High-Throughput production and characterization of Carbohydrate-Active enZYmes for animal nutrition

Tese de Doutoramento em Ciências Veterinárias na especialidade Produção AnimalThe biodegradation of plant cell wall (PCW) carbohydrates is performed by microbial enzymes that are generally referred to as CAZymes. In animal nutrition, it is now well established that the monogastric animals produce a limited repertoire of CAZymes and as such cannot use efficiently some dietary ingredients that sometimes display antinutritional properties. The dietary supplementation with exogenous CAZymes improves the nutritive value of diets and increases animal’s performance. In particular, this study demonstrated that 1,3-1,4-β-glucanases and not cellulases improve the nutritive value of β-glucan-containing diets for monogastric animals. In addition, it was revealed that exogenous enzyme supplementation with β-xylanases improved the nutritive value of diets incorporating wheat lots with high viscosity and low endogenous endo-1,4-β-xylanase activity. In contrast, when the wheat lot showed lower viscosity and higher levels of endogenous endo-1,4-β-xylanase activity, broiler response was clearly diminished. Moreover, the data revealed that xylo-oligosaccharides released by xylanases acting on cereal arabinoxylans display a pre-biotic and positive effect in broiler chicks. However, although we observe an exponential accumulation of genomic and metagenomic information, knowledge on CAZYmes with potential to be used in animal nutrition is limited. This work also aimed to develop high-throughput (HTP) methodologies to isolate and characterize potentially important enzymes for animal nutrition. Thus, 1476 recombinant enzymes were selected and produced recombinantly. The data revealed that 79% of recombinant proteins were produced in the soluble form in Escherichia coli. Factors, such as, organism of origin, gene production strategy, fusion with solubility tags, protein molecular weight and amino acids composition of primary sequences may be used to justify and predict levels of solubility. The establishment of a high-throughput pipeline for recombinant enzyme production was used to obtain a library of feruloyl esterases (FAEs) and glucuronoyl esterases (GEs), enzymes which remove the side chains and break crosslinks between hemicellulosic carbohydrates and lignin. Thus 480 putative FAEs and 20 GEs were produced and biochemically characterized. Following gene isolation, 372 FAEs and 11 GEs were produced in a soluble form in E. coli. Activity results showed that 50% of the enzymes produced retained significant levels of activity and stability. The library of innovative FAEs and GEs produced during this project will be used to develop a novel generation of enzymes for animal nutrition, in particular to exploit the release of cellulose and hemicellulose from lignin.RESUMO - Na natureza, a biodegradação dos hidratos de carbono da parede celular vegetal é realizada por enzimas microbianas, geralmente conhecidas como CAZymes. Os animais monogástricos produzem um reportório limitado de enzimas para degradação destes hidratos de carbono, não conseguindo usar eficientemente alguns ingredientes da dieta, que muitas vezes manifestam propriedades anti nutritivas. Assim, é sabido que a suplementação com CAZymes exógenas melhora o valor nutritivo das dietas e aumenta o desempenho produtivo dos animais. Este trabalho revelou que as enzimas mais apropriadas para suplementar dietas ricas em 1,3-1,4-β-glucanos são as 1,3-1,4-β-glucanases e não as celulases. Além disso, verificou-se que a suplementação com β-xilanases melhorou o valor nutritivo de dietas que continham variedades de trigo com maior viscosidade e menor atividade endógena de endo-1,4-β-xilanase. Em oposição, quando o lote de trigo apresentou menor viscosidade e maiores níveis de atividade endógena de endo-1,4-β-xilanase, a resposta dos animais à adição das enzimas foi menor. Este trabalho mostra, igualmente, que os xilo-oligossacarídeos, resultantes da degradação de arabinoxilanos por xilanases exógenas, possuem uma ação pré-biótica na alimentação de frangos, promovendo a melhoria do desempenho zootécnico. Contudo, apesar de estarem descritas uma grande diversidade CAZymes, poucas são as estudadas com potencial para serem usadas em alimentação animal. Portanto, este trabalho pretendeu, também, desenvolver metodologias para isolar e caracterizar enzimas potencialmente importantes em larga escala. Foram selecionadas, produzidas e expressas na forma recombinante 1476 CAZymes. Os dados revelaram que 79% das proteínas recombinantes foram produzidas na forma solúvel em Escherichia coli. Verificou-se, ainda, que fatores como o organismo de origem, a estratégia de produção, a fusão com marcadores de solubilidade, o peso molecular da proteína e composição de aminoácidos das sequências primárias, parecem justificar os resultados da solubilidade. Estes ensinamentos foram utilizados para produzir enzimas, tais como ferulolil esterases (FAEs) e glucuronil esterases (GEs), que removem as cadeias laterais e quebram as ligações cruzadas entre hidratos de carbono hemicelulósicos e a lenhina. Assim sendo, foram selecionadas 480 FAEs e 20 GEs para produção recombinante e caracterização bioquímica. Cerca de 372 FAEs e 11 GEs foram produzidos em forma solúvel em E. coli e aproximadamente 50% das enzimas produzidas mantiveram níveis significativos de atividade e estabilidade. Com isto, foi possível identificar e produzir um número significativo de FAEs e GEs com potencial para alimentação animal, em especial as que libertam celulose e hemicelulose da lenhina.N/

Structure and functional studies of plant cell wall degrading enzymes

Presently, plant biomass is considered as one of the major future renewable sources for the production of second-generation biofuels. While the first generation biofuels essentially are based on starch and sucrose rich feed stocks and which production may compete with food production, the second-generation biofuels may be based on lignocellulose as feedstock, which is less problematic from an ethical point of view. The degradation of carbohydrates in plant biomass to fermentable sugars requires the concerted action of several diverse classes of carbohydrate active enzymes (CAZymes) for a total and efficient conversion of the plant biomass. Through a carefully balanced synergism mechanistically different CAZymes are able to degrade the stable and recalcitrant pol- ymers in the plant cell walls, such as cellulose, to soluble and fermentable monosaccharides. It is crucial to study the properties and function of these enzymes if we want to strive for a sustainable production of chemicals and biofuels, as they serve as a reservoir of environmentally friendly molecular tools. The main focus of the research work presented in this thesis is biochemical and structure-function characterizations of two classes of CAZymes: fungal glycoside hydrolase family 3 (GH3) β-glucosidases, and bacterial lytic polysaccharide monooxygenases, often referred to as LPMOs. GH3 β- glucosidases catalyse the conversion of disaccharides, produced by other CAZymes e.g. cellulases, to glucose. H. jecorina Cel3A, R. emersonii Cel3A and N. crassa NcGH3-3 are three industrially relevant fungal GH3 β-glucosidases for which the structures have been determined using X-ray crystallographic methods. The H. jecorina Cel3A, R. em- ersonii Cel3A enzymes has also been characterized biochemically. The LPMOs act in the very initial stage of plant cell wall degradation and cleave glycosidic bonds in crys- talline polysaccharides via an oxidative mechanism, which facilitates access to new chain ends for other CAZymes. To elucidate the structural and biochemical properties of LPMOs with bacterial origin, the structure of an AA10 LPMO the LPMO10A from Enterococcus faecalis was determined using X-ray crystallography. Furthermore, structural changes of the active site metal configuration by so-called X-ray induced photoreduction, were determined. During this reduction process, which mimics the active enzyme, the bound active site copper atom is reduced from Cu(I) to Cu(II), which causes changes in the ligation configuration

The battle for chitin recognition in plant-microbe interactions

Fungal cell walls play dynamic functions in interaction of fungi with their surroundings. In pathogenic fungi, the cell wall is the first structure to make physical contact with host cells. An important structural component of fungal cell walls is chitin, a well-known elicitor of immune responses in plants. Research into chitin perception has sparked since the chitin receptor from rice was cloned nearly a decade ago. Considering the widespread nature of chitin perception in plants, pathogens evidently evolved strategies to overcome detection, including alterations in the composition of cell walls, modification of their carbohydrate chains and secretion of effectors to provide cell wall protection or target host immune responses. Also non-pathogenic fungi contain chitin in their cell walls and are recipients of immune responses. Intriguingly, various mutualists employ chitin-derived signaling molecules to prepare their hosts for the mutualistic relationship. Research on the various types of interactions has revealed different molecular components that play crucial roles and, moreover, that various chitin-binding proteins contain dissimilar chitin-binding domains across species that differ in affinity and specificity. Considering the various strategies from microbes and hosts focused on chitin recognition, it is evident that this carbohydrate plays a central role in plant-fungus interaction

Carbohydrate binding modules: diversity of domain architecture in amylases and cellulases from filamentous microorganisms

Microbial Biotechnolog

Tracing the molecular and evolutionary determinants of novel functions in protein families

This thesis explores the limits of homology-based inference of protein function and evolution, where overall similarity between sequences can be a poor indicator of functional similarity or evolutionary relationships. Each case presented has undergone different patterns of evolutionary change due to differing selective pressures. Surface adaptations and regulatory (e.g., gene expression) divergence are examined as molecular determinants of novel functions whose patterns are easily missed by assessments of overall sequence similarity. Following this, internal repeats and mosaic sequences are investigated as cases in which key evolutionary events involving fragments of protein sequences are masked by overall comparison. Lastly, virulence factors, which cannot be unified based on sequence, are predicted by analysis of elevated host-mimicry patterns in pathogenic versus non-pathogenic bacterial genomes. These patterns have resulted from unique co-evolutionary pressures that apply to bacterial pathogens, but may be lacking in their close relatives. A recurring theme in the proteins/genes/genomes analyzed is an involvement in microbial pathogenesis or pathogen-defense. Due to the ongoing "evolutionary arms race" between hosts and pathogens, virulence and defense proteins have undergone—and will likely continue to generate—evolutionary novelties. Thus, they demonstrate the necessity to look beyond overall sequence comparison, and assess multiple dimensions of functional innovation in proteins

Structure and function relationships in novel cellulosomal enzymes and cohesindockerin complexes

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e BiomédicasPlant cell walls are the most abundant source of organic carbon on earth, providing an extraordinary supply of energy for various microorganisms. The energetic constrains posed by anaerobic ecosystems lead to the evolution of highly efficient multi-enzymatic complexes, termed cellulosomes, which orchestrate the deconstruction of structural carbohydrates. Clostridium thermocellum cellulosome has been extensively studied as the bacterium exhibits one of the highest growth rates on cellulose. Cellulosomes are assembled by a large non-catalytic multi-modular scaffoldin which contains repeated type I cohesins. Type I dockerin modules, usually located at the C-terminus of enzymes, bind tenaciously to type I cohesins. Scaffoldins may contain a type II dockerin which specifically recognizes type II cohesins located at the cell envelope, allowing the cell surface attachment of cellulosomes. Here a combination of methodologies was applied to study the structure and function relationships of novel cellulosomal enzymes and cohesin-dockerin complexes. Innovative molecular biology and biochemical protocols that can be applied to crystallize and solve the structure of cohesin-dockerin complexes are described in chapter 2. In addition, the crystal structures of two novel type I cohesin-dockerin complexes (CtCohOlpC-Doc124A and CtCohOlpA-Doc918) are described here. They revealed that the two dockerins are unusual since they lack the structural symmetry that supports the dual binding mode typical of type I modules. Thus, these dockerins present a single binding mode and seem to bind preferentially to cohesins located at the bacterium cell surface and not to cellulosomes (chapter 3). Doc124A is the dockerin of CtCel124A, an endoacting cellulase with a superhelical fold that acts in synergy with the major cellulosomal exo-cellulase, Cel48S, during cellulose hydrolysis. The crystal structure of CtCel124A in complex with two cellotriose molecules suggests that the enzyme may target the interface between crystalline and amorphous cellulose (chapter 5). In addition, the structure of a novel type II cohesin-dockerin complex (CtCohScaC2-XDocCipB) was solved. The functional importance of specific dockerin residues was determined. Type II dockerins are suggested to present two different cohesin-binding faces that express different specificities (chapter 4). Finally, the crystal structure of a penta-modular cellulosomal protein (CtXyl5A), previously of unknown function, was assessed (chapter 6). This protein is one of the largest cellulosomal components and comprises a GH5, two CBMs from families 6 and 13, a fibronectin type III-like module, a CBM from family 62 and a type I dockerin. CtGH5 has a canonical (α/β)8-barrel fold and displays specificity for arabinoxylans and as such, is defined as an arabinoxylanase. CtCBM6 adopts a β-sandwich fold and displays affinity for the reaction products generated by CtGH5 and for undecorated xylooligosaccharides. In addition, the penta-modular structure revealed a great flexibility for the CtCBM62 domain.RESUMO - Estrutura e função de novas enzimas celulossomais e de novos complexos coesina-doquerina - A parede celular vegetal constitui uma das principais fontes de carbono do planeta, sendo por isso um extraordinário recurso energético para muitos microrganismos. As limitações energéticas características dos ecossistemas anaeróbios conduziram à evolução de complexos multi-enzimáticos de elevada eficiência, denominados celulossomas, os quais coordenam a degradação dos hidratos de carbono da parede celular vegetal. O Clostridium thermocellum produz um celulossoma relativamente bem caracterizado já que a bactéria apresenta uma das maiores taxas de crescimento em celulose. A organização do celulossoma é efectuada por uma proteína não-catalítica multi-modular. Esta proteína de integração possui uma série de módulos repetidos, denominadas coesinas do tipo I. Na extremidade C-terminal das enzimas celulosomais existem módulos doquerina do tipo I, os quais se ligam fortemente às coesinas do tipo I. As proteínas de integração celulossomal podem conter ainda uma doquerina do tipo II que reconhece especificamente coesinas do tipo II localizadas no envelope celular permitindo, assim, a fixação dos celulossomas à superfície da bactéria. No presente trabalho foram desenvolvidas várias metodologias inovadoras com o intuito de determinar a estrutura e a função de novas enzimas celulossomais e de novos complexos coesina-doquerina. Os protocolos de biologia molecular e bioquímicos aqui descritos (capítulo 2) permitem ultrapassar as dificuldades inerentes à cristalização e, por conseguinte, à resolução da estrutura de complexos coesina-doquerina. Com base nestas metodologias, foram elucidadas as estruturas tridimensionais de dois novos complexos coesina-doquerina do tipo I (CtCohOlpCDoc124A e CtCohOlpA-Doc918). A análise destas estruturas revelou que as suas doquerinas são atípicas, uma vez que não possuem a simetria estrutural característica dos módulos doquerina do tipo I que lhes confere um modo de ligação duplo. Com efeito, estas novas doquerinas apresentam um modo de ligação simples e parecem ligar-se preferencialmente a coesinas localizadas na superfície da bactéria (capítulo 3). A Doc124A é a doquerina da CtCel124A, uma endo-celulase que possui um enrolamento super-helicoidal e que atua em sinergia com a principal exo-celulase celulossomal, a Cel48S, na hidrólise da celulose. A estrutura tridimensional da CtCel124A em complexo com duas moléculas de celotriose sugere que a enzima pode ter como substrato alvo a interface entre as formas cristalina e amorfa da celulose (capítulo 5). Para além da elucidação destas estruturas, foi também resolvida a estrutura de um novo complexo coesina-doquerina do tipo II (CtCohScaC2-XDocCipB). Os resultados sugerem que as doquerinas do tipo II apresentam duas interfaces de ligação a coesinas que expressam diferentes especificidades (capítulo 4). Por último, é apresentada a estrutura tridimensional de uma proteína celulossomal penta-modular (CtXyl5A) e é elucidada a sua função (capítulo 6). Esta proteína é um dos componentes celulossomais de maiores dimensões e compreende uma GH5, dois módulos de ligação a hidratos de carbono (CBMs) das famílias 6 e 13, um módulo de fibronectina do tipo III, um CBM da família 62 e ainda uma doquerina do tipo I. A GH5 apresenta um enrolamento canónico em barril (α/β)8 e possui especificidade para os arabinoxilanos, tendo sido por isso definida como uma arabinoxilanase. O CBM6 apresenta um enrolamento em β-sanduiche e possui afinidade para os produtos de reação gerados pela GH5 e para xilo-oligossacáridos nãodecorados. A estrutura penta-modular desta proteína revelou uma grande flexibilidade no domínio CBM62

NMR insights into molecular recognition: structure and interactions of peptides and proteins

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular I, leída el 16-12-2016La investigación de las bases moleculares que gobiernan los procesos biológicos es esencial para comprenderlos, así como para entender sus implicaciones y el impacto que tienen en el organismo de los seres vivos. En dichos procesos, los eventos de reconocimiento biomolecular son cruciales y es necesario lograr una descripción detallada a nivel atómico para conocer las particularidades de los mecanismos, su regulación, selectividad, especificidad… En este contexto, la espectroscopía de RMN es una técnica que proporciona gran cantidad de información fiable que permite estudiar las características de los eventos de reconocimiento biomolecular con resolución atómica, lo que es clave para deducir y clarificar los fundamentos de los relevantes procesos biológicos. En este trabajo se ha empleado la espectroscopía de RMN, en combinación con diversas técnicas bioquímicas, fisicoquímicas y computacionales, para estudiar varios sistemas biológicos de interés en los que están presentes algunos eventos de reconocimiento biomolecular representativos, tales como las interacciones péptido–membrana, las interacciones carbohidrato–proteína, o las interacciones proteína–proteína. El objeto de esta tesis es profundizar en el conocimiento sobre varios casos de los citados eventos de reconocimiento biomolecular y reforzar el papel de la espectroscopía de RMN como una potente herramienta para abordar la caracterización de las interacciones biomoleculares. Esta tesis está estructurada en seis capítulos. El capítulo 2 describe el estudio de una serie de péptidos derivados del dominio de unión a colina de la autolisina de pneumococo, LytA. Este estudio está dirigido a averiguar si dichos péptidos mantienen su estructura nativa y su capacidad de unir colina cuando se encuentran aislados de la proteína completa. Utilizando espectroscopía de RMN en disolución, dicroísmo circular (CD) y técnicas de fluorescencia, se han descubierto tres péptidos que adoptan conformaciones de horquilla β nativas en disolución acuosa, tal y como se pretendía, y que sufren una inesperada transición reversible de estructura β a estructura α en presencia de micelas de detergente. Se ha propuesto una explicación para la interacción que tiene lugar entre estos péptidos y las micelas de detergente, y se han caracterizado las bases fisicoquímicas de la transición estructural observada mediante el estudio de variantes de uno de estos péptidos...The investigation of the molecular bases governing biological processes is essential to understand them, as well as their implications and impact in the organism of living creatures. In these processes, biomolecular recognition events are pivotal and the search for a comprehensive description at atomic level is necessary to know the details of mechanisms, regulation, selectivity, specificity, etc. In this context, NMR spectroscopy is a technique that provides a vast diversity of reliable information allowing the study of the characteristics of biomolecular recognition events at atomic resolution, which is the key to infer and clarify the fundamentals of relevant biological processes. In this work, NMR spectroscopy combined with diverse biochemical, physicochemical, and computational techniques have been utilised to study several interesting biological systems involving some representative biomolecular recognition events, such as peptide–membrane interactions, carbohydrate–protein interactions, and protein–protein interactions. The aim of this thesis is to go deeper in the knowledge of some biomolecular recognition events, and reinforce that NMR spectroscopy as a powerful tool to address the characterization of biomolecular interactions. This thesis has been structured in six chapters. Chapter 2 describes the study of a series of peptides derived from the choline–binding domain of pneumococcal autolysin LytA, aimed to find out whether they maintain their native structure and the ability to bind choline when isolated from the full–length protein. Using solution NMR, CD and fluorescence techniques, three peptides were found to show native–like, β–hairpin conformations in aqueous solution, as intended, and undergo an unexpected, reversible β–to–α transition in the presence of detergent micelles. An explanation to the interaction between these peptides and detergent micelle has been proposed, and the physicochemical bases of the observed structural transition were characterised by studying variants of one of these peptides...Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEunpu