Genetically encodable bioluminescent system from fungi

Advanced Biology

2020

Mushroom-forming fungi in the order Agaricales represent an independent origin of bioluminescence in the tree of life, yet the diversity, evolutionary history, and timing of the origin of fungal luciferases remain elusive. We sequenced the genomes and transcriptomes of five bonnet mushroom species (Mycena spp.), a diverse lineage comprising the majority of bioluminescent fungi. Two species with haploid genome assemblies ∼150Mb are amongst the largest in Agaricales, and we found that a variety of repeats between Mycena species were differentially mediated by DNA methylation. We show that bioluminescence evolved in the last common ancestor of mycenoid and the marasmioid clade of Agaricales and was maintained through at least 160 million years of evolution. Analyses of synteny across genomes of bioluminescent species resolved how the luciferase cluster was derived by duplication and translocation, frequently rearranged and lost in most Mycena species, but conserved in the Armillaria li...

Journal of Biological Chemistry, 2020

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/l. McH3H was found to be a single component monomeric NAD(P)Hdependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxy-hispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydro-peroxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.

Nature Biotechnology, 2020

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. Bioluminescent reporters have not been broadly applied in plants because exogenous addition of luciferin is expensive and can be toxic. Although bacterial bioluminescence genes can be targeted to plastids to engineer autoluminescence, it is technically cumbersome and fails to produce sufficient light 1. The caffeic acid cycle, which is a metabolic pathway responsible for luminescence in fungi, was recently characterized 2. We report light emission in Nicotiana tabacum and Nicotiana benthamiana plants without the addition of any exogenous substrate by engineering fungal bioluminescence genes into the plant nuclear genome. Caffeic acid is an intermediate in the phenylpropanoid pathway, which produces lignin and other metabolites in vascular plants. We reasoned that it might be feasible to integrate the fungal caffeic acid cycle into plant metabolism. Moreover, the green luminescence produced by the caffeic acid cycle fits well with the optical transparency window of pigmented plant tissues (Fig. 1a). Although caffeic acid is not native to animals, autonomous luminescence could also be enabled in animals by including two additional enzymes needed for its biosynthesis from tyrosine-tyrosine ammonia lyase and coumarate 3-hydroxylase-or their functional equivalents (Fig. 1b and Supplementary Fig. 1) 3. We engineered autonomously glowing N. tabacum plants by random-site genome integration using Agrobacterium-mediated transformation of DNA cassettes comprising codon-optimized versions of four Neonothopanus nambi bioluminescence genes 2 : nnluz (luciferase), nnhisps (hispidin synthase), nnh3h (hispidin-3-hydroxylase) and nncph (caffeoyl pyruvate hydrolase) (Fig. 1, Methods, Supplementary Fig. 2 and Supplementary Note 1).

Scientific Reports, 2021

Using the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the ...

International Journal of Molecular Sciences, 2021

Bioluminescence, the emission of light catalysed by luciferases, has evolved in many taxa from bacteria to vertebrates and is predominant in the marine environment. It is now well established that in animals possessing a nervous system capable of integrating light stimuli, bioluminescence triggers various behavioural responses and plays a role in intra- or interspecific visual communication. The function of light emission in unicellular organisms is less clear and it is currently thought that it has evolved in an ecological framework, to be perceived by visual animals. For example, while it is thought that bioluminescence allows bacteria to be ingested by zooplankton or fish, providing them with favourable conditions for growth and dispersal, the luminous flashes emitted by dinoflagellates may have evolved as an anti-predation system against copepods. In this short review, we re-examine this paradigm in light of recent findings in microorganism photoreception, signal integration and...

Journal of Fungi

During nocturnal field expeditions in the Brazilian Atlantic Rainforest, an unexpected bioluminescent fungus with reduced form was found. Based on morphological data, the taxon was first identified as belonging to the cyphelloid genus Maireina, but in our phylogenetic analyses, Maireina was recovered and confirmed as a paraphyletic group related to genera Merismodes and Cyphellopsis. Maireina filipendula, Ma. monacha, and Ma. subsphaerospora are herein transferred to Merismodes. Based upon morphological and molecular characters, the bioluminescent cyphelloid taxon is described as the new genus Eoscyphella, characterized by a vasiform to urceolate basidiomata, subglobose to broadly ellipsoid basidiospores, being pigmented, weakly to densely encrusted external hyphae, regularly bi-spored basidia, unclamped hyphae, and an absence of both conspicuous long external hairs and hymenial cystidia. Phylogenetic analyses based on ITS rDNA and LSU rDNA support the proposal of the new genus and ...

Proceedings of the National Academy of Sciences of the United States of America, 2020

Journal of Fungi

There is a large number of bioactive polyketides well-known for their anticancer, antibiotic, cholesterol-lowering, and other therapeutic functions, and hispidin is among them. It is a highly abundant secondary plant and fungal metabolite, which is investigated in research devoted to cancer, metabolic syndrome, cardiovascular, neurodegenerative, and viral diseases. This review summarizes over 20 years of hispidin studies of its antioxidant, anti-inflammatory, anti-apoptotic, antiviral, and anti-cancer cell activity.

International Journal of Molecular Sciences

Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fun...