Enzymes of luciferin biosynthesis and use thereof

Abstract

The invention is directed to new enzymes of fungal luciferin biosynthesis, the nucleic acids that encode them, and the use of proteins as enzymes catalyzing the stages of fungal luciferin biosynthesis. The invention also provides the use of nucleic acids to obtain said enzymes in a cell or an organism. Methods are also provided for obtaining chemical compounds that are fungal luciferins and preluciferins in in vitro and in vivo systems. Vectors are also provided which comprise the nucleic acid described in the present invention. Furthermore, this invention provides expression cassettes comprising the nucleic acid of the present invention and regulatory elements needed for the expression of the nucleic acid in the chosen host cell. Furthermore, cells, stable cell lines and transgenic organisms (for example, plants, animals, fungi, microorganisms) comprising the nucleic acids, vectors, or expression cassettes of the present invention are provided. Combinations of nucleic acids are also provided for obtaining autonomously fluorescing cells, cell lines or transgenic organisms. In preferred embodiments, cells and transgenic organisms are capable of producing fungal luciferin from precursors. In some embodiments, cells and transgenic organisms are capable of producing fungal preluciferin from precursors. In some embodiments, cells and transgenic organisms are capable of bioluminescence in the presence of a fungal luciferin precursor. In some embodiments, cells and transgenic organisms are capable of autonomous bioluminescence. Combinations of proteins are also provided for obtaining luciferin and its precursors from simpler chemical compounds. A set is also provided which comprises nucleic acids or vectors or expression cassettes of the present invention and which is intended for receiving luminescent cells, cell lines or transgenic organisms.

Description

[0001] field of invention [0002] The invention relates to the fields of biotechnology and genetic engineering. In particular, the invention relates to enzymes of fungal bioluminescence systems. [0003] Background of the invention [0004] Enzymes that can catalyze the oxidation of low molecular weight compounds of luciferin with concomitant luminescence or bioluminescence are known as "luciferases". Oxidation of luciferin results in the release of oxidized luciferin from its complex with luciferase. [0005] Luciferase is widely used as a reporter gene in many biomedical applications and biotechnology. For example, in carcinogenicity studies in animal models, in methods for the detection of microorganisms or toxic substances in media, luciferase is used to determine the viability of cells and the activity of promoters or other components of living systems, as Indicators of substance concentrations to visualize transmission of signaling cascades, etc. [Scott et al., Annua...

AI Technical Summary

Problems solved by technology

However, the low level of bioluminescent intensity, which is only 12 times higher than the signal emitted by non-bioluminescent cells, precludes the use of systems developed to solve most application problems [Close et al., PloS One, 2010, 5(8) :e12441)

Attempts to increase the intensity of emitted light were unsuccessful due to the toxicity of components of the bacterial system to eukaryotic cells [Hollis et al. EFBS Letters, 2001, 506(2): 140-42]

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