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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Boundaries in metagenomic screenings using lacZ alpha-based vectors

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Alves, Luana de Fatima [1, 2] ; Borelli, Tiago Cabral [2] ; Westmann, Caua Antunes [3] ; Silva-Rocha, Rafael [3] ; Guazzaroni, Maria-Eugenia [2]
Total Authors: 5
[1] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, Ribeirao Preto, SP - Brazil
[2] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, Av Bandeirantes 3-900, BR-14049901 Ribeirao Preto, SP - Brazil
[3] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Biol Celular & Mol, Ribeirao Preto, SP - Brazil
Total Affiliations: 3
Document type: Journal article
Source: GENETICS AND MOLECULAR BIOLOGY; v. 43, n. 1 2020.
Web of Science Citations: 0

Metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from a soil sample by using the lacZ alpha -based plasmid pSEVA232. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye for detection of both enzymes, as previously reported in literature. Although we effectively identified positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZ gene present in the original vector. Analyses of the thermodynamic stability of mRNA secondary structures indicated that recovering of positive clones was probably due to higher expression levels of the chimeric lacZ alpha-genes in respect to the original from empty vector. We concluded that this method has a higher tendency for recovery false positive clones, when used in combination with a lacZ alpha-based vector. As these vectors are massively used in functional metagenomic screenings, we highlight the importance of reporting boundaries in established metagenomic screenings methodologies. (AU)

FAPESP's process: 15/04309-1 - Novel approaches to improve functional screening of biocatalysts in metagenomic libraries
Grantee:María Eugenia Guazzaroni
Support Opportunities: Research Grants - Young Investigators Grants
FAPESP's process: 16/05472-6 - Synthetic biology approaches to engineer/mine novel regulatory elements in bacteria
Grantee:Cauã Antunes Westmann
Support Opportunities: Scholarships in Brazil - Master
FAPESP's process: 17/20818-9 - Characterisation of new metagenomics regulatory elements
Grantee:Tiago Cabral Borelli
Support Opportunities: Scholarships in Brazil - Scientific Initiation
FAPESP's process: 16/06323-4 - New tools for improving metagenomic cellulase screenings
Grantee:Luana de Fátima Alves
Support Opportunities: Scholarships in Brazil - Doctorate