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Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases

Grant number: 12/20549-4
Support type:Program for Research on Bioenergy (BIOEN) - Young Investigators Grants
Duration: November 01, 2013 - June 30, 2018
Field of knowledge:Biological Sciences - Microbiology
Principal Investigator:André Ricardo de Lima Damasio
Grantee:André Ricardo de Lima Damasio
Home Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Associated grant(s):18/01840-6 - 40th symposium on biotechnology for fuels and chemicals, AR.EXT
Associated scholarship(s):17/26315-9 - Influence of transcription factors on the secretion of enzymes in Aspergillus nidulans, BP.MS
16/16306-0 - Functional and biological analyses of LPMO (Lytic Polysaccharide Monooxigenases) and accessory proteins (non-CAZymes) for degradation of sugarcane straw by filamentous fungi, BP.PD
14/23051-2 - Comparative analysis of the heterologous protein secretion mechanisms in Aspergillus nidulans, BP.MS
+ associated scholarships 14/15403-6 - Aspergillus nidulans as a model to manipulate unfolded protein response-related genes, BP.DR
13/24988-5 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases, BP.DD
13/18910-3 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases, BP.JP.BIOEN - associated scholarships

Abstract

Only a third of the biomass of sugarcane is processed into ethanol, while the other two thirds are dropped as bagasse and straw. Cellulose and hemicellulose are the most abundant polymers in sugar cane bagasse, and have a very complex arrangement in the cell wall. Cellulases and hemicellulases are glycoside hydrolases responsible for the complete hydrolysis of plants cell-wall polysaccharides. Filamentous fungi are the largest source for cell-wall degrading enzymes prospection, especially those of the genus Aspergillus. Aspergillus and Trichoderma are extraordinary in their ability to secrete large amounts of proteins into the culture medium. Most fungal proteins undergoes glycosylation co- or posttranslational in the endoplasmic reticulum (ER) and/or Golgi complex, and therefore in essence are glycoproteins. However, this proposal will focus on two major objectives: 1) Effect of N-glycans on functional properties of glycoside hydrolases: investigate possible functional changes of a GH11 endo-xylanase variants, non-glycosylated, hypo- and hyper-glycosylated, cloned from Penicillium funiculosum. These changes include increased or reduced activity, abnormal pattern of hydrolysis products, changes in kinetic constants (Km and Kcat), changes in secondary structure and variations in specificity and affinity to different substrates; 2) Constitutive expression of hacA and cpcA (genes involved in ER stress and unfolded protein response) in A. nidulans: we propose the construction of strains expressing constitutively hacAi and gcn4p/cpcA, systematically checking the heterologous production of a target protein, GH12 xyloglucanase (XegA) from Aspergillus clavatus (Acla_029940). (AU)

Scientific publications (13)
(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)
RUBIO, MARCELO VENTURA; FANCHINI TERRASAN, CESAR RAFAEL; CONTESINI, FABIANO JARES; ZUBIETA, MARIANE PALUDETTI; GERHARDT, JAQUELINE ALINE; OLIVEIRA, LEANDRO CRISTANTE; DE SOUZA SCHMIDT GONCALVES, ANY ELISA; ALMEIDA, FAUSTO; SMITH, BRADLEY JOSEPH; MARTINS FERREIRA DE SOUZA, GUSTAVO HENRIQUE; SAMPAIO DIAS, ARTUR HERMANO; SKAF, MUNIR; DAMASIO, ANDRE. Redesigning N-glycosylation sites in a GH3 beta-xylosidase improves the enzymatic efficiency. BIOTECHNOLOGY FOR BIOFUELS, v. 12, n. 1 NOV 14 2019. Web of Science Citations: 0.
ZUBIETA, MARIANE PALUDETTI; CONTESINI, FABIANO JARES; RUBIO, MARCELO VENTURA; DE SOUZA SCHMIDT GONCALVES, ANY ELISA; GERHARDT, JAQUELINE ALINE; PRADE, ROLF ALEXANDER; DE LIMA DAMASIO, ANDRE RICARDO. Protein profile in Aspergillus nidulans recombinant strains overproducing heterologous enzymes. MICROBIAL BIOTECHNOLOGY, v. 11, n. 2, p. 346-358, MAR 2018. Web of Science Citations: 4.
CONTESINI, FABIANO JARES; LIBERATO, MARCELO VIZONA; RUBIO, MARCELO VENTURA; CALZADO, FELIPE; ZUBIETA, MARIANE PALUDETTI; RIANO-PACHON, DIEGO MAURICIO; SQUINA, FABIO MARCIO; BRACHT, FABRICIO; SKAF, MUNIR S.; DAMASIO, ANDRE RICARDO. Structural and functional characterization of a highly secreted alpha-L-arabinofuranosidase (GH62) from Aspergillus nidulans grown on sugarcane bagasse. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1865, n. 12, p. 1758-1769, DEC 2017. Web of Science Citations: 4.
DE LIMA DAMASIO, ANDRE RICARDO; RUBIO, MARCELO VENTURA; GONCALVES, THIAGO AUGUSTO; PERSINOTI, GABRIELA FELIX; SEGATO, FERNANDO; PRADE, ROLF ALEXANDER; CONTESINI, FABIANO JARES; DE SOUZA, AMANDA PEREIRA; BUCKERIDGE, MARCOS SILVEIRA; SQUINA, FABIO MARCIO. Xyloglucan breakdown by endo-xyloglucanase family 74 from Aspergillus fumigatus. Applied Microbiology and Biotechnology, v. 101, n. 7, p. 2893-2903, APR 2017. Web of Science Citations: 7.
DE VRIES, RONALD P.; RILEY, ROBERT; WIEBENGA, AD; AGUILAR-OSORIO, GUILLERMO; AMILLIS, SOTIRIS; UCHIMA, CRISTIANE AKEMI; ANDERLUH, GREGOR; ASADOLLAHI, MOJTABA; ASKIN, MARION; BARRY, KERRIE; BATTAGLIA, EVY; BAYRAM, OZGUR; BENOCCI, TIZIANO; BRAUS-STROMEYER, SUSANNA A.; CALDANA, CAMILA; CANOVAS, DAVID; CERQUEIRA, GUSTAVO C.; CHEN, FUSHENG; CHEN, WANPING; CHOI, CINDY; CLUM, ALICIA; CORREA DOS SANTOS, RENATO AUGUSTO; DE LIMA DAMASIO, ANDRE RICARDO; DIALLINAS, GEORGE; EMRI, TAMAS; FEKETE, ERZSEBET; FLIPPHI, MICHEL; FREYBERG, SUSANNE; GALLO, ANTONIA; GOURNAS, CHRISTOS; HABGOOD, ROB; HAINAUT, MATTHIEU; LAURA HARISPE, MARIA; HENRISSAT, BERNARD; HILDEN, KRISTIINA S.; HOPE, RYAN; HOSSAIN, ABEER; KARABIKA, EUGENIA; KARAFFA, LEVENTE; KARANYI, ZSOLT; KRASEVEC, NADA; KUO, ALAN; KUSCH, HARALD; LABUTTI, KURT; LAGENDIJK, ELLEN L.; LAPIDUS, ALLA; LEVASSEUR, ANTHONY; LINDQUIST, ERIKA; LIPZEN, ANNA; LOGRIECO, ANTONIO F.; MACCABE, ANDREW; MAKELA, MIIA R.; MALAVAZI, IRAN; MELIN, PETTER; MEYER, VERA; MIELNICHUK, NATALIA; MISKEI, MARTON; MOLNAR, AKOS P.; MULE, GIUSEPPINA; NGAN, CHEW YEE; OREJAS, MARGARITA; OROSZ, ERZSEBET; OUEDRAOGO, JEAN PAUL; OVERKAMP, KARIN M.; PARK, HEE-SOO; PERRONE, GIANCARLO; PIUMI, FRANCOIS; PUNT, PETER J.; RAM, ARTHUR F. J.; RAMON, ANA; RAUSCHER, STEFAN; RECORD, ERIC; RIANO-PACHON, DIEGO MAURICIO; ROBERT, VINCENT; ROEHRIG, JULIAN; RULLER, ROBERTO; SALAMOV, ASAF; SALIH, NADHIRA S.; SAMSON, ROB A.; SANDOR, ERZSEBET; SANGUINETTI, MANUEL; SCHUTZE, TABEA; SEPCIC, KRISTINA; SHELEST, EKATERINA; SHERLOCK, GAVIN; SOPHIANOPOULOU, VICKY; SQUINA, FABIOM.; SUN, HUI; SUSCA, ANTONIA; TODD, RICHARD B.; TSANG, ADRIAN; UNKLES, SHIELA E.; VAN DE WIELE, NATHALIE; VAN ROSSEN-UFFINK, DIANA; DE CASTRO OLIVEIRA, JULIANA VELASCO; VESTH, TAMMI C.; VISSER, JAAP; YU, JAE-HYUK; ZHOU, MIAOMIAO; ANDERSEN, MIKAEL R.; ARCHER, DAVID B.; BAKER, SCOTT E.; BENOIT, ISABELLE; BRAKHAGE, AXEL A.; BRAUS, GERHARD H.; FISCHER, REINHARD; FRISVAD, JENS C.; GOLDMAN, GUSTAVO H.; HOUBRAKEN, JOS; OAKLEY, BERL; POCSI, ISTVAN; SCAZZOCCHIO, CLAUDIO; SEIBOTH, BERNHARD; VANKUYK, PATRICIA A.; WORTMAN, JENNIFER; DYER, PAUL S.; GRIGORIEV, IGOR V. Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus. Genome Biology, v. 18, FEB 14 2017. Web of Science Citations: 99.
TRAMONTINA, ROBSON; FRANCO CAIRO, JOAO PAULO L.; LIBERATO, MARCELO V.; MANDELLI, FERNANDA; SOUSA, AMANDA; SANTOS, SAMANTHA; RABELO, SARITA CANDIDA; CAMPOS, BRUNA; IENCZAK, JACIANE; RULLER, ROBERTO; DAMASIO, ANDRE R. L.; SQUINA, FABIO MARCIO. The Coptotermes gestroi aldo-keto reductase: a multipurpose enzyme for biorefinery applications. BIOTECHNOLOGY FOR BIOFUELS, v. 10, JAN 3 2017. Web of Science Citations: 5.
CALZADO, FELIPE; PRATES, ERICA T.; GONCALVES, THIAGO A.; RUBIO, MARCELO V.; ZUBIETA, MARIANE P.; SQUINA, FABIO M.; SKAF, MUNIR S.; DAMASIO, ANDRE R. L. Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases. Biotechnology and Bioengineering, v. 113, n. 12, p. 2577-2586, DEC 2016. Web of Science Citations: 5.
VITCOSQUE, GABRIELA LEAL; RIBEIRO, LILIANE FRAGA COSTA; DE LUCAS, ROSYMAR COUTINHO; DA SILVA, TONY MARCIO; RIBEIRO, LUCAS FERREIRA; DE LIMA DAMASIO, ANDRE RICARDO; FARINAS, CRISTIANE SANCHEZ; LOPES GONCALVES, ALINE ZORZETTO; SEGATO, FERNANDO; BUCKERIDGE, MARCOS SILVEIRA; JORGE, JOAO ATILIO; POLIZELI, MARIA DE LOURDES T. M. The functional properties of a xyloglucanase (GH12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation. Applied Microbiology and Biotechnology, v. 100, n. 21, p. 9133-9144, NOV 2016. Web of Science Citations: 3.
GOLDBECK, ROSANA; GONCALVES, THIAGO A.; DAMASIO, ANDRE R. L.; BRENELLI, LIVIA B.; WOLF, LUCIA D.; PAIXAO, DOUGLAS A. A.; ROCHA, GEORGE J. M.; SQUINA, FABIO M. Effect of hemicellulolytic enzymes to improve sugarcane bagasse saccharification and xylooligosaccharides production. JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, v. 131, p. 36-46, SEP 2016. Web of Science Citations: 6.
RUBIO, MARCELO VENTURA; ZUBIETA, MARIANE PALUDETTI; LOURENO FRANCO CAIRO, JOAO PAULO; CALZADO, FELIPE; PAES LEME, ADRIANA FRANCO; SQUINA, FABIO MARCIO; PRADE, ROLF ALEXANDER; DE LIMA DAMASIO, ANDRE RICARDO. Mapping N-linked glycosylation of carbohydrate-active enzymes in the secretome of Aspergillus nidulans grown on lignocellulose. BIOTECHNOLOGY FOR BIOFUELS, v. 9, AUG 8 2016. Web of Science Citations: 10.
LIMA, MATHEUS S.; DAMASIO, ANDRE R. DE L.; CRNKOVIC, PAULA M.; PINTO, MARCELO R.; DA SILVA, ANA M.; DA SILVA, JEANC. R.; SEGATO, FERNANDO; DE LUCAS, ROSYMAR C.; JORGE, JOAO A.; POLIZELI, MARIA DE L. T. DE M. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment. FRONTIERS IN MICROBIOLOGY, v. 7, APR 28 2016. Web of Science Citations: 7.
FURTADO, GILVAN P.; SANTOS, CAMILA R.; CORDEIRO, ROSA L.; RIBEIRO, LUCAS F.; DE MORAES, LUIZ A. B.; DAMASIO, ANDR R. L.; POLIZELI, MARIA DE LOURDES T. M.; LOURENZONI, MARCOS R.; MURAKAMI, MARIO T.; WARD, RICHARD J. Enhanced xyloglucan-specific endo-beta-1,4-glucanase efficiency in an engineered CBM44-XegA chimera. Applied Microbiology and Biotechnology, v. 99, n. 12, p. 5095-5107, JUN 2015. Web of Science Citations: 10.
SEGATO, FERNANDO; DAMASIO, ANDRE R. L.; DE LUCAS, ROSYMAR C.; SQUINA, FABIO M.; PRADE, ROLF A. Genomics Review of Holocellulose Deconstruction by Aspergilli. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, v. 78, n. 4, p. 588-613, DEC 2014. Web of Science Citations: 36.

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