Big trouble with little inserts: boundaries in metagenomic screenings usinglacZα based vectors

Abstract

21The vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening 22 and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been 23 of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, 24 which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable 25resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities 26 from microbial communities inhabiting a soil sample by using the lacZα-based plasmid pSEVA232 in the generation 27 of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH 28 indicator dye as previously reported in literature. Although we effectively identified nine positive clones in the 29 screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded 30 in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within 31 the coding region of the lacZα gene present in the original vector. We concluded that the current method has a higher 32 tendency for false positive clones' recovery, when used in combination with a lacZα-based vector. Finally, we discuss 33 the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in 34 metagenomic screenings methodologies. 35