Bioprospecting Novel Luciferase Genes from Museum Coleoptera

Abstract

Museum collections of Coleoptera contain genetic material of potential interest to biotechnology, and non-destructive DNA extraction enables the preservation of important specimens with concomitant release of mitochondrial and genomic DNA. Mini-barcoding of regions of the mitochondrial cytochrome oxidase subunit I (MT-COI) gene helps identify and eliminate known species from further investigation. Here we identify a novel luciferase gene, using Consensus Degenerate Hybrid Oligonucleotide (CODEHOP) primers targeting the region of the luciferase gene spanning the fourth exon, intron, and fifth exon to detect luciferase gene content and eliminate samples containing known luciferase sequences. Biotinylated luciferase gene probes from the firefly Photinus pyralis enabled the enrichment of potential luciferase gene fragments for next-generation sequencing. A bioinformatic analysis suite was then used to identify a luciferase gene sequence from a previously unidentified firefly originally collected in Costa Rica in 2012. We demonstrate that this newly discovered luciferase, termed CRLuc, catalyses a bioluminescent reaction and we determined its emission spectra, Km for the substrates ATP and D-luciferin, and pH stability.