Continuous hypermutation and evolution of luciferase variants

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Luciferases are crucial for imaging and biosensing, but current optimization methods are inefficient.
• Existing workflows involve laborious, manual generation and screening of mutant libraries.

Purpose of the Study:

• To develop a streamlined platform for luciferase engineering using continuous hypermutation.
• To eliminate the need for manual library generation in iterative optimization cycles.

Main Methods:

• Utilized an orthogonal DNA replication (OrthoRep) system for continuous hypermutation of the GeNL luciferase.
• Implemented short cycles of culturing and screening for enzyme evolution.
• Characterized novel luciferase variants in cell models.

Main Results:

• Identified new GeNL variants with enhanced light output using an inexpensive, non-cognate luciferin.
• Demonstrated the efficiency of the OrthoRep system in evolving luciferases without manual library construction.
• Successfully characterized novel luciferases in cellular contexts.

Conclusions:

• Established OrthoRep and continuous hypermutation as a viable strategy for luciferase engineering.
• The developed platform significantly accelerates the development of bioluminescent reporters.
• This approach facilitates rapid engineering of enzymes for diverse biosensing applications.