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Novel approaches to improve functional screening of biocatalysts in metagenomic libraries

Grant number: 15/04309-1
Support Opportunities:Research Grants - Young Investigators Grants
Duration: October 01, 2015 - September 30, 2021
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal Investigator:María Eugenia Guazzaroni
Grantee:María Eugenia Guazzaroni
Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated researchers:Rafael Silva Rocha ; Raquel Fonseca Maldonado
Associated scholarship(s):19/25432-7 - Searching for new molecular tools to enhance Pseudomonas putida robustness to abiotic stresses, BP.DR
20/01656-0 - Functional screening of antibiotic resistance genes in bacteria of clinical importance, BP.IC
20/02228-2 - Evaluation of cultivable microorganisms in risky sites for horizontal gene transfer of antibiotic resistance genes, BP.IC
+ associated scholarships 19/18789-6 - Determination of tolerance to various bacterial stress conditions, BP.TT
19/18783-8 - Characterization of acidity resistance in Pseudomonas putida using synthetic circuits, BP.IC
19/00390-0 - Resistome and mobilome in genomes of bacteria from diverse Brazilian regions, BP.MS
18/18158-3 - Search for determinants of antibiotic resistance in the interior of São Paulo using functional metagenomics, BP.MS
18/21160-0 - Identification of antimicrobial resistance genes in metagenomic data, BP.IC
18/18296-7 - Identification of genes encoding chaperones in metagenomic databases, BP.IC
18/19188-3 - Construction of a repertoire of metagenomic libraries in E. coli from DNA isolated from diverse environmental samples, BP.TT
18/11926-5 - Characterization of translation levels of variants of synthetic bacterial RBSs, BP.IC
18/07261-8 - Expanding acid resistance in bacteria using synthetic circuits, BP.MS
17/20818-9 - Characterisation of new metagenomics regulatory elements, BP.IC
16/18827-7 - Integrating protein engineering and synthetic biology to develop new high-throughput selection systems, BP.PD
16/06323-4 - New tools for improving metagenomic cellulase screenings, BP.DR
16/06922-5 - Functional screening of new proteases and glycosyl hydrolases in metagenomic libraries, BP.IC
16/01330-2 - Construction of a repertoire of metagenomic libraries in E. coli from DNA isolated from diverse environmental samples, BP.TT - associated scholarships

Abstract

Biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Currently, there is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in microbial genomes found in environmental samples and provides a set of new technologies directed towards screening of new catalytic activities with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal metagenomic screening strategies generally results in an extremely low success rate for enzymes identification. In this sense, the present proposal aims to perform three main approaches in order to develop new tools to improve target gene recovery. Firstly, two different strategies will be used to overcome the biased expression of foreign proteins in the screenings: (i) Construction of metagenomic libraries both in Pseudomonas putida and E. coli and; (ii) Utilization of the optimized and synthetic pSEVA broad host-range vector for library production. At present, there is no reported work in literature using a vector with similar features in functional metagenomic screenings. On the other hand, a third strategy comprising the engineering of a bacterial host will be developed using a synthetic biology approach. In particular, the strategy aims to improve the recovery rate of enzymes by exploiting the synergistic interaction between the metagenomic enzyme (or other accessory/complementary protein) and an enzyme expressed from the chromosome of the engineered host strain. Together, these approaches would provide a new, high efficient platform for the construction and functional screening of metagenomic libraries. (AU)

Articles published in Pesquisa FAPESP Magazine about the research grant:
Las bacterias en la UTI 
Bacteria in the ICU 
Articles published in Agência FAPESP Newsletter about the research grant:
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Scientific publications (27)
(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)
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, . (16/06323-4, 17/20818-9, 16/05472-6, 15/04309-1)
WESTMANN, CAUA ANTUNES; GUAZZARONI, MARA-EUGENIA; SILVA-ROCHA, RAFAEL. Engineering Complexity in Bacterial Regulatory Circuits for Biotechnological Applications. MSYSTEMS, v. 3, n. 2, SI, . (15/04309-1, 16/05472-6, 12/22921-8)
AMORES, GERARDO RUIZ; GUAZZARONI, MARIA-EUGENIA; ARRUDA, LETICIA MAGALHAES; SILVA-ROCHA, RAFAEL. Recent Progress on Systems and Synthetic Biology Approaches to Engineer Fungi As Microbial Cell Factories. CURRENT GENOMICS, v. 17, n. 2, p. 85-98, . (15/04309-1, 12/22921-8)
FONSECA, BRUNA CONSTANTE; GUAZZARONI, MARIA-EUGENIA; REGINATTO, VALERIA. Fermentative production of H-2 from different concentrations of galactose by the new isolate Clostridium beijerinckii Br21. INTERNATIONAL JOURNAL OF HYDROGEN ENERGY, v. 41, n. 46, p. 21109-21120, . (15/04309-1, 15/06074-1)
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, . (16/05472-6, 15/04309-1, 16/06323-4, 16/17582-0)
DE FRIAS, ULYSSES AMANCIO; BONIFACIO PEREIRA, GREICY KELLY; GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL. Boosting Secondary Metabolite Production and Discovery through the Engineering of Novel Microbial Biosensors. BIOMED RESEARCH INTERNATIONAL, . (15/04309-1, 12/22921-8)
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, . (16/06323-4, 15/04309-1, 16/05472-6, 17/20818-9)
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, . (16/19179-9, 17/17924-1, 16/06323-4, 18/04810-0, 15/04309-1, 12/22921-8)
RIBEIRO, LUCAS FERREIRA; LOPES, ERICA M.; KISHI, LUCIANO T.; COSTA RIBEIRO, LILIANE FRAGA; MENEGUETI, MAYRA GONCALVES; GASPAR, GILBERTO GAMBERO; SILVA-ROCHA, RAFAEL; GUAZZARONI, MARIA-EUGENIA. Microbial Community Profiling in Intensive Care Units Expose Limitations in Current Sanitary Standards. FRONTIERS IN PUBLIC HEALTH, v. 7, . (15/04309-1, 16/20358-5, 16/18827-7)
NORA, LUISA CZAMANSKI; WESTMANN, CAUA ANTUNES; GUAZZARONI, MARIA-EUGENIA; SIDDAIAH, CHANDRANAYAKA; GUPTA, VIJAI KUMAR; SILVA-ROCHA, RAFAEL. Recent advances in plasmid-based tools for establishing novel microbial chassis. BIOTECHNOLOGY ADVANCES, v. 37, n. 8, . (16/03763-3, 15/04309-1, 16/05472-6, 12/22921-8)
KIKUTI MANCILIO, LUCCA BONJY; RIBEIRO, GUILHERME AUGUSTO; RODRIGUES DE ALMEIDA, ERICA JANAINA; VIANA DE SIQUEIRA, GUILHERME MARCELINO; ROCHA, RAFAEL SILVA; GUAZZARONI, MARIA-EUGENIA; DE ANDRADE, ADALGISA RODRIGUES; REGINATTO, VALERIA. Adding value to lignocellulosic byproducts by using acetate and p-coumaric acid as substrate in a microbial fuel cell. INDUSTRIAL CROPS AND PRODUCTS, v. 171, . (18/05454-3, 19/25432-7, 18/15528-4, 14/50945-4, 18/12471-1, 19/15675-0, 15/04309-1)
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., . (16/05472-6, 16/06323-4, 17/17924-1, 16/19179-9, 16/03763-3, 12/22921-8, 15/04309-1)
RIBEIRO, LUCAS F.; AMARELLE, VANESA; RIBEIRO, LILIANE F. C.; GUAZZARONI, MARIA-EUGENIA. Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion. BIOMED RESEARCH INTERNATIONAL, v. 2019, p. 15-pg., . (16/18827-7, 16/20358-5, 15/04309-1)
WESTMANN, CAUA ANTUNES; GUAZZARONI, MARA-EUGENIA; SILVA-ROCHA, RAFAEL. Engineering Complexity in Bacterial Regulatory Circuits for Biotechnological Applications. MSYSTEMS, v. 3, n. 2, p. 4-pg., . (16/05472-6, 12/22921-8, 15/04309-1)
DE SIQUEIRA, V, GUILHERME M.; SILVA-ROCHA, RAFAEL; GUAZZARONI, MARIA-EUGENIA. Turning the Screw: Engineering Extreme pH Resistance in Escherichia coli through Combinatorial Synthetic Operons. ACS SYNTHETIC BIOLOGY, v. 9, n. 6, p. 1254-1262, . (15/04309-1, 18/07261-8)
FONSECA, BRUNA CONSTANTS; RIANO-PACHON, DIEGO MAURICIO; GUAZZARONI, MARIA-EUGENIA; REGINATTO, VALERIA. Genome sequence of the H-2-producing Clostridium beijerinckii strain Br21 isolated from a sugarcane vinasse treatment plant. GENETICS AND MOLECULAR BIOLOGY, v. 42, n. 1, p. 139-144, . (15/04309-1, 15/06074-1)
RIBEIRO, LUCAS F.; AMARELLE, VANESA; RIBEIRO, LILIANE F. C.; GUAZZARONI, MARIA-EUGENIA. Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion. BIOMED RESEARCH INTERNATIONAL, . (16/20358-5, 15/04309-1, 16/18827-7)
AMARELLE, VANESA; SANCHES-MEDEIROS, ANANDA; SILVA-ROCHA, RAFAEL; GUAZZARONI, MARIA-EUGENIA. Expanding the Toolbox of Broad Host-Range Transcriptional Terminators for Proteobacteria through Metagenomics. ACS SYNTHETIC BIOLOGY, v. 8, n. 4, p. 647-654, . (18/04810-0, 15/04309-1)
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, . (16/19179-9, 17/17924-1, 16/06323-4, 16/05472-6, 16/03763-3, 15/04309-1, 12/22921-8)
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, . (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, . (15/04309-1, 16/05472-6, 17/20818-9, 16/06323-4)
KIKUTI MANCILIO, LUCCA BONJY; RIBEIRO, GUILHERME AUGUSTO; LOPES, ERICA MENDES; KISHI, LUCIANO TAKESHI; MARTINS-SANTANA, LEONARDO; VIANA DE SIQUEIRA, GUILHERME MARCELINO; ANDRADE, ADALGISA RODRIGUES; GUAZZARONI, MARIA-EUGENIA; REGINATTO, VALERIA. Unusual microbial community and impact of iron and sulfate on microbial fuel cell ecology and performance. CURRENT RESEARCH IN BIOTECHNOLOGY, v. 2, p. 10-pg., . (18/00789-7, 18/15528-4, 15/04309-1, 16/18827-7)
CAMPOS ANTONIETO, AMANDA CRISTINA; VIEIRA NOGUEIRA, KAROLINE MARIA; DE PAULA, RENATO GRACIANO; NORA, LUFSA CZAMANSKI; ANZOLINI CASSIANO, MURILO HENRIQUE; GUAZZARONI, MARIA-EUGENIA; ALMEIDA, FAUSTO; DA SILVA, THIAGO APARECIDO; RIES, LAURE NICOLAS ANNICK; DE ASSIS, LEANDRO JOSE; et al. A Novel Cys2His2 Zinc Finger Homolog of AZF1 Modulates Holocellulase Expression in Trichoderma reesei. MSYSTEMS, v. 4, n. 4, . (16/23233-9, 16/03322-7, 15/04309-1, 17/04217-5, 16/03763-3, 18/03766-8, 15/09553-8, 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, . (16/18827-7, 15/04309-1, 18/18158-3, 18/07261-8, 19/00390-0, 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., . (17/20818-9, 16/05472-6, 16/06323-4, 15/04309-1)
DE FRIAS, ULYSSES AMANCIO; BONIFACIO PEREIRA, GREICY KELLY; GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL. Boosting Secondary Metabolite Production and Discovery through the Engineering of Novel Microbial Biosensors. BIOMED RESEARCH INTERNATIONAL, v. 2018, p. 11-pg., . (12/22921-8, 15/04309-1)
BORELLI, TIAGO CABRAL; LOVATE, GABRIEL LENCIONI; SCARANELLO, ANA FLAVIA TONELLI; RIBEIRO, LUCAS FERREIRA; ZARAMELA, LIVIA; PEREIRA-DOS-SANTOS, FELIPE MARCELO; SILVA-ROCHA, RAFAEL; GUAZZARONI, MARIA-EUGENIA. Combining Functional Genomics and Whole-Genome Sequencing to Detect Antibiotic Resistance Genes in Bacterial Strains Co-Occurring Simultaneously in a Brazilian Hospital. ANTIBIOTICS-BASEL, v. 10, n. 4, . (19/00390-0, 15/04309-1, 19/06672-7, 16/18827-7, 19/15675-0, 18/18158-3)

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