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

• Grant number: 13/24988-5
• Support type: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): January 01, 2014
• Effective date (End): June 30, 2018
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal researcher: André Ricardo de Lima Damasio
• Grantee: Marcelo Ventura Rubio
• Home Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 12/20549-4 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases, AP.BIOEN.JP

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

(5)

(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)

ZUBIETA, MARIANE P.; GERHARDT, JAQUELINE A.; RUBIO, MARCELO V.; TERRASAN, CESAR R. F.; PERSINOTI, GABRIELA F.; ANTONIEL, EVERTON P.; CONTESINI, FABIANO J.; PRADE, ROLF A.; DAMASIO, ANDRE. Improvement of homologous GH10 xylanase production by deletion of genes with predicted function in the Aspergillus nidulans secretion pathway. MICROBIAL BIOTECHNOLOGY, v. 13, n. 4 MAR 2020. Web of Science Citations: 2.

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.

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: 6.

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: 3.

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: 8.