Directed molecular evolution reveals Gaussia luciferase variants with enhanced light output stability

Gaussia Luciferase (Gluc) has proven to be a powerful mammalian cell reporter for monitoring numerous biological processes in immunology, virology, oncology, and neuroscience. Current limitations of Gluc as a reporter include its emission of blue light, which is absorbed by mammalian tissues, limiting its use in vivo, and a flash-type bioluminescence reaction, making it unsuited for high-throughput applications. To overcome these limitations, a library of Gluc variants was generated using directed molecular evolution and screened for relative light output, a shift in emission spectrum, and glow-type light emission kinetics. Several variants with a 10-15 nm shift in their light emission peak were found. Further, a Gluc variant that catalyzes a glow-type bioluminescence reaction, suited for high-throughput applications, was also identified. These results indicate that molecular evolution could be used to modulate Gluc bioluminescence reaction characteristics.