ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant. Issue 1 (23rd October 2017)

• Record Type: Journal Article
• Authors: Halliwell, Lisa M.; Jathoul, Amit P.; Bate, Jack P.; Worthy, Harley L.; Anderson, James C.; Jones, D. Dafydd; Murray, James A.H.
• Is Part Of: Biotechnology and bioengineering. Volume 115:Issue 1(2018)
• Page Start: 50
• Page End: 59
• Publication Date: 2017-10-23
• Subjects: bioimaging -- bioluminescence -- bioreporter -- deletion mutagenesis -- firefly luci ferase -- infraluciferi; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26451
• Languages: English
• ISSNs: 0006-3592

Abstract

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. Abstract : Halliwell et al identify brighter firefly luciferase mutants in a thermostable Photinus pyralis background (×11 Fluc) by surveying single amino acid deletion mutations in solvent‐exposed loops. They find that deletions are largely tolerated in terms of activity throughout the structure, but deletions in a surface‐based omega loop lead to two‐ to threefold increases in activity. These variants display high activity at 37°C and could be applied as bioreporters in bioluminescence imaging with D‐luciferin or DL‐infraluciferin.