RESUMEN
Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.
Asunto(s)
Hongos/genética , Proteínas Luminiscentes/genética , Secuencia de Aminoácidos , Animales , Vías Biosintéticas/genética , Ácidos Cafeicos , Línea Celular , Línea Celular Tumoral , Femenino , Duplicación de Gen/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Alineación de Secuencia , Xenopus laevisRESUMEN
Odontosyllis undecimdonta is a marine syllid polychaete that produces bright internal and exuded bioluminescence. Despite over fifty years of biochemical investigation into Odontosyllis bioluminescence, the light-emitting small molecule substrate and catalyzing luciferase protein have remained a mystery. Here we describe the discovery of a bioluminescent protein fraction from O. undecimdonta, the identification of the luciferase using peptide and RNA sequencing, and the in vitro reconstruction of the bioluminescence reaction using highly purified O. undecimdonta luciferin and recombinant luciferase. Lastly, we found no identifiably homologous proteins in publicly available datasets. This suggests that the syllid polychaetes contain an evolutionarily unique luciferase among all characterized luminous taxa.
Asunto(s)
Luciferasas/química , Luciferasas/metabolismo , Poliquetos/enzimología , Secuencia de Aminoácidos , Animales , Evolución Molecular , Japón , Luciferasas/genética , Luminiscencia , Poliquetos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants.
Asunto(s)
Luciferina de Luciérnaga/metabolismo , Nicotiana/genética , Plantas Modificadas Genéticamente/metabolismo , Ácidos Cafeicos/metabolismo , Hongos/genética , Hongos/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.