Biology subjects

Westmann, C. A.

Publications and source records attributed to Westmann, C. A..

2 recordsLinked to original sources

bioRxiv2017

Assessing and characterising the repertoire of constitutive promoter elements in soil metagenomic libraries in Escherichia coli

Although functional metagenomics has been widely employed for the discovery of genes relevant to biotechnology and biomedicine, its potential for assessing the diversity of transcriptional regulatory elements of microbial communities has remained poorly explored. Here, we have developed a novel framework for prospecting, characterising and estimating the accessibility of promoter sequences in metagenomic libraries by combining a bi-directional reporter vector, high-throughput fluorescence assays and predictive computational methods. Using the expression profiling of fluorescent clones from two independent libraries from soil samples, we directly analysed the regulatory dynamics of novel promoter elements, addressing the relationship between the \"metaconstitutome\" of a bacterial community and its environmental context. Through the construction and screening of plasmid-based metagenomic libraries followed by in silico analyses, we were able to provide both (i) a consensus exogenous promoter elements recognizable by Escherichia coli and (ii) an estimation of the accessible promoter sequences in a metagenomic library, which was close to 1% of the whole set of available promoters. The results presented here should provide new directions for the exploration through functional metagenomics of novel regulatory sequences in bacteria, which could expand the Synthetic Biology toolbox for novel biotechnological and biomedical applications.

microbiology↗

bioRxiv2017

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

The vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from microbial communities inhabiting a soil sample by using the lacZ-based plasmid pSEVA232 in the generation of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye as previously reported in literature. Although we effectively identified nine 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 alpha gene present in the original vector. We concluded that the current method has a higher tendency for false positive recovery of clones, when used in combination with a lacZ-based vector. Finally, we discuss the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in metagenomic screenings methodologies.

molecular biology↗