Ferrer González, Edgar F.

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    Biocatalytic activity screening for the advanced glycation end product (Nε-Carboxymethyl-Lysine) in metagenomics libraries
    (2015) Ferrer González, Edgar F.; Ríos Velázquez, Carlos; College of Arts and Sciencies - Sciences; Martínez Cruzado, Juan C.; Ortiz Bermúdez, Patricia; Department of Biology; López Moreno, Martha L.
    Throughout the years, our body tends to accumulate a great variety of small molecules that are incapable of being metabolized or eradicated from our system. As the concentration of these molecules increases, they start having ill effects on our health and are often related to a great variety of aging diseases. For instance, the Advanced Glycation End product known as Nε- (carboxymethyl)-Lysine (CML) plays an important role in the development of secondary pathologies associated with diabetes. The CML has also been found to induce steatosis in non-alcoholic fatty liver. Even though the production of CML can be inhibited, the small molecule remains present in the affected tissue and crossed-linked proteins. Thus, the development of enzymatic based treatments that could remove substantially the accumulated CML would be highly beneficial to our society. In order to find a biocatalytic agent for CML, we implemented functional metagenomics by screening on minimal medium supplemented with CML. Four metagenomic libraries were screened using this method: one from the sub-tropical rainforest, El Yunque, another one from the dry forest at Guánica, other one from benthic microbial mat and the last one from an ephemeral microbial mat found at Cabo Rojo. A total of 178 clones were isolated from all the libraries. The study was focused on the RBI-3 clone from El Yunque metagenomic library. Transposition mutagenesis was performed with Tn5 transposon in order to generate random mutants that could knock out the activity. Each mutant was sequenced using Sanger sequencing. The clone was fully sequenced using Illumina Mi-Seq platform and assembled by 𝘥𝘦 𝘯𝘰𝘷𝘰 assembly and hybrid Sanger-Illumina template assembly. Automated annotation was performed by the RAST pipeline, and manual annotations were made near the Tn5 insertion sites. Four predicted candidate genes were characterized via phylogenetic protein analysis due to the proximity to the Tn5 insertion site. A predicted sulfite oxidase was used on a 𝘵𝘳𝘢𝘯𝘴 complementation assay, as result the experimental data demonstrated that the activity of this gene was rescued and granting 𝘌. 𝘤𝘰𝘭𝘪 EPI300 the ability to use CML as sole carbon source. These findings could lead to the development of 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 assays and further on being applied to industrial applications to lower the levels of CML on food content or a clinical approach to medical bioremediation.