Exploring novel strategies for depolymerization of plant cell-wall polysaccharides: from structure, function and rational design of glycosyl hydrolases to biological implications and potential biotechnological applications

• Grant number: 15/26982-0
• Support Opportunities: Research Projects - Thematic Grants
• Duration: February 01, 2017 - November 30, 2022
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Mário Tyago Murakami
• Grantee: Mário Tyago Murakami
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovações (Brasil). Campinas , SP, Brazil
• Pesquisadores principais: Roberto Ruller
• Associated researchers: Camila Ramos dos Santos ; Celso Eduardo Benedetti ; Gabriela Felix Persinoti ; Leticia Maria Zanphorlin ; Paulo Sergio Lopes de Oliveira ; Priscila Oliveira de Giuseppe
• Associated grant(s): 22/04606-0 - 18th International Symposium on Microbial Ecology, AR.EXT; 18/18991-7 - XIII Brazilian Seminar of Enzyme Techonology - ENZITEC 2018, AR.BR; 18/04505-3 - Innovations applied to enzymes, plant polysaccharides, carbohydrate chemistry and biochemistry, AV.EXT; 17/14310-2 - Cellulases & Other Carbohydrate-Active Enzymes, AR.EXT
• Associated scholarship(s): 19/10754-9 - Production, purification and structural studies of accessory enzymes for xylan degradation, BP.TT; 18/02865-2 - Molecular investigation of new xylose isomerases for application to lignocellulosic materials fermentation, BP.DD; 17/14253-9 - Evaluation of the biotechnological potential of the cellulolytic system of Xanthomonas axonopodis pv. citri: a structural, functional and applied approach, BP.PD; + associated scholarships 16/19995-0 - Analysis of structural and functional diversity of GH43 enzymes from Xanthomonas axonopodis pv. citri: biological implications and potential biotechnological applications, BP.PD; 17/00203-0 - Studies of the molecular basis of mannan degradation and utilization system from the phytopathogen Xanthomonas axonopodis pv. citri, BP.DD; 16/06509-0 - Understanding the enzymatic system involved in the degradation and utilization of xyloglucans from the plant pathogen Xanthomonas axonopodis pv. citri, BP.PD - associated scholarships

Abstract

The plant cell wall is an important physical barrier to pathogens, and to chemical and physical stress for plants and it is essentially composed of polysaccharides of great biological and industrial importance. However, the challenge to deconstruct the plant cell wall into simple sugars is not a fully consolidated and economically viable process for many industrial applications as, for example, in the production of biofuels. Thus, develop and discover new strategies and processes biochemically more effective in dismantling the plant cell wall are of great economic interest. Furthermore, there is a strong biological appeal over the glycoside hydrolases (GHs) due to their direct involvement in a number of physio(patho)logical events including plant-pathogen interaction, the carbon cycle and homeostasis of plants. Despite great advances in the field Carbohydrate Enzymology, our knowledge is still limited on the vast universe of GHs. Only a very small fraction of the nature´s repertoire is known and this is evidenced by low rate of characterized emzymes in relation to the total of known sequences. Of the more than 330,000 GHs classified in CAZy database, only 2% have been biochemically studied and even less from a structural view. Among the various niches virtually unexplored, it highlights the broad and diverse GH arsenal of some plant pathogens and enzymes belonging to uncharacterized subfamilies. The bacterium that causes citrus canker (Xanthomonas axonopodis pv citri; Xac), for example, has in its genome more than 100 GH genes spread into 45 families, although it does not perform massive maceration of the plant cell wall. Compared to other species of the same genus as Xanthomonas albinieans (56 GHs, 32 families), there is an expansion in both the quantity and the diversity of GHs in Xac, suggesting an important role for these enzymes in their evolutive adaptation to citrus plants. However, the biochemical and physiological function of this vast enzymatic repertoire is still poorly understood and our research group has been a pioneer in demonstrating their biotechnological potential. In a recent study, we developed a highly efficient chimeric endoxylanase from the characterization and the rational redesign of a Xac exo-enzyme, which had its patent applied for industrial uses. In addition to the bias of functional and structural studies to GHs of certain species, a literature survey clearly shows the random and redundant way that these studies have been conducted within the various families of glycoside hydrolases. According to phylogenetic and similarity network analyses, a number of isofunctional clusters remain unknown at both functional and structural view. These isofunctional clusters, as demonstrated for the families 5, 13, 16 and 43, exhibit a range of activities and modes of action. However, the characterization of few members of each family is insufficient to reveal all functional diversity contained in the subfamilies, which usually have sequence identity lower than 30% of each other. Therefore, in this project we aim to explore and understand, at the mechanistic level, the functional diversity of GHs of unexplored niches such as the Xac pathogen and uuncharacterized subfamilies of the families 5 and 128. To accomplish with these objectives, we will use a multidisciplinary approach that integrates evolutionary, biochemical and structural studies to provide results that potentially will reveal novel strategies for depolymerization of plant cell wall and will generate instrumental data for the rational and optimized use of GHs for industrial purposes. Furthermore, using Xac pathogen as a model organism, we expect to better understand the role of GHs in the complex mechanisms mediating plant-pathogen interactions, thus contributing to assess the potential of these metabolic routes as targets for the development of molecular strategies employed to combat agricultural diseases economically relevant to the country. (AU)

Scientific publications

(19)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

CABRAL, LUCELIA; PERSINOTI, GABRIELA F.; PAIXAO, DOUGLAS A. A.; MARTINS, MARCELE P.; MORAIS, MARIANA A. B.; CHINAGLIA, MARIANA; DOMINGUES, MARIANE N.; SFORCA, MAURICIO L.; PIROLLA, RENAN A. S.; GENEROSO, WESLEY C.; et al. ut microbiome of the largest living rodent harbors unprecedented enzymatic systems to degrade plant polysaccharide. NATURE COMMUNICATIONS, v. 13, n. 1, FEB 2 2022. (15/26982-0, 16/19995-0)

ZANPHORLIN, LETICIA MARIA; BUENO DE MORAIS, MARIANA ABRAHAO; DIOGO, JOSE ALBERTO; DOMINGUES, MARIANE NORONHA; MOREIRA DE SOUZA, FLAVIO HENRIQUE; RULLER, ROBERTO; MURAKAMI, MARIO TYAGO. Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43. Biotechnology and Bioengineering, v. 116, n. 4, p. 734-744, APR 2019. (16/19995-0, 13/24622-0, 15/26982-0)

SANTOS, CLELTON A.; MORAIS, MARIANA A. B.; TERRETT, OLIVER M.; LYCZAKOWSKI, JAN J.; ZANPHORLIN, LETICIA M.; FERREIRA, JAIRE A.; TONOLI, CELISA C. C.; MURAKAMI, MARIO T.; DUPREE, PAUL; SOUZA, ANETE P.. An engineered GH1 ss-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials. SCIENTIFIC REPORTS, v. 9, MAR 20 2019. (16/19995-0, 15/26982-0, 17/17390-7, 15/09202-0)

TOYAMA, DANYELLE; BUENO DE MORAIS, MARIANA ABRAHAO; RAMOS, FELIPE CARDOSO; ZANPHORLIN, LETICIA MARIA; COSTA TONOLI, CELISA CALDANA; BALULA, AUGUSTO FURIO; DE MIRANDA, FERNANDO FELTON; ALMEIDA, VITOR MEDEIROS; MARANA, SANDRO ROBERTO; RULLER, ROBERTO; et al. A novel beta-glucosidase isolated from the microbial metagenome of Lake Poraque (Amazon, Brazil). BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1866, n. 4, p. 569-579, APR 2018. (15/26982-0)

SANTOS, CAMILA R.; COSTA, PEDRO A. C. R.; VIEIRA, PLINIO S.; GONZALEZ, SINKLER E. T.; CORREA, THAMY L. R.; LIMA, EVANDRO A.; MANDELLI, FERNANDA; PIROLLA, RENAN A. S.; DOMINGUES, MARIANE N.; CABRAL, LUCELIA; et al. Structural insights into beta-1,3-glucan cleavage by a glycoside hydrolase family. Nature Chemical Biology, v. 16, n. 8, MAY 2020. (13/08293-7, 15/26982-0)

RIBEIRO CORREA, THAMY LIVIA; TOMAZINI JUNIOR, ATILIO; WOLF, LUCIA DANIELA; BUCKERIDGE, MARCOS SILVEIRA; DOS SANTOS, LEANDRO VIEIRA; MURAKAMI, MARIO TYAGO. An actinobacteria lytic polysaccharide monooxygenase acts on both cellulose and xylan to boost biomass saccharification. BIOTECHNOLOGY FOR BIOFUELS, v. 12, MAY 10 2019. (15/26982-0)

DE SOUSA, AMANDA S.; DE MELO, RICARDO R.; MIYAMOTO, RENAN Y.; MORAIS, MARIANA A. B.; ANDRADE, LILIANE P.; MILAN, NATALIA; DE AVILA, MAYARA C.; DE SOUZA, CLAUDIA M.; ADAO, REGINA C.; SCARPASSA, JOSIANE A.; et al. A rationally identified marine GH1 beta-glucosidase has distinguishing functional features for simultaneous saccharification and fermentation. BIOFUELS BIOPRODUCTS & BIOREFINING-BIOFPR, AUG 2020. (16/19995-0, 17/14253-9, 18/02865-2, 15/26982-0, 16/06509-0, 19/08855-1)

VIEIRA, PLINIO S.; BONFIM, ISABELA M.; ARAUJO, EVANDRO A.; MELO, RICARDO R.; LIMA, AUGUSTO R.; FESSEL, MELISSA R.; PAIXAO, DOUGLAS A. A.; PERSINOTI, GABRIELA F.; ROCCO, SILVANA A.; LIMA, TATIANI B.; et al. Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors. NATURE COMMUNICATIONS, v. 12, n. 1, JUN 30 2021. (17/14253-9, 15/26982-0, 16/06509-0, 15/13684-0, 18/03724-3, 19/13936-0, 17/00203-0, 16/19995-0)

BERTO, GABRIELA LEILA; VELASCO, JOSMAN; CABOS RIBEIRO, CAIO TASSO; ZANPHORLIN, LETICIA MARIA; DOMINGUES, MARIANE NORONHA; MURAKAMI, MARIO TYAGO; POLIKARPOV, IGOR; DE OLIVEIRA, LEANDRO CRISTANTE; FERRAZ, ANDRE; SEGATO, FERNANDO. Functional characterization and comparative analysis of two heterologous endoglucanases from diverging subfamilies of glycosyl hydrolase family 45. Enzyme and Microbial Technology, v. 120, p. 23-35, JAN 2019. (14/06923-6, 14/18714-2, 15/13684-0, 15/26982-0)

TRAMONTINA, ROBSON; BRENELLI, LIVIA BEATRIZ; SOUSA, AMANDA; ALVES, RAFAEL; ZETTY ARENAS, ANA MARIA; NASCIMENTO, VIVIANE MARCOS; RABELO, SARITA CANDIDA; FREITAS, SINDELIA; RULLER, ROBERTO; SQUINA, FABIO MARCIO. Designing a cocktail containing redox enzymes to improve hemicellulosic hydrolysate fermentability by microorganisms. Enzyme and Microbial Technology, v. 135, APR 2020. (15/50590-4, 13/03061-0, 17/15477-8, 15/26982-0, 16/07926-4)

NARESSI SCAPIN, SANDRA MARA; MOREIRA SOUZA, FLAVIO HENRIQUE; ZANPHORLIN, LETICIA MARIA; DE ALMEIDA, THAMYRES SILVA; SADE, YOUSSEF BACILA; CARDOSO, ALEXANDER MACHADO; PINHEIRO, GUILHERME LUIZ; MURAKAMI, MARIO TYAGO. Structure and function of a novel GH8 endoglucanase from the bacterial cellulose synthase complex of Raoultella ornithinolytica. PLoS One, v. 12, n. 4, APR 27 2017. (15/26982-0)

DOS SANTOS, CAMILA RAMOS; DE GIUSEPPE, PRISCILA OLIVEIRA; MOREIRA DE SOUZA, FLAVIO HENRIQUE; ZANPHORLIN, LETICIA MARIA; DOMINGUES, MARIANE NORONHA; SIQUEIRA PIROLLA, RENAN AUGUSTO; HONORATO, RODRIGO VARGAS; COSTA TONOLI, CELISA CALDANA; BUENO DE MORAIS, MARIANA ABRAHAO; DE MATOS MARTINS, VANESA PEIXOTO; et al. The mechanism by which a distinguishing arabinofuranosidase can cope with internal di-substitutions in arabinoxylans. BIOTECHNOLOGY FOR BIOFUELS, v. 11, AUG 11 2018. (13/13309-0, 14/17264-3, 15/26982-0, 14/07135-1)

DE MELO, RICARDO RODRIGUES; DE LIMA, EVANDRO ANTONIO; PERSINOTI, GABRIELA FELIX; VIEIRA, PLINIO SALMAZO; DE SOUSA, AMANDA SILVA; ZANPHORLIN, LETICIA MARIA; DE GIUSEPPE, PRISCILA OLIVEIRA; RULLER, ROBERTO; MURAKAMI, MARIO TYAGO. Identification of a cold-adapted and metal-stimulated beta-1,4-glucanase with potential use in the extraction of bioactive compounds from plants. International Journal of Biological Macromolecules, v. 166, p. 190-199, JAN 1 2021. (15/26982-0, 17/14253-9)

MORAIS, MARIANA A. B.; COINES, JOAN; DOMINGUES, MARIANE N.; PIROLLA, RENAN A. S.; TONOLI, CELISA C. C.; SANTOS, CAMILA R.; CORREA, JESSICA B. L.; GOZZO, FABIO C.; ROVIRA, CARME; MURAKAMI, MARIO T.. Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations. NATURE COMMUNICATIONS, v. 12, n. 1, JAN 14 2021. (18/22138-8, 16/19995-0, 15/26982-0)

DE MORAIS, MARIANA ABRAHAO BUENO; POLO, CARLA CRISTINA; DOMINGUES, MARIANE NORONHA; PERSINOTI, GABRIELA FELIX; PIROLLA, RENAN AUGUSTO SIQUEIRA; DE SOUZA, FLAVIO HENRIQUE MOREIRA; CORREA, JESSICA BATISTA DE LIMA; DOS SANTOS, CAMILA RAMOS; MURAKAMI, MARIO TYAGO. Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 beta-Xylosidases. FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, v. 8, MAY 15 2020. (16/19995-0, 13/24622-0, 15/26982-0)

RADE, LETICIA L.; DA SILVA, MELQUE N. P.; VIEIRA, PLINIO S.; MILAN, NATALIA; DE SOUZA, CLAUDIA M.; DE MELO, RICARDO R.; KLEIN, BRUNO C.; BONOMI, ANTONIO; DE CASTRO, HEIZIR F.; MURAKAMI, MARIO T.; et al. A Novel Fungal Lipase With Methanol Tolerance and Preference for Macaw Palm Oil. FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, v. 8, MAY 6 2020. (19/08855-1, 16/50403-2, 15/26982-0, 18/04897-9, 18/10517-4)

DOMINGUES, MARIANE NORONHA; MOREIRA SOUZA, FLAVIO HENRIQUE; VIEIRA, PLINIO SALMAZO; BUENO DE MORAIS, MARIANA ABRAHAO; ZANPHORLIN, LETICIA MARIA; DOS SANTOS, CAMILA RAMOS; SIQUEIRA PIROLLA, RENAN AUGUSTO; HONORATO, RODRIGO VARGAS; LOPES DE OLIVEIRA, PAULO SERGIO; GOZZO, FABIO CESAR; et al. Structural basis of exo--mannanase activity in the GH2 family. Journal of Biological Chemistry, v. 293, n. 35, p. 13636-13649, AUG 31 2018. (16/06509-0, 15/26982-0, 16/19995-0, 13/24622-0)

CORDEIRO, ROSA LORIZOLLA; SIQUEIRA PIROLLA, RENAN AUGUSTO; PERSINOTI, GABRIELA FELIX; GOZZO, FABIO CESAR; DE GIUSEPPE, PRISCILA OLIVEIRA; MURAKAMI, MARIO TYAGO. N-glycan Utilization by Bifidobacterium Gut Symbionts Involves a Specialist beta-Mannosidase. Journal of Molecular Biology, v. 431, n. 4, p. 732-747, FEB 15 2019. (16/00740-2, 15/26982-0)

MANDELLI, FERNANDA; DE MORAIS, MARIANA ABRAHAO BUENO; DE LIMA, EVANDRO ANTONIO; OLIVEIRA, LEANE; PERSINOTI, GABRIELA FELIX; MURAKAMI, MARIO TYAGO. Spatially remote motifs cooperatively affect substrate preference of a ruminal GH26-type endo-?-1,4-mannanase. Journal of Biological Chemistry, v. 295, n. 15, p. 5012-5021, APR 10 2020. (16/19995-0, 15/26982-0)