Bioluminescence is a unique feature of living organisms to emit light. It is based on the biochemical oxidation reaction of the luciferin catalyzed by the enzyme luciferase. There are about 40 different bioluminescent systems known to date, but the chemical nature of light has been studied only for some of them. The bioluminescent systems of firefly (McElroy 1947), click beetles (Yu and Liu 2020), bacteria (Brodl, Winkler, and Macheroux 2018), marine organisms (O. Shimomura and Johnson
1975), dinoflagellates (Dunlap, Hastings, and Shimomura 1980), etc. were most thoroughly studied. It is important to note that bioluminescent systems have become the basis of many bioimaging instruments in both prokaryotic and eukaryotic organisms. Compared, for example, to fluorescence approaches, bioluminescence-based methods have several advantages: external light excitation is not required, the sample is not heated during the analysis, light scattering and autofluorescence are absent. In addition, small amounts of both luciferin and luciferase are required for analysis. Luminescent approaches are very sensitive, provide good spatial resolution, wide dynamic range and simple signal quantification.

Thus, luminescence-based reporters are widely used in both basic and applied research. Luciferin-luciferase systems are used as reporters for studying gene expression, post-translational modifications, protein distribution in tissues (using tissue-specific promoters), protein-protein interactions, etc. (Badr and Tannous 2011). Applied bioluminescence methods include quantitative metabolite analysis (Turman and Mathews 1996), high-throughput drug screening (Che et al. 2012), diagnosis of
autoimmune diseases (Burbelo, Lebovitz, and Notkins 2015), infectious diseases (Van Reet et al. 2014) and cancer (Andruska et al. 2012). Additionally, there is an approach of the multicolor luciferase system consisting of three different luciferases catalyzing oxidation reactions of the same substrate, accompanied by green, orange, and red light emission (Branchini et al. 2018; Nakajima et al. 2005). This method allows to analyze the expression of several genes at the same time and compare
the transcriptional activities in one population of cells or tissues.

However, despite the fact that luminescence-based approaches have been widely used in basic and applied research, they still have several disadvantages. To catalyze the reaction, all known luciferases require exogenous administration of the luciferin substrate (except for the bacterial bioluminescent system), which makes it impossible to conduct non-invasive imaging in vivo. Biosensors, based on bacterial bioluminescent system, are mainly used for applications in bacteria due to the toxicity of the luciferin to eukaryotic cells, even though bacterial bioluminescence can be genetically encoded and has been optimized for expression in eukaryotic cells.

At the same time, the bioluminescent system of fungi N. nambi, which was studied in our laboratory, is currently the only genetically encoded system suitable for use in eukaryotic organisms (Kotlobay et al. 2018). Today it is obvious that further study of the biosynthetic pathways of luciferins from other eukaryotic organisms is necessary in order to develop the possibilities of implementing a variety of new visualization tools, even more advanced. Among other goals, this will also ensure the transition to multi-color imaging for simultaneous tracking of several events or molecules in the studied cells, tissues or whole organisms.

Therefore, the study of the bioluminescent system of another eukaryotic organism — polychaeta Odontosyllis undecimdonta — seems to be a very promising and fundamental task. This system has a very bright green-blue luminescence, and its study began back in 1952 (Harvey, n.d.). It turned out that Odontosyllis luminescence is very different from previously studied in terms of the structure of the main components and their properties (Mitani et al. 2018). In 2019 our laboratory managed to
elucidate the structure of Odontosyllis luciferin, which is an unusual tricyclic sulfur-containing heterocycle (Kotlobay et al. 2019). In addition, based on the data obtained, a biochemical pathway for the enzymatic and non-specific oxidation of the luciferin was proposed. However, the data on the biosynthetic precursors of the luciferin and the enzymes involved in its biosynthesis are still missing.

Thus, the goal of this project is to establish the structures of intermediates for O. undecimdonta luciferin biosynthetic pathway and to identify the enzymes involved in this pathway, to develop and optimize the chemical synthesis of Odontosyllis luciferin in order to create a basis for the future development of new luminescent analytical systems for high-throughput screening and optical control. The novelty of the expected results of the project will allow the expansion of the possibilities of non-invasive luminescence imaging and ensure the development of new domestic analytical methods for biomedicine.