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=<p>Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550 nm) and red (ca.600 nm), including intermediate colors such as yellow and orange, allowing up to 3 simultaneous parameters to be resolved <italic>in vitro</italic> with natural luciferin (<italic>D</italic>-LH<sub>2</sub>). Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH<sub>2</sub>). We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH<sub>2,</sub> including in human cells. iLH<sub>2</sub> 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) and for robust dual parameter imaging <italic>in vivo</italic> (including in systemic hematological models). Here, we report the properties of different enzymes with iLH<sub>2</sub>: Lampyrid wild-type (WT) <italic>Photinus pyralis</italic> (<italic>Ppy</italic>) firefly luciferase, <italic>Ppy</italic>-based derivatives previously engineered to be thermostable with <italic>D</italic>-LH<sub>2</sub>, and also color-shifted Elaterid-based enzymes: blue-shifted <italic>Pyrearinus termitilluminans</italic> derivative Eluc (reported D-LH<sub>2</sub> λmax = 538 nm) and red-shifted <italic>Pyrophorus plagiopthalamus</italic> derivative click beetle red (CBR) luciferase (<italic>D</italic>-LH<sub>2</sub> λmax = 618 nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (λmax = 536 nm) with <italic>DL</italic>-iLH<sub>2</sub> whereas <italic>Ppy</italic> Fluc (λmax = 689 nm), x2 Fluc (λmax = 704 nm), x5 Fluc (λmax = 694 nm), x11 Fluc (λmax = 694 nm) and CBR (λmax = 721 nm) produced far-red to nIR peak wavelengths. Therefore, with iLH<sub>2,</sub> enzyme λmaxes 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 iLH<sub>2</sub> and engineer an improved infraluciferase (iluc) based on this mutant.</p>