ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant

Abstract

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an "omega-loop" motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin , altered emission colors, and altered substrate specificity for redshifted analog DL-infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω-loop impacts on active site hydrophobicity to increase light yields.This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox. K E Y W O R D Sbioimaging, bioluminescence, bioreporter, deletion mutagenesis, firefly luci ferase, infraluciferi

Portable light detectors for bioluminescence biosensing applications: A comprehensive review from the analytical chemist's perspective

“…N-terminal domain of FLuc and the C-terminal domain of Luciola italica luciferase to further improve thermal and pH stability with threefold higher activity than the original FLuc [20][21].…”

Development and Applications of Bioluminescent and Chemiluminescent Reporters and Biosensors

“…FLuc was further codon-optimized for mammalian expression (e.g., the luc2 gene). Recently, consecutive single amino acid deletion mutants of FLuc, ΔFlucs, have been reported for higher activities and lower K M toward D-luciferin (9). To further improve the thermal and pH stability of FLuc, a chimeric luciferase was developed by fusing the Nterminal domain of FLuc and the C-terminal domain of Luciola italica luciferase, thereby resulting in a codon-optimized PLG2 that shows ~threefold higher activity than the original FLuc (10).…”

Lessons Learned from Luminous Luciferins and Latent Luciferases

“…, through rotational restriction). We anticipate that many of these features can be tuned by mutagenesis to further broaden the scope and selectivity of luminogenic substrates. ,, Furthermore, both emissive and nonemissive analogues have value for the design of turn-ON or turn-OFF luminescent sensors, and the increasing variety of luminogenic scaffolds offer new strategies for their construction. , One can thus expect many more luciferin analogues and luciferin-based probes to be reported in the coming years, with useful properties for selective imaging in live cells, tissues, and animals.…”