Enzymatic oxidation of sugarcane bagasse: discovery, characterization and new application of oxidative enzymes active in carbohydrates, applied to the enhancement of a fungal cell factory

• Grant number: 14/18714-2
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants
• Duration: May 01, 2015 - December 31, 2019
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Fernando Segato
• Grantee: Fernando Segato
• Host Institution: Escola de Engenharia de Lorena (EEL). Universidade de São Paulo (USP). Lorena , SP, Brazil
• Associated grant(s): 21/06679-1 - Enzymatic oxidation of sugarcane bagasse 2: exploring the interaction between LPMOs and its redox partner CDHs aiming for the development of more efficient enzymes for insertion into an engineered cell factory, AP.BIOEN.JP2; 17/50025-0 - Development of lignin active enzyme mixture to produce sugars from lignocellulosic biomass for production of fuels, chemicals and bio based materials, AP.R SPRINT
• Associated scholarship(s): 16/09997-6 - Heterologous expression, characterization and comparison of two endoglucanases from family GH45 obtained from the ascomycete Myceliophthora thermophila and the basidiomycete Gloeophyllum trabeum genomes, BP.IC; 15/08079-0 - Myceliophthora thermophila as a chassis to the development of an efficient cell factory to secrete enzymes active in lignocellulosic materials, BP.IC

Abstract

Lignocellulosic biomass is an abundant source of polysaccharides that can be used as a renewable raw material to produce biofuels and other compounds. The conversion of these polysaccharides into fermentable sugars by enzymatic pathway still is a slow and expensive process, which a fast and efficient process still remains to be discovered. Until recently, the available commercial preparations had in your repertoire enzymes that the mechanism to hydrolyze components of lignocellulose were based in acid-base mechanism, which are not efficient to hydrolyze crystalline cellulose and lignin. Recently, proteins with oxidative mechanisms working as auxiliary enzymes acting directly on crystalline cellulose called LPMOs have been discovered. Since then, those enzymes were added to the available cocktails improving it’s efficient. Analyzing the secretome of thermophilic fungi Aspergillus niveus and Myceliophthora thermophila, we found that these organisms have different mechanisms (hydrolytic and oxidative, respectively), to degrade lignocellulosic materials. When the enzymatic extracts of the two species were mixed, the activity was increased five-fold. Therefore, this proposal will aim to 1) discover new oxidative protein working as electron acceptor obtained from the thermophilic fungus M. thermophila, participating in combination with acid-base mechanisms in the degradation of lignocellulosic biomass, followed for its biochemical and structural characterization; 2) Development of a multigenic expression system for A. nidulans and or A. niveus based in BioBricks combination and using Gibson Assembly method 3) combine the synergistic actions of oxidative and hydrolytic mechanism of two thermophilic microorganisms in a single cell with thermophilic characteristics and study the effects of this complementation during the time course of the lignocellulosic biomass breakdown. (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)

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)

OLIVEIRA, DYONI M.; MOTA, THATIANE R.; OLIVA, BIANCA; SEGATO, FERNANDO; MARCHIOSI, ROGERIO; FERRARESE-FILHO, OSVALDO; FAULDS, CRAIG B.; DOS SANTOS, WANDERLEY D.. Feruloyl esterases: Biocatalysts to overcome biomass recalcitrance and for the production of bioactive compounds. Bioresource Technology, v. 278, p. 408-423, APR 2019. (14/18714-2)

LIU, ENSHI; SEGATO, FERNANDO; PRADE, ROLF A.; WILKINS, MARK R.. xploring lignin depolymerization by a bi-enzyme system containing aryl alcohol oxidase and lignin peroxidase in aqueous biocompatible ionic liquid. Bioresource Technology, v. 338, OCT 2021. (14/18714-2, 17/50025-0)

QUEIROZ, SARAH S.; OLIVA, BIANCA; SILVA, TATIANE F.; SEGATO, FERNANDO; FELIPE, MARIA G. A.. Integrated bioinformatics, modelling, and gene expression analysis of the putative pentose transporter from Candida tropicalis during xylose fermentation with and without glucose addition. Applied Microbiology and Biotechnology, v. N/A, p. 20-pg., 2022-06-16. (19/22284-7, 19/27092-9, 14/18714-2, 16/22179-0, 19/06663-8)

ASCENCIO, JESUS J.; PHILIPPINI, RAFAEL R.; GOMES, FABRICIO M.; PEREIRA, FELIX M.; DA SILVA, SILVIO S.; KUMAR, VINOD; CHANDEL, ANUJ K.. Comparative Highly Efficient Production of beta-glucan by Lasiodiplodia theobromae CCT 3966 and Its Multiscale Characterization. FERMENTATION-BASEL, v. 7, n. 3, SEP 2021. (16/10636-8, 16/14852-7, 14/18714-2, 08/57926-4)

ABDELLA, ASMAA; SEGATO, FERNANDO; WILKINS, MARK R.. Optimization of nutrient medium components for production of a client endo-beta-1,4-xylanase from Aspergillus fumigatus var. niveus using a recombinant Aspergillus nidulans strain. BIOCATALYSIS AND AGRICULTURAL BIOTECHNOLOGY, v. 20, JUL 2019. (14/06923-6, 14/18714-2)

LIU, ENSHI; SEGATO, FERNANDO; WILKINS, MARK R.. Fed-batch production of Thermothelomyces thermophilus lignin peroxidase using a recombinant Aspergillus nidulans strain in stirred-tank bioreactor. Bioresource Technology, v. 325, APR 2021. (14/18714-2, 17/50025-0)

SEGATO, FERNANDO; DIAS, BRUNO; BERTO, GABRIELA L.; DE OLIVEIRA, DYONI M.; DE SOUZA, FLAVIO H. M.; CITADINI, ANA PAULA; MURAKAMI, MARIO T.; DAMASIO, ANDRE R. L.; SQUINA, FABIO MARCIO; POLIKARPOV, IGOR. Cloning, heterologous expression and biochemical characterization of a non-specific endoglucanase family 12 from Aspergillus terreus NIH2624. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1865, n. 4, p. 395-403, APR 2017. (09/52840-7, 14/18714-2, 13/21545-5)

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. (11/02169-7, 08/58037-9, 12/20549-4, 14/18714-2)

LIU, ENSHI; SEGATO, FERNANDO; PRADE, ROLF A.; WILKINS, MARK R.. Exploring lignin depolymerization by a bi-enzyme system containing aryl alcohol oxidase and lignin peroxidase in aqueous biocompatible ionic liquids. APPLIED GEOCHEMISTRY, v. 131, AUG 2021. (14/18714-2, 17/50025-0)

VELASCO, JOSMAN; OLIVA, BIANCA; GONCALVES, ALINE LARISSA; LIMA, AWANA SILVA; FERREIRA, GISLENE; FRANCA, BRUNO ALVES; MULINARI, EVANDRO JOSE; GONCALVES, THIAGO AUGUSTO; SQUINA, FABIO MARCIO; SEIKI KADOWAKI, MARCO ANTONIO; et al. Functional characterization of a novel thermophilic exo-arabinanase fromThermothielavioides terrestris. Applied Microbiology and Biotechnology, v. 104, n. 19, AUG 2020. (14/18714-2, 17/16089-1, 14/06923-6, 19/01165-0, 15/50590-4, 19/06663-8)

BARRIOS-GUTIERREZ, SOLANGE GRACE; VELEZ-MERCADO, MARTHA INES; ORTEGA, JULIA RODRIGUES; LIMA, AWANA DA SILVA; SARAIVA, ANA LUIZA DA ROCHA FORTES; BERTO, GABRIELA LEILA; SEGATO, FERNANDO. Oxidative Machinery of basidiomycetes as potential enhancers in lignocellulosic biorefineries: A lytic polysaccharide monooxygenases approach. Bioresource Technology, v. 386, p. 11-pg., 2023-10-01. (22/00539-6, 22/04227-9, 14/18714-2, 19/22284-7, 22/04713-0, 21/06679-1)

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; et al. 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. (10/10184-3, 14/18714-2, 12/20549-4)

VIEIRA, F. R.; PECCHIA, J. A.; SEGATO, F.; POLIKARPOV, I.. Exploring oyster mushroom (Pleurotus ostreatus) substrate preparation by varying phase I composting time: changes in bacterial communities and physicochemical composition of biomass impacting mushroom yields. Journal of Applied Microbiology, v. 126, n. 3, p. 931-944, MAR 2019. (13/21545-5, 14/18714-2, 15/13684-0, 09/52840-7)

MACHADO, ANGELA S.; VALADARES, FERNANDA; SILVA, TATIANE F.; MILAGRES, ADRIANE M. F.; SEGATO, FERNANDO; FERRAZ, ANDRE. The Secretome ofPhanerochaete chrysosporiumandTrametes versicolorGrown in Microcrystalline Cellulose and Use of the Enzymes for Hydrolysis of Lignocellulosic Materials. FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, v. 8, JUL 17 2020. (14/18714-2, 14/06923-6)

VIEIRA, MATHEUS MAITAN; KADOGUCHI, ELEN; SEGATO, FERNANDO; DA SILVA, SILVIO S.; CHANDEL, ANUJ K.. Production of cellulases byAureobasidium pullulansLB83: optimization, characterization, and hydrolytic potential for the production of cellulosic sugars. PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, v. 51, n. 2, AUG 2020. (14/18714-2, 16/14852-7, 16/10636-8, 08/57926-4)

VELASCO, JOSMAN; PELLEGRINI, VANESSA DE OLIVEIRA ARNOLDI; SEPULCHRO, ANA GABRIELA VEIGA; KADOWAKI, MARCO ANTONIO SEIKI; SANTO, MELISSA CRISTINA ESPIRITO; POLIKARPOV, IGOR; SEGATO, FERNANDO. Comparative analysis of two recombinant LPMOs from Aspergillus fumigatus and their effects on sugarcane bagasse saccharification. Enzyme and Microbial Technology, v. 144, MAR 2021. (19/22284-7, 15/13684-0, 14/06923-6, 14/18714-2, 19/01165-0, 17/50025-0)

VELASCO, JOSMAN; SEPULCHRO, ANA GABRIELA V.; HIGASI, PAULA M. R.; PELLEGRINI, VANESSA O. A.; CANNELLA, DAVID; DE OLIVEIRA, LEANDRO CRISTANTE; POLIKARPOV, IGOR; SEGATO, FERNANDO. Light Boosts the Activity of Novel LPMO from Aspergillus fumigatus Leading to Oxidative Cleavage of Cellulose and Hemicellulose. ACS SUSTAINABLE CHEMISTRY & ENGINEERING, v. 10, n. 50, p. 16-pg., 2022-12-08. (19/22284-7, 18/22300-0, 19/01165-0, 21/06679-1, 14/18714-2, 15/13684-0)

CAMARGO, SUELEN; MULINARI, EVANDRO J.; DE ALMEIDA, LEONARDO R.; BERNARDES, AMANDA; PRADE, ROLF A.; GARCIA, WANIUS; SEGATO, FERNANDO; MUNIZ, JOAO R. C.. Functional and structural characterization of an alpha-L-arabinofuranosidase from Thermothielavioides terrestris and its exquisite domain-swapped beta-propeller fold crystal packing. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1868, n. 12, p. 12-pg., 2020-12-01. (17/16291-5, 16/09152-6, 14/18714-2, 14/50897-0, 19/22284-7, 17/50025-0)