New tools for improving metagenomic cellulase screenings

• Grant number: 16/06323-4
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: October 01, 2016
• End date: April 30, 2021
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: María Eugenia Guazzaroni
• Grantee: Luana de Fátima Alves
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 15/04309-1 - Novel approaches to improve functional screening of biocatalysts in metagenomic libraries, AP.JP
• Associated scholarship(s): 18/16191-3 - Engineering Saccharomyces cerevisiae strains to improve acid- and lignocellulosic inhibitor-tolerance, BE.EP.DR

Abstract

Conversion of lignocellulosic biomass into biofuels is a promissory alternative to replace fossil fuels derived from nonrenewable energy sources. Lignocellulose consists primarily of cellulose, hemicellulose and lignin. Cellulose is the most recalcitrant material present in plant cell walls, but has a substantial potential as a bioethanol production source. Currently, industry requires enzymes with tolerance to process-specific parameters or improved catalytic performance. In this way, metagenomics allows the identification of new biocatalysts with specific activities without the need of previous isolation and cultivation of microorganisms. Several examples reported in literature showed the identification of new enzymes and other bio-molecules from different environments through functional metagenomic screening. However, the discovery of new genes by metagenomic approaches has been limited. The probability of identifying a target gene depends on many factors that are intrinsically linked. For instance, the efficiency of heterologous gene expression of a target gene from a metagenomic DNA fragment is restricted by the molecular machinery of the heterologous host organism employed in the screenings and the use of non-optimized vectors resulting in low enzyme identification rates. Previous studies suggest that about 40% of the enzymatic activities may be readily recovered by random cloning in Escherichia coli, the most frequently bacteria used for functional metagenomic screenings. In order to circumvent these limitations, the present proposal aims to develop new tools to improve target gene recovery in functional metagenomic screenings. For this, it is proposed (i) construct metagenomic libraries in broad-host-range vectors (pSEVA) and (ii) developof a new vector pointing to improve the screening strategy, the pCELsyn vector which is derived from a pSEVA vector. In this sense, pCELsyn plasmid harboring the Cel5A endoglucanase from Bacillus subtilis will be used in synergistic screenings in order to increase celullase activities identification. The screenings will be performed in two hosts: E. coli and Pseudomonas putida, aiming to sort out the insufficient or biased expression of foreign genes in E. coli, as described by several studies. The metagenomic libraries will be generated from biomass-rich soil samples since it probably will be enriched in genes encoding for cellulases. Functional screenings will be perform using cellulase screening methodologies well-established in literature. Thus, it is expected to improve the efficiency of enzyme recovery in metagenomic screenings.

Scientific publications

(11)

(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)

ALVES, LUANA DE FATIMA; WESTMANN, CAUA ANTUNES; LOVATE, GABRIEL LENCIONI; VIANA DE SIQUEIRA, GUILHERME MARCELINO; BORELLI, TIAGO CABRAL; GUAZZARONI, MARIA-EUGENIA. Metagenomic Approaches for Understanding New Concepts in Microbial Science. INTERNATIONAL JOURNAL OF GENOMICS, 2018. (16/06323-4, 15/04309-1, 16/05472-6, 17/20818-9)

LUANA DE FÁTIMA ALVES; TIAGO CABRAL BORELLI; CAUÃ ANTUNES WESTMANN; RAFAEL SILVA-ROCHA; MARÍA-EUGENIA GUAZZARONI. Boundaries in metagenomic screenings using lacZα-based vectors. GENETICS AND MOLECULAR BIOLOGY, v. 43, n. 1, 2020-03-06. (16/06323-4, 17/20818-9, 16/05472-6, 15/04309-1)

ALVES, LUANA DE FATIMA; MELEIRO, LUANA PARRAS; SILVA, ROBERTO N.; WESTMANN, CAUA ANTUNES; GUAZZARONI, MARIA-EUGENIA. Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated beta-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome. FRONTIERS IN MICROBIOLOGY, v. 9, OCT 29 2018. (16/05472-6, 15/04309-1, 16/06323-4, 16/17582-0)

OLIVEIRA MONTEIRO, LUMMY MARIA; ARRUDA, LETICIA MAGALHAES; SANCHES-MEDEIROS, ANANDA; MARTINS-SANTANA, LEONARDO; DE FATIMA ALVES, LUANA; DEFELIPE, LUCAS; GUSTAVO TURJANSKI, ADRIAN; GUAZZARONI, MARIA-EUGENIA; DE LORENZO, VICTOR; SILVA-ROCHA, RAFAEL. Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli. ACS SYNTHETIC BIOLOGY, v. 8, n. 8, p. 1890-1900, AUG 2019. (16/19179-9, 17/17924-1, 16/06323-4, 18/04810-0, 15/04309-1, 12/22921-8)

NORA, LUISA CZAMANSKI; WESTMANN, CAUA ANTUNES; MARTINS-SANTANA, LEONARDO; ALVES, LUANA DE FATIMA; OLIVEIRA MONTEIRO, LUMMY MARIA; GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL. The art of vector engineering: towards the construction of next-generation genetic tools. MICROBIAL BIOTECHNOLOGY, v. 12, n. 1, p. 23-pg., 2019-01-01. (16/05472-6, 16/06323-4, 17/17924-1, 16/19179-9, 16/03763-3, 12/22921-8, 15/04309-1)

ALVES, LUANA DE FATIMA; BORTOLUCCI, JONATA; REGINATTO, VALERIA; GUAZZARONI, MARIA-EUGENIA; MUSSATTO, SOLANGE I.. Improving Saccharomyces cerevisiae acid and oxidative stress resistance using a prokaryotic gene identified by functional metagenomics. HELIYON, v. 9, n. 4, p. 12-pg., 2023-04-01. (21/01748-5, 16/06323-4, 20/03168-3, 18/16191-3)

NORA, LUISA CZAMANSKI; WESTMANN, CAUA ANTUNES; MARTINS-SANTANA, LEONARDO; ALVES, LUANA DE FATIMA; OLIVEIRA MONTEIRO, LUMMY MARIA; GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL. The art of vector engineering: towards the construction of next-generation genetic tools. MICROBIAL BIOTECHNOLOGY, v. 12, n. 1, p. 125-147, JAN 2019. (16/19179-9, 17/17924-1, 16/06323-4, 16/05472-6, 16/03763-3, 15/04309-1, 12/22921-8)

RIBEIRO, LUCAS FERREIRA; AMARELLE, VANESA; ALVES, LUANA DE FATIMA; VIANA DE SIQUEIRA, GUILHERME MARCELINO; LOVATE, GABRIEL LENCIONI; BORELLI, TIAGO CABRAL; GUAZZARONI, MARIA-EUGENIA. Genetically Engineered Proteins to Improve Biomass Conversion: New Advances and Challenges for Tailoring Biocatalysts. Molecules, v. 24, n. 16, AUG 2019. (16/18827-7, 15/04309-1, 18/18158-3, 18/07261-8, 19/00390-0, 16/06323-4)

WESTMANN, CAUA A.; ALVES, LUANA DE FATIMA; SILVA-ROCHA, RAFAEL; GUAZZARONI, MARIA-EUGENIA. Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli. FRONTIERS IN MICROBIOLOGY, v. 9, JUN 20 2018. (16/06323-4, 15/04309-1, 16/05472-6)

ALVES, LUANA DE FATIMA; BORELLI, TIAGO CABRAL; WESTMANN, CAUA ANTUNES; SILVA-ROCHA, RAFAEL; GUAZZARONI, MARIA-EUGENIA. Boundaries in metagenomic screenings using lacZ alpha-based vectors. GENETICS AND MOLECULAR BIOLOGY, v. 43, n. 1, 2020. (15/04309-1, 16/05472-6, 17/20818-9, 16/06323-4)

ALVES, LUANA DE FATIMA; WESTMANN, CAUA ANTUNES; LOVATE, GABRIEL LENCIONI; VIANA DE SIQUEIRA, GUILHERME MARCELINO; BORELLI, TIAGO CABRAL; GUAZZARONI, MARIA-EUGENIA. Metagenomic Approaches for Understanding New Concepts in Microbial Science. INTERNATIONAL JOURNAL OF GENOMICS, v. 2018, p. 15-pg., 2018-01-01. (17/20818-9, 16/05472-6, 16/06323-4, 15/04309-1)