An improved pathway for autonomous bioluminescence imaging in eukaryotes.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• The bioluminescence pathway from Neonothopanus nambi fungus allows for self-sustained light emission in engineered eukaryotes.
• Native fungal enzymes in heterologous hosts exhibit limited performance, restricting luminescence brightness.

Purpose of the Study:

• To engineer optimized versions of the fungal bioluminescence pathway.
• To significantly enhance bioluminescence intensity in diverse eukaryotic hosts.
• To enable advanced imaging applications, including longitudinal video-rate imaging.

Main Methods:

• Enzyme engineering and directed evolution of key components of the Neonothopanus nambi bioluminescence pathway.
• Heterologous expression and characterization of optimized pathways in plant, fungal, and mammalian cell systems.
• Assessment of luminescence output and imaging capabilities using advanced microscopy techniques.

Main Results:

• Optimized pathway variants demonstrated a one-to-two order of magnitude increase in bioluminescence brightness compared to native enzymes.
• Sustained and bright luminescence was achieved across plant, fungal, and mammalian host systems.
• The enhanced bioluminescence enabled high-resolution, longitudinal video-rate imaging of biological processes.

Conclusions:

• Engineered bioluminescence pathways offer a substantial improvement in light output for various eukaryotic hosts.
• This advancement provides a powerful tool for non-invasive, real-time biological imaging and monitoring.
• The optimized system has broad applications in research, diagnostics, and biotechnology.