Super RLuc8: A novel engineered Renilla luciferase with a red-shifted spectrum and stable light emission

• A novel engineered Renilla luciferase, super RLuc8 namely, was developed by directed evolution.
• Super RLuc8 has a red-shifted spectrum and shows stable light emission.
• The relationship between the protein structure, stability and emission spectrum has been studied in super RLuc8 using MD simulations.

Renilla luciferase is a bioluminescent enzyme which is broadly used as a reporter protein in molecular biosensors. In this study, a novel luciferase with desired light emission wavelength and thermostability is reported. The results indicated that the new luciferase, namely super RLuc8, had a red-shifted spectrum and showed stable light emission. Super RLuc8 showed a 10-fold (p-value = 0.0084) increase in the thermostability at 37 °C after 20 min incubation, in comparison to the native enzyme. The optimum temperature of the mutant increased from 30 to 37 °C. Molecular dynamics simulation analysis indicated that the increased thermostability was most probably caused by a better structural compactness and more local rigidity in the regions out of the emitter site.