AUTHOR=Jathoul Amit P. , Branchini Bruce R. , Anderson James C. , Murray James A. H. 

TITLE=A higher spectral range of beetle bioluminescence with infraluciferin

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 10 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.897272

DOI=10.3389/fbioe.2022.897272

ISSN=2296-4185

ABSTRACT=Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550nm) and red (ca.600nm) including intermediate colors such as yellow and orange, allowing up-to 3 simultaneous parameters to be resolved in vitro with natural luciferin (D-LH2) . Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH2) ,  . We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH2, including in human cells. iLH2 was designed for dual color far-red to nIR bioluminescence imaging (BLI) in small animals and has been utilized in different mouse models of cancer (including a metastatic hepatic model showing detailed hepatic morphology)2 and for robust dual parameter imaging in vivo (including in systemic hematological models) . Here, we report properties of different enzymes with iLH2: Lampyrid wild-type (WT) Photinus pyralis (Ppy) firefly luciferase, Ppy-based derivatives previously engineered to be thermostable with D-LH2, and also color-shifted Elaterid-based enzymes: blue-shifted Pyrearinus termitilluminans derivative Eluc (D-LH2 ?max = 538nm) and red-shifted Pyrophorus plagiopthalamus derivative Click Beetle Red (CBR) luciferase (D-LH2 ?max = 618nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (?max 536nm) with DL-iLH2 whereas Ppy Fluc (?max = 689nm), x2 Fluc (?max = 704nm), x5 Fluc (?max = 694nm), x11 Fluc (?max = 694nm) and CBR (?max = 721nm) produced far-red to nIR peak wavelengths. Therefore, with iLH2, enzyme ?maxes can be separated by ca.185nm, giving almost non-overlapping spectra. This is the first report of single-substrate bioluminescence color emission ranging from visible green to nIR in cells and may help shed light on the color tuning mechanism of beetle luciferases.  We also report on the reason for the improvement in activity of x11 Fluc with iLH2 and engineer an improved infraluciferase (iluc) based on this mutant.