We'll make anyone glow
Russian biologists have deciphered the genetic "secret" of glowing mushrooms
Daria Spasskaya, N+1
Russian biologists have identified all the genes responsible for the bioluminescence of the glowing mushroom. Recreating the synthesis pathways of the necessary components – luciferase and luciferin – in yeast cells caused them to emit light visible to the naked eye. In addition, the authors of an article in the Proceedings of the National Academy of Sciences (Kotlobay et al., Genetically encoded bioluminescent system from fungi) showed that a new luciferase from a fungus works perfectly as a reporter protein in bacteria, frog embryos and tumor cells when a substrate is added to the medium.
Many species of living organisms are able to emit visible light due to bioluminescence. They are allowed to glow by the enzyme luciferase, which oxidizes the substrate luciferin. These main components can be different in structure – so, in total, about 40 bioluminescent systems are known, including seven different families of luciferases. However, a complete description of the luminescence system, that is, the identification of the genes encoding luciferase and the pathways of luciferin synthesis, the determination of the structure of these components, was done only for bacteria.
Luciferase is actively used in biotechnology as a reporter protein, since its glow is convenient to detect. Most often, firefly luciferase is used for this. However, in these cases, the substrate, that is, luciferin, must be added from the outside every time.
Scientists from The Institute of Bioorganic Chemistry of the Russian Academy of Sciences, under the leadership of Ilya Yampolsky, is studying bioluminescence systems that could be recreated in model organisms and make them glow independently without adding a substrate (for example, among the authors of the article are the founders of the company Planta, which is engaged in the cultivation of genetically engineered luminous plants). Bacterial systems are not suitable for this.
"Bacteria are prokaryotes, not eukaryotes, so attempts to "stuff" the whole prokaryotic system into the eukaryotic system did not work. In order to sew a bacterial system into a eukaryotic being, a plant, you need to change a lot of things so that they learn how to synthesize the necessary enzymes and proteins. In experiments, a ready–made label is injected, and it lights up when necessary," explains N+1 Egor Zadereev, whose colleagues from the Institute of Biophysics of the Krasnoyarsk Scientific Center SB RAS participated in the study.
Two years ago, scientists managed to decipher the chemical structure of the components of the luciferin synthesis pathway from the Vietnamese glowing mushroom Neonotopanus nambi and establish that mushroom luciferin is 3-hydroxygispidin, which after several intermediate stages is formed from caffeic acid, a common metabolite of plants. Nevertheless, in order to recreate the synthesis pathway in other organisms, it was necessary to identify the genes encoding synthesis enzymes and the mushroom luciferase itself.
To solve the last problem, a library of all genes Neonotopanus nambi was expressed in yeast, and luciferin was sprayed on the grown colonies. DNA was isolated from the glowing colonies and the sequence of the mushroom gene responsible for the glow was determined. It turned out that mushroom luciferase is encoded by the nnLuz gene and is not similar to other luciferases, that is, it represents a new family.
The researchers also completely sequenced the genome Neonotopanus nambi and looked at which genes are located next to nnLuz. Among the luciferase neighbors, they found two genes presumably encoding enzymes for the biosynthesis of 3-hydroxygispidin from caffeic acid. When these genes, together with the luciferase gene and the gene of another already known enzyme, were expressed in yeast, such yeast turned out to be able to glow in the dark (provided that caffeic acid was added to the medium, since yeast itself does not synthesize it). At the next stage, three genes for the synthesis of caffeic acid from tyrosine were additionally embedded in the modified yeast, as a result of which they were already able to glow independently, without the addition of substrates.
"All plants have caffeic acid, it is one of the intermediate products of wood biosynthesis. Now, to obtain luminous plants, only one step is required – you need to "branch" caffeic acid to hispidin, and it to luciferin, and also make a system with luciferase so that luciferin and luciferase meet and the plant lights up. This is a much closer and understandable alteration, not all the metabolism needs to be changed in the plant, it is not necessary to turn the plant into a bacterium," says Zadereev.
The glow of fungal luciferase in yeast (A), in human cells (B), in a mouse tumor (C), in a frog embryo (D). From an article in PNAS.
To test whether fungal luciferase can be used as a reporter protein in other cells, the researchers tested its work in bacteria, spur frog embryos and human cells. In addition, it was compared with the firefly luciferase already used in biology for its ability to "mark" tumor cells in the mouse body, and it was found out that the mushroom luciferase works no worse. Thus, scientists not only revealed the genetic basis of fungal bioluminescence, but also showed the applicability of the found system in biotechnology and biomedicine.
The study of fungal bioluminescence began at the Krasnoyarsk Institute of Biophysics SB RAS with the participation of Nobel laureate Osamu Shimomura, who discovered the green fluorescent protein of jellyfish. It was already possible to bring the work to its logical conclusion under the leadership of Yampolsky at the IBH RAS. Scientists from Austria, Spain, Brazil, England and Japan also took part in the work.
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