. In another experimental set we tested the direct effect of GH2, GH5, GH11 treatment on A. thaliana seedling 7 days old. In this contest, it has been evaluated both the expression of the five selected genes and the effects on the enzymes due to plants exudates. By and large, this work describes new experimental findings that open new interesting scenarios to better understand some physiological aspects of the plant defence mechanisms, and provides insight of the structural basis of GHIPs inhibitory activity, specificity and repercussions on cellular responses to pathogens attacks

A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

Butyrivibrio proteoclasticus B316T is a Gram-positive, lignocellulose degrading bacterium that is prevalent in the rumen of animals grazing pasture, and is one of only a few rumen microbes known to degrade and utilise xylan in vitro. Xylan is a hemicellulose that comprises up to 45% of the polysaccharide component of ruminant forages. Often as little as 30% of the total energy content of forages is utilised by the ruminant due to poor hemicellulose degradation by the fibrolytic rumen microbes. An opportunity exists to improve forage degradation in the rumen, which is predicted to improve the productivity of forage fed ruminants. A clearer understanding of the strategies employed by fibrolytic rumen microbes to degrade and utilise lignocellulose is important in realising this goal. Almost 10% of the B. proteoclasticus genome encodes proteins involved in polysaccharide metabolism and transport, which includes 134 fibrolytic enzymes that are active upon plant fibre. Many of these are clustered into one of 36 polysaccharide utilisation loci that also contain transmembrane transporters, transcriptional regulators, environmental sensors and genes involved in further polysaccharide metabolism. Gel-based and gel-free proteomic analyses of the cytosolic, cell-associated, and secreted fractions of cells grown on xylan were used to identify proteins involved in the degradation, assimilation, and metabolism of hemicellulose. A set of 416 non-redundant proteins were identified, which included 12 extracellular and 24 cytosolic polysaccharidases, and 59 proteins involved in the uptake and further metabolism of polysaccharide degradation products, many of which were substrate-binding protein components of ATP-driven transporter systems. In cells grown on xylan, several of these proteins displayed significant protein abundance changes relative to cells grown on the monomeric sugar xylose, in a pattern that reflected the growth substrates used. A model of xylan degradation by B. proteoclasticus based on these results hypothesises that B. proteoclasticus attacks the xylan backbone and main substituent groups of hemicellulose in the extracellular space, assimilates the xylooligosaccharides and performs the final stages of degradation within the cell. These results provide insight into a xylan degrading enzyme system that has evolved to efficiently degrade and utilise hemicellulose, extend our understanding of the enzymes that are likely to play important roles in hemicellulose degradation, and support the notion that Butyrivibrio species are important contributors to rumen fibre degradation

The cell wall microstructures of syncytia induced by cyst nematodes

Plant parasitic cyst nematodes induce the formation of specialised feeding structures, termed syncytia, from which they feed within the host roots. The multinucleate syncytium initiates from a single host root cell and expands by the local cell wall dissolution of neighbouring cells. In this study, a set of monoclonal antibodies were applied to reveal the microstructures of syncytial cell walls induced by four economically important cyst nematode species, Globodera pallida, Heterodera glycines, Heterodera avenae and Heterodera filipjevi, in their respective potato, soybean and wheat host roots. In situ fluorescence analysis revealed that cell walls of syncytia induced by G. pallida and H. glycines share high structural similarity. Both consisted of abundant xyloglucans, methyl-esterified homogalacturonan and pectic arabinans. In contrast, the walls of syncytia induced in wheat roots by H. avenae and H. filipjevi contain much less xyloglucan but are rich in feruloylated and substituted heteroxylans and arabinans, with variable levels of mixed-linkage glucans and galactans. Further investigations were implemented using a range of cell wall related Arabidopsis xyloglucan and pectic arabinan mutants. In situ analysis was applied on those H. schachtii induced syncytia. The results indicated the strong adaptions during the induction and formation of the syncytia while the cell wall composition of the syncytium was stable. Besides, the syncytial wall pectin methyl-esterification status was shown to fluctuate along with the syncytium development in addition to coping with induced PEG-simulated drought stress. Further analysis was carried out on selected pectic homogalacturonan related mutants, and the fluorescence-based quantifications revealed the complexity of the forming and regulating pectin methyl esterification. Transgenic wheat lines with a root-cap-specific promoter were made via biolistics, in the hope of using this system to further investigate the syncytia formed in wheat, which were shown to be different from the other syncytia analysed

Cell wall engineering for better conversion of lignocellulosic biomass

The objective of our research was to use the flexibility of the cell wall to its extremes by modifying its composition while avoiding deleterious effects on plant physiology. One of the strategies to achieve this is the heterologous expression of genes coding for cell wall degrading (CWD) enzymes in plants. These can alter the cell wall structure during plant growth and might improve saccharification yield of the lignocellulosic biomass. In this PhD study 29 genes coding for cell wall degrading enzymes (66 different constructs) were introduced in Arabidopsis and one of the enzymes (a galactanase) was studied in detail. Interestingly, galactanase expression improved the saccharification efficiency without causing a dramatic yield penalty. In parallel, an attempt was made to modify cell wall properties by the expression of the A. caulinodans NodC gene in Arabidopsis. The obtained transgenic lines accumulated GlcNAc mono- and oligosaccharides in their apoplast, which influenced the overall cell wall architecture and modified the cell wall properties. Furthermore, since lignin is major limiting factor that affect saccharification yield, reducing lignin content became a potential strategy to improve the saccharification yield. Here, we have generated Arabidopsis lines with an altered PIRIN2 and PIRIN4 expression. The lines had an altered lignin composition, reduced lignin content and an improved saccharification yield. Despite their modified cell wall, no dramatic effect on plant growth and development was detected

Influence of the ethylene on the grape berry development and related-gene expression

Le raisin est un fruit non climactérique dont la maturation semble ne pas nécessiter l'éthylène. Ici, il est confirmé que l’accumulation d’anthocyanes est liée à l’accumulation d’une glucoslyltransferase (UFGT), dont le promoteur a été cloné. Il a été trouvé 7 cis -éléments éthylène-dépendants. Cette étude a montré la stimulation d'expression de l'ufgt par l’éthylène n'est pas dépendant de MybA, un des régulateurs de la transcription de l'ufgt. Des expériences ont été conçues pour étudier l’ensemble des gènes affecté par éthylène au début de la maturation du raisin. Parmi eux certains sont en relation avec les variations de diamètre de la baie. Ces gènes sont impliqués dans la circulation de l'eau : plusieurs aquaporines, et dans la structure de la paroi cellulaire : polygalactoronases, xyloglucan endotransglucosylases, pectine méthylesterase, cellulose synthase et expansines. L’éthylène stimule l'accumulation de la plupart de leurs transcrits entre 1 heure et 24 heures d’incubation. ABSTRACT : The grape is a non-climacteric fruit which maturation apparently does not require ethylene. Here, it is confirmed that the accumulation of anthocyanins is linked to the accumulation of a glucoslyltransferase (UFGT), whose promoter was cloned. We found 7 cis-elements ethylene-dependent. This study showed the stimulation of ufgt expression by ethylene is not dependent to MybA, transcription regulators of the ufgt. Experiments were designed to investigate all genes affected by ethylene in early ripening grapes. Among them some are in relation to variations in berry diameter. These genes are involved in the movement of water : several aquaporins, and the structure of the cell wall : polygalactoronases, xyloglucan endotransglucosylases, méthylesterase pectin, cellulose synthase and expansines. Ethylene stimulates the accumulation of most of their transcripts between 1 hour and 24 hours of incubation

Isolation and characterization of novel enzymatic activities from gut metagenomes to support lignocellulose breakdown

The agricultural sector produces a large amount of organic waste as by-products (crop remains, foliage, seed pods, straw, etc.). Currently, these materials are not properly treated and their uncontrolled disposal can lead to many problems. In many countries crop remains are burned on the field, sometimes causing severe air pollution as well as damage health. This contributes to climate change and could impact the climate further to the point of irreversible damage. To realize a sustainable agriculture, organic wastes should not be disposed or burned but used as a cheap source for biomaterials. In line with the philosophy of the bio-based economy, agricultural waste is recycled and used as raw material in the chemical industry, replacing fossil fuels. However, the process of converting agricultural waste into useful products is not efficient and can be improved further for optimization. One of the dominant components of agricultural waste is lignocellulose, a complex biomaterial that is difficult to handle. To break down this complex structure, large amounts of energy or chemicals for treatment are required. This thesis aims to explore novel natural biological catalysts that can help to degrade lignocellulose and deliver useful compounds for the bio-based chemical industry. To discover such novel catalysts, the digestive systems of a number of different animals: goats, springtails, isopods and termites were investigated. Animal guts are mini ecosystems that contain many unknown interesting bacteria. These microorganisms are adapted to the host and might have interesting properties that can be explored. By looking at these organisms and their catalysts it is possible to identify and mine novel genes that can be used to breakdown biomass. This process creates substrates that can be used for many other procedures. In this thesis my main focus was on enzymes that can break down carbohydrates. The various bonds in complex carbohydrate molecules are cleaved by different enzymes. Every bacterium has a suit of carbohydrate-active enzymes, called CAZymes. Using metagenomics and bioinformatic tools to explore the genomes of microbial communities in search of novel CAZymes. Unlike traditional culturing methods, metagenomics is aimed at the whole genome of the communities involved, that is, all bacteria jointly. In addition, genes encoding antibiotic resistance, and the production of secondary metabolites were investigated since these two gene categories greatly contribute to the survival of bacteria in complex microbial communities. The work on this thesis shows the possibilities of using bioinformatic tools to investigate the microbiome communities and mine for interesting enzymes from metagenomes. The bacterial community appears to be very diverse and different between hosts. However, at the enzymatic level there is a core group of carbohydrate enzymes and antibiotic resistances. Some of the studied enzymes show the potential for bio-applications. The method could be further tested on different animal metagenomes. With the expansion of sequencing and bioinformatic tools as well as advances in computing and machine learning, it is possible to use and understand more about the natural environment. These tools could help to mine enzymes with interesting properties. Together with enzyme characterization, the bioinformatic tools could be improved further. When the whole process is streamlined and part of a pipeline, a large number of enzymes can be identified and tested to find optimal conditions for their action. These enzymes can be combined together to create cocktails, which can efficiently breakdown biomass. Since the enzymes are natural and the products are used as substrates, little energy and resources are wasted. Beneficial enzymes and bacteria are also preserved and promoted. By creating a recycling plant, agricultural waste can turn into new substrates and products, which in turn can help to improve the environment