Superluminescent Variants of Gaussia Luciferase in Living Animal Deep-tissue Imaging

Gaussia luciferase (Gluc) is widely recognized as a powerful bioluminescent tool for biosensing and molecular imaging. However, its utility in mammalian deep tissue imaging and high-throughput applications is hampered by two primary limitations: the emission of blue light and the transient nature of its bioluminescence reaction. In this study, we have engineered two novel luciferases, BeM and SuM, by incorporating known amino acid mutations that have demonstrated the ability to stabilize, enhance, or shift the wavelength of Gluc-catalyzed luminescence towards the red spectrum. The BeM and SuM-coelenterazine systems exhibited a more than 100-fold increase in bioluminescence intensity in vitro, and over a 10-fold enhancement in vivo, compared to the conventional firefly luciferase-D-luciferin system. This significant improvement enables the noninvasive deep tissue imaging of a small number of cells within living mice. BeM and SuM emerge as the most efficient Gaussia luciferase variants to date, showcasing their immense potential as bioengineered light sources. Their applications could span various fields, ranging from fundamental scientific research to clinical diagnostics and therapeutic interventions, marking a significant leap forward in bioluminescence technology.