Genetically encodable bioluminescent system from fungi

Kotlobay, Alexey A.; Sarkisyan, Karen S.; Mokrushina, Yuliana A.; Marcet‐Houben, Marina; Serebrovskaya, Ekaterina O.; Markina, Nadezhda M.; Somermeyer, Louisa Gonzalez; Gorokhovatsky, Andrey Yu.; Vvedensky, A. V.; Пуртов, К. В.; Petushkov, Valentin N.; Родионова, Н. С.; Chepurnyh, Tatiana V.; Fakhranurova, Liliia I.; Guglya, Elena B.; Ziganshin, Rustam H.; Tsarkova, Aleksandra S.; Kaskova, Zinaida M.; Shender, Victoria О.; Abakumov, Maxim A.; Abakumova, Tatiana O.; Povolotskaya, Inna; Eroshkin, Fedor M.; Zaraisky, Andrey G.; Mishin, Alexander S.; Dolgov, Sergey; Mitiouchkina, Tatiana; Kopantzev, E. P.; Waldenmaier, Hans Eugene; Oliveira, Adriana Gonçalves; Oba, Yuichi; Barsova, Ekaterina V.; Богданова, Е. А.; Gabaldón, Toni; Stevani, Cassius V.; Lukyanov, Sergey; Смирнов, Иван; Gitelson, J. I.; Kondrashov, Fyodor A.; Yampolsky, Ilia V.

doi:10.1073/pnas.1803615115

Cited by 139 publications

(147 citation statements)

References 28 publications

(28 reference statements)

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“…To test if the fungal bioluminescence pathway described by Kotlobay and Sarkisyan et al 12 would function in planta, we attempted to transiently reconstitute this pathway in the leaves of Nicotiana benthamiana. We chose to use N. benthamiana because Agrobacterium infiltration provides an easy way to transiently express genes.…”

Section: Reconstitution Of the Fungal Bioluminescence Pathway In Planmentioning

confidence: 99%

“…While auto-luminescence was achieved, this approach is extremely challenging to extend to other plants due to the technical challenges of transforming plastids. In their 2018 paper, Kotlobay and Sarkisyan et al identify a fungal bioluminescence pathway (FBP) consisting of a set of three genes that are sufficient to convert caffeic acid, a common plant metabolite 12 , into a luciferin molecule and a paired luciferase, Luz, that can oxidize it to create a bioluminescent signal 12 . This pathway can be nuclear encoded, as it is derived from a eukaryotic fungus, overcoming the challenges associated with plastid transformation.…”

Section: Introductionmentioning

confidence: 99%

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Building customizable auto-luminescent luciferase-based reporters in plants

Khakhar

Starker

Chamness

et al. 2019

Preprint

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Bioluminescence is a powerful biological signal that scientists have repurposed to design reporters for gene expression in plants and animals. However, there are some downsides associated with the need to provide a substrate to these reporters, such as its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using an easily composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for the future construction of programmable auto-luminescent plant traits, such as creating light driven plant-pollinator interactions or light emitting plant-based sensors.

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Section: Reconstitution Of the Fungal Bioluminescence Pathway In Planmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Building customizable auto-luminescent luciferase-based reporters in plants

Khakhar

Starker

Chamness

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…In addition to the luciferase, several other enzymes participate in the process of bioluminescence by synthesizing the luciferin from cellular metabolites and by converting it back into its reduced form. For most bioluminescence systems, these proteins have not fully been identified so far, with the exception of the bacterial (1)(2)(3)(4)(5) and the recently solved fungal system (6). Knowledge of all enzymes participating in the reaction cascade means that they can in principle be heterologously expressed in any cell type in order to produce autonomous bioluminescence for imaging applications.…”

Section: Abstract Bioluminescence | Luciferase | Lux | Microscopymentioning

confidence: 99%

“…One obstacle for this is that fungal luciferase loses its activity at temperatures above 30 °C (6). The bacterial bioluminescence system has been implemented in mammalian cells (11,12), but the resulting bioluminescence is much dimmer than that produced by other luciferases which require an external luciferin (13).…”

Section: Abstract Bioluminescence | Luciferase | Lux | Microscopymentioning

confidence: 99%

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

Gregor

Pape

Gwosch

et al. 2019

Preprint

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Bioluminescence based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has so far not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells. Significance statementBioluminescence is generated by luciferases that oxidize a specific luciferin. The enzymes involved in the synthesis of the luciferin from widespread cellular metabolites have so far been identified for only two bioluminescence systems, those of bacteria and fungi. In these cases, the complete reaction cascade is genetically encodable, meaning that heterologous expression of the corresponding genes can potentially produce autonomous bioluminescence in cell types other than the bacterial or fungal host cells. However, the light levels achieved in mammalian cells so far are not sufficient for single-cell applications. Here we present, for the first time, autonomous bioluminescence images of single mammalian cells by coexpression of the genes encoding the six enzymes from the bacterial bioluminescence system.

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“…In a tour-de-force, Yampolsky et. al discovered a set of genes required for expressing both the fungal luciferase and luciferin [37]. However, the fungal bioluminescence system requires a total of seven genes assembled from multiple organisms and it can be challenging to express efficiently in eukaryotic systems.…”

Section: Introductionmentioning

confidence: 99%

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

Srinivasan

Griffin

Joshi

et al. 2019

Preprint

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1.AbstractGenetically encoded reporters have greatly increased our understanding of biology, especially in neuroscience. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach we have engineered Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential. Unlike other luciferase-luciferin bioluminescent systems, AMBER encodes the genes to express both the luciferase and luciferin. AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When AMBER is co-expressed with the luciferin producing genes it reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods we show that AMBER modulates its enzymatic activity as a function of the membrane potential. AMBER shows several-fold increase in the luminescent (ΔL/L) signal upon switching from the off to on state when the cell is depolarized. In vivo expression of AMBER in C. elegans allowed detecting pharyngeal pumping action and mechanosensory neural activity from multiple worms simultaneously. AMBER reports neural activity of multiple animals at the same time and can be used in social behavior assays to elucidate the role of membrane potential underlying behavior.2.Significance StatementThere have been many exciting advances in the development of genetically encoded voltage indicators to monitor intracelluar voltage changes. Most sensors employ fluorescence, which requires external light, potentially causing photobleaching or overheating. Consequently, there has been interest in developing luminescence reporters. However, they require addition of an exogenous substrate to produce light intracellularly. Here, we engineered a genetically encoded bioluminescent voltage indicator, AMBER, which unlike other bioluminescent activity indicators, does not require addition of an exogenous substrate. AMBER allows a large differential signal, a high signal-to-noise ratio, and causes minimal metabolic demand on cells. We used AMBER to record voltage activity in freely-moving C. elegans, demonstrating that AMBER is a important new tool for monitoring neuronal activity during social behavior.

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Genetically encodable bioluminescent system from fungi

Cited by 139 publications

References 28 publications

Building customizable auto-luminescent luciferase-based reporters in plants

Building customizable auto-luminescent luciferase-based reporters in plants

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

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