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.
Highlights
IntroductionRenewable resources, such as plant biomass (essentially lignocellulose), have a significant potential for the production of biofuels and other biotech-produced industrial chemicals due to their higher abundancy and lower price in comparison to other commercial substrates (Simmons et al, 2010)
Renewable resources, such as plant biomass, have a significant potential for the production of biofuels and other biotech-produced industrial chemicals due to their higher abundancy and lower price in comparison to other commercial substrates (Simmons et al, 2010)
To the screening for enzymes in the selected skimmed milk agar (SMA)-PR media (Figure 1A), we carried out controls for testing the phenotype of clones carrying pSEVA232, the minimal and modular vector used in the construction of the metagenomic library (Figure 1B)
Summary
Renewable resources, such as plant biomass (essentially lignocellulose), have a significant potential for the production of biofuels and other biotech-produced industrial chemicals due to their higher abundancy and lower price in comparison to other commercial substrates (Simmons et al, 2010). Strong vector expression signals (e.g., promoter and ribosome binding site) are used to guarantee that small DNA fragments (2-10 kb) cloned in the vector reach a good chance of being expressed and detected by activity screens (Ferrer et al, 2008; Guazzaroni et al, 2015) At this point, it is of particular relevance mentioning that lacZa-based vectors are frequently used in different screenings, with high prevalence in small-insert expression metagenomic libraries (Lämmle et al, 2007; Mirete et al, 2007; Guazzaroni et al, 2013; Morgante et al, 2015; Gao et al, 2016; Zhou et al, 2016). The blue/white screening, inherent of a-based vectors is one of the most common molecular techniques that allows detecting the successful ligation, and subsequently expressing the gene of interest in a vector (Zamenhof and Villarejo, 1972; Langley et al, 1975; Ausubel et al, 2003)
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