Genetically encodable bioluminescent system from fungi.

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.

Identification of hispidin as a bioluminescent active compound and its recycling biosynthesis in the luminous fungal fruiting body.

We previously showed that luminous fungi share a common mechanism in bioluminescence, and identified hispidin as a luciferin precursor in Neonothopanus nambi mycelium. Here we showed the presence of hispidin as a bioluminescent active compound at 25-1000 pmol g-1 in the fruiting bodies of Mycena chlorophos, Omphalotus japonicus, and Neonothopanus gardneri. These results suggest that luminous mushrooms contain hispidin as a luciferin precursor. We also found that non-luminous "young" fruiting bodies exhibited luminescence by hispidin treatment. Furthermore, we observed a gradual luminescence enhancement of the cell-free fruiting body extract by the addition of hispidin biosynthetic components, namely caffeic acid, ATP and malonyl-CoA. These findings suggest that continuous weak glow of luminous mushrooms is regulated by slow recycling biosynthesis of hispidin.

"Lab of the Future"─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics.

Here we describe a state-of-the-art, integrated, multi-instrument automated system designed to execute methods involved in mass spectrometry characterization of biotherapeutics. The system includes liquid and microplate handling robotics and utilities, integrated LC-MS, along with data analysis software, to perform sample purification, preparation, and analysis as a seamless integrated unit. The automated process begins with tip-based purification of target proteins from expression cell-line supernatants, which is initiated once the samples are loaded onto the automated system and the metadata are retrieved from our corporate data aggregation system. Subsequently, the purified protein samples are prepared for MS, including deglycosylation and reduction steps for intact and reduced mass analysis, and proteolytic digestions, desalting, and buffer exchange via centrifugation for peptide map analysis. The prepared samples are then loaded into the LC-MS instrumentation for data acquisition. The acquired raw data are initially stored on a local area network storage system that is monitored by watcher scripts that then upload the raw MS data to a network of cloud-based servers. The raw MS data are processed with the appropriately configured analysis workflows such as database search for peptide mapping or charge deconvolution for undigested proteins. The results are verified and formatted for expert curation directly in the cloud. Finally, the curated results are appended to sample metadata in the corporate data aggregation system to accompany the biotherapeutic cell lines in subsequent processes.

Mechanism and color modulation of fungal bioluminescence.

Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

Circadian control sheds light on fungal bioluminescence.

Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi-only 71 species, all within the ∼ 9,000 fungi of the temperate and tropical Agaricales order-are reported from among ∼ 100,000 described fungal species [6, 7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a byproduct of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature-compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millennia [8-15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin "mushrooms," internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy, where wind flow is greatly reduced.

Current status of research on fungal bioluminescence: biochemistry and prospects for ecotoxicological application.

Over the last half decade the study of fungal bioluminescence has regained momentum since the involvement of enzymes has been confirmed after over 40 years of controversy. Since then our laboratory has worked mainly on further characterizing the substances involved in fungal bioluminescence and its mechanism, as well as the development of an ecotoxicological bioluminescent assay with fungi. Previously, we proved the involvement of a NAD(P)H-dependent reductase and a membrane-bound luciferase in a two-step reaction triggered by addition of NAD(P)H and molecular oxygen to generate green light. The fungal luminescent system is also likely shared across all lineages of bioluminescent fungi based on cross-reaction studies. Moreover, fungal bioluminescence is inhibited by the mycelium exposure to toxicants. The change in light emission under optimal and controlled conditions has been used as endpoint in the development of toxicological bioassays. These bioassays are useful to better understand the interactions and effects of hazardous compounds to terrestrial species and to assist the assessment of soil contaminations by biotic or abiotic sources. In this work, we present an overview of the current state of the study of fungal luminescence and the application of bioluminescent fungi as versatile tool in ecotoxicology.