Boundaries in metagenomic screenings using lacZα-based vectors

Author(s)

Luana de Fátima Alves ; Tiago Cabral Borelli ^[2] ; Cauã Antunes Westmann ^[3] ; Rafael Silva-Rocha ^[4] ; María-Eugenia Guazzaroni ^[5]

• Document type: Journal article
• Source: GENETICS AND MOLECULAR BIOLOGY; v. 43, n. 1 2020-03-06.

Abstract

Abstract 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 lacZα -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 lacZα-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 lacZα-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: 16/06323-4 - New tools for improving metagenomic cellulase screenings
• Grantee: Luana de Fátima Alves
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 17/20818-9 - Characterisation of new metagenomics regulatory elements
• Grantee: Tiago Cabral Borelli
• Support Opportunities: Scholarships in Brazil - Scientific Initiation