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

Halliwell, Lisa M., Jathoul, Amit P. ORCID: https://orcid.org/0000-0002-6767-7775, Bate, Jack P., Worthy, Harley L., Anderson, James C., Jones, D. Dafydd ORCID: https://orcid.org/0000-0001-7709-3995 and Murray, James Augustus Henry ORCID: https://orcid.org/0000-0002-2282-3839 2018. ΔFlucs: brighter photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant. Biotechnology and Bioengineering 115 (1) , pp. 50-59. 10.1002/bit.26451

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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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Advanced Research Computing @ Cardiff (ARCCA) Biosciences
Publisher:	Wiley
ISSN:	0006-3592
Date of First Compliant Deposit:	21 September 2017
Date of Acceptance:	11 September 2017
Last Modified:	08 Nov 2024 20:30
URI:	https://orca.cardiff.ac.uk/id/eprint/104917

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